Leonardo Dos Santos Corrêa Amorim Avaliação De

UNIVERSIDADE FEDERAL FLUMINENSE INSTITUTO DE BIOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS E BIOTECNOLOGIA

LEONARDO DOS SANTOS CORRÊA AMORIM

AVALIAÇÃO DE SUBSTÂNCIAS NATURAIS EXTRAÍDAS DAS ALGAS MARINHAS Dictyota friabilis E Osmundaria obtusiloba E SUBSTÂNCIAS SINTÉTICAS DERIVADOS DE NAFTOQUINONAS E QUINOLINAS FRENTE AOS VÍRUS DE IMPORTÂNCIA CLÍNICA (HIV-1, ZIKV, HSV-1 e MAYV).

Tese de Doutorado submetida à Universidade Federal Fluminense como requisito parcial visando à obtenção do grau de Doutor em Ciências e Biotecnologia.

Orientador(es): Izabel Christina Nunes de Palmer Paixão

Claudio Cesar Cirne dos Santos

NITERÓI

2020

LEONARDO DOS SANTOS CORRÊA AMORIM

Trabalho desenvolvido no Laboratório de Virologia Molecular do Departamento de Biologia Celular e Molecular do Instituto de Biologia, Programa de Pós- Graduação em Ciências e Biotecnologia, Universidade Federal Fluminense. Apoio Financeiro: CAPES, CNPq, FAPERJ, UFF-FOPESQ.

Tese de Doutorado submetida à Universidade Federal Fluminense como requisito parcial visando à obtenção do grau de Doutor em Ciências e Biotecnologia.

Orientador(es): Izabel Christina Nunes de Palmer Paixão

Claudio Cesar Cirne dos Santos

Ficha catalográfica automática - SDC/BCV Gerada com informações fornecidas pelo autor

C824a Corrêa amorim, Leonardo dos Santos AVALIAÇÃO DE SUBSTÂNCIAS NATURAIS EXTRAÍDAS DAS ALGAS MARINHAS Dictyota friabilis E Osmundaria obtusiloba E SUBSTÂNCIAS SINTÉTICAS DERIVADOS DE NAFTOQUINONAS E QUINOLINAS FRENTE AOS VÍRUS DE IMPORTÂNCIA CLÍNICA (HIV-1, ZIKV, HSV-1 e MAYV). / Leonardo dos Santos Corrêa amorim ; Izabel Christina Nunes de Palmer Paixão, orientadora ; Cláudio César Cirne dos Santos, coorientadora. Niterói, 2020. 157 f. : il.

Tese (doutorado)-Universidade Federal Fluminense, Niterói, 2020.

DOI: http://dx.doi.org/10.22409/PPBI.2020.d.11344133789

1. Antivirais. 2. Vírus de Importância Clínica. 3. Substâncias Naturais. 4. Substâncias Sintéticas. 5. Produção intelectual. I. Paixão, Izabel Christina Nunes de Palmer, orientadora. II. Cirne dos Santos, Cláudio César, coorientadora. III. Universidade Federal Fluminense. Instituto de Biologia. IV. Título.

CDD -

Bibliotecário responsável: Sandra Lopes Coelho - CRB7/3389 LEONARDO DOS SANTOS CORRÊA AMORIM

Tese de Doutorado submetida à Universidade Federal Fluminense como requisito parcial visando à obtenção do grau de Doutor em Ciências e Biotecnologia.

Banca Examinadora

______Viveca Antonia Giongo Galvão da Silva – Universidade Estácio de Sá ______Juliana Eymara Fernandes Barbosa Paula – Hospital Universitário Gafree Guinle – UNIRIO. ______Ana Maria Viana Pinto – Instituto Biomédico – UFF ______Patrícia Burth – Instituto de Biologia – UFF. ______Helena Carla Castro – Instituto de Biologia – UFF. ______Juliana Silva Novais – Instituto de Biologia – UFF. ______Rosa Teixeira de Pinho – Instituto Oswaldo Cruz – Fiocruz. ______Izabel Christina Nunes de Palmer Paixão – Instituto de Biologia – UFF. ______Claudio Cesar Cirne dos Santos – Instituto de Biologia – UFF.

III

Dedico este meu trabalho aos meus avós, pais e irmão que sempre fizeram de tudo para que eu realizasse o meu sonho, aos meus orientadores a todos os amigos, por me ajudarem a torná-lo possível.

AGRADECIMENTOS Acho justo começar este agradecimento citando um trecho de uma música da qual raramente ouço, porém, nunca se fez tão literal em minha vida. “Você não sabe o quanto eu caminhei, para chegar até aqui...”, nenhuma frase poderia exprimir melhor minha trajetória.

Agradeço à Deus, e a toda e qualquer força astral superior que possa ter me auxiliado de alguma forma a atingir meus objetivos.

A esta universidade, sеu corpo docente, direção е administração que oportunizaram а janela que hoje vislumbro um horizonte superior, eivado pela acendrada confiança no mérito е ética aqui presentes.

Agradeço а todos os professores por me proporcionar о conhecimento não apenas racional, mas а manifestação do caráter е afetividade da educação no processo de formação profissional, por tanto que se dedicaram а mim, não somente por terem me ensinado, mas por terem me feito aprender. А palavra mestre, nunca fará justiça aos professores dedicados aos quais sem nominar terão os meus eternos agradecimentos.

A minha orientadora, pelo emprenho dedicado à elaboração deste trabalho. Aos momentos dispensados a mim, seja para uma conversa científica ou simplesmente pelos momentos de amizades, ambos sempre muito gratificantes e construtivos.

Agradeço aos meus pais e irmão por todo amor, incentivo e apoio incondicional! Se me tornei o homem que sou hoje, com certeza foi pelo esforço dedicado a mim desde sempre. Por não medirem esforços e sempre me incentivarem a continuar lutando por meus sonhos e objetivos, mesmo e principalmente quando eu tinha vontade de desistir.

Agradeço também aos meus familiares que sempre confiaram em minha capacidade e sempre proferiram palavras de incentivo e orgulho.

Aos meus amigos, serei eternamente grato! Pois sempre estiveram ao meu lado e me ajudaram a superar as adversidades mesmo não havendo obrigação. Fizeram-se presentes em momentos únicos da minha trajetória e

V mostraram, que a amigos de verdade são raros, mas quando encontrado, são extremamente valiosos!

Agradeço também aos meus amigos de trabalho, que se fizeram importantes em várias etapas da minha vida acadêmica, me auxiliando em análises, experimentos e na manutenção da sanidade mental que muitas vezes perdemos entre uma tarefa e outra.

Para terminar, parafraseio um dos maiores educadores e pensadores brasileiros: “...para mim, é impossível existir sem sonho. A vida na sua totalidade me ensinou como grande lição que é impossível assumí-la sem risco.” (Paulo Freire).

O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Código de Financiamento 001.

SUMÁRIO

AGRADECIMENTOS ...... V SUMÁRIO ...... VII LISTA DE ABREVIATURAS ...... IX LISTA DE ILUSTRAÇÕES ...... XII LISTA DE TABELA ...... XIII RESUMO...... XIV ABSTRACT ...... XV 1. ANTIVIRAIS ...... 1 1.1. HISTÓRICO ...... 1 1.1.1. ANÁLOGOS DE NUCLEOSÍDEO ...... 2 1.1.2. ANÁLOGOS DE PIROFOSFATO ...... 5 1.1.3. INIBIDORES NUCLEOSÍDEOS DA TRANSCRIPTASE RESERVA 6 1.1.4. INIBIDORES NÃO NUCLEOSÍDEOS DA TRANSCRIPTASE REVERSA ...... 7 1.1.5. INIBIDORES DE PROTEASE ...... 7 1.1.6. INIBIDORES DE INTEGRASE ...... 9 1.1.7. INIBIDORES DE ENTRADA ...... 10 1.1.8. INIBIDORES ANÁLOGOS DA GUANOSINA ACÍCLICOS ...... 13 1.1.9. INIBIDORES ANÁLOGOS FOSFANATOS DE NUCLEOSÍDEO ACÍCLICO ...... 15 1.1.10. INIBIDORES DO NS5A/NS5B DO HCV ...... 18 1.1.11. INIBIDORES DOS VÍRUS INFLUENZA ...... 20 1.1.12. INIBIDORES A BASE DE INTERFERONS, IMUNOESTIMULADORES, OLIGONUCLEOTÍDEOS E ANTIMITÓTICOS. 23 1.2. IMPORTÂNCIA BIOTECNOLÓGICA ...... 25 1.3. A IMPORTÂNCIA DA ESTRUTURA E REPLICAÇÃO VIRAL PARA OS ANTIVIRAIS...... 28 2. DISCUSSÃO ...... 36 ATIVIDADE ANTIVIRAL DAS ALGAS MARINHAS Dictyota friabilis E Osmundaria obtusiloba...... 36 Diterpene From Marine Brown Alga Dictyota friabilis As A Potential Microbicide Against Hiv-1 In Tissue Explants ...... 36

VII

Antiviral Effect Of The Seaweed Osmundaria obtusiloba Against The Zika Vírus ...... 38 ATIVIDADE ANTIVIRAL DOS DERIVADOS AMINONAFTOQUINONAS E HIDROXOQUINOLINOS...... 40 Aminomethylnaphthoquinones And Hsv-1 - In Vitro And In Silico Evaluations Of Potential Antivirals ...... 40 Synthesis of 4-Oxoquinoline Acyclonucleoside Phosphonate Analogs And Anti-MAYV Evaluation...... 44 3. CONCLUSÃO ...... 47 4. REFERÊNCIAS BIBLIOGRÁFICAS ...... 49 5. APÊNDICE ...... 67 5.1. ARTIGOS ...... 67 5.1.1. DITERPENE FROM MARINE BROWN ALGAE Dictyota friabilis AS A POTENTIAL MICROBICIDE AGAINST HIV-1 IN TISSUE EXPLANTS ...... 68 5.1.2. ANTIVIRAL EFFECT OF THE SEAWEED Osmundaria obtusiloba AGAINST THE ZIKA VIRUS ...... 75 5.1.3. AMINOMETHYLNAPHTHOQUINONES AND HSV-1: IN VIVO AND IN SILICO EVALUATIONS OF POTENTIAL ANTIVIRALS ...... 85 5.1.4. SYNTHESIS OF 4-OXOQUINOLINE ACYCLONUCLEOSIDE PHOSPHONATE ANALOGS AND ANTI-MAYV EVALUATION ...... 95 5.1.5. HIV EPIDEMIC - THE IMPORTANCE OF SEXUAL EDUCATION AND AIDING PROGRAMS ...... 116 5.2. PATENTES ...... 122 5.2.1. USO DE DERIVADOS QUILÔNICOS NA PREVENÇÃO DO HIV-1 E SUA AÇÃO VIRUCIDA IN VITRO...... 123

VIII

LISTA DE ABREVIATURAS SIGLA (-)FTC Emtricibatina AADS Antivirais de Ação Direta AASLD Associação Americana de Estudos de Doenças do Fígado ACV Aciclovir ADV Adefovir Dipivoxil AIDS Síndrome da Imunodeficiência Adquirida ANP Fosfanatos de Nucleosídeo Acíclicos AZT Zidovudina BVDU Brivudina CCR5 Receptor de Quimiocina Tipo 5 CD4 Grupo de Diferenciação 4 CDV Cidofovir COBI Cobicistate CXCR4 Receptor de Quimiocina X-C-X Tipo 4 DENV Vírus da Dengue DNA Ácido Desoxiribonucléico EASL Associação Européia de Estudos do Fígado EBOV Vírus Ebola EBV Vírus Epstein-Barr EFV Efavirenz ETV Entecavir EVG Elvitegravir FDA Food and Drug Administration Gag Antígeno Grupo Específico H7N9 Vírus da Influenza HAART Terapia Antiviral Altamente Ativa HBV Vírus da Hepatite B HCMV Citomegalovírus Humano

HCV Vírus da Hepatite C HHV Herpes Vírus Humano HIV Vírus da Imunodeficiência Humana HPV Papilomavírus Humano HSV-1 Vírus Herpes Simples Tipo-1 HSV-2 Vírus Herpes Simples Tipo-2 IDU Idoxuridina IFNα Interferon Alfa IgG Imunoglobulina G IMP Iosina-5’-monofosfato NNRTI Inibidores Não Nucleosídeos da Transcriptase Reversa NRTI Inibidores Nucleosídeos da Transcriptase Reversa NS3A Proteína Não Estrutural 3A NS4A Proteína Não Estrutural 4A NS5A Proteína Não Estrutural 5A NS5B Proteína Não Estrutural 5B OMS Organização Mundial de Saúde PegIFNα-2a Interferon Alfta Peguilado 2a PegIFNα-2b Interferon Alfa Peguilado 2b RNA Ácido Ribonucléico RNAm Ácido Ribonucléico Mensageiro RPV Rilpivirina RSV Vírus Sincicial Respiratório RSV-IGIV Globulina Imunológica para Vírus Sincicial Respiratório por via Intravenosa SARS Síndrome Respiratória Aguda Grave TAF Tenofovir Alafenamida TDF Fumarato de Tenofovir Disoproxila UE União Européia VariZIG Globulina Imune à Varicela-Zoster

VZV Vírus Varicela-Zoster XMP Xantosina Monofosfato

LISTA DE ILUSTRAÇÕES Figura 1 – Grupos de medicamentos antivirais para o tratamento de 9 doenças infecciosas...... 2

Figura 2 – Estrutura química da Idoxuridina (5-iodo-2'-desoxiuridina)...... 3

Figura 3 – Estrutura química da Trifluridina (5-trifluorometil-2'-desoxitimidina). 3

Figura 4 – Estrutura química da Brivudina [(E)-5-(2-bromovinil)-2'-deoxirudina]...... 4

Figura 5 – Estruturas químicas dos Análogos de Nucleosídeos...... 5

Figura 6 – Estrutura química do Foscarnet...... 5

Figura 7 – Estrutura química do 3’-azido-2’,3’-didesoxitimidina. Zidovudina. .... 6

Figura 8 – Estrutura química do Saquinavir...... 9

Figura 9 – Estruturas químicas dos Inibidores de Integrase utilizados no tratamento da infecção pelo HIV...... 9

Figura 10 – Estrutura química do Enfuvirtida...... 11

Figura 11 – Estrutura química do Maraviroc...... 12

Figura 12 – Estrutura química do Doconasol...... 13

Figura 13 – Estrutura química dos Inibidores Análogos da Guanosina Acíclicos ...... 14

Figura 14 – Inibidores Análogos Fosfanatos de Nucleosídeo Acíclico...... 16

Figura 15 – Inibidores do NS5A/NS5B do HCV...... 19

Figura 16 – Inibidores Não Nucleosídeos da polimerase NS5B...... 20

Figura 17 – Inibidores do Vírus Influenza...... 21

Figura 18 – Ciclo replicativo viral representando todas as etapas da replicação e possíveis alvos farmacológicos dos antivirais. 30

Figura 19 – Imagem demonstrativa dos vírus humanos mais comuns com seu tamanho relativo...... 32

XII

LISTA DE TABELA Tabela 1 – Grupo de inibidores de protease para o HIV e HSV NS3/A4 e seus respectivos status perante o FDA...... 8

Tabela 2 – Lista dos fármacos análogos de fosfanato de nucleosídeo acíclico aprovados pelo FDA...... 15

XIII

RESUMO

Como a maioria das doenças virais não possui um fármaco específico para seu tratamento ou até mesmo a cura, é essencial utilizar a história dos antivirais como base para busca de novos tratamentos. Neste trabalho, avaliamos a atividade citotóxica e atividade antiviral de algas marinhas e de derivados das naftoquinonas e quinolinas através do método de MTT, ensaio de redução de placa, inativação da partícula viral, tempo de adição de fármacos e avaliação in silico contra vírus de importância clínica. Nossos resultados mostraram que o dolabelladienotriol, isolado da alga parda Dictyota friabilis apresentou inibição da replicação do HIV-1 variando entre 60 e 80% e demonstrou perfil inibitório elevado quando avaliado em relação ao tempo de adição de fármacos além da manutenção da viabilidade do explante da cérvice uterina em cultura por 13 dias. O extrato da alga vermelha Osmundaria obtusiloba apresentou excelentes valores de viabilidade celular e atividade anti-

ZIKV (CC50=525μg/mL e EC50=1,82μg/mL). Quando avaliado o mecanismo de ação do extrato de O. obtusiloba, o mesmo apresentou atividade virucida de 80% e inibição da produção de partículas virais de 60% a 90% dependendo da fase de tratamento em relação ao ciclo de replicação. Os derivados de naftoquinonas foram testados contra o HSV-1 e apresentaram valores de CC50 variando entre 964µM a 2,654µM e EC50 variando entre 0,83µM e 2,13µM. A análise conformacional destas moléculas mostrou que os compostos 1 e 2 apresentaram um perfil mais promissor que o composto 3. Todos os compostos apresentaram baixa atividade virucida, aproximadamente 40%, na concentração de 50µM. Em relação ao tempo de adição dos compostos, estes se mostraram efetivos na inibição da fase L do ciclo replicativo do HSV-1, contudo, os compostos 1 e 2 também inibiram as outras duas fases, principalmente o composto 2 com quase 100% de eficácia. Os derivados de quinolina foram testados contra o vírus Mayaro. O composto 7a apresentou valores de CC50 e EC50 = 956.720µM e 0,83µM, respectivamente. No estudo do tempo de adição das substâncias em relação ao ciclo de replicação viral o composto 7a demonstrou inibição nos eventos precoces da replicação viral, variando entre 40% e 60% nas etapas de pré-tratamento e tempo 0. A avaliação in silico da interação deste composto com o Mayaro, revelou uma ligação do composto ao complexo de proteínas do envelope viral. Como pudemos observar, diante de todo o contexto histórico e evidências científicas, todas as substâncias estudadas neste trabalho são promissoras como novos fármacos antivirais.

XIV

ABSTRACT

Most viral diseases do not have a specific cure and/or pharmacological treatment. Therefore, it is essential to use the history of antivirals as a basis for the research of new treatments. In this work, we evaluated the cytotoxicity and antiviral activity from marine algae, naphthoquinones derivatives and quinoline derivatives through the MTT assay, plaque reduction assay, inactivation of the viral particle, time of addition of drugs and in silico evaluation against viruses of clinical importance. Our results have shown that the dolabelladienotriol, isolated from the brown algae Dictyota friabilis showed an inhibitory profile of HIV-1 replication varying between 60 and 80% and demonstrated higher inhibitory profile when evaluated concerning the time of addition of drugs beyond the maintenance of viability of explant in cell culture for 13 days. The extract from the red algae Osmundaria obtusiloba showed an excellent value of cell viability and anti-ZIKV activity (CC50=525μg/mL e EC50=1,82μg/mL). When the mechanism of action of O. obtusiloba's extract was evaluated, it has presented the virucidal activity of 80% and inhibition of viral production of 60% to 90% depending on the treatment level regarding the replication. The naphthoquinones derivatives were tested against the HSV-1 and presented

CC50 values varying between 964µM a 2,654µM and the EC50 varying between 0,83µM e 2,13µM. The conformational analysis of these molecules showed that compounds 1 and 2 presented a more promissory profile than compound 3. All the compounds had presented low virucidal activity, approximately 40% at 50µM concentration. Regarding the time of addition, these compounds proved to be effective on the inhibition of phase L of replicative cycle of HSV-1, however, the compounds 1 and 2 also have inhibited the other two phases, mainly the compound 2 with almost 100% of efficacy. The quinoline derivatives were tested against Mayaro vírus. The compound 7a presented CC50 and EC50 of 956.720µM e 0,83µM respectively. In the evaluation of the time of addition, the compound 7a demonstrated inhibition in early events of viral replication, varying between 40% to 60% on pre-treatment steps and in zero time. The in silico evaluation from the interaction from this compound with Mayaro, revealed a bind of the compound to the protein complex of the viral envelope. As we could observe, in the presence of every historical and scientific context, all substances studied in this work are promising as new antiviral drugs.

1. ANTIVIRAIS 1.1. HISTÓRICO Durante toda a história da civilização humana as infecções virais representam a maior ameaça à saúde mundial. Nos últimos 50 anos, avanços consideráveis no desenvolvimento de antivirais foram conquistados devido a um esforço significativo, levando a uma terapêutica eficaz para alguns vírus. No entanto, outras infecções virais continuam a se espalhar globalmente e novas ameaças continuam a surgir de vírus emergentes e reemergentes, assim como a resistência a medicamentos(DE CLERCQ E LI, 2016; TAKIZAWA E YAMASAKI, 2017).

Desde que a primeira substância antiviral, a 5-iodo-2´-desoxiuridina (Idoxuridina – IDU), foi aprovada,marcando a década de 60 como o início da busca de antivirais,aproximadamente 90 substâncias antivirais aprovadas para o tratamento de 9 vírus, como o Vírus da Imunodeficiência Humana (HIV), os Vírus da Hepatite B e C (HBV e HCV), Vírus da Herpes (HSV), Influenza (H7N9), Citomegalovírus Humano (HCMV), Varicela-Zoster (VZV), Vírus sincicial Respiratório (RSV) e Papilomavírus Humano (HPV) (DE CLERCQ E LI, 2016) (Figura 1).

Figura 1 – Grupos de medicamentos antivirais para o tratamento de 9 doenças infecciosas. Os medicamentos antivirais aprovados são agrupados para vírus RNA (HCV, RSV e vírus influenza), vírus DNA (HCMV, HBV, HPV, HSV e VZV) e retrovírus (HIV). Os nomes dos medicamentos antivirais atualmente em uso estão entre oblongos laranjas. Os nomes dos medicamentos antivirais descontinuados ou abandonados estão entre oblongos cinzentos. Os medicamentos que inibem mais de um vírus são mostrados na sobreposição regiões entre grupos de vírus. Para medicamentos para HCV, um símbolo de adição é usado para indicar a combinação de medicamentos aprovados (simeprevir mais sofosbuvir, sofosbuvir mais daclatasvir, daclatasvir mais asunaprevir e ribavirina mais PegIFNα-2b). (DE CLERCQ E LI, 2016) 1.1.1. ANÁLOGOS DE NUCLEOSÍDEO Historicamente, a Era da quimioterapia antiviral teve seu marco inicial no ano de 1959, com a descrição da Idoxuridina (Figura 2) (5-iodo-2´- desoxiuridina). Inicialmente, a IDU foi relatada como um potente agente antitumoral, o que mais tarde veio a ser tornar o primeiro fármaco antiviral a ser

2 utilizado clinicamente no tratamento tópico da infecção ocular ocasionada pelo HSV, tendo sido descrito em 1961 sua atividade antiviral contra o vírus vaccinia (DE CLERCQ E LI, 2016).

Figura 2 – Estrutura química da Idoxuridina (5-iodo-2'-desoxiuridina). A IDU é um análogo da timidina que atua saturando a timidina quinase das células queiscentes além de bloquear a DNA polimerase viral, responsável pela replicação do genoma. Assim como os demais análogos de nucleosídeos, a IDU só se torna ativa após ser fosforilada 3 vezes, o que a torna não específica para células infectadas e explica sua alta toxicidade no epitélio da córnea, já que estas fosforilações podem ser realizadas pelas quinases virais ou celulares (LABETOULLE, 2004).

Posteriormente, Kaufman e Heidelberger descreveram a eficácia da Trifluridina (Figura 3) (5-trifluorometil-2 = desoxitimidina) contra as infecções causadas pelo HSV. Desde então, a Idoxuridina e Trifluridina começaram a ser utilizadas no tratamento tópico da queratite epitelial causada pelo HSV (DE CLERCQ E LI, 2016).

Figura 3 – Estrutura química da Trifluridina (5-trifluorometil-2'-desoxitimidina).

No ano de 1970, a descoberta de análogos de nucleosídeos acíclicos, que são capazes de inibir a replicação do DNA do vírus herpes simples (HSV) em concentrações bem mais baixas que outros análogos que afetavam a

3 síntese de DNA celular, deu início a uma nova era de quimioterapia de antivirais (DE CLERCQ, 2007).

A Brivudina (Figura 4) (BVDU) [(E)-5-(2-bromovinil)2’-deoxirudina] foi originalmente sintetizada em 1976 pelo Departamento de Química da Universidade de Birmingham. Foi descrito como um potente e seletivo fármaco com alta especificidade contra o Herpes Simplex Tipo 1 (HSV-1) (DE CLERCQ, 2004).

Figura 4 – Estrutura química da Brivudina [(E)-5-(2-bromovinil)-2'-deoxirudina]. A BVDU foi aprovada em diversos países para a utilização no tratamento oral das infecções pelo HSV e VZV, contudo, os Estados Unidos e Reino Unido não aprovaram sua utilização (DE CLERCQ E LI, 2016).

Durante a década de 70, três fármacos análogos a nucleosídeos foram aprovados. O grupo de análogos de nucleosídeos inclui três fármacos aprovados pelo Food and Drug Administration (FDA): Vidarabina, Entecavir (ETV) e Telbivudina (Figura 5). Historicamente, os análogos do nucleosídeo arabinosil foram isolados pela primeira vez a partir de esponjas (BERGMANN E FEENEY, 1950). Antes de Schabel relatar seu potencial antiviral, a arabinosil adenina foi primeiramente considerada um potencial agente anticâncer (COHEN, 1966). Demonstrando uma elevada atividade contra as infecções causadas pelo HSV e VZV, a Vidarabina, que possui como alvo a DNA Polimerase viral. Foi o primeiro análogo de nucleosídeo aprovado pelo FDA e administrado sistematicamente em clínicas (DE CLERCQ E LI, 2016).

Figura 5 – Estruturas químicas dos Análogos de Nucleosídeos. A) Vidarabina; B) Entecavir e C) Telbivudina Elion et al, em 1978 descreveu a ação anti-herpes do Aciclovir, mesmo já existindo alguns outros fármacos antivirais disponíveis. Através de estudos mais aprofundados a cerca da estrutura da molécula, foram feitas alterações químicas as quais permitiam uma maior solubilidade aquosa o que gerou novos fármacos análogos (DE CLERCQ, 2011a).

1.1.2. ANÁLOGOS DE PIROFOSFATO Em 1978 foi descoberto um novo fármaco antiviral, o Fosfonoformato trissódico, conhecido como Foscarnet (Figura 6). Apesar de ser o único fármaco aprovado no grupo dos análogos de pirofosfatos, ele não foi o primeiro, pois havia sido precedido pelo ácido fosfonoacético (SHIPKOWITZ et al., 1973). O diferencial do foscarnet e do ácido fosfonoacético para os demais antivirais clássicos se deve ao fato deles não necessitarem ser fosforilados antes de se ligarem à molécula alvo (TCHESNOKOV et al., 2006).

Figura 6 – Estrutura química do Foscarnet. Apesar de alguns estudos relatarem o amplo espectro do Foscarnet contra HSV-1, HSV-2, HCMV, EBV, HIV e HBV visando as DNA polimerases virais. No entanto, o foscarnet não inibiu a atividade das RNAs Polimerases virais, nem inibiu a replicação de vírus RNA, com exceção de retrovírus (OBERG, 1989).

Apesar de sua atividade inibitória contra especificamente aos vírus DNA e aos retrovírus, o foscarnet atua de maneira única, pois se liga diretamente,

5 como análogo de pirofosfato às DNA polimerases virais. O foscarnet é diferente dos análogos de nucleotídeos que devem ser fosforilados em suas formas trifosfatos (nucleosídeos) ou em suas formas difosfato (nucleotídeos) antes de ocorrer sua ligação à DNA polimerase viral (DE CLERCQ E LI, 2016)

Foscarnet é exclusivamente utilizado no tratamento de HCMV ou HSV que tenham se tornado resistente aos análogos de nucleosídeos clássicos como o aciclovir (DE CLERCQ E LI, 2016).

1.1.3. INIBIDORES NUCLEOSÍDEOS DA TRANSCRIPTASE RESERVA No ano de 1980 foram descritos os efeitos antivirais e metabólicos de diversos análogos de 2’,3’-didesoxinucleósidos, incluindo 3’-azido-2’,3’- didesoxitimidina (AZT) (Figura 7) (DE CLERCQ et al., 1980). Entretanto, o HIV não foi incluído nos ensaios simplesmente pelo fato de tanto o vírus como sua patologia não haviam sido identificadas na época (DE CLERCQ, 2011a).

Figura 7 – Estrutura química do 3’-azido-2’,3’-didesoxitimidina. Zidovudina. Portanto, logo após a descoberta de seu potencial Anti-HIV em 1985 (MITSUYA et al., 1985; WILLIAMS, 2010), a Zidovudina (AZT) foi licenciada para a utilização clínica em 1987. O AZT não foi apenas o primeiro fármaco aprovado para o tratamento, mas também o primeiro fármaco a pertencer ao grupo de Inibidores Nucleosídeos da Transcriptase Reversa (NRTIs) (DE CLERCQ E LI, 2016).

Devido ao grande sucesso da Zidovudina em combater o progresso da Síndrome da Imunodeficiência Adquirida (AIDS), seis fármacos pertencentes ao grupo dos NRTIs foram subsequentemente aprovados para o tratamento das infecções pelo HIV e HBV. Todos os fármacos pertencentes ao grupo dos

NRTIs são conhecidos como análogos de 2’,3’-didesoxinucleósidos, possuindo mecanismos de ação similar (DE CLERCQ E LI, 2016).

1.1.4. INIBIDORES NÃO NUCLEOSÍDEOS DA TRANSCRIPTASE REVERSA No final da década de 80, o grupo de Inibidores Não Nucleosídeos da Transcriptase Reversa (NNRTI) incluía cinco fármacos aprovados com atividade Anti-HIV (DE CLERCQ E LI, 2016). O termo NNRTI foi criado em meados dos anos 90, para distinguir do grupo de substâncias que não agiam no sítio alostérico da transcriptase reversa os NRTIs (DE CLERCQ, 2011a).

Historicamente os NNRTIs foram originados de duas classes de compostos descobertas independente uma da outra, os análogos de 1-[(2- hidroxi-etoxi)metil]-6-feniltiotimina (HEPT) (BABA et al., 1989; MIYASAKA et al., 1989; CHEN et al., 2012; DE CLERCQ, 2013c) e os análogos de tetra-hidro- imidazo[4,5,1-jk][1,4]-benzodiazepina-2 (1H) -ona e -tiona (TIBO) (PAUWELS et al., 1990; CHEN et al., 2012; DE CLERCQ, 2013c). Na prática clínica, os NNRTIs são largamente utilizados como agentes de primeira linha. Eles podem ser combinados com o Tenofovir disoproxil fumarato e com a Rilpivirina para fornecer tratamentos completos de infecções por HIV com uma pílula diária, Complera nos EUA ou Eviplera na União Européia (UE) (DE CLERCQ E LI, 2016).

1.1.5. INIBIDORES DE PROTEASE

Por duas décadas, os inibidores de protease (PI) virais vêm sendo considerados componentes chave da terapia antirretroviral. Desde sua introdução na Terapia Antiviral Altamente Ativa (HAART) em 1996 (AGBOWURO et al., 2018).

No grupo dos inibidores de protease, atualmente existem 12 fármacos contra o HIV e 14 fármacos contra HCV NS3/4A, sendo que destes, 04 foram descontinuados, 7 se encontram em fases distintas da triagem clínica e apenas 3 estão aprovados para utilização no momento (Tabela 1) (DE CLERCQ E LI, 2016; AGBOWURO et al., 2018).

Tabela 1–Grupo de inibidores de protease para o HIV e HSV NS3/A4 e seus respectivos status perante o FDA. Adaptado de(DE CLERCQ E LI, 2016; AGBOWURO et al., 2018).

Nome do Fármaco Status Clínico HIV Saquinavir Aprovado Ritonavir Aprovado Indinavir Aprovado Nelfinavir Aprovado Amprenavir Lopinavir-ritonavir Aprovado Atazanavir Aprovado Fosamprenavir Aprovado Tipranavir Aprovado Durunavir Aprovado Durunavir + cobicistat Atazanavir + cobicistat PPL-100 Fase 1 Ensaios clínicos

HCV NS3/A4 Boceprevir Descontinuado Telaprevir Descontinuado Simeprevir Aprovado Paritaprevir Aprovado Grazoprevir Aprovado Faldaprevir Descontinuado Narlaprevir Descontinuado Asunaprevir Fase 3 Ensaios Clínicos Sovaprevir Fase 2 Ensaios Clínicos Danoprevir Fase 2 Ensaios Clínicos Vaniprevir Fase 2 Ensaios Clínicos Vedroprevir Fase 2 Ensaios Clínicos Glecaprevir Fase 2 Ensaios Clínicos

Em 1995, o Saquinavir (Figura 8) foi aprovado como o primeiro inibidor de protease, consolidando assim o início de uma era para os novos antivirais anti- HIV.Os inibidores de protease do HIV são baseados no mesmo princípio, no qual a ligação hidroxietileno atua como o suporte peptidomimético. Quando os inibidores de protease competem com os substratos naturais da protease do

HIV, como o suporte peptidomimético, variações de aminoácidos próximas a esse suporte e nos locais de clivagem dos substratos da protease (ou seja, Gag e Gag-Pol) podem ter sido selecionadas durante a evolução do vírus para causar resistência aos inibidores de protease (DE CLERCQ E LI, 2016).

Figura 8 – Estrutura química do Saquinavir. 1.1.6. INIBIDORES DE INTEGRASE

Desde que o primeiro inibidor de integrase de HIV foi aprovado em 2007 pela FDA, três inibidores (Raltegravir, Elvitegravir e Dolutegravir) (Figura 9) têm sido utilizados no HAART (DE CLERCQ E LI, 2016).

Figura 9 – Estruturas químicas dos Inibidores de Integrase utilizados no tratamento da infecção pelo HIV. A) Raltegravir; B) Elvitegravir e C) Dolutegravir.

O Raltegravir, foi o primeiro inibidor de integrase a ser aprovado em 2007, a utilização do raltegravir foi eficaz particularmente no tratamento de pacientes infectados pelo HIV com altos níveis de RNA do HIV-1, baixa contagem de células CD4 e baixo escore de sensibilidade genotípica ou fenotípica (DE CLERCQ E LI, 2016; WONG et al., 2016).

O Ralvitegravir foi aprovado em 2012 e está disponível como produto combinado que contem elvitegravir, cobicistat, entricitabina e tenofovir (WONG et al., 2016). Estudos in vitro indicaram que o elvitegravir inibia não somente diversas cepas do HIV-1, mas também um amplo espectro de vírus como HIV- 2, vírus da leucemia murina e vírus da imunodeficiência símia (ROQUEBERT et al., 2008; SHIMURA et al., 2008).

O Dolutegravir foi aprovado em 2013 e é indicado por o uso em pacientes que são HIV-1 positivos, sem tratamento ou com experiência (WONG et al., 2016). Embora o dolutegravir e o raltegravir compartilhem eficácias e perfis de segurança semelhantes, o dolutegravir exibe uma barreira genética mais alta ao desenvolvimento da resistência aos medicamentos (DE CLERCQ E LI, 2016).

1.1.7. INIBIDORES DE ENTRADA

O grupo de inibidores de entrada é composto atualmente por sete fármacos aprovados pelo FDA, incluindo um fármaco anti-HSV (docosanol), dois fármacos anti-HIV (enfuvirtida e maraviroc), dois fármacos anti-RSV (palivizumabe e imunoglobulina do vírus respiratório sincicial), e dois anticorpos como fármacos anti-VZV (imunoglobulina varicela-zoster [VariZIG] e imunoglobulina varicela-zoster [VZIG]) (DE CLERCQ E LI, 2016).

O Enfuvirtida (figura 10), também conhecido como T20, é o primeiro inibidor de peptídeo aprovado pelo FDA, sendo um polipeptídeo homólogo à região de repetição do heptado da GP41 do HIV-1 (MATTHEWS et al., 2004). Para bloquear a fusão do HIV-1 com a membrana extracelular da célula hospedeira, o Enfuvirtida mimetiza uma estrutura do HIV-1 que previne a interação entre as estruturas HR-1 e HR-2 (WILD et al., 1993; CILLIERS et al., 2005; BERKHOUT et al., 2012). Kilby e colaboradores demonstraram a eficácia do Enfuvirtida como agente inibidor de entrada do HIV-1 em células humanas e de linhagem (KILBY et al., 1998; BRITO, 2011). Aprovado pela FDA em março de 2003, o Enfuvirtida é ainda o único fármaco anti-HIV que necessita ser aplicado através de injeção subcutânea duas vezes ao dia (DE CLERCQ E LI, 2016).

Figura 10 – Estrutura química do Enfuvirtida.

O Maraviroc é o primeiro antagonista do receptor de quimiocina CCR5, ou inibidor da CCR5 aprovado pela FDA (figura 11). O receptor de quimiocina CCR5 se encontra na superfície das células TCD4 e macrófagos (KURITZKES, 2009). Inicialmente, Ed Berger e colaboradores foram os primeiros a demonstrar a importância dos receptores de quimiocina CCR5 durante a entrada do HIV nas células alvo (ALKHATIB et al., 1996). É necessário salientar que durante a infecção precoce pelo HIV, os vírus R5 utilizam predominantemente os receptores CCR5 para a entrada do vírus, enquanto os vírus R4 utilizam os receptores CXCR4, o que geralmente ocorre nos estágios finais da progressão da doença (DOLIN, 2008).

Figura 11 – Estrutura química do Maraviroc.

O RSV-IGIV, aprovado pela FDA em janeiro de 1996, é uma imunoglobulina humana estéril produzida através do plasma de adultos com alto índice de anticorpos neutralizantes ao RSV. Tais anticorpos neutralizantes são capazes de impedir que as glicoproteínas de superfície F e G do RSV consigam se ligar às células hospedeiras (ROBINSON E NAHATA, 2000).

Apesar de o RSV-IGIV diminuir de forma eficiente o número de hospitalizações e dias de internação dos pacientes (EDWARD CONNOR et al., 1997; WU et al., 2008). O elevado custo e as rígidas diretrizes sobre sua utilização continuam sendo uma questão problemática (BLACK, 2003). Como uma melhor alternativa e um melhor custo-eficiência (PRESCOTT et al., 2010) o Palivizumabe (Synagis) foi central na descontinuação do RespiGam em 2004 (ROBINSON E NAHATA, 2000). Tendo sido aprovado pela FDA em junho de 1998, o Palivizumabe é uma imunoglobulina anticorpo monoclonal de rato humanizada que possui como alvo direto um epítopo conservado do sítio antigênico A da proteína de fusão do SRV (MICHAEL T. BRADY et al., 2014). Mesmo apresentando resultados promissores em ensaios clínicos, algumas revisões sistemáticas apontam que os benefícios clínicos e sociais quando levados em consideração são poucos para justificar o alto custo da profilaxia do Palivizumabe (JOSEPH A. BOCCHINI JR et al., 2009; WANG et al., 2011; MICHAEL T. BRADY et al., 2014).

Historicamente, o VZIG foi descoberto em 1969 quando concentrados de imunoglobulina foram extraídos de pacientes infectados por VZV. Alguns estudos sorológicos e clínicos concluíram que o VZIG poderia diminuir os riscos de complicações e reduzir a doença em pacientes imunocomprometidos

(ZAIA et al., 1983; CDC, 1984; BATE et al., 2019). Após ter sido utilizado por duas décadas, VZIG foi descontinuado em outubro de 2004 e posteriormente substituído por um produto chamado VariZIG. Aprovado pela FDA em dezembro de 2012, o VariZIG é uma preparação de IgG purificado isolado de plasma humano contendo altos níveis de anticorpos anti-VZV (CDC, 1996). Sendo licenciado para profilaxia pós-exposição para as infecções de VZV, o VariZIG é administrado intramuscular apenas em pacientes de alto risco que não possuem evidência de imunidade contra o VZV e não são elegíveis para a vacinação (CDC, 2012).

O Docosanol é um inibidor de amplo espectro contra vírus envelopados (ex.: HSV, RSV, HCMV e VZV) com evidências em experimentos in vitro (figura 12) (KATZ et al., 1991; SACKS et al., 2001). Evidências clínicas relatam que o doconasol tópico é efetivo e seguro no que diz respeito à redução do tempo de cura e na duração dos sintomas de pacientes em tratamento de herpes labial recorrente causado por infecções por HSV-1 ou HSV-2 (SACKS et al., 2001). Considera-se que docosanol atue inibindo a entrada do vírus, interferindo assim na interação entre as proteínas do envelope viral e os receptores da célula alvo (KATZ et al., 1991; POPE et al., 1998; VERGARA et al., 2015). Aprovado pelo FDA em julho de 2020, o docosanol permanece sendo o único fármaco utilizado que não necessita de receita em uso clínico para herpes labial e febre (DE CLERCQ E LI, 2016).

Figura 12 – Estrutura química do Doconasol. 1.1.8. INIBIDORES ANÁLOGOS DA GUANOSINA ACÍCLICOS

Historicamente o Aciclovir foi o primeiro análogo acíclico da guanosina descrito por Nick Oliver em 1974 (DE CLERCQ E FIELD, 2006). As propriedades antivirais do Aciclovir foram descritas inicialmente por Peter Colins e John Bauer nos Laboratórios Wellcome em Beckenham, Reino Unido, tendo sido originalmente projetada como um inibidor de adenosina desaminase para aumentar a atividade antiviral da vidarabina (DE CLERCQ, 2008). Segundo Elion e colaboradores (ELION et al., 1977) o aciclovir apresentava

13 seletividade contra o HSV devido à fosforilação específica das timidinas- quinases virais. Logo depois, foi relatada a potente atividade antiviral contra herpes vírus (HSV-1 e HSV-2) (SCHAEFFER et al., 1978).

De todos os análogos acíclicos de guanosina descobertos posteriormente, o Penciclovir foi utilizado no tratamento das infecções pelo VZV, e o Ganciclovir tornou-se o fármaco utilizado no tratamento das infecções causadas pelo HCMV (WILHELMUS, 2015).

Com o objetivo de aumentar a biodisponibilidade oral, foi utilizada a metodologia de pré-fármaco para todos os três análogos acíclicos de nucleosídeo, contribuindo para o desenvolvimento do Fanciclovir, Valaciclovir e Valganciclovir (Figura 13) (DE CLERCQ E FIELD, 2006).

O Aciclovir, o Ganciclovir e o Penciclovir atuam de forma similar e todos são fosforilados (Figura 13). O Ganciclovir é fosforilado especificamente pelas quinases do hospedeiro (SULLIVAN et al., 1992; YABIKU et al., 2011), enquanto o Aciclovir e o Penciclovir são ambas fosforiladas pela timidina quinase viral (ELION et al., 1977; HODGE, 1993).

Figura 13 – Estrutura química dos Inibidores Análogos da Guanosina Acíclicos: A) Fanciclovir, B) Valaciclovir, C) Valganciclovir, D) Aciclovir, E) Ganciclovir e F) Penciclovir.

Até os dias atuais, o Aciclovir permanece sendo o fármaco padrão ouro para o tratamento das infecções por HSV. Mesmo apresentando uma excelente

14 eficiência, o nível de mortalidade nos pacientes com encefalite causada pelo HSV que receberam o Aciclovir como tratamento varia de 14 a 19% (GNANN et al., 2015). Mesmo o Valaciclovir não oferecendo um benefício adicional em comparação ao placebo em testes de acompanhamento de tratamento por 3 meses seguidos, o Valaciclovir substituiu o aciclovir no tratamento das infecções por HSV ou VZV devido a sua biodisponibilidade oral aumentada (O'BRIEN E CAMPOLI-RICHARDS, 1989; GNANN et al., 2015). Estudos realizados mostraram que o Valganciclovir foi mais econômico que o Valaciclovir utilizado no primeiro ano após transplante renal. Por outro lado, o Fanciclovir apresentou benefícios mais significativos, tendo as cicatrizações das lesões mais rápidas. Por essa razão, o Fanciclovir agora é amplamente utilizado na terapêutica das infecções por HSV e VZV (M.G. GOPAL et al., 2013; KACER et al., 2015).

1.1.9. INIBIDORES ANÁLOGOS FOSFANATOS DE NUCLEOSÍDEO ACÍCLICO

No grupo dos análogos fosfanatos de nucleosídeo acíclico (ANP), existem 10 combinações farmacológicas aprovadas pelo FDA (Tabela 2).

Tabela 2 – Lista dos fármacos análogos de fosfanato de nucleosídeo acíclico aprovados pelo FDA. Modificado de: (DE CLERCQ E LI, 2016)

Nome dos Abreviação Utilização Data de Fármacos Clínica Aprovação

Cidofovir CDV Retinite por HCMV Junho de 1996 (Pacientes com AIDS) Fumarato de TDF HIV e HBV Outubro de 2001 Tenofovir Disoproxila Adefovir Dipivoxil ADV HBV Setembro de 2002 Fumarato de TDF + (-)FTC HIV Agosto de 2004 Tenofovir Disoproxila + Emtricitabina Fumarato de TDF + EFV + (-)FTC HIV Julho de 2006 Tenofovir Disoproxila + Efavirenz + Emtricitabina Fumarato de TDF + RPV + (-)FTC HIV Agosto de 2011 Tenofovir Disoproxila + Rilpivirina + Emtricitabina Fumarato de TDF + COBI + (-)FTC HIV Agosto de 2012 Tenofovir Disoproxila + EVG

+ Cobicistate + Emtricitabina + Elvitegravir Tenofovir TAF + COBI + (-)FTC HIV Novembro de 2015 Alafenamida + + EVG Cobicistate + Emtricitabina + Elvitegravir Tenofovir TAF + RPV + (-) FTC HIV Março de 2016 Alafenamida + Rilpivirina + Emtricitabina Tenofovir TAF + (-) FTC HIV Abril de 2016 Alafenamida + Emtricitabina

Os ANP que possuem atividade inibitória contra DNA polimerases, derivam de uma hibridização da (S)-DHPA [(S) -9- (2,3-di-hidroxipropil)adenina] com ácido fosfonoacético, gerando assim (S)-HPMPA [(S) -9- (3-hidroxi-2- fosfonilmetoxipropil) adenina] (DE CLERCQ E LI, 2016). Historicamente, a atividade antiviral de amplo espectro do (S)-DHPA foi inicialmente descrita por De Clercq et al. (DE CLERCQ et al., 1978; CTRNÁCTÁ et al., 2010), pouco tempo depois do aciclovir ter sido relatado como um agente anti-herpético específico (SCHAEFFER et al., 1978). O (S)-HPMPA teve sua primeira descrição como um novo agente antiviral de amplo espectro para vírus de DNA em 1986 por De Clercq et al. (DE CLERCQ et al., 1986; BEADLE et al., 2006). Apesar de o próprio (S)-HPMPA não ter sido comercializado pra utilização clínica, o mesmo pode ser considerado o ANP percursor dos análogos de ANP como o cidofovir, adefovir e tenofovir (Figura 14) (DE CLERCQ E HOLY, 2005; DE CLERCQ, 2009b, 2013a).

Figura 14 – Inibidores Análogos Fosfanatos de Nucleosídeo Acíclico: A) Cidofovir, B) Adefovir e C) Tenofovir.

Em junho de 1996, o cidofovir foi aprovado para o tratamento de retinite por HCMV em pacientes com AIDS. O cidofovir também tem sido utilizado como um fármaco off-label para tratar muitas infecções por vírus de DNA, como o HSV e infecções por adenovírus, varíola, polioma e papiloma (DE CLERCQ, 2003). Posteriormente, o adefovir foi comercializado na sua forma pró-fármaco oral, adefovir dipivoxil, para o tratamento da infecção pelo HBV, assim como o tenofovir, na sua forma pró-fármaco TDF, para o tratamento da infecção pelo HIV e /ou HBV (DE CLERCQ E LI, 2016)

Devido à sua potência e alta barreira à resistência, a monoterapia de TDF é frequentemente indicada como tratamento de primeira linha em casos de hepatite B crônica, seguindo os protocolos estabelecidos pela Associação Americana de Estudos de Doenças do Fígado (AASLD) (TERRAULT et al., 2016), pela Associação Européia de Estudos do Fígado (EASL) (EUROPEAN ASSOCIATION FOR THE STUDY OF THE LIVER, 2012) e pela Organização Mundial de Saúde (OMS) (WORLD HEALTH ORGANIZATION, 2015).

A principal caracterização dos ANPs está baseada na presença de uma ligação fosfonato ao contrário de uma ligação fosfato, sendo uma característica dos análogos de nucleotídeos que apresentam atividade contra as polimerases virais (DE CLERCQ, 2011b).

Em novembro de 2015, o TAF (GS-7340) foi aprovado em combinação com o cobicistate, emtricitabina e elvitegravir (Genvoya) para o tratamento de infecções pelo HIV. Este medicamento encontra-se no mercado com uma dose fixa de TAF (10mg), cobicistate (150mg), entricitabina (200mg) e elvitegravir (150mg). Estudos clínicos em fase três sugerem que esta combinação de fármacos pode alcançar um sucesso no tratamento em cerca de 94,9% dos pacientes em 48 semanas (SAX et al., 2015; MILLS et al., 2016).

Aprovadas pela FDA em 2016, o TAF é agora utilizado em duas combinações: tenofovir alafenamida, rilpivirina e emtricitabina (Odefsey) ou tenofovir alafenamida e emtricitabina (Descovy). Esses fármacos combinados com o TAF possuem diversas vantagens distintas por que são utilizados especificamente por células linfóides, com isso, suas dosagens podem ser

17 diminuída em 10 vezes. Tal vantagem reduz significativamente o risco toxicidade, como distúrbios renais e/ou desmineralização óssea (WYATT E BAETEN, 2015; RAY et al., 2016).

1.1.10. INIBIDORES DO NS5A/NS5B DO HCV

Até abril de 2016, existiam 8 combinações de fármacos inibidores do HCV NS5A/NS5B aprovados. Os Antivirais de Ação Direta (AAD) licenciados para o tratamento de infecções por HCV contemplam, de modo geral, quatro classes: (i) Inibidores da Protease NS3/4A, (ii) Inibidores da Proteína NS5A, (iii) Inibidores da Polimerase NS5B do tipo Nucleosídeo e/ou nucleotídeo e (iv) Inibidores da Polimerase NS5B do tipo Não Nucleosídeo (AASLD/IDSA HCV GUIDANCE PANEL, 2015)

Estes medicamentos estão sendo os substitutos da combinação de interferons peguilados e ribarina, padrão de atendimento para o tratamento de infecções crônicas por HCV previamente utilizado (DE CLERCQ, 2012a).

Em abril de 2016 quatro inibidores da NS5A foram aprovados: daclatasvir, ledipasvir, ombitasvir e o elbasvir (Figura 15). O daclatasvir pode se ligar especificamente à proteína não estrutural NS5A do HCV (NETTLES et al., 2014). O mecanismo de ação desta substância ainda é discutido, especialmente no que se refere a sua potencial inibição da estabilidade estrutural, dimerização ou distribuição subcelular da NS5A (BERGER et al., 2014; LIU et al., 2015).

Figura 15 – Inibidores do NS5A/NS5B do HCV: A) Daclatasvir, B) Ledipasvir, C) Ombitasvir e o D) Elbasvir.

Em algumas pesquisas realizadas com a adição de asunaprevir ao tratamento utilizando o daclatasvir, mostraram-se eficazes no que diz respeito à terapêutica, este estudo mostrou números promissores de SVR 12 (> 80%) para o daclatasvir (60mg, uma vez ao dia) mais a adição do Asunaprevir (100mg, duas vezes ao dia) em três grupos distintos de pacientes (MANNS et al., 2014).

Em janeiro de 2016 a terapia baseada na combinação do elbasvir e grazoprevir (Zepatier) foi aprovado pela FDA a ser utilizado no tratamento da infecção pelo HCV genótipo 1 ou 4. Essa junção de fármacos inibe a proteína não estrutural NS5A do HCV e a protease NS3/4A, respectivamente. Alguns ensaios clínicos de diferentes etapas, elegendo pacientes em diferentes situações e foram realizados e demonstraram altas taxas de resposta ao vírus se estendendo por até 12 – 18 semanas; baixa taxa de eventos adversos e taxas promissoras de SVR12, revelando assim uma taxa de sucesso de SVR12 de até 95,8%. Nestes estudos, os efeitos colaterais mais comuns foram náusea, dor de cabeça e fadiga (LAWITZ et al., 2015; ROTH et al., 2015; ZEUZEM et al., 2015).

No entanto, a eficácia dessa combinação farmacológica na infecção pelo HCV genótipo 6 ainda não foi totalmente esclarecida (ZEUZEM et al., 2015). 19

Com tudo, a combinação do grazoprevir e elbasvir tem demonstrado ser efetivo contra infecções pelo HCV genótipos 1 e 4.

Para os Inibidores Não Nucleosídeos da polimerase NS5B, o sofosbuvir e o dasabuvir (Figura 16) foram aprovados pelo FDA enquanto um número considerado de inibidores experimentais tem sido identificados para atingir alvos alostéricos da polimerase NS5B do HCV (DE CLERCQ, 2014, 2015a).

Figura 16 – Inibidores Não Nucleosídeos da polimerase NS5B: A) Sofosbuvir e B) Dasabuvir.

O Repertório de inibidores nucleosídeos da polimerase NS5B é escassa devido aos seus efeitos colaterais indesejáveis. O sofosbuvir, no entanto, é uma exceção neste grupo, já que revelou baixa toxicidade ou resistência medicamentosa, além de poder ser administrado juntamente com outros medicamentos para o tratamento do HCV em um período total de 12 semanas, garantindo assim um alto nível de manutenção da resposta antiviral (BUTI et al., 2015).

1.1.11. INIBIDORES DOS VÍRUS INFLUENZA

Até a data de abril de 2016, haviam 8 fármacos aprovados para o tratamento de infecções por Influenza. Tais fármacos podem ser classificados como Inibidores de Matrix 2 (amantadina e rimantadina), Inibidores da Neuraminidase (zanamivir, oseltamivir, peramivi e octanoato de laninamivir) e os Inibidores da Polimerase (ribavirina e favupiravir) (Figura 17) (DE CLERCQ E LI, 2016).

Figura 17 – Inibidores do Vírus Influenza. Inibidores de Matrix 2: A) Amantadina e B) Rimantadina; Inibidores da Neuraminidase: C) Zanamivir, D) Oseltamivir, E) Peramivi e F) Octanoato de Laninamivir) e os Inibidores da Polimerase: G) Ribavirina e H) Favupiravir).

A amantadina (1-adamantanamine) foi o primeiro composto antiviral aprovado em 1966 para o tratamento da Influenza A (DAVIES et al., 1964). Esse composto bloqueia o transporte do íon H+ através dos canais de proteína Matrix 2 (M2) para o interior das partículas virais, impossibilitando assim o desnudamento das partículas virais de influenza nos endossomas (CADY et al., 2009; LIANG et al., 2014).

Após a descoberta da amantadina, a rimantadina, e um grande número de derivados de Amantadina foram posteriormente sintetizados (DE CLERCQ, 2006, 2009a), porém não obtiveram sucesso no mercado terapêutico, com exceção da amantadina e rimantadina. Embora tenham sido aprovadas para a utilização em pacientes adultos, a amantadina e a rimantadina não auxiliam na

21 prevenção, tratamento ou redução da infecção pelo vírus Influenza A em crianças e idosos (ALVES GALVÃO et al., 2012). Devido a sua alta taxa de resistência, a amantadia teve sua utilização descartada para o tratamento de infecções por Influenza apesar do aumento de sua utilização para o tratamento de outras doenças como Doença de Parkinson (RAZONABLE, 2011; HUBSHER et al., 2012; RODNITZKY E NARAYANAN, 2014).

O desenho computacional que deu origem ao Zanamivir, marcou uma nova era no desenvolvimento de medicamentos antiviriais (DE CLERCQ, 2006). Sendo um inibidor de neuramidases da Influenza A e B altamente seletivo, o Zanamivir previne novas infecções por Influenza, impedindo a liberação do vírus, ao invés de inibir a entrada do vírus ou os demais estágios do ciclo de multiplicação viral (RUSSELL et al., 2006).

Foi observado que quando o zanamivir era administrado por inalação junto à administração oral do oseltamivir, juntos apresentavam benefícios como: redução da mortalidade e diminuição na duração dos sintomas e complicações da infecção pela Influenza (MCKIMM-BRESCHKIN et al., 2003; HSU et al., 2012).

Em seguida ao sucesso do zanamivir e oseltamivir, dois Inibidores da Neuraminidase foram lançados para o tratamento de infecções por Influenza (DE CLERCQ, 2013b), peramivir, que pode ser administrado com uma única injeção intravenosa (MCLAUGHLIN et al., 2015), e octanoato de laninamivir, que é administrado com uma inalação única (WATANABE et al., 2010).

A ribavirina (1-β-D-ribofuranosil-1,2,4-triazole-3-carboxamida) é o primeiro análogo de nucleosídeo sintético tendo sido referenciado como ativo contra uma vasta quantidade de vírus RNA (HCV, RSV e Vírus Influenza) (SIDWELL, 1972). Seu principal mecanismo de ação foi estabelecido rapidamente após sua descoberta (STREETER et al., 1973), tratando-se da inibição da inosina-5’- monofosfato (IMP) desidrogenase, que converte IMP em xantosina monofosfato (XMP) e, portanto, é responsável pela biossíntese de novo GTP (WRAY et al., 1985). A atividade inibitória da ribavirina na IMP desidrogenase pode contribuir para efeitos imunossupressores da ribavirina (POTTER et al.,

1976). Por sua vez, isso contribui para o sucesso significativo obtido pela ribavirina, em combinação com o peginterferon alfa 2ª, no tratamento da infecção pelo HCV (FRIED et al., 2002; TORRIANI et al., 2004).

A potencial atividade da ribavirina frente a vírus de RNA é demonstrada na busca por candidatos antivirais contra alguns vírus causadores de doenças infecciosas emergentes como: Dengue Vírus (DENV) (KAUR E CHU, 2013) Norovírus (KIM et al., 2015; KOCHER E YUAN, 2015), Marbug Vírus (MARV) (MEHEDI et al., 2011) e os Vírus Hendra e Nipah (BRODER et al., 2013)

O favipiravir (6-fluoro-3-hidroxi-2-pirazina carboxamida) foi utilizada principalmente para o tratamento de infecções por Influenza (FURUTA, 2002; KISO et al., 2010; DE CLERCQ, 2013d). De acordo com o mecanismo de ação descrito por Furuta et. al. 2005 (FURUTA, 2005), o favipiravir é convertido intracelularmente à sua forma ribofuranosil monofosfato pela fosforibosil transferase. Posteriormente, ocorrem duas fosforilações que convertem a forma ribofuranosil monofosfato na forma trifosfato, o metabólito ativo do favipiravir. É importante salientar que o trifosfato de favipiravir mostra atividades inibidoras de amplo espectro contra as polimerases de RNA dos vírus influenza A, incluindo o vírus altamente patogênico H5N1 (SMEE et al., 2009; KISO et al., 2010) e muitos outros vírus de RNA de sentido positivo e RNA de sentido negativo (DE CLERCQ, 2013d). Recentemente, o favipiravir teve a utilização proposta no combate a infecção pelo Vírus Ebola (EBOV) (VAN HERP et al., 2015). Resultados preliminares indicam que o favipiravir inibiu eficientemente, o vírus Ebola em modelos murinos (OESTEREICH, 2014; SMITHER, 2014), mas investigações mais aprofundadas ainda são necessárias (DE CLERCQ, 2015b).

1.1.12. INIBIDORES A BASE DE INTERFERONS, IMUNOESTIMULADORES, OLIGONUCLEOTÍDEOS E ANTIMITÓTICOS.

No grupo dos fármacos de interferons, imunoestimuladores, oligonucleotídeos e inibidores antimitóticos, existem 08 medicamentos aprovados pela FDA: (i) interferons para infecções para HBV e HCV; (ii) fomivirsen (um oligonucleotídeo antisense) apara infecções por HCMV; (iii)

23 podofilox (um inibidor antiaminiótico), imiquimod (um imunoestimulador), e sinecatechins (um fármaco natural) para o tratamento de infecção genital externa causada por HPV. Todas essas substâncias aprovadas compartilham de algo em comum, elas possuem efeitos inibitórios específicos sem ter proteínas virais como alvos diretos (DE CLERCQ E LI, 2016).

Para tratar infecções por HBV e HCV, três interferons foram licenciados: Interferon Alfacon 1, Interferon Alfa Peguilado 2a (PegIFNα-2a e PegINFα-2b). Devido a seus graves efeitos adversos, o Interferon alfacon 1 foi descontinuado desde setembro de 2013. Atualmente, os regimes baseados em PegINF são preferencialmente utilizados para tratamento das infecções por HBV, mas não para infecções por HCV, pois os medicamentos sem inteferon se mostraram eficazes contra infecções por HCV (HEIM, 2013). O Interferon alfa (IFNα), predominantemente secretado pelas células hematopoiéticas, é um Interferon do tipo I bem definido que estimula o sistema imunológico a uma defesa antiviral (IVASHKIV E DONLIN, 2014; CROUSE et al., 2015; HOFFMANN et al., 2015). Com a intenção de aumentar a meia vida dos Interferons Inibidores no soro, polímeros de polietileno glicol são ligados convalentemente aos INF-α para a produção de PegIFNα. Em relação ao mecanismo de ação do fármaco, o PegIFNα-2a e o PegIFNα-2b interferem principalmente na replicação viral em dois aspectos. Primeiramente, eles estimulam as células da imunidade a melhorar a via não citolítica dos vírus pelas citocinas ou citólise das células infectadas, posteriormente eles estimulam a expressão de genes e proteínas antivirais inatos para bloquear a replicação viral (ZOULIM E DURANTEL, 2015).

Na prática clínica, tratamentos a base de interferons são utilizados com pouca frequência devido aos seus múltiplos efeitos colaterais, altos custos e inconveniência na administração (PERRILLO, 2009). Além do mais, permanece discutível se os interferons devem ou não ser combinados com outros fármacos antivirais (BROUWER et al., 2015; LAMPERTICO, 2015; DE NIET et al., 2016).

Aprovado pela FDA, em fevereiro de 1997, o imiquimod (1-isobutil-1H- imidazo[4,5-c]quinolina-4-amina) também conhecida por (R-837 e S26308) é uma amina heterocíclica não nucleosídea de formulação cremosa a 5%. Seu

24 mecanismo de ação está baseado em estimular macrófagos a produzirem e secretarem citocinas para regressão de verrugas e reações inflamatórias (BEUTNER et al., 1998a; BEUTNER et al., 1998b; WILEY et al., 2002).

Ensaios clínicos demonstraram que a formulação cremosa de Imiquimod a 5% é segura e apresenta boa atividade no tratamento das verrugas genitais externas (BEUTNER et al., 1998a; BEUTNER et al., 1998b; BAKER et al., 2011).

A pomada de sinecatecina a 15% (Veregen) foi o primeiro fármaco botânico aprovado pela FDA para tratamento tópico das verrugas vaginais causadas pelas infecções do HPV (CHEN et al., 2008). O veregen é um produto purificado a partir de folhas de chá verde chinês contendo 80% de catequinas e polifenóis. As catequinas são conhecidas por apresentarem atividade antiangiogênica, atividades antiinflamatórias e imunoestimuladoras além de um potencial antimicrobiano (SCHNEIDER E SEGRE, 2009).

A podofilotoxina (Condylox) é um composto antimitótico extraído e purificado da resina bruta do podophyllum (TYRING et al., 1998). A podofilotoxina é seguro e efetivo no tratamento de verrugas vaginais externas (361,362). Em vez de ter como alvo direto as proteínas do HPV, a podofilotoxina é um medicamento citotóxico, ou seja, com ações farmacológicas contra a formação do fuso mitótico na metáfase, levando à interrupção da divisão celular (GREENBERG et al., 1991; GUNTER, 2003).

O fomivirse (Vitravene) é o primeiro oligonucleotídeo anti-senso aprovado pela FDA, possuindo oligonucleotídeos de fosforotioato de 21 nucleotídeos (5’- GCG TTTGCTCTT CTT CTT GCG-3’) (DE SMET et al., 1999). Segundo seu mecanismo de sentido contrário, o fomivirsen é complementar a uma sequência de RNAm que codifica a principal região imediata 2 do HCMV, assim, a ligação do fomivirsen a esta região inibe a expressão gênica de proteínas essenciais do HCMV (GRILLONE E LANZ, 2001). Embora possuísse boa tolerância e um perfil de segurança favorável, o fomivirsen foi interrompido por razões comerciais (VITRAVENE STUDY GROUP., 2002).

1.2. IMPORTÂNCIA BIOTECNOLÓGICA

O elevado custo no processo de desenvolvimento de medicamentos vem limitando o número de tratamento de doenças virais a uma lista relativamente curta. O fato de substâncias antivirais serem extremamente específicas para um único agente infeccioso faz necessário que o diagnóstico preciso e rápido deva ser realizado antes de se iniciar a terapia antiviral (LITTLER E OBERG, 2005).

A falta de fármacos antivirais se deve, principalmente, ao fato de que os vírus são absolutamente dependentes das vias metabólicas das células hospedeiras para sua replicação. Assim, a maioria dos agentes que bloqueiam a replicação viral é letal ou prejudicial às células (DE CLERCQ, 2002).

Cada vez mais grupos de pesquisa se dedicam aos estudos de novas substâncias antivirais, sejam elas naturais ou sintéticas. Dentro do grupo de substâncias sintéticas, os análogos de nucleosídeos e os derivados de quinolinas, pirimidinas e pirazol são intensamente estudados. A descoberta do aciclovir (ACV) como primeiro potente e seletivo inibidor do herpes simples tipo 1 (HSV-1) tem estimulado a contínua investigação e avaliação de novos análogos de nucleosídeos (DE CLERCQ, 2010).

Entretanto, até o momento não existem fármacos licenciados para diversos vírus de grande importância como, por exemplo, a maioria dos vírus RNA emergentes ou altamente patogênicos que podem causar quadros clínicos severos, como síndromes respiratórias graves, febres hemorrágicas e encefalites (DE CLERCQ, 2007).

O primeiro passo na busca por novos fármacos é identificar a estrutura e função das moléculas que hipoteticamente possuem atividade. Além disso, é importante escolher os alvos terapêuticos, identificando assim, etapas críticas no ciclo de multiplicação viral, genes e proteínas essenciais para a replicação (EVERTS et al., 2017).

A utilização de alvos em partículas virais resulta em fármacos com poucos efeitos colaterais e maior especificidade devido à maior probabilidade do medicamento inibir apenas a replicação viral, sem afetar os processos naturais das células hospedeiras (EVERTS et al., 2017).

Contudo, o desafio desta abordagem é que tais alvos podem não se manter preservados em vírus relacionados, mesmo apresentando sorotipos ou genótipos do mesmo vírus, limitando assim o espectro de atividade desta substância (ASSELAH et al., 2016).

Atualmente o tratamento para as infecções existentes se baseia na terapia farmacológica. No entanto, com o gradativo aumento da resistência aos fármacos, aliados ao desafio da utilização de combinações terapêuticas não ser de fácil acesso em países de baixa renda devido aos elevados custos. Adicional a este problema, os custos e tempo necessários para o estudo e desenvolvimento de novos medicamentos efetivos podem dificultar o controle de doenças no futuro (WAHEED et al., 2016).

Uma das maiores conquistas no campo da pesquisa farmacêutica, nos últimas três décadas, foi à inserção de sistemas biológicos in vitro para estudos em grande escala em curto prazo de tempo (HOUGHTON, 2000).

A identificação de novos alvos terapêuticos não necessariamente ocorre de maneira simultânea com o desenvolvimento e licenciamento de novos compostos bioativos. A OMS precisa lidar com vírus críticos reemergentes, caracterizados por potencial pandemia e responsáveis por surtos alarmantes nos últimos anos, que ainda carecem de tratamento específico, como o ZIKV, o EBOV, o Coronavírus da Síndrome respiratória do Oriente Médio (MERS-CoV) (MERCORELLI et al., 2018).

Uma solução alternativa mais rápida e menos dispendiosa para este problema é o reaproveitamento ou reposicionamento de medicamentos. Em vez de se buscar tratamentos que ajam especificamente em um tipo viral, O reaproveitamento e reposicionamento de fármacos foca em encontrar tratamentos que possuam amplo-espectro para tais infecções emergentes, porém focando as vias das células hospedeiras em vez do agente infeccioso diretamente. As formas de auxiliar o uso de antivirais com fármacos que aumentam os mecanismos de resistência às células hospedeiras tornaram-se assim uma área ativa e promissora, como tem sido o caso da pandemia pelo novo coronavírus (SARS-CoV-2). Ensaios clínicos vêm sendo realizados para avaliar a eficiência de vários fármacos já existentes para o tratamento da

COVID-19. Em 7 de março de 2020, as terapias antivirais mais frequentemente avaliadas foram lovinapir/ritonavir (LVP/r) (n = 15), cloroquina (n = 11), arbidol (n = 9), hidroxicloroquina (n = 7), favipiravir (n = 7) e remdesivir (n = 5). A maioria destes fármacos demonstrou atividade antiviral in vitro contro o coronavírus (GARCÍA-SERRADILLA et al., 2019; DELANG E NEYTS, 2020; SCHEIN, 2020).

É importante salientar que a maioria dos novos conhecimentos sobre replicação viral, patogênese e epidemiologia provém da pesquisa acadêmica de base. Um possível passo para melhorar a rapidez no processo de descoberta de medicamentos é formar possíveis parcerias com as indústrias biotecnológicas ou farmacêuticas, visando à fusão do conhecimento acadêmico em virologia e a experiência na descoberta e desenvolvimento de fármacos (EVERTS et al., 2017).

1.3. A IMPORTÂNCIA DA ESTRUTURA E REPLICAÇÃO VIRAL PARA OS ANTIVIRAIS Infecções causadas por vírus como o vírus herpes simples tipo 1(HSV-1), HSV-2, vírus da imunodeficiência humana tipo 1 (HIV-1), papilomavírus humanos (HPVs), entre outros são comuns e sua incidência continua crescendo apesar da ampla gama de terapias disponíveis e/ou experimentais (WU et al., 2005).

Frequentemente, novos agentes infecciosos são transmitidos de outras espécies para a espécie humana, se estabelecendo na população. Infecções virais ainda são a principal causa de doenças e mortalidade em humanos (STRAUSS, 2008).

Na maioria dos casos, os antivirais existentes são para prevenir ou tratar essas doenças, pois ainda não há cura para elas. Apesar da variedade de drogas antivirais, novas estratégias de tratamento são necessárias devido a mutações virais e desenvolvimento de cepas resistentes aos atuais tratamentos (WU et al., 2005).

Algumas doenças virais como hepatite A e B, poliomielite, sarampo, rubéola, entre outras, podem ser prevenidas através de vacinas. Entretanto, há

28 muitas infecções causadas por vírus que atualmente não podem ser prevenidas pela vacinação. Isso inclui infecções crônicas como vírus da imunodeficiência humana (HIV), e hepatite C; infecções comuns em regiões endêmicas, que é o caso da dengue, febre amarela e hepatite E e infecções recém-descobertas, como por exemplo, febre do Nilo ocidental e síndrome respiratória aguda grave (SARS), síndrome respiratória do Oriente Médio (MERS) e a pandemia pela nova síndrome respiratória aguda grave (SARS- CoV-2), que até abril de 2020 já haviam ocorrido cerca de 1.285,257 casos de COVID-19 com taxa de mortalidade de aproximadamente 5,4%, ou seja, 70.344 óbitos (MAGDEN et al., 2005; TU et al., 2020).

A busca por novos antivirais e estratégias de combates a infecções por vírus continua sendo essencial, visto que há uma constante necessidade de produzir compostos que preencham as lacunas deixadas pelos atuais fármacos antivirais (MAGDEN et al., 2005).

A pesquisa por antivirais depende principalmente da compreensão detalhada das interações dos compostos antivirais com os vírus. Cada etapa do ciclo replicativo viral é um potencial local para intervenção de um agente antiviral (Figura 18). Dessa forma, o conhecimento da estrutura dos vírus é importante para sua identificação e possibilidade de desenvolvimento de ações contra ele (MAGDEN et al., 2005).

Figura 18 – Ciclo replicativo viral representando todas as etapas da replicação e possíveis alvos farmacológicos dos antivirais. Adaptado de: (DE CLERCQ E LI, 2016) O vírus é um parasita intracelular obrigatório e não se reproduz fora da célula. Quando o vírus reconhece um hospedeiro susceptível, ele integra seu genoma ao genoma celular, se apropriando da maquinaria da célula a fim de sintetizar componentes virais para montagem e liberação da progênie viral (LEIMAN et al., 2003; ROSSMANN, 2013).

Os vírus possuem DNA ou RNA como material genético e seu ácido nucléico pode ser fita simples ou fita dupla. Uma partícula viral infecciosa completa é chamada de vírion e consiste de ácido nucléico e um capsídeo, isto é, um envoltório protéico que envolve o material genético dos vírus. Por vezes, o genoma está envolto em uma camada de proteínas distinta do capsídeo (LODISH, 2000).

De maneira geral, os vírus apresentam algumas características importantes: (1) são pequenos, da ordem de nanômetros; (2) são totalmente dependentes de células vivas para sua replicação e existência; (3) possuem somente um tipo de ácido nucléico, DNA ou RNA; e (4) apresentam glicoproteínas - proteínas de ligação ao receptor celular - que acoplam às células e assim, consigam comandar as células como fábricas de produção de vírus. (FIELDS, 2007)

O capsídeo viral pode se apresentar de 3 formas diferentes, organizando os vírus em três grupos estruturais baseados em microscopia eletrônica. São eles: (a) icosaédrico, (b) forma de bastão e (c) complexo.

Vírus icosaédricos, como o picornavírus, herpesvírus e adenovírus, possuem os capsômeros (unidade proteica que constitui o capsídeo) organizados em 20 triângulos equiláteros. Vírus helicoidais possuem uma conformação em que o capsídeo apresenta forma uma estrutura parecida com um bastão. É o caso do rhabdovírus, filovírus e bunyavírus, E vírus complexos são aqueles que não apresentam uma morfologia característica do capsídeo, como por exemplo podovírus, myovírus e siphovírus (Figura 19) (FIELDS, 2007).

Figura 19 – Imagem demonstrativa dos vírus humanos mais comuns com seu tamanho relativo. Os ácidos nucléicos não estão em escala real. Adaptado de https://viralzone.expasy.org/5216 acesso em 19/06/2020. A classificação de Baltimore estabelece algumas categorias virais de acordo com o material genético contido no vírion: vírus de RNA sentido positivo (ex.: Rinovírus, Vírus da Hepatite C); vírus de RNA sentido negativo (vírus influenza, vírus ebola); vírus de RNA dupla fita (rotavírus, Vírus da Doença Infecciosa da Bursa,); Retrovírus (HIV, Vírus, Linfotrópico das Células T Humanas); Para-retrovírus (Vírus da Hepatite B); Vírus de DNA fita simples (Parvovirose, Bacteriófagos ɸX174); e Vírus de DNA fita dupla (Papiloma Vírus, Herpesvírus, Adenovírus e Poxvírus) (SANJUÁN E DOMINGO-CALAP, 2016).

Cada uma das 83 famílias virais possui uma estratégia de replicação diferente, que exige proteínas e enzimas únicas (HULO et al., 2011). Podemos citar como exemplo a replicação do HSV-1 e do EBOV, não apresentam similaridade. O HSV é um vírus dsDNA que codifica 73 proteínas e sua replicação acontece no núcleo da célula hospedeira onde novos genomas virais são encapsulados antes de realizar o brotamento através do RE e depois em vesículas que liberam o vírus para o exterior da célula (BOEHMER E NIMONKAR, 2003). O EBOV por sua vez é um vírus ssRNA que codifica oito proteínas, sua replicação ocorre no citoplasma da células hospedeira utilizando

32 seu próprio complexo de RNA polimerase dependente de RNA e seu brotamento ocorre diretamente na membrana da célula (ASCENZI et al., 2008).

O conhecimento da estrutura do vírus é fundamental para sua identificação e auxilia no entendimento de propriedades importantes e particularidades dos vírus. Os processos do ciclo replicativo como adsorção, penetração e, posteriormente maturação e liberação podem variar de acordo com o tipo viral, pois está ligada intrinsecamente à sua morfologia. Um exemplo é o fato de o vírus possuir ou não envelope viral. Vírus envelopados conseguem se ligar à membrana plasmática e entrar nas células hospedeiras. O envelope viral age como uma barreira de permeabilidade e protege o genoma do vírus, enquanto que vírus não envelopados tendem a ser mais resistentes ao calor e medidas clássicas de higiene como usar agentes desinfetantes (COLLIER L, 2016).

O ciclo replicativo viral é compreendido por diversas etapas como adsorção, penetração e desnudamento, transcrição e replicação do genoma viral. regulação gênica e montagem das novas partículas virais (CHINCHAR, 1999).

A infecção viral tem seu início pela ligação do vírion, através do capsídeo e/ou suas proteínas de envelope (macromoléculas específicas na superfície celular). A especificidade dessa interação, vírus/célula, é determinada pela presença do receptor viral na superfície das células hospedeiras (CHINCHAR, 1999).

A natureza de alguns desses receptores são de conhecidas, como macromoléculas celulares que estão diretamente envolvidas na interação do ligante, endocitose e reconhecimento celular (MILLARD et al., 2006). Contudo, alguns ainda não são descritos para infecções virais. Em geral, são glicoproteínas – modificadas pela fixação de açúcares para torná-los mais hidrofílicos, tornando-os assim mais funcionais em ambientes aquosos. Este processo usualmente ocorre em várias etapas, sendo a ligação inicial relativamente específica, seguida a uma segunda interação de receptores altamente específicos, como é o caso do HIV (HARPER, 2012; KHEDKAR E PATZAK, 2020).

Após a etapa de ligação, se inicia a penetração e desnudamento viral. Depois da ligação entre receptores, os vírus podem ser internalizados por uma variedade de processos. O processo mais comumente utilizado é denominado endocitose mediada por receptor, e é o mesmo mecanismo utilizado pela célula para o transporte de macromoléculas não permeáveis à membrana. Os vírus envelopados podem se fundir com a membrana celular ou podem ser absorvidos por vesículas, onde o processo de acidificação é crucial para desencadear o desnudamento do nucleocapsídeo no citoplasma celular (CHINCHAR, 1999; MILLARD et al., 2006; HARPER, 2012). Os vírus não envelopados também utilizam a estratégia da endocitose mediada por receptor, contudo o desnudamento não envolve a fusão com a membrana celular (CHINCHAR, 1999).

Seguindo o processo de multiplicação do vírus, ocorre a transcrição viral, onde a síntese dos ácidos nucléicos deste vírus é produzida através da utilização de enzimas celulares, cuja contribuição relativa vai depender do tipo de vírus e da molécula específica a ser transcrita (CHINCHAR, 1999). Em muitos casos, são produzidas proteínas específicas que desativam as funções celulares ou as adaptam à replicação viral. Tais proteínas são reconhecidas como proteínas precoces, pois são produzidas com a infecção precoce e também estão relacionadas com a produção do genoma viral da nossa geração de vírus a ser liberada pelas células infectadas (HARPER, 2012).

Durante a replicação viral ocorre a regulação gênica. Os vírus estão envolvidos em diversos processos, controlando mecanismos para controlar a expressão gênica da célula infectada e com isso maximizar a eficiência desta infecção. Em algumas infecções virais, a expressão gênica é dividida em fases, dentre as quais proteínas catalíticas e reguladoras são sintetizadas no início da infecção, enquanto a síntese de proteínas estruturais é limitada aos períodos tardios da infecção (CHINCHAR, 1999).

Apesar de todos componentes necessários para a produção de novas partículas virais terem sido produzidas na fase de replicação, o processo de montagem pode ser complexo. Pouco se sabe a cerca dos mecanismos moleculares que estão envolvidos e controlam a montagem. É fundamental

34 agregar os genomas virais com todas as proteínas envolvidas e garantir que todas se associem corretamente. Em alguns vírus simples, suas próprias proteínas orientam esta montagem, porém em vírus mais complexos faz-se necessário proteínas chaperonas específicas para a condução deste processo (HARPER, 2012).

Apesar do grande número de famílias de vírus, apenas três tipos de nucleocapsídeos são encontrados: complexo, helicoidal e icosaédrico (esférico). Durante o processo a montagem do vírus, os nucleocapsídeos migram para as membranas celulares onde as glicoproteínas virais se concentram. Então, após a interação entre as glicoproteínas virais e o nucleocapsídeo, este é envolvido pela membrana celular em um processo denominado brotamento. As proteínas das células hospedeiras são então excluídas da membrana e o envelope resultante contém apenas glicoproteínas codificadas pelo vírus em questão (CHINCHAR, 1999).

2. DISCUSSÃO O paradigma na obtenção de novos protótipos bioativos que sejam, ao mesmo tempo, eficientes como antivirais e de baixa toxicidade celular e sistêmica, aliado ao surgimento de resistência às terapias atuais, se revela um problema de difícil solução (LOLIS et al., 2008; ARORA E DIXIT, 2009; HAZUDA et al., 2009; KOZAL, 2009; DE CLERCQ, 2011b, 2012b)

No presente trabalho, estudamos um grupo de substâncias de origem natural, extraídas e isoladas das algas marinhas e substâncias sintéticas derivadas das naftoquinonas e das oxoquinolinas.

As substâncias de origem natural, plantas ou algas, apresentam-se como importante fonte de novas moléculas com diferentes atividades biológicas. Muitos terpenos têm elevada atividade antiviral, como já foi reportado em trabalhos prévios desenvolvidos em nosso laboratório, onde substâncias extraídas de algas apresentaram atividade anti-HSV-1 e anti-HIV anti-ZIKV e anti-CHIKV (BARBOSA et al., 2004; PEREIRA et al., 2005; CIRNE-SANTOS et al., 2006; SOUZA et al., 2007; CIRNE-SANTOS et al., 2008; ABRANTES et al., 2010; FERREIRA et al., 2010; CIRNE-SANTOS et al., 2019; ESTEVES et al., 2019; OLIVEIRA et al., 2019; CIRNE-SANTOS et al., 2020)

As exigências necessárias para que uma substância seja considerada um fármaco antiviral são sua efetividade inibitória e o mínimo de toxicidade sobre as células (FLINT, 2000; MELO et al., 2000).

ATIVIDADE ANTIVIRAL DAS ALGAS MARINHAS Dictyota friabilis E Osmundaria obtusiloba.

Diterpene From Marine Brown Alga Dictyota friabilis As A Potential Microbicide Against Hiv-1 In Tissue Explants

Pudemos observar que o pré-tratamento de células mononucleares do sangue periférico (PBMC) com dolabelladienetriol (Figura 1 do artigo 1 em anexo) resultou em significativa atividade inibitória da replicação do HIV-1 (Figura 2a do artigo 1 em anexo), com uma inibição variando entre 60 e 80% para ambos os isolados do HIV-1, X4-trópico (CXCR4) e R5-trópico (CCR5).

Nos macrófagos, o pré-tratamento com dolabelladienetriol por 1 ou 5 dias resultou em um forte bloqueio na replicação do HIV-1 (Figura 2b do artigo 1 em anexo). O mesmo resultado foi encontrado quando o dolabelladienetriol foi adicionado após a infecção das células. Os resultados mostram que este composto, dolabelladienetriol, que não é adicionado novamente após a infecção, tem um forte efeito supressor na replicação do HIV-1. De acordo com (BARROS et al., 2016) Diterpenos isolados da alga Canistrocarpus cervicornis apresentaram atividade inibitória da replicação do HIV-1 em células MT-2 na mesma concentração (DINESH et al., 2016) mostraram que diferentes frações da alga parda Sargassum swartzii inibiram entre 50% na menor concentração utilizada e atingiram uma inibição de 95,6% na maior concentração utilizada. (THUY et al., 2015) Constataram que os fucoidanos devirados das três algas pardas S. mcclurei, S. polycystum e Turbinara ornate, também demonstraram atividades anti-HIV com um IC50 médio variando de 0,33 a 0,7g/mL. Estes fucoidanos demonstraram inibição da infecção pelo HIV-1 quando pré- incubados em sistema acelular com o vírus, mostrando que foram capazes de bloquear as etapas iniciais da infecção pelo HIV, entretanto, diferente de nosso resultado, não demonstraram inibição no tratamento pós-infecção (LUTHULI et al., 2019).

Nossos resultados a cerca da viabilidade do explante em condições normais de cultivo demonstraram que a cultura dos explantes diminuiu o tecido escamoso estratificado (ectocérvice), com perda de celularidade nas primeiras 72 horas. A partir deste momento, a redução foi lenta e gradual até o final do período de cultivo (13 dias) (Figura 3 do artigo 1 em anexo). Em contrapartida, a redução de estruturas do tecido conjuntivo foi lenta até o final deste período. Observamos a presença de um pequeno número de microvasculatura e celularidade estromal em comparação com a fase inicial. Esse padrão histológico se assemelha ao relatado previamente por (CUMMINS et al., 2007)

A utilização das culturas de explantes do colo uterino e da glande do pênis atende aos requisitos das etapas dos estudos pré-clínicos para o estudo e desenvolvimento de novos microbicidas. Contemplando um cenário de inexistência de vacinas e medicamentos que possuem efeitos colaterais

37 graves, o desenvolvimento de novos microbicidas é uma estratégia muito importante para a prevenção de doenças (CUMMINS et al., 2007).

Microbicidas são possibilidades que, devido à sua baixa toxicidade e baixo custo de desenvolvimento, podem ser úteis para a prevenção da infecção pelo HIV (MORIN et al., 2012; OPOKU-ANANE et al., 2012; SHATTOCK E ROSENBERG, 2012). Nossos estudos envolvendo o composto extraído da D. fribialis, o dolabelladienotriol, demonstrou uma clara inibição dose-dependente da replicação do HIV-1. Ademais, nosso grupo determinou o efeito e o tempo de proteção promovido pelo composto, além da preservação da cultura de explantes.

De acordo com (LUTHULI et al., 2019) o microbicida Griffitsin extraído das algas vermelhas Griffithsia SP., é considerado o microbicida com o maior poder inibidor de penetração do HIV até o presente momento. Griffitsin demonstrou sua segurança em várias linhas celulares, incluindo as células do canal cervical, fibroblastos e linhas celulares dendríticas (BESEDNOVA et al., 2019).

Antiviral Effect Of The Seaweed Osmundaria obtusiloba Against The Zika Vírus

Neste estudo, os resultados demonstram que o extrato de O. obtusiloba inibiu significativamente a replicação viral quando as células foram tratadas com diferentes concentrações do extrato apresentando uma inibição dose dependente, resultando num valor de EC50 de 1,82μg/mL (Figura 1 do artigo 2 em anexo).

Outros trabalhos desenvolvidos em nosso grupo demonstraram valores de EC50 similares. A mesma alga O. obtusiloba quando testada contra o vírus

Chikungunya evidenciou um EC50 de 1,25 μg/mL (CIRNE-SANTOS et al., 2006). Em contra partida, a alga Canistrocarpus cervicornis quando testada sua atividade antiviral para Zika e Chikungunya apresentou EC50 = 0,95 μM e 1,3 μM, respectivamente (CIRNE-SANTOS et al., 2020).

Souza e colaboradores em 2012 testaram o potencial inibitório da O. obtusiloba contra o HSV-1 e HSV-2 e seus resultados demonstraram valores de EC50 de 42μg/mL e 12μg/mL (DE SOUZA et al., 2012).

Além disso, o extrato demonstrou ser menos citotóxico em células Vero, com um CC50 de 525μg/mL, evidenciando assim um índice de seletividade (SI) de 288 que, como demonstrado por diversos estudos, compostos que apresentem SI superiores a 100 são considerados compostos promissores (SILVA et al., 2011; ZANDI et al., 2011; DE SOUZA et al., 2012).

De acordo com a literatura, existe um número relevante de estudos antivirais para Dengue com resultados bastante promissores (ZANDI et al., 2011; LOW et al., 2017; LIM, 2019). Contudo, poucos são os estudos realizados para os demais arbovírus com resultados tão significativos quanto os existentes para a Dengue. O foco dos estudos para ZIKA, têm sido associado a síndromes graves (ALVARADO-SOCARRAS et al., 2018; BARBI et al., 2018; CIRNE-SANTOS et al., 2019; CIRNE-SANTOS et al., 2020).

Extratos de algas anteriormente foram relatados apresentando baixa citotoxicidade (ALENCAR et al., 2014), mas com considerável atividade antiviral, por exemplo, em estudos com HIV (NOGUEIRA et al., 2016; LUTHULI et al., 2019; WITTINE et al., 2019) e contra Herpes (DE SOUZA et al., 2012; DE SOUZA BARROS et al., 2017).

Neste trabalho, avaliamos inicialmente o potencial virucida deste extrato de alga marinha que demonstrou inativar as partículas de Zika (Figura 2 do artigo 2 em anexo). Os estudos demonstraram que o extrato de O. obtusiloba inativou as partículas virais em até 80% em concentrações de até 10μg/mL. Deste modo, estudos adicionais sobre o mecanismo de ação são necessários para o desenvolvimento de novas estratégias para a preparação de possíveis medidas preventivas.

Analisando características específicas acerca do mecanismo de ação do extrato em questão, ensaios como o tempo de adição dos extratos (Time of Addition Experiment) mostraram que o O. obtusiloba utilizado em diferentes momentos no tratamento, tanto pré-infecção como pós-infecção, possuem grande potencial para inibir a replicação do ZIKV, cerca de 60% após o tratamento até 3h de infecção. No tempo 0, no entanto, a adição do extrato foi concomitante com a infecção das células e apresentou inibição da replicação viral de 90%. Mesmo quando o extrato foi adicionado até 3 horas após a

39 infecção, foi possível observar 80% de inibição viral (Figura 3 do artigo 2 em anexo). Com a finalidade de obter a mesma avaliação, Zmurko et al. (2016) realizaram o ensaio de tempo de adição de substâncias contra o ZIKV com o inibidor da polimerase viral 7 DMA, mas sem pré-tratamento e com tempos de pós tratamentos de até 24h, diferentes dos utilizados por nosso grupo. O grupo de pesquisa anteriormente citado, observou que a adição do composto às células infectadas pode ser adiada até aproximadamente 10h após a infecção sem muita perda da potencia antiviral (ZMURKO et al., 2016).

Existe muito interesse em procurar combinações de medicamentos para a inibição da replicação de diversos vírus, como descrito para Dengue (YEO et al., 2015), HSV-1 (MANCINI et al., 2009), Chikungunya (MISHRA et al., 2016) e HIV (ALAM et al., 2019). Tais análises são realizadas com o objetivo de reduzir a concentração de substâncias utilizadas e otimizar os tratamentos, tornando- os mais eficazes e menos tóxicos. Os resultados aqui apresentados demonstraram um importante efeito sinérgico pela ação combinada da Ribavirina e extrato de O. obtusiloba (Figura 4 do artigo 2 em anexo), uma vez que o uso de ambos combinados em sub-doses foi capaz de inibir a replicação viral três vezes mais do que ambos avaliados separadamente.

ATIVIDADE ANTIVIRAL DOS DERIVADOS AMINONAFTOQUINONAS E HIDROXOQUINOLINOS.

Aminomethylnaphthoquinones And Hsv-1 - In Vitro And In Silico Evaluations Of Potential Antivirals

Neste trabalho, realizamos a avaliação da atividade anti-HSV-1 de três aminonaftoquinonas (1-3) que demonstrou uma alta atividade antiviral de maneira dose-dependente. Interessantemente, o composto 2 exibiu o menor

EC50 (EC50 = 0,83µM) comparado não apenas aos compostos 1 e 3, mas também quando comparado ao ACV e outros compostos como, PMEO-DAPym (6-fosfonilmetoxietoxi-2,4-diaminopirimidina), um análogo de fosfato nucleosídeo acíclico, cuja atividade anti-HSV é menor que a do ACV (tabela 1 do artigo 3 em anexo). Os resultados apresentados por PINTO et al., 2014, que estudou a atividade de doze aminometilnaftoquinonas contra o Vírus da Herpes

Bovina 5, tendo EC50 variando entre 3,2µM a 3,8µM (BALZARINI et al., 2013; PINTO et al., 2014)

A análise inicial da relação entre estrutura-atividade (SAR) mostrou que modificações estruturais no composto 2 (EC50 = 0,83µM) envolvendo a substituição do benzil no átomo de nitrogênio por um grupo n-butil (composto 1

EC50 = 1,7µM) ou os dois átomos de cloro no anel fenil para um grupo nitro

(composto 3, EC50 = 2,13µM) resultou em uma diminuição da atividade. Esses dados apontam, portanto, tanto para o substituinte benzil no átomo de nitrogênio quanto para os dois substituintes cloro no grupo fenil como importantes características estruturais para a modulação positiva da atividade antiviral da aminonaftoquinona.

A toxicidade in vitro e in silico dessas aminonaftoquinonas também foram avaliadas de forma teórica, utilizando uma abordagem de modelagem molecular. Entre as diferentes propriedades estereoletrônicas e físico-químicas, exploramos a reatividade química teórica dessas aminonaftoquinonas analisando a energia e a distribuição de seus HOMO e/ou LUMO. Consequentemente, quanto maior a diferença entre os níveis de energia HOMO e LUMO, maior o perfil de atividade dessa pequena série. Embora não tenha sido encontrada nenhuma relação reconhecível entre a distribuição HOMO e a atividade, quanto menor a distribuição LUMO no anel de naftoquinona, maior a atividade antiviral (figura 2 do artigo em anexo 3).

A análise conformacional também demonstrou que a orientação mais ampla do composto 3 pode ter comprometido seu perfil antiviral devido a impedimentos estéricos. Como os compostos 1 e 2 apresentaram um perfil promissor como moléculas protótipos, tais aspectos estruturais necessitam ser levados em consideração no planejamento de novas moléculas baseadas nelas. É imperativo ressaltar que a análise da potencialidade teórica para se tornar elegível a novos medicamentos comercializados (escore de drogas e semelhança de medicamentos) fortalece nossa proposta de que os compostos 1 e 2 são boas moléculas, sendo igualmente comparáveis ou inclusive melhores que o ACV para estudos contínuos, em contraste com algumas novas moléculas relatadas na literatura que ainda precisam ser aprimoradas para

41 uma investigação mais aprofundada (BALZARINI et al., 2013). Após elucidar os verdadeiros alvos desses compostos, proteínas virais ou proteínas da célula hospedeira, a relação estrutura-atividade quantitativa (QSAR) será significativa para estabelecer a relação direta dessas moléculas.

A concentração in vitro que inibe 50% da atividade mitocondrial celular (CC50) variou de 964 a 2,654µM para esses compostos, o que é maior que o do ACV. Outros estudos a cerca da citotoxicidade de análogos de naftoquinonas mostraram alguns compostos apresentaram valores de CC50 variando entre 12,8µM e 132,2µM (NGOC et al., 2019; ROA-LINARES et al.,

2019). O maior valor de CC50 encontrado para o composto 1 (CC50 = 2,654µM) sugere que é um protótipo antiviral promissor. Supostamente o perfil de citotoxicidade in vitro dessas moléculas depende a natureza do substituinte no grupo fenil e no átomo de nitrogênio. Esses resultados estão de acordo com nossos estudos anteriores de citotoxicidade de outras bases análogas de Mannich derivadas do lawsone, que mostraram aumento da citotoxicidade ao variar o comprimento da cadeia de carbono do substituinte no átomo de nitrogênio (C4H9

Atualmente, estudos in silico de parâmetros de toxicidade são realizados nos estágios preliminares do processo de desenvolvimento de medicamentos para economizar tempo, dinheiro e reduzir o uso de animais (VAN DE WATERBEEMD E GIFFORD, 2003). De acordo cocm a análise de citotoxicidade in vitro, nossos dados de toxicidade in silico sugerem que esses compostos apresentam baixo risco de toxicidade relacionado a efeitos de mutagenicidade e tumorigênese. Deve-se enfatizar que o baixo perfil teórico de toxicidade atribuído a esses compostos não significa ausência de efeitos tóxicos, mas aponta para o perfil promissor dessa pequena série para continuar a avaliação usando ensaios in vivo.

O valor de SI expressa a segurança de utilização de uma substância e é calculada usando as relações entre CC50 e EC50. Quanto maior o valor de SI, mais promissora é a substância para estudos in vivo e in vitro. Os compostos 1 e 2 apresentaram valores de SI (1.525,29 e 1.161,45 respectivamente) superiores ao ACV (SI= 880,73). Gonzaga e colaboradores em 2019 testaram

42 uma nova série de Bis-Naftoquinonas contra o vírus Zika, onde o composto mais promissor apresentou valor de SI = 1664, corroborando assim com nossos dados e o que reforça o potencial perfil de nossas substâncias para estudos posteriores (GONZAGA et al., 2019). Essas aminonaftoquinonas têm um potencial antiviral mais do que algumas moléculas descritas na literatura, incluindo produtos naturais e sintéticos, como as flavonas de Ficus benjamina (SI = 100 – 666) (YARMOLINSKY et al., 2012) e a 4’-fenilflavona (SI = 213,35) (HAYASHI et al., 2012), o Sulfonoquinovosildiacilglicerídeo do Azadirachta indica (SI = 12,4) (BHARITKAR et al., 2014) e o análogo tricíclico do ACV e do Ganciclovir portador do sistema 3,9-di-hidro-9-oxo-5H-imidazo[1,2-a]purina (SI = 1.000) (GOSLINSKI et al., 2003).

Para começar a entender o mecanismo de ação dessas aminonaftoquinonas, primeiramente avaliamos seu perfil virucida, que mostra se os compostos podem inativar a partícula viral. De acordo com (SCHUHMACHER et al., 2003), compostos que apresentam não apenas um padrão dose-dependente, mas também um padrão tempo-dependente, mostram atividade virucida significativa. Nossos resultados mostraram que, apesar do padrão dose-dependente dos compostos, as aminonaftoquinonas 1- 3 não possuem um perfil dependente do tempo, e todos eles apresentaram baixa atividade virucida a 50µM (menos de 40% da inibição de partículas virais). Estudos com outras classes de aminonaftoquinonas revelaram que tais compostos mostraram uma pequena inibição do ensaio virucida, sugerindo uma inativação da partícula viral pouco expressiva, corroborando assim com nossos dados (PINTO et al., 2014).

A maioria dos medicamentos comercializados para o tratamento de infecções por HSV (análogos de nucleosídeos) tem como alvo a DNA polimerase viral, o que aumenta o risco de pressão de seleção de cepas resistentes (DE CLERCQ, 2011b). Portanto, a busca de novos agentes antivirais com diferentes mecanismos de ação é um objetivo fundamental para o tratamento futuro de infecções causadas por cepas resistentes. Entre os novos alvos possíveis dos estágios do ciclo replicativo do HSV estão: a) proteínas que regulam a replicação viral – Proteínas IE (por exemplo ICP27); b) proteínas que sintetizam e empacotam DNA – Proteína E (por exemplo UL42);

43 e outras proteínas do vírion – Proteína L (por exemplo gD). Moléculas direcionadas a qualquer uma dessas proteínas podem interromper a replicação do HSV e apresentar perfil como potencial drogas antivirais. Curiosamente, todas as aminonaftoquinonas aqui testadas foram capazes de inibir a fase L do ciclo replicativo do HSV-1. De fato, os compostos 1 e 2 também inibiram as outras duas fases, principalmente o composto 2 com quase 100% de eficácia. É importante ressaltar que outros ensaios de mecanismo revelaram que apenas as aminonaftoquinonas 1 e 2 foram capazes de inibir a expressão da proteína gD. É possível que este efeito na expressão de gD possa ser causado por uma inibição precoce no ciclo de replicação do HSV-1, mas não para o composto 3, uma vez que o ensaio de tempo de adição deste composto mostrou inibição apenas na fase tardia. Além disso, o composto 1 também influenciou o peso molecular da gD, indicando uma possível inibição da glicosilação. Assim, a substituição dos dois átomos de cloro no anel fenil dos compostos 1 e 2 por um grupo nitro no composto 3, parece ter afetado sua orientação ao alvo. No geral, esses resultados biológicos reforçaram o perfil promissor desses compostos para análises adicionais in vitro e in vivo e como possíveis novos medicamentos antivirais.

Synthesis of 4-Oxoquinoline Acyclonucleoside Phosphonate Analogs And Anti-MAYV Evaluation.

Os resultados deste estudo mostraram que o composto 7a possui uma potente atividade anti-Mayaro, demonstrando um efeito potente na replicação viral com valor de EC50 de 0,83 µM e um índice de seletividade SI acima de 1000, o que parece ser considerado um excelente valor para um antiviral. Além disso, como mostrado na Tabela 1 (artigo 4 em anexo), essa parece ser uma característica exclusiva desse composto, pois não observamos efeitos anti- MAYV nos outros compostos testados. Recentemente, vários estudos mostraram compostos com efeitos na replicação de Mayaro, no entanto, os valores de EC50 dessas substâncias foram maiores aos demonstrados em nosso estudo (AMORIM et al., 2017; FERRAZ et al., 2019).

Em geral, o que diferencia um antiviral é seu mecanismo de ação, portanto, a busca e a compreensão dos possíveis efeitos que esse composto

44 pode causar nas etapas do ciclo replicativo devem ser realizadas de forma estratégica e cuidadosamente avaliada. No estudo do tempo de adição das substâncias em relação ao ciclo de replicação viral, nossos dados sugerem que, na concentração testada do composto 7a, seus efeitos parecem ocorrer em eventos muito precoces de replicação viral, uma vez que em um pré- tratamento de 2 horas já era possível observar um efeito maior que 40% de inibição na replicação viral. E com maior efeito sobre a concentração utilizada quando realizamos o tratamento simultâneo da infecção, levando a uma inibição de aproximadamente 60% (Figura 1 do artigo 4 em anexo), demonstrando comportamento diferente de outros estudos apresentados para o vírus Mayaro ou para diferentes vírus (KAUR et al., 2013; WINTACHAI et al., 2015; AMORIM et al., 2017). Curiosamente, essas porcentagens de inibições diminuem levemente no tratamento desde a primeira hora após a infecção, demonstrando um efeito duradouro que, embora seja menor, mas é mantido até a terceira hora.

Nesse contexto, empregamos ferramentas computacionais para investigar o alvo antiviral putativo de 7a de acordo com os dados experimentais, observamos que 7a pode se ligar teoricamente ao complexo de proteínas do envelope do MAYV, especialmente no sítio 3. É importante ressaltar que a ligação de 7a entre as proteínas E1 e E2 pode impedir alterações conformacionais das proteínas do envelope, o que, por sua vez, pode levar a o efeito virucida desse composto, bem como a inibição de eventos precoces na replicação viral (RASHAD E KELLER, 2013). Além disso, esse efeito estrutural também pode afetar as etapas pós-entrada, como montagem e brotamento de vírus (BYRD E KIELIAN, 2017; CHEN et al., 2018), o que pode explicar a manutenção da atividade antiviral de 7a quando adicionada algumas horas após a infecção (embora tenha sido observada uma atividade mais reduzida).

Questões farmacocinéticas e toxicológicas são as principais preocupações no processo de desenvolvimento de medicamentos e uma das principais razões para a falha de medicamentos em ensaios clínicos (WANG et al., 2015; DONG et al., 2018). Consequentemente, empregamos ferramentas in silico para avaliar o perfil semelhante ao medicamento do composto 7a. Curiosamente, este composto mostrou farmacocinética teórica promissora e

45 propriedades toxicológicas. Por exemplo, este composto é compatível com a administração oral, pois mostrou boa biodisponibilidade oral e absorção intestinal, bem como baixa probabilidade de apresentar toxicidade farmacocinética e pré-clínica de acordo com as regras da indústria farmacêutica. Os riscos de toxicidade previstos sugeriram um perfil de segurança para a ribavirina comparável ao observado para 7a. Por outro lado, um alerta de hepatotoxicidade foi gerado para este composto, provavelmente associado ao grupo fosfonato. É importante ressaltar que a cloroquina apresenta risco de hepatotoxicidade, além de riscos de cardiotoxicidade e genotoxicidade. De fato, muitos desses efeitos tóxicos têm sido relacionados experimentalmente à cloroquina (TRAEBERT et al., 2004; WIELGO-POLANIN et al., 2005; OLSSON, 2007; FANG et al., 2013; THANACOODY, 2016) e, ainda assim, chegou ao mercado. Portanto, nossos resultados reforçam o potencial do composto 7a como candidato a medicamentos anti-MAYV que merece investigações futuras.

3. CONCLUSÃO A busca de antivirais para o tratamento de doenças virais de importância clínica sempre foi uma grande questão de saúde pública no que tange a sociedade moderna. A importância de conhecermos os diversos passos da replicação viral auxilia a resposta aos desafios de se encontrar moléculas bioativas e por conseqüência elucidar seus possíveis mecanismos de ação.

Neste trabalho pudemos analisar quatro diferentes moléculas e suas ações frente a quatro diferentes vírus de importância clínica, mostrando assim, a diversidade e complexidade no estudo da virologia e na busca e obtenção por novos antivirais, sendo eles de origem natural ou sintética.

A primeira substância utilizada foi o dolabelladienotriol, isolada da alga parda Dictyota friabilis. Em experimentos realizados com o HIV-1, pudemos observar que o pré-tratamento de PBMCs e macrófagos com a substância dolabelladienotriol mostrou uma alta inibição da replicação viral e após utilizarmos o modelo ex vivo de explante de cérvix uterina, pudemos constatar o potencial microbicida desta substância em diferentes concentrações.

A segunda substância utilizada foi o extrato da alga vermelha Osmundaria obtusiloba. Nossos resultados demonstraram que tal extrato apresentou atividade anti-ZIKV com significante efeito virucida, além de ter demonstrado em ensaios sobre sinergismo uma boa combinação com o fármaco de referência, a ribavirina.

Em nosso terceiro trabalho utilizamos três derivados de naftoquinonas frente ao HSV-1. Nossos resultados demonstraram que estes derivados possuem baixo perfil citotóxico e que todos inibiram a fase-L do ciclo lítico de replicação. Com base em análises estruturais e distribuição do perfil de energia pudemos concluir que estas novas moléculas são promissoras por serem funcionalmente e estruturalmente diferentes do fármaco de referência, porém mais estudos ainda são necessários.

Nosso último trabalho abordou a atividade anti-MAYV de uma nova classe de moléculas derivadas das quinolinas. A molécula 7ª mostrou ter um perfil mais potente e seguro do que o fármaco comercial cloroquina. Em estudos

47 acerca do mecanismo de ação, a substância 7a mostrou inibição dos eventos primários da replicação viral, e após análise in silico foi demonstrado que este composto se liga ao complexo E1-E2 e pode provocar mudanças conformacionais importantes para a interação com a célula hospedeira ou do processo de fusão.

Apesar de alguns destes vírus em questão já possuírem tratamento, é de notório saber que muito dos fármacos utilizados ainda produzem efeitos colaterais fortes e muitas vezes resistência. A busca por fármacos cada vez menos tóxicos se faz necessário em um cenário onde o número de casos de doenças virais continua crescendo sem a previsão de uma cura concreta.

Como pudemos demonstrar, a biodiversidade marinha é de suma importância para o estudo e desenvolvimento de antivirais, assim como a síntese de novas moléculas baseadas em moléculas bioativas. Porém, diante de um cenário pandêmico, como o atual, é necessário avaliar a utilização de substâncias já previamente aprovadas pelos órgãos vigentes como possíveis novas fontes de moléculas ativas para doenças reemergentes.

4. REFERÊNCIAS BIBLIOGRÁFICAS AASLD/IDSA HCV Guidance Panel. Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus. Hepatology. 2015; 62 (3):932-54. Abrantes JL, Barbosa J, Cavalcanti D, Pereira RC, Frederico Fontes CL, Teixeira VL, Moreno Souza TL, Paixão IC. The effects of the diterpenes isolated from the Brazilian brown algae Dictyota pfaffii and Dictyota menstrualis against the herpes simplex type-1 replicative cycle. Planta Med. 2010; 76 (4):339-44. Agbowuro AA, Huston WM, Gamble AB, Tyndall JDA. Proteases and protease inhibitors in infectious diseases. 2018; 38 (4):1295-331. Alam MM, Kuwata T, Tanaka K, Alam M, Takahama S, Shimura K, Matsuoka M, Fukuda N, Morioka H, Tamamura H, Matsushita S. Synergistic inhibition of cell-to-cell HIV-1 infection by combinations of single chain variable fragments and fusion inhibitors. Biochem Biophys Rep. 2019; 20:100687. Alencar DB, Silva SR, Pires-Cavalcante KM, Lima RL, Pereira Júnior FN, Sousa MB, Viana FA, Nagano CS, Nascimento KS, Cavada BS, Sampaio AH, Saker-Sampaio S. Antioxidant potential and cytotoxic activity of two red seaweed species, Amansia multifida and Meristiella echinocarpa, from the coast of Northeastern Brazil. An Acad Bras Cienc. 2014; 86 (1):251-63. Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, Berger EA. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science. 1996; 272 (5270):1955-8. Alvarado-Socarras JL, Idrovo Á J, Contreras-García GA, Rodriguez-Morales AJ, Audcent TA, Mogollon-Mendoza AC, Paniz-Mondolfi A. Congenital microcephaly: A diagnostic challenge during Zika epidemics. Travel Med Infect Dis. 2018; 23:14-20. Alves Galvão MG, Rocha Crispino Santos MA, Alves da Cunha AJ. Amantadine and rimantadine for influenza A in children and the elderly. Cochrane Database Syst Rev. 2012; 1:Cd002745. Amorim R, de Meneses MDF, Borges JC, da Silva Pinheiro LC, Caldas LA, Cirne-Santos CC, de Mello MVP, de Souza AMT, Castro HC, de Palmer Paixão ICN, Campos RM, Bergmann IE, Malirat V, Bernardino AMR, Rebello MA, Ferreira DF. Thieno[2,3-b]pyridine derivatives: a new class of antiviral drugs against Mayaro virus. Arch Virol. 2017; 162 (6):1577- 87. Arora P, Dixit NM. Timing the emergence of resistance to anti-HIV drugs with large genetic barriers. PLoS Comput Biol. 2009; 5 (3):e1000305. Ascenzi P, Bocedi A, Heptonstall J, Capobianchi MR, Di Caro A, Mastrangelo E, Bolognesi M, Ippolito G. Ebolavirus and Marburgvirus: insight the Filoviridae family. Mol Aspects Med. 2008; 29 (3):151-85. Asselah T, Boyer N, Saadoun D, Martinot-Peignoux M, Marcellin P. Direct- acting antivirals for the treatment of hepatitis C virus infection: optimizing current IFN-free treatment and future perspectives. Liver Int. 2016; 36 Suppl 1:47-57.

Baba M, Tanaka H, De Clercq E, Pauwels R, Balzarini J, Schols D, Nakashima H, Perno CF, Walker RT, Miyasaka T. Highly specific inhibition of human immunodeficiency virus type 1 by a novel 6-substituted acyclouridine derivative. Biochem Biophys Res Commun. 1989; 165 (3):1375-81. Baker DA, Ferris DG, Martens MG, Fife KH, Tyring SK, Edwards L, Nelson A, Ault K, Trofatter KF, Liu T, Levy S, Wu J. Imiquimod 3.75% cream applied daily to treat anogenital warts: combined results from women in two randomized, placebo-controlled studies. Infect Dis Obstet Gynecol. 2011; 2011:806105. Balzarini J, Andrei G, Balestra E, Huskens D, Vanpouille C, Introini A, Zicari S, Liekens S, Snoeck R, Holý A, Perno CF, Margolis L, Schols D. A multi- targeted drug candidate with dual anti-HIV and anti-HSV activity. PLoS Pathog. 2013; 9 (7):e1003456. Barbi L, Coelho AVC, Alencar LCA, Crovella S. Prevalence of Guillain-Barré syndrome among Zika virus infected cases: a systematic review and meta-analysis. Braz J Infect Dis. 2018; 22 (2):137-41. Barbosa JP, Pereira RC, Abrantes JL, Cirne dos Santos CC, Rebello MA, Frugulhetti IC, Texeira VL. In vitro antiviral diterpenes from the Brazilian brown alga Dictyota pfaffii. Planta Med. 2004; 70 (9):856-60. Barros CdS, Cirne-Santos CC, Garrido V, Barcelos I, Stephens PRS, Giongo V, Teixeira VL, de Palmer Paixão ICN. Anti-HIV-1 activity of compounds derived from marine alga Canistrocarpus cervicornis. Journal of Applied Phycology. 2016; 28 (4):2523-7. Bate J, Baker S, Breuer J, Chisholm JC, Gray J, Hambleton S, Houlton A, Jit M, Lowis S, Makin G, O'Sullivan C, Patel SR, Phillips R. PEPtalk2: results of a pilot randomised controlled trial to compare VZIG and aciclovir as postexposure prophylaxis (PEP) against chickenpox in children with cancer. 2019; 104 (1):25-9. Beadle JR, Wan WB, Ciesla SL, Keith KA, Hartline C, Kern ER, Hostetler KY. Synthesis and antiviral evaluation of alkoxyalkyl derivatives of 9-(S)-(3- hydroxy-2-phosphonomethoxypropyl)adenine against cytomegalovirus and orthopoxviruses. J Med Chem. 2006; 49 (6):2010-5. Berger C, Romero-Brey I, Radujkovic D, Terreux R, Zayas M, Paul D, Harak C, Hoppe S, Gao M, Penin F, Lohmann V, Bartenschlager R. Daclatasvir- like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication. Gastroenterology. 2014; 147 (5):1094-105.e25. Bergmann W, Feeney RJ. THE ISOLATION OF A NEW THYMINE PENTOSIDE FROM SPONGES1. Journal of the American Chemical Society. 1950; 72 (6):2809-10. Berkhout B, Eggink D, Sanders RW. Is there a future for antiviral fusion inhibitors? Curr Opin Virol. 2012; 2 (1):50-9. Besednova NN, Zvyagintseva TN, Kuznetsova TA, Makarenkova ID, Smolina TP, Fedyanina LN, Kryzhanovsky SP, Zaporozhets TS. Marine Algae Metabolites as Promising Therapeutics for the Prevention and Treatment of HIV/AIDS. Metabolites. 2019; 9 (5). Beutner KR, Spruance SL, Hougham AJ, Fox TL, Owens ML, Douglas JM, Jr. Treatment of genital warts with an immune-response modifier (imiquimod). J Am Acad Dermatol. 1998a; 38 (2 Pt 1):230-9.

Beutner KR, Tyring SK, Trofatter KF, Jr., Douglas JM, Jr., Spruance S, Owens ML, Fox TL, Hougham AJ, Schmitt KA. Imiquimod, a patient-applied immune-response modifier for treatment of external genital warts. Antimicrob Agents Chemother. 1998b; 42 (4):789-94. Bharitkar YP, Bathini S, Ojha D, Ghosh S, Mukherjee H, Kuotsu K, Chattopadhyay D, Mondal NB. Antibacterial and antiviral evaluation of sulfonoquinovosyldiacylglyceride: a glycolipid isolated from Azadirachta indica leaves. Lett Appl Microbiol. 2014; 58 (2):184-9. Black CP. Systematic review of the biology and medical management of respiratory syncytial virus infection. Respir Care. 2003; 48 (3):209-31; discussion 31-3. Boehmer PE, Nimonkar AV. Herpes virus replication. IUBMB Life. 2003; 55 (1):13-22. Brito MA. Fármacos recentes usados para o tratamento da infecção pelo HIV-1: enfuvirtida, maraviroc, raltegravir e etravirina. Revista de Ciências Farmacêuticas Básica e Aplicada. 2011; 32 (2):159-68. Broder CC, Xu K, Nikolov DB, Zhu Z, Dimitrov DS, Middleton D, Pallister J, Geisbert TW, Bossart KN, Wang LF. A treatment for and vaccine against the deadly Hendra and Nipah viruses. Antiviral Res. 2013; 100 (1):8-13. Brouwer WP, Xie Q, Sonneveld MJ, Zhang N, Zhang Q, Tabak F, Streinu- Cercel A, Wang JY, Idilman R, Reesink HW, Diculescu M, Simon K, Voiculescu M, Akdogan M, Mazur W, Reijnders JG, Verhey E, Hansen BE, Janssen HL. Adding pegylated interferon to entecavir for hepatitis B e antigen-positive chronic hepatitis B: A multicenter randomized trial (ARES study). Hepatology. 2015; 61 (5):1512-22. Buti M, Riveiro-Barciela M, Esteban R. Management of direct-acting antiviral agent failures. J Hepatol. 2015; 63 (6):1511-22. Byrd EA, Kielian M. An Alphavirus E2 Membrane-Proximal Domain Promotes Envelope Protein Lateral Interactions and Virus Budding. 2017; 8 (6). Cady SD, Mishanina TV, Hong M. Structure of amantadine-bound M2 transmembrane peptide of influenza A in lipid bilayers from magic-angle- spinning solid-state NMR: the role of Ser31 in amantadine binding. J Mol Biol. 2009; 385 (4):1127-41. CDC CfDC. Varicella-zoster immune globulin for the prevention of chickenpox. Recommendations of the Immunization Practices Advisory Committee, Centers for Disease Control. Ann Intern Med. 1984; 100 (6):859-65. CDC CfDC. Prevention of varicella: Recommendations of the Advisory Committee on Immunization Practices (ACIP). Centers for Disease Control and Prevention. MMWR Recomm Rep. 1996; 45 (Rr-11):1-36. CDC CfDC. FDA approval of an extended period for administering VariZIG for postexposure prophylaxis of varicella. MMWR Morb Mortal Wkly Rep. 2012; 61 (12):212. Chen L, Wang M, Zhu D. Implication for alphavirus host-cell entry and assembly indicated by a 3.5Å resolution cryo-EM structure. 2018; 9 (1):5326. Chen ST, Dou J, Temple R, Agarwal R, Wu KM, Walker S. New therapies from old medicines. Nat Biotechnol. 2008; 26 (10):1077-83.

Chen W, Zhan P, Wu J, Li Z, Liu X. The development of HEPT-type HIV non- nucleoside reverse transcriptase inhibitors and its implications for DABO family. Curr Pharm Des. 2012; 18 (27):4165-86. Chinchar VG. Replication of Viruses. Encyclopedia of Virology. 1999:1471-8. Cilliers T, Moore P, Coetzer M, Morris L. In vitro generation of HIV type 1 subtype C isolates resistant to enfuvirtide. AIDS Res Hum Retroviruses. 2005; 21 (9):776-83. Cirne-Santos C, Barros C, Gomes M, Gomes R, Cavalcanti D, Obando J, Ramos C, Villaça R, Teixeira V, Paixão I. In Vitro Antiviral Activity Against Zika Virus From a Natural Product of the Brazilian Brown Seaweed Dictyota menstrualis. Nat Prod Commun. 2019; 14:1934578X85912. Cirne-Santos C, Barros C, Oliveira M, Rabelo V, Azevedo R, Teixeira V, Ferreira D, Paixão I. In vitro Studies on The Inhibition of Replication of Zika and Chikungunya Viruses by Dolastane Isolated from Seaweed Canistrocarpus cervicornis. Sci Rep. 2020; 10. Cirne-Santos CC, Souza TM, Teixeira VL, Fontes CF, Rebello MA, Castello- Branco LR, Abreu CM, Tanuri A, Frugulhetti IC, Bou-Habib DC. The dolabellane diterpene Dolabelladienetriol is a typical noncompetitive inhibitor of HIV-1 reverse transcriptase enzyme. Antiviral Res. 2008; 77 (1):64-71. Cirne-Santos CC, Teixeira VL, Castello-Branco LR, Frugulhetti IC, Bou-Habib DC. Inhibition of HIV-1 replication in human primary cells by a dolabellane diterpene isolated from the marine algae Dictyota pfaffii. Planta Med. 2006; 72 (4):295-9. Cohen SS. Introduction to the biochemistry of D-arabinosyl nucleosides. Prog Nucleic Acid Res Mol Biol. 1966; 5:1-88. Collier L KP, Oxford J. 2016. Human Virology. 5th edition. . 5th ed: Oxford University Press. Crouse J, Kalinke U, Oxenius A. Regulation of antiviral T cell responses by type I interferons. Nat Rev Immunol. 2015; 15 (4):231-42. Ctrnáctá V, Fritzler JM, Surinová M, Hrdý I, Zhu G, Stejskal F. Efficacy of S- adenosylhomocysteine hydrolase inhibitors, D-eritadenine and (S)- DHPA, against the growth of Cryptosporidium parvum in vitro. Exp Parasitol. 2010; 126 (2):113-6. Cummins JE, Jr., Guarner J, Flowers L, Guenthner PC, Bartlett J, Morken T, Grohskopf LA, Paxton L, Dezzutti CS. Preclinical testing of candidate topical microbicides for anti-human immunodeficiency virus type 1 activity and tissue toxicity in a human cervical explant culture. Antimicrob Agents Chemother. 2007; 51 (5):1770-9. Davies WL, Grunert RR, Haff RF, McGahen JW, Neumayer EM, Paulshock M, Watts JC, Wood TR, Hermann EC, Hoffmann CE. ANTIVIRAL ACTIVITY OF 1-ADAMANTANAMINE (AMANTADINE). Science. 1964; 144 (3620):862-3. De Clercq E. Strategies in the design of antiviral drugs. Nat Rev Drug Discov. 2002; 1 (1):13-25. De Clercq E. Clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of DNA virus and retrovirus infections. Clinical microbiology reviews. 2003; 16 (4):569-96.

De Clercq E. Discovery and development of BVDU (brivudin) as a therapeutic for the treatment of herpes zoster. Biochem Pharmacol. 2004; 68 (12):2301-15. De Clercq E. Antiviral agents active against influenza A viruses. Nat Rev Drug Discov. 2006; 5 (12):1015-25. De Clercq E. Three decades of antiviral drugs. Nature Reviews Drug Discovery. 2007; 6 (12):941-. De Clercq E. The discovery of antiviral agents: ten different compounds, ten different stories. Med Res Rev. 2008; 28 (6):929-53. De Clercq E. Another ten stories in antiviral drug discovery (part C): "Old" and "new" antivirals, strategies, and perspectives. Med Res Rev. 2009a; 29 (4):611-45. De Clercq E. Antiviral drug discovery: ten more compounds, and ten more stories (part B). Med Res Rev. 2009b; 29 (4):571-610. De Clercq E. Antiviral therapy: quo vadis? Future Med Chem. 2010; 2 (7):1049- 53. De Clercq E. A 40-year journey in search of selective antiviral chemotherapy. Annu Rev Pharmacol Toxicol. 2011a; 51:1-24. De Clercq E. The clinical potential of the acyclic (and cyclic) nucleoside phosphonates: the magic of the phosphonate bond. Biochem Pharmacol. 2011b; 82 (2):99-109. De Clercq E. The race for interferon-free HCV therapies: a snapshot by the spring of 2012. Rev Med Virol. 2012a; 22 (6):392-411. De Clercq E. Ten paths to the discovery of antivirally active nucleoside and nucleotide analogues. Nucleosides Nucleotides Nucleic Acids. 2012b; 31 (4):339-52. De Clercq E. The acyclic nucleoside phosphonates (ANPs): Antonin Holy's legacy. Med Res Rev. 2013a; 33 (6):1278-303. De Clercq E. Antivirals: past, present and future. Biochem Pharmacol. 2013b; 85 (6):727-44. De Clercq E. Dancing with chemical formulae of antivirals: a personal account. Biochem Pharmacol. 2013c; 86 (6):711-25. De Clercq E. Highlights in antiviral drug research: antivirals at the horizon. Med Res Rev. 2013d; 33 (6):1215-48. De Clercq E. Current race in the development of DAAs (direct-acting antivirals) against HCV. Biochem Pharmacol. 2014; 89 (4):441-52. De Clercq E. Development of antiviral drugs for the treatment of hepatitis C at an accelerating pace. Rev Med Virol. 2015a; 25 (4):254-67. De Clercq E. Ebola virus (EBOV) infection: Therapeutic strategies. Biochem Pharmacol. 2015b; 93 (1):1-10. De Clercq E, Balzarini J, Descamps J, Eckstein F. Antiviral, antimetabolic and antineoplastic activities of 2'- or 3'-amino or -azido-substituted deoxyribonucleosides. Biochem Pharmacol. 1980; 29 (12):1849-51. De Clercq E, Descamps J, P DES, Holyacute A. (S)-9-(2,3- Dihydroxypropyl)adenine: An Aliphatic Nucleoside Analog with Broad- Spectrum Antiviral Activity. Science. 1978; 200 (4341):563-5. De Clercq E, Field HJ. Antiviral prodrugs - the development of successful prodrug strategies for antiviral chemotherapy. Br J Pharmacol. 2006; 147 (1):1-11.

De Clercq E, Holy A. Acyclic nucleoside phosphonates: a key class of antiviral drugs. Nat Rev Drug Discov. 2005; 4 (11):928-40. De Clercq E, Holy A, Rosenberg I, Sakuma T, Balzarini J, Maudgal PC. A novel selective broad-spectrum anti-DNA virus agent. Nature. 1986; 323 (6087):464-7. De Clercq E, Li G. Approved Antiviral Drugs over the Past 50 Years. Clinical microbiology reviews. 2016; 29 (3):695-747. de Niet A, Stelma F, Jansen L, Sinnige MJ, Remmerswaal EB, Takkenberg RB, Kootstra NA, Reesink HW, van Lier RA, van Leeuwen EM. Restoration of T cell function in chronic hepatitis B patients upon treatment with interferon based combination therapy. J Hepatol. 2016; 64 (3):539-46. de Smet MD, Meenken CJ, van den Horn GJ. Fomivirsen - a phosphorothioate oligonucleotide for the treatment of CMV retinitis. Ocul Immunol Inflamm. 1999; 7 (3-4):189-98. de Souza Barros C, Garrido V, Melchiades V, Gomes R, Gomes MWL, Teixeira VL, de Palmer Paixão ICN. Therapeutic efficacy in BALB/C mice of extract from marine alga Canistrocarpus cervicornis (Phaeophyceae) against herpes simplex virus type 1. Journal of Applied Phycology. 2017; 29 (2):769-73. de Souza LM, Sassaki GL, Romanos MT, Barreto-Bergter E. Structural characterization and anti-HSV-1 and HSV-2 activity of glycolipids from the marine algae Osmundaria obtusiloba isolated from Southeastern Brazilian coast. Mar Drugs. 2012; 10 (4):918-31. Delang L, Neyts J. Medical treatment options for COVID-19. Eur Heart J Acute Cardiovasc Care. 2020; 9 (3):209-14. Dinesh S, Menon T, Hanna LE, Suresh V, Sathuvan M, Manikannan M. In vitro anti-HIV-1 activity of fucoidan from Sargassum swartzii. Int J Biol Macromol. 2016; 82:83-8. Dolin R. A new class of anti-HIV therapy and new challenges. N Engl J Med. 2008; 359 (14):1509-11. Dong J, Wang NN, Yao ZJ, Zhang L, Cheng Y, Ouyang D, Lu AP, Cao DS. ADMETlab: a platform for systematic ADMET evaluation based on a comprehensively collected ADMET database. 2018; 10 (1):29. Edward Connor M, Franklin Top M, Andrew Kramer, PhD, Miriam Schneider, Joni, Love R, David Carlin, PhD, MedImmune, Inc, Gaithersburg,, MD; George Siber M, Jeanne Leszczynski, DrPH, James McIver,, PhD MPHBL, Boston,, MA; Val Hemming M, Sally Henson, Uniformed Services, Health University B, MD; Susan Spruill, MS, Christine, Colven PPD, Wilmington, NC;, Janet Wittes P, Statistics Collaborative, Washington, DC; Dharmapuri Vidyasagar, MD, C. Lucy Park, MD, University of Illinois, at Chicago C, IL; John F. Modlin, MD, Torunn Rhodes, MD,, Dartmouth-Hitchcock Medical Center L, NH; Arun K., Pramanik M, Sue Jue, MD, Ed Gustavson, MD, Martic Smith,, RN LSUMC, Shreveport, LA;, Gerard P. Rabalais M, Sofia Franco, MD Shirley Wilkerson MD,, Karen Bibb M, Kosair Children’s Hospital, Louisville, KY; Major, Bruce E. Pichoff I, MD, Richard Blaszak, MD, Shon Remich, MD,, Matthew Rettke M, William Beaumont Army Medical Center, El, Paso TLEW, MD, Meghan C. McDonald, MD,, Karen E. Johnson M, Karen Adams, RN, Baylor College of, Medicine H, TX; Stephen A. Chartrand, MD, Mark C., Wilson M, Creighton School of

Medicine, Omaha, NE; Gregory, J. Redding M, Dennis Mayock, MD, Heather J. S. Hanna, RN,, University of Washington School of Medicine S, WA; David, K. Stevenson M, Ronald L. Ariagno, MD, Marian M. Adams,, MD SM, BS, Packard Children’s Hospital at Stanford, Palo, Alto CKNSS, MD, Yolande A. Smith, MD,, Deborah A. Hoy M, Ildiko Kunos, MD, Pamela Angelus, RN,, Georgetown University Medical Center W, DC; Ram, Yogev M, Melissa Davis, RN, Dietra D. Millard, MD, Children’s, Memorial Hospital C, IL; Jean J. Steichen, MD, Tari Gratton, PA, Children’s Hospital Medical Center, Cincinnati, OH; Judy, Bernbaum M, Beverly Banks, MD, Sharon Zirin, R.N, Jane, Fricko R, Linda Corcoran, RN, Children’s Hospital of Philadelphia, Philadelphia, PA; Cynthia T. Barrett, MD, Nancy L. Pusser,, MD ERS, MD, UCLA School of Medicine, Los, Angeles CERC, MD, Alan Fujii, MD, Boston City, Hospital B, MA; Laurence B. Givner, MD, T. Michael, O’Shea M, Beth Norman, RN, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC; Terese L. Jennings, MD, Robert E. Kimura, MD, Ushanalini Vasan, MD, Amy, Hennessy R, Rush Presbyterian Medical College, Chicago, IL;, Andrea Kovacs M, Robert deLemos, MD, Margaret Khoury,, MD LUMC, CA Women’s & Children’s, Hospital LSAN, MD, State University of New, York SB, NY; Paul E. Stobie, MD, Judy Coggins, RN,, Kathy M. Perea R, Presbyterian Hospital, Albuquerque, NM;, Stephen C. Aronoff M, Susan Lynch, MD, Mark Polak, MD,, Linda S. Baer R, West Virginia University Health Sciences Center, Morgantown, WV; Caroline B. Hall, MD, Christine Long,, MPH UoRMC, Rochester, NY;, Richard Inwood M, Excelsis Antonio, MD, Bellipady C. Rai,, MD MG, MD, Lorraine E. Solecki, BSN, Newark Beth, Israel Medical Center N, NJ; Martha L. Lepow, MD,Michael Horgan, MD, Holly Swanson, MD, Albany Medical College, Albany, NY; Roy C. Maynard, MD, David Brasel, MD, Kathy, Werner B, Children’s Health Care Hospital, Minneapolis, MN;, David G. Oelberg M, Eastern Virginia Medical School, Children’s Hospital of the King’s Daughter, Norfolk, VA; Pablo J., Sanchez M, R. Sue Broyles, MD, Fiker Zeray, RN University of, Texas Southwestern Medical Center at Dallas D, TX; Elaine S., Barefield M, Monica V. Collins, RN, MEd, University of Alabama at Birmingham, Birmingham, AL; Shobhana A. Desai, MD,, Thomas E. Wiswell M, James E. Cullen, RN, BSN, Jefferson, Medical College P, PA; William C. Gruber, MD,, Thomas A. Hazinski M, Jane Baker, RN, Stefanie Freeman, RN,, MSN OS, RN, MP, Vanderbilt University Medical, Center N, TN; Jeffrey S. Gerdes, MD, Reyin Lien, MD,, Jean Ann Cieplinski R, Pennsylvania Hospital, Philadelphia,, PA; J.Owen Hendley M, Robert J. Boyle, MD, Melinda Robinson, RN, University of Virginia Health Sciences Center, Charlottesville, VA; Kathleen D. Pfeffer, MD, Dale Robertson, MD,, Chris Portelli R, University of Utah Medical Center, Salt Lake, City UEAS, MD, Grace Fabillar, RN, Deidre, Bloom M, Sam Zinner, MD, Kaiser Foundation Hospital, Los, Angeles CRCW, MD, Debra A. Tristram, MD,, Cynthia Shuff R, Kathy Alessi, RN Children’s Hospital of Buffalo, Buffalo, NY; Richard Lemen, MD, Leslie Barton, MD, Nahid, Maleniazi R, University of Arizona Health Sciences Center,, Tucson AMJR, MD, Kathy Goei, RN, MSN, San, Antonio Pediatric Pulmonary & Critical Care Associates S, Antonio TJWS, MD, MPH, Harvard Medical

School,, Boston MRBT, MD, Medical University of South, Carolina C, SC; Ellen R. Wald, MD, Marian Michaels,, MD M, Michael Green, MD, MPH, Karen Smail, RN, MSN,, CRNP CsHoP, Pittsburgh, PA; Andrew, M. Davey M, David G. Rupar, MD, Hortense Turner, RN, MSN,, Carolinas Medical Center C, NC; Penelope H. Dennehy,, MD RIH, Providence, RI; Donald M. Null, Jr,, MD AGH, Pittsburgh, PA; Juan A. Dumois,, MD GT, RN, All Children’s Hospital, St. Petersburg,, FL; Karen Hardy M, Julie Bushnell, PNP, Kayla Harvey, PNP,, California Pacific Medical Center SF, CA; Brian P., O’Sullivan M, Rhoda Spaulding, MSN, FNP, University of Massachusetts Medical Center, Worcester, MA; Peter D. Reuman, MD,, MPH IG, MD, Connie Nixon, RN, University of Florida,, Gainesville FMLK, MD, Candice E. Johnson, MD,, PhD MC, MD, MetroHealth Medical Center, Cleveland,, OH; Susan Sniderman M, Nancy Newton, RN, BSN, Jean Millar,, RN B, Kimberly Champawat, RN, BSN, University of California-San Francisco, San Francisco, CA; Russell B. Van Dyke, MD,, Benjamin Estrada M, Tulane University School of Medicine,, New Orleans LJEB, MD, Robert Hostoffer, MD, Susan, W. Aucott M, Avroy A. Fanaroff, MB, BCh, Rainbow Babies and, Children’s Hospital C, OH; Stephen C. Eppes, MD,, Judith A. Childs P, RN, Alfred I. duPont Institute, Wilmington,, DE; Joseph Marc Majure M, Duke University Medical Center,, Durham NMMSM, Donald K. Murphey, MD,, Mary L. Jackson R, Cook-Fort Worth Children’s Medical Center,, Fort Worth T. Reduction of respiratory syncytial virus hospitalization among premature infants and infants with bronchopulmonary dysplasia using respiratory syncytial virus immune globulin prophylaxis. The PREVENT Study Group. Pediatrics. 1997; 99 (1):93-9. Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, Schaeffer HJ. Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine. Proc Natl Acad Sci U S A. 1977; 74 (12):5716-20. Esteves PO, de Oliveira MC, de Souza Barros C, Cirne-Santos CC, Laneuvlille VT, Palmer Paixão IC. Antiviral Effect of Caulerpin Against Chikungunya. Nat Prod Commun. 2019; 14 (10):1934578X19878295. European Association for the Study of the Liver. EASL clinical practice guidelines: Management of chronic hepatitis B virus infection. J Hepatol. 2012; 57 (1):167-85. Everts M, Cihlar T, Bostwick JR, Whitley RJ. Accelerating Drug Development: Antiviral Therapies for Emerging Viruses as a Model. Annu Rev Pharmacol Toxicol. 2017; 57:155-69. Fang H, Liu A, Dahmen U, Dirsch O. Dual role of chloroquine in liver ischemia reperfusion injury: reduction of liver damage in early phase, but aggravation in late phase. Cell Death Dis. 2013; 4 (6):e694. Ferraz AC, Moraes TFS, Nizer W, Santos MD, Tótola AH, Ferreira JMS, Vieira- Filho SA, Rodrigues VG, Duarte LP, de Brito Magalhães CL, de Magalhães JC. Virucidal activity of proanthocyanidin against Mayaro virus. Antiviral Res. 2019; 168:76-81. Ferreira WJ, Amaro R, Cavalcanti DN, de Rezende CM, da Silva VA, Barbosa JE, Paixão IC, Teixeira VL. Anti-herpetic activities of chemical components from the Brazilian red alga Plocamium brasiliense. Nat Prod Commun. 2010; 5 (8):1167-70.

Fields BNK, D.M.; Howley, P.M. . 2007. Virology. 6th ed: Philadelphia: Wolters Kluwer Health / Lippincott Williams & Wilkins. Flint SJE, L.W.; Krug, R.M.; Racaniello, V.R.; Skalka, A.M. 2000. Principles of virology: molecular, biology, pathogenesis and control.: American Society for Microbiology. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Gonçales FL, Jr., Häussinger D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A, Hoffman J, Yu J. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med. 2002; 347 (13):975-82. Furuta Y. In vitro and in vivo activities of anti-influenza virus compound T-705. Antimicrob. Agents Chemother. 2002; 46. Furuta Y. Mechanism of action of T-705 against influenza virus. Antimicrob. Agents Chemother. 2005; 49. García-Serradilla M, Risco C, Pacheco B. Drug repurposing for new, efficient, broad spectrum antivirals. Virus Res. 2019; 264:22-31. Gnann JW, Jr., Skoldenberg B, Hart J, Aurelius E, Schliamser S, Studahl M, Eriksson BM, Hanley D, Aoki F, Jackson AC, Griffiths P, Miedzinski L, Hanfelt-Goade D, Hinthorn D, Ahlm C, Aksamit A, Cruz-Flores S, Dale I, Cloud G, Jester P, Whitley RJ. Herpes Simplex Encephalitis: Lack of Clinical Benefit of Long-term Valacyclovir Therapy. Clin Infect Dis. 2015; 61 (5):683-91. Gonzaga DTG, Gomes RSP, Marra RKF, Silva FCd, Gomes MWL, Ferreira DF, Santos RMA, Pinto AMV, Ratcliffe NA, Cirne-Santos CC, Barros CS, Ferreira VF, Paixão ICNP. Inhibition of Zika Virus Replication by Synthetic Bis-Naphthoquinones. Journal of the Brazilian Chemical Society. 2019; 30:1697-706. Goslinski T, Wenska G, Golankiewicz B, Balzarini J, De Clercq E. Synthesis and fluorescent properties of 6-(4-biphenylyl)-3,9-dihydro-9-oxo-5H- imidazo[1,2-a]purine analogues of acyclovir and ganciclovir. Nucleosides Nucleotides Nucleic Acids. 2003; 22 (5-8):911-4. Greenberg MD, Rutledge LH, Reid R, Berman NR, Precop SL, Elswick RK, Jr. A double-blind, randomized trial of 0.5% podofilox and placebo for the treatment of genital warts in women. Obstet Gynecol. 1991; 77 (5):735-9. Grillone LR, Lanz R. Fomivirsen. Drugs Today (Barc). 2001; 37 (4):245-55. Gunter J. Genital and perianal warts: new treatment opportunities for human papillomavirus infection. Am J Obstet Gynecol. 2003; 189 (3 Suppl):S3- 11. Harper D. 2012. Virus Replication. In eLS. Hayashi K, Iinuma M, Sasaki K, Hayashi T. In vitro and in vivo evaluation of a novel antiherpetic flavonoid, 4'-phenylflavone, and its synergistic actions with acyclovir. Arch Virol. 2012; 157 (8):1489-98. Hazuda D, Iwamoto M, Wenning L. Emerging pharmacology: inhibitors of human immunodeficiency virus integration. Annu Rev Pharmacol Toxicol. 2009; 49:377-94. Heim MH. 25 years of interferon-based treatment of chronic hepatitis C: an epoch coming to an end. Nat Rev Immunol. 2013; 13 (7):535-42. Hodge RAV. Famciclovir and Penciclovir. The Mode of Action of Famciclovir Including Its Conversion to Penciclovir. Antiviral Chemistry and Chemotherapy. 1993; 4 (2):67-84.

Hoffmann HH, Schneider WM, Rice CM. Interferons and viruses: an evolutionary arms race of molecular interactions. Trends Immunol. 2015; 36 (3):124-38. Houghton PJ. Use of small scale bioassays in the discovery of novel drugs from natural sources. Phytother Res. 2000; 14 (6):419-23. Hsu J, Santesso N, Mustafa R, Brozek J, Chen YL, Hopkins JP, Cheung A, Hovhannisyan G, Ivanova L, Flottorp SA, Saeterdal I, Wong AD, Tian J, Uyeki TM, Akl EA, Alonso-Coello P, Smaill F, Schünemann HJ. Antivirals for treatment of influenza: a systematic review and meta-analysis of observational studies. Ann Intern Med. 2012; 156 (7):512-24. Hubsher G, Haider M, Okun MS. Amantadine: the journey from fighting flu to treating Parkinson disease. Neurology. 2012; 78 (14):1096-9. Hulo C, de Castro E, Masson P, Bougueleret L, Bairoch A, Xenarios I, Le Mercier P. ViralZone: a knowledge resource to understand virus diversity. Nucleic Acids Res. 2011; 39 (Database issue):D576-82. Ivashkiv LB, Donlin LT. Regulation of type I interferon responses. Nat Rev Immunol. 2014; 14 (1):36-49. Joseph A. Bocchini Jr M, Chairperson, Henry H. Bernstein D, John S. Bradley M, Michael T. Brady M, Carrie L. Byington M, Margaret C. Fisher M, Mary P. Glode M, Mary Anne Jackson M, Harry L. Keyserling M, David W. Kimberlin M, Walter A. Orenstein M, Gordon E. Schutze M, Rodney E. Willoughby M, FAAP. Modified Recommendations for Use of Palivizumab for Prevention of Respiratory Syncytial Virus Infections. Pediatrics. 2009; 124 (6):1694-701. Kacer M, Kielberger L, Bouda M, Reischig T. Valganciclovir versus valacyclovir prophylaxis for prevention of cytomegalovirus: an economic perspective. Transplant Infectious Disease. 2015; 17 (3):334-41. Katz DH, Marcelletti JF, Khalil MH, Pope LE, Katz LR. Antiviral activity of 1- docosanol, an inhibitor of lipid-enveloped viruses including herpes simplex. Proc Natl Acad Sci U S A. 1991; 88 (23):10825-9. Kaur P, Chu JJ. Chikungunya virus: an update on antiviral development and challenges. Drug Discov Today. 2013; 18 (19-20):969-83. Kaur P, Thiruchelvan M, Lee RC, Chen H, Chen KC, Ng ML, Chu JJ. Inhibition of chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression. Antimicrob Agents Chemother. 2013; 57 (1):155-67. Khedkar PH, Patzak A. SARS-CoV-2: What do we know so far? 2020; 229 (2):e13470. Kilby JM, Hopkins S, Venetta TM, DiMassimo B, Cloud GA, Lee JY, Alldredge L, Hunter E, Lambert D, Bolognesi D, Matthews T, Johnson MR, Nowak MA, Shaw GM, Saag MS. Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nature Medicine. 1998; 4 (11):1302-7. Kim Y, Galasiti Kankanamalage AC, Chang KO, Groutas WC. Recent Advances in the Discovery of Norovirus Therapeutics. J Med Chem. 2015; 58 (24):9438-50. Kiso M, Takahashi K, Sakai-Tagawa Y, Shinya K, Sakabe S, Le QM, Ozawa M, Furuta Y, Kawaoka Y. T-705 (favipiravir) activity against lethal H5N1 influenza A viruses. Proc Natl Acad Sci U S A. 2010; 107 (2):882-7.

Kocher J, Yuan L. Norovirus vaccines and potential antinorovirus drugs: recent advances and future perspectives. Future Virol. 2015; 10 (7):899-913. Kozal MJ. Drug-resistant human immunodefiency virus. Clin Microbiol Infect. 2009; 15 Suppl 1:69-73. Kuritzkes DR. HIV-1 entry inhibitors: an overview. Curr Opin HIV AIDS. 2009; 4 (2):82-7. Labetoulle M. [The latest in herpes simplex keratitis therapy]. J Fr Ophtalmol. 2004; 27 (5):547-57. Lampertico P. The royal wedding in chronic hepatitis B: The haves and the have-nots for the combination of pegylated interferon and nucleos(t)ide therapy. Hepatology. 2015; 61 (5):1459-61. Lawitz E, Gane E, Pearlman B, Tam E, Ghesquiere W, Guyader D, Alric L, Bronowicki JP, Lester L, Sievert W, Ghalib R, Balart L, Sund F, Lagging M, Dutko F, Shaughnessy M, Hwang P, Howe AY, Wahl J, Robertson M, Barr E, Haber B. Efficacy and safety of 12 weeks versus 18 weeks of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin for hepatitis C virus genotype 1 infection in previously untreated patients with cirrhosis and patients with previous null response with or without cirrhosis (C-WORTHY): a randomised, open-label phase 2 trial. Lancet. 2015; 385 (9973):1075-86. Leiman PG, Kanamaru S, Mesyanzhinov VV, Arisaka F, Rossmann MG. Structure and morphogenesis of bacteriophage T4. Cell Mol Life Sci. 2003; 60 (11):2356-70. Liang R, Li H, Swanson JMJ, Voth GA. Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel. Proc. Natl Acad. Sci. USA. 2014; 111. Lim SP. Dengue drug discovery: Progress, challenges and outlook. Antiviral Res. 2019; 163:156-78. Littler E, Oberg B. Achievements and challenges in antiviral drug discovery. Antivir Chem Chemother. 2005; 16 (3):155-68. Liu D, Ji J, Ndongwe TP, Michailidis E, Rice CM, Ralston R, Sarafianos SG. Fast hepatitis C virus RNA elimination and NS5A redistribution by NS5A inhibitors studied by a multiplex assay approach. Antimicrob Agents Chemother. 2015; 59 (6):3482-92. Lodish HB, A.; Zipursky, S.L.; Matsudaira, P.; Baltimore, D.; Darnell, J. 2000. Molecular Cell Biology. 4th ed: New York: W. H. Freeman & Co. Lolis MS, González L, Cohen PJ, Schwartz RA. Drug-resistant herpes simplex virus in HIV infected patients. Acta Dermatovenerol Croat. 2008; 16 (4):204-8. Low JG, Ooi EE, Vasudevan SG. Current Status of Dengue Therapeutics Research and Development. J Infect Dis. 2017; 215 (suppl_2):S96-s102. Luthuli S, Wu S, Cheng Y, Zheng X, Wu M, Tong H. Therapeutic Effects of Fucoidan: A Review on Recent Studies. Mar Drugs. 2019; 17 (9). M.G. Gopal, Shannoma, Sharath Kumar B.C., Ramesh M., Nandini A.S., Manjunath NC. A Comparative Study to Evaluate the Efficacy and Safety of Acyclovir and Famciclovir in the Management of Herpes Zoster. JCDR - Journal of Clinical and Diagnostic Research. 2013; 7 (12):2904. Magden J, Kääriäinen L, Ahola T. Inhibitors of virus replication: recent developments and prospects. Appl Microbiol Biotechnol. 2005; 66 (6):612-21.

Mancini DAP, Torres RP, Pinto JR, Mancini-Filho J. Inhibition of DNA virus: Herpes-1 (HSV-1) in cellular culture replication, through an antioxidant treatment extracted from rosemary spice. Brazilian Journal of Pharmaceutical Sciences. 2009; 45:127-33. Manns M, Pol S, Jacobson IM, Marcellin P, Gordon SC, Peng CY, Chang TT, Everson GT, Heo J, Gerken G, Yoffe B, Towner WJ, Bourliere M, Metivier S, Chu CJ, Sievert W, Bronowicki JP, Thabut D, Lee YJ, Kao JH, McPhee F, Kopit J, Mendez P, Linaberry M, Hughes E, Noviello S. All-oral daclatasvir plus asunaprevir for hepatitis C virus genotype 1b: a multinational, phase 3, multicohort study. Lancet. 2014; 384 (9954):1597- 605. Matthews T, Salgo M, Greenberg M, Chung J, DeMasi R, Bolognesi D. Enfuvirtide: the first therapy to inhibit the entry of HIV-1 into host CD4 lymphocytes. Nat Rev Drug Discov. 2004; 3 (3):215-25. McKimm-Breschkin J, Trivedi T, Hampson A, Hay A, Klimov A, Tashiro M, Hayden F, Zambon M. Neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir. Antimicrob Agents Chemother. 2003; 47 (7):2264-72. McLaughlin MM, Skoglund EW, Ison MG. Peramivir: an intravenous neuraminidase inhibitor. Expert Opin Pharmacother. 2015; 16 (12):1889- 900. Mehedi M, Groseth A, Feldmann H, Ebihara H. Clinical aspects of Marburg hemorrhagic fever. Future Virol. 2011; 6 (9):1091-106. Melo PS, Maria SS, Vidal BC, Haun M, Durán N. Violacein cytotoxicity and induction of apoptosis in V79 cells. In Vitro Cell Dev Biol Anim. 2000; 36 (8):539-43. Mercorelli B, Palù G, Loregian A. Drug Repurposing for Viral Infectious Diseases: How Far Are We? Trends Microbiol. 2018; 26 (10):865-76. Michael T. Brady M, FAAP – Chairperson, Red, Editor BA, Carrie L. Byington M, FAAP, H. Dele Davies M, FAAP, Kathryn M. Edwards M, FAAP, Mary Anne Jackson M, FAAP – Red Book, Editor A, Yvonne A. Maldonado M, FAAP, Dennis L. Murray M, FAAP, Walter A. Orenstein M, FAAP, Mobeen H. Rathore M, FAAP, Mark H. Sawyer M, FAAP, Gordon E. Schutze M, FAAP, Rodney E. Willoughby M, FAAP, Theoklis E. Zaoutis M, FAAP. Updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection. Pediatrics. 2014; 134 (2):e620-38. Millard PJ, Bickerstaff LE, LaPatra SE, Kim CH. Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplification. J Fish Dis. 2006; 29 (4):201-13. Mills A, Arribas JR, Andrade-Villanueva J, DiPerri G, Van Lunzen J, Koenig E, Elion R, Cavassini M, Madruga JV, Brunetta J, Shamblaw D, DeJesus E, Orkin C, Wohl DA, Brar I, Stephens JL, Girard PM, Huhn G, Plummer A, Liu YP, Cheng AK, McCallister S. Switching from tenofovir disoproxil fumarate to tenofovir alafenamide in antiretroviral regimens for virologically suppressed adults with HIV-1 infection: a randomised, active-controlled, multicentre, open-label, phase 3, non-inferiority study. Lancet Infect Dis. 2016; 16 (1):43-52. Mishra P, Kumar A, Mamidi P, Kumar S, Basantray I, Saswat T, Das I, Nayak TK, Chattopadhyay S, Subudhi BB, Chattopadhyay S. Inhibition of

Chikungunya Virus Replication by 1-[(2-Methylbenzimidazol-1-yl) Methyl]-2-Oxo-Indolin-3-ylidene] Amino] Thiourea(MBZM-N-IBT). Sci Rep. 2016; 6:20122. Mitsuya H, Weinhold KJ, Furman PA, St Clair MH, Lehrman SN, Gallo RC, Bolognesi D, Barry DW, Broder S. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc Natl Acad Sci U S A. 1985; 82 (20):7096-100. Miyasaka T, Tanaka H, Baba M, Hayakawa H, Walker RT, Balzarini J, De Clercq E. A novel lead for specific anti-HIV-1 agents: 1-[(2- hydroxyethoxy)methyl]-6-(phenylthio)thymine. J Med Chem. 1989; 32 (12):2507-9. Morin SF, Yamey G, Rutherford GW. HIV pre-exposure prophylaxis. Bmj. 2012; 345:e5412. Nettles JH, Stanton RA, Broyde J, Amblard F, Zhang H, Zhou L, Shi J, McBrayer TR, Whitaker T, Coats SJ, Kohler JJ, Schinazi RF. Asymmetric binding to NS5A by daclatasvir (BMS-790052) and analogs suggests two novel modes of HCV inhibition. J Med Chem. 2014; 57 (23):10031-43. Neves AP, Pereira MX, Peterson EJ, Kipping R, Vargas MD, Silva FP, Jr., Carneiro JW, Farrell NP. Exploring the DNA binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3- (aminomethyl)-1,4-naphthoquinone Mannich bases and their platinum(II) complexes. J Inorg Biochem. 2013; 119:54-64. Ngoc TM, Phuong NTT, Khoi NM, Park S, Kwak HJ, Nhiem NX, Trang BTT, Tai BH, Song JH, Ko HJ, Kim SH. A new naphthoquinone analogue and antiviral constituents from the root of Rhinacanthus nasutus. Nat Prod Res. 2019; 33 (3):360-6. Nogueira CCR, Paixão ICNP, Cirne-Santos CC, Stephens PRS, Villaça RC, Pereira HS, Teixeira VL. Anti-HIV-1 activity in human primary cells and Anti-HIV-1 RT inhibitory activity of extracts from the red seaweed Acanthophora spicifera. Journal of Medicinal Plants Research. 2016; 10 (35):621-5. O'Brien JJ, Campoli-Richards DM. Acyclovir. An updated review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy. Drugs. 1989; 37 (3):233-309. Oberg B. Antiviral effects of phosphonoformate (PFA, foscarnet sodium). Pharmacol Ther. 1989; 40 (2):213-85. Oestereich L. Successful treatment of advanced Ebola virus infection with T- 705 (favipiravir) in a small animal model. Antiviral Res. 2014; 105. Oliveira I, Fonseca RR, Cirne-Santos CC, Barros CS, V.L.; T, Paixao ICNP. Antiviral Activity of Extracts of Plocamium Brasiliense and Plocamium Cartilagineum at in Vitro Replication of Human Immunodeficiency Virus Type 1. Archives in Biomedical Engneering & Biotechnology. 2019; 3 (1). Olsson JM. Pharmacokinetics. Pediatr Rev. 2007; 28 (10):398. Opoku-Anane J, Diouf K, Nour NM. New Success With Microbicides and Pre- Exposure Prophylaxis for Human Immunodeficiency Virus (HIV): Is Female-Controlled Prevention the Answer to the HIV Epidemic? Rev Obstet Gynecol. 2012; 5 (1):50-5. Pauwels R, Andries K, Desmyter J, Schols D, Kukla MJ, Breslin HJ, Raeymaeckers A, Van Gelder J, Woestenborghs R, Heykants J, et al.

Potent and selective inhibition of HIV-1 replication in vitro by a novel series of TIBO derivatives. Nature. 1990; 343 (6257):470-4. Pereira HS, Leão-Ferreira LR, Moussatché N, Teixeira VL, Cavalcanti DN, da Costa LJ, Diaz R, Frugulhetti IC. Effects of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis on HIV-1 reverse transcriptase. Planta Med. 2005; 71 (11):1019-24. Perrillo R. Benefits and risks of interferon therapy for hepatitis B. Hepatology. 2009; 49 (5 Suppl):S103-11. Pinto AM, Leite JP, Neves AP, da Silva GB, Vargas MD, Paixão IC. Synthetic aminomethylnaphthoquinones inhibit the in vitro replication of bovine herpesvirus 5. Arch Virol. 2014; 159 (7):1827-33. Pope LE, Marcelletti JF, Katz LR, Lin JY, Katz DH, Parish ML, Spear PG. The anti-herpes simplex virus activity of n-docosanol includes inhibition of the viral entry process. Antiviral Res. 1998; 40 (1-2):85-94. Potter CW, Phair JP, Vodinelich L, Fenton R, Jennings R. Antiviral, immunosuppressive and antitumour effects of ribavirin. Nature. 1976; 259 (5543):496-7. Prescott WA, Jr., Doloresco F, Brown J, Paladino JA. Cost effectiveness of respiratory syncytial virus prophylaxis: a critical and systematic review. Pharmacoeconomics. 2010; 28 (4):279-93. Rashad AA, Keller PA. Structure based design towards the identification of novel binding sites and inhibitors for the chikungunya virus envelope proteins. J Mol Graph Model. 2013; 44:241-52. Ray AS, Fordyce MW, Hitchcock MJ. Tenofovir alafenamide: A novel prodrug of tenofovir for the treatment of Human Immunodeficiency Virus. Antiviral Res. 2016; 125:63-70. Razonable RR. Antiviral drugs for viruses other than human immunodeficiency virus. Mayo Clin Proc. 2011; 86 (10):1009-26. Roa-Linares VC, Miranda-Brand Y, Tangarife-Castaño V, Ochoa R. Anti- Herpetic, Anti-Dengue and Antineoplastic Activities of Simple and Heterocycle-Fused Derivatives of Terpenyl-1,4-Naphthoquinone and 1,4- Anthraquinone. 2019; 24 (7). Robinson RF, Nahata MC. Respiratory syncytial virus (RSV) immune globulin and palivizumab for prevention of RSV infection. Am J Health Syst Pharm. 2000; 57 (3):259-64. Rodnitzky RL, Narayanan NS. Amantadine's role in the treatment of levodopa- induced dyskinesia. Neurology. 2014; 82 (4):288-9. Roquebert B, Damond F, Collin G, Matheron S, Peytavin G, Benard A, Campa P, Chene G, Brun-Vezinet F, Descamps D. HIV-2 integrase gene polymorphism and phenotypic susceptibility of HIV-2 clinical isolates to the integrase inhibitors raltegravir and elvitegravir in vitro. J Antimicrob Chemother. 2008; 62 (5):914-20. Rossmann MG. Structure of viruses: a short history. Q Rev Biophys. 2013; 46 (2):133-80. Roth D, Nelson DR, Bruchfeld A, Liapakis A, Silva M, Monsour H, Jr., Martin P, Pol S, Londoño MC, Hassanein T, Zamor PJ, Zuckerman E, Wan S, Jackson B, Nguyen BY, Robertson M, Barr E, Wahl J, Greaves W. Grazoprevir plus elbasvir in treatment-naive and treatment-experienced patients with hepatitis C virus genotype 1 infection and stage 4-5 chronic

kidney disease (the C-SURFER study): a combination phase 3 study. Lancet. 2015; 386 (10003):1537-45. Russell RJ, Haire LF, Stevens DJ, Collins PJ, Lin YP, Blackburn GM, Hay AJ, Gamblin SJ, Skehel JJ. The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature. 2006; 443 (7107):45-9. Sacks SL, Thisted RA, Jones TM, Barbarash RA, Mikolich DJ, Ruoff GE, Jorizzo JL, Gunnill LB, Katz DH, Khalil MH, Morrow PR, Yakatan GJ, Pope LE, Berg JE. Clinical efficacy of topical docosanol 10% cream for herpes simplex labialis: A multicenter, randomized, placebo-controlled trial. J Am Acad Dermatol. 2001; 45 (2):222-30. Sanjuán R, Domingo-Calap P. Mechanisms of viral mutation. Cell Mol Life Sci. 2016; 73 (23):4433-48. Sax PE, Wohl D, Yin MT, Post F, DeJesus E, Saag M, Pozniak A, Thompson M, Podzamczer D, Molina JM, Oka S, Koenig E, Trottier B, Andrade- Villanueva J, Crofoot G, Custodio JM, Plummer A, Zhong L, Cao H, Martin H, Callebaut C, Cheng AK, Fordyce MW, McCallister S. Tenofovir alafenamide versus tenofovir disoproxil fumarate, coformulated with elvitegravir, cobicistat, and emtricitabine, for initial treatment of HIV-1 infection: two randomised, double-blind, phase 3, non-inferiority trials. Lancet. 2015; 385 (9987):2606-15. Schaeffer HJ, Beauchamp L, de Miranda P, Elion GB, Bauer DJ, Collins P. 9-(2- hydroxyethoxymethyl) guanine activity against viruses of the herpes group. Nature. 1978; 272 (5654):583-5. Schein CH. Repurposing approved drugs on the pathway to novel therapies. Med Res Rev. 2020; 40 (2):586-605. Schneider C, Segre T. Green tea: potential health benefits. Am Fam Physician. 2009; 79 (7):591-4. Schuhmacher A, Reichling J, Schnitzler P. Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro. Phytomedicine. 2003; 10 (6-7):504-10. Shattock RJ, Rosenberg Z. Microbicides: topical prevention against HIV. Cold Spring Harb Perspect Med. 2012; 2 (2):a007385. Shimura K, Kodama E, Sakagami Y, Matsuzaki Y, Watanabe W, Yamataka K, Watanabe Y, Ohata Y, Doi S, Sato M, Kano M, Ikeda S, Matsuoka M. Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor elvitegravir (JTK-303/GS- 9137). J Virol. 2008; 82 (2):764-74. Shipkowitz NL, Bower RR, Appell RN, Nordeen CW, Overby LR, Roderick WR, Schleicher JB, Von Esch AM. Suppression of herpes simplex virus infection by phosphonoacetic acid. Appl Microbiol. 1973; 26 (3):264-7. Sidwell RW. Broad-spectrum antiviral activity of virazole: 1-f8- D-ribofuranosyl- 1,2,4-triazole- 3-carboxamide. Science. 1972; 177. Silva A, Morais S, Marques M, Lima D, Santos S, Almeida R, Vieira I, Guedes M. Antiviral activities of extracts and phenolic components of two Spondias species against dengue virus. Journal of Venomous Animals and Toxins including Tropical Diseases. 2011; 17:406-13. Smee DF, Hurst BL, Egawa H, Takahashi K, Kadota T, Furuta Y. Intracellular metabolism of favipiravir (T-705) in uninfected and influenza A (H5N1) virus-infected cells. J Antimicrob Chemother. 2009; 64 (4):741-6.

Smither SJ. Post-exposure efficacy of Oral T-705 (Favipiravir) against inhalational Ebola virus infection in a mouse model. Antiviral Res. 2014; 104. Souza TM, Abrantes JL, de AER, Leite Fontes CF, Frugulhetti IC. The alkaloid 4-methylaaptamine isolated from the sponge Aaptos aaptos impairs Herpes simplex virus type 1 penetration and immediate-early protein synthesis. Planta Med. 2007; 73 (3):200-5. Strauss JHS, E.G. . 2008. Viruses and human disease. . 2nd ed: San Diego: Academic, . Streeter DG, Witkowski JT, Khare GP, Sidwell RW, Bauer RJ, Robins RK, Simon LN. Mechanism of action of 1- -D-ribofuranosyl-1,2,4-triazole-3- carboxamide (Virazole), a new broad-spectrum antiviral agent. Proc Natl Acad Sci U S A. 1973; 70 (4):1174-8. Sullivan V, Talarico CL, Stanat SC, Davis M, Coen DM, Biron KK. A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells. Nature. 1992; 358 (6382):162-4. Takizawa N, Yamasaki M. Current landscape and future prospects of antiviral drugs derived from microbial products. J Antibiot (Tokyo). 2017. Tchesnokov EP, Gilbert C, Boivin G, Gotte M. Role of helix P of the human cytomegalovirus DNA polymerase in resistance and hypersusceptibility to the antiviral drug foscarnet. J Virol. 2006; 80 (3):1440-50. Terrault NA, Bzowej NH, Chang KM, Hwang JP, Jonas MM, Murad MH. AASLD guidelines for treatment of chronic hepatitis B. Hepatology. 2016; 63 (1):261-83. Thanacoody H. Quinine and chloroquine. Medicine. 2016; 44. Thuy TT, Ly BM, Van TT, Quang NV, Tu HC, Zheng Y, Seguin-Devaux C, Mi B, Ai U. Anti-HIV activity of fucoidans from three brown seaweed species. Carbohydr Polym. 2015; 115:122-8. Torriani FJ, Rodriguez-Torres M, Rockstroh JK, Lissen E, Gonzalez-García J, Lazzarin A, Carosi G, Sasadeusz J, Katlama C, Montaner J, Sette H, Jr., Passe S, De Pamphilis J, Duff F, Schrenk UM, Dieterich DT. Peginterferon Alfa-2a plus ribavirin for chronic hepatitis C virus infection in HIV-infected patients. N Engl J Med. 2004; 351 (5):438-50. Traebert M, Dumotier B, Meister L, Hoffmann P, Dominguez-Estevez M, Suter W. Inhibition of hERG K+ currents by antimalarial drugs in stably transfected HEK293 cells. Eur J Pharmacol. 2004; 484 (1):41-8. Tu YF, Chien CS, Yarmishyn AA, Lin YY, Luo YH, Lin YT, Lai WY, Yang DM, Chou SJ, Yang YP, Wang ML, Chiou SH. A Review of SARS-CoV-2 and the Ongoing Clinical Trials. Int J Mol Sci. 2020; 21 (7). Tyring S, Edwards L, Cherry LK, Ramsdell WM, Kotner S, Greenberg MD, Vance JC, Barnum G, Dromgoole SH, Killey FP, Toter T. Safety and efficacy of 0.5% podofilox gel in the treatment of anogenital warts. Arch Dermatol. 1998; 134 (1):33-8. van de Waterbeemd H, Gifford E. ADMET in silico modelling: towards prediction paradise? Nat Rev Drug Discov. 2003; 2 (3):192-204. Van Herp M, Declerck H, Decroo T. Favipiravir--a prophylactic treatment for Ebola contacts? Lancet. 2015; 385 (9985):2350. Vergara M, Olivares A, Altamirano C. Antiproliferative evaluation of tall-oil docosanol and tetracosanol over CHO-K1 and human melanoma cells. Electronic Journal of Biotechnology. 2015; 18:291-4.

Vitravene Study Group. A randomized controlled clinical trial of intravitreous fomivirsen for treatment of newly diagnosed peripheral cytomegalovirus retinitis in patients with AIDS. Am J Ophthalmol. 2002; 133 (4):467-74. Waheed MT, Sameeullah M, Khan FA, Syed T, Ilahi M, Gottschamel J, Lössl AG. Need of cost-effective vaccines in developing countries: What plant biotechnology can offer? Springerplus. 2016; 5:65. Wang D, Bayliss S, Meads C. Palivizumab for immunoprophylaxis of respiratory syncytial virus (RSV) bronchiolitis in high-risk infants and young children: a systematic review and additional economic modelling of subgroup analyses. Health Technol Assess. 2011; 15 (5):iii-iv, 1-124. Wang Y, Xing J, Xu Y, Zhou N, Peng J, Xiong Z, Liu X, Luo X, Luo C, Chen K, Zheng M, Jiang H. In silico ADME/T modelling for rational drug design. Q Rev Biophys. 2015; 48 (4):488-515. Watanabe A, Chang SC, Kim MJ, Chu DW, Ohashi Y. Long-acting neuraminidase inhibitor laninamivir octanoate versus oseltamivir for treatment of influenza: A double-blind, randomized, noninferiority clinical trial. Clin Infect Dis. 2010; 51 (10):1167-75. Wielgo-Polanin R, Lagarce L, Gautron E, Diquet B, Lainé-Cessac P. Hepatotoxicity associated with the use of a fixed combination of chloroquine and proguanil. Int J Antimicrob Agents. 2005; 26 (2):176-8. Wild C, Greenwell T, Matthews T. A synthetic peptide from HIV-1 gp41 is a potent inhibitor of virus-mediated cell-cell fusion. AIDS Res Hum Retroviruses. 1993; 9 (11):1051-3. Wiley DJ, Douglas J, Beutner K, Cox T, Fife K, Moscicki AB, Fukumoto L. External genital warts: diagnosis, treatment, and prevention. Clin Infect Dis. 2002; 35 (Suppl 2):S210-24. Wilhelmus KR. Antiviral treatment and other therapeutic interventions for herpes simplex virus epithelial keratitis. Cochrane Database Syst Rev. 2015; 1:Cd002898. Williams SD. A turning point in the fight against HIV. ChemMedChem. 2010; 5 (11):1799-801. Wintachai P, Kaur P, Lee RC, Ramphan S, Kuadkitkan A, Wikan N, Ubol S, Roytrakul S, Chu JJ, Smith DR. Activity of andrographolide against chikungunya virus infection. Sci Rep. 2015; 5:14179. Wittine K, Saftić Martinović L, Peršurić Ž, Pavelić S. Novel Antiretroviral Structures from Marine Organisms. Molecules. 2019; 24:3486. Wong E, Trustman N, Yalong A. HIV pharmacotherapy: A review of integrase inhibitors. Jaapa. 2016; 29 (2):36-40. World Health Organization. Guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection 2015 [cited. Available from https://apps.who.int/iris/bitstream/handle/10665/154590/9789241549059 _eng.pdf;jsessionid=1FC1DEB66FAC7A8B1E718E201F0D02B1?seque nce=1. Wray SK, Gilbert BE, Noall MW, Knight V. Mode of action of ribavirin: effect of nucleotide pool alterations on influenza virus ribonucleoprotein synthesis. Antiviral Res. 1985; 5 (1):29-37. Wu H, Pfarr DS, Losonsky GA, Kiener PA. Immunoprophylaxis of RSV infection: advancing from RSV-IGIV to palivizumab and motavizumab. Curr Top Microbiol Immunol. 2008; 317:103-23.

Wu JJ, Pang KR, Huang DB, Tyring SK. Advances in antiviral therapy. Dermatol Clin. 2005; 23 (2):313-22. Wyatt C, Baeten JM. Tenofovir alafenamide for HIV infection: is less more? Lancet. 2015; 385 (9987):2559-60. Yabiku ST, Yabiku MM, Bottós KM, Araújo AL, Freitas D, Belfort R, Jr. [Ganciclovir 0.15% ophthalmic gel in the treatment of adenovirus keratoconjunctivitis]. Arq Bras Oftalmol. 2011; 74 (6):417-21. Yarmolinsky L, Huleihel M, Zaccai M, Ben-Shabat S. Potent antiviral flavone glycosides from Ficus benjamina leaves. Fitoterapia. 2012; 83 (2):362-7. Yeo KL, Chen YL, Xu HY, Dong H, Wang QY, Yokokawa F, Shi PY. Synergistic suppression of dengue virus replication using a combination of nucleoside analogs and nucleoside synthesis inhibitors. Antimicrob Agents Chemother. 2015; 59 (4):2086-93. Zaia JA, Levin MJ, Preblud SR, Leszczynski J, Wright GG, Ellis RJ, Curtis AC, Valerio MA, LeGore J. Evaluation of varicella-zoster immune globulin: protection of immunosuppressed children after household exposure to varicella. J Infect Dis. 1983; 147 (4):737-43. Zandi K, Teoh BT, Sam SS, Wong PF, Mustafa MR, Abubakar S. Antiviral activity of four types of bioflavonoid against dengue virus type-2. Virol J. 2011; 8:560. Zeuzem S, Ghalib R, Reddy KR, Pockros PJ, Ben Ari Z, Zhao Y, Brown DD, Wan S, DiNubile MJ, Nguyen BY, Robertson MN, Wahl J, Barr E, Butterton JR. Grazoprevir-Elbasvir Combination Therapy for Treatment- Naive Cirrhotic and Noncirrhotic Patients With Chronic Hepatitis C Virus Genotype 1, 4, or 6 Infection: A Randomized Trial. Ann Intern Med. 2015; 163 (1):1-13. Zmurko J, Marques RE, Schols D, Verbeken E, Kaptein SJ, Neyts J. The Viral Polymerase Inhibitor 7-Deaza-2'-C-Methyladenosine Is a Potent Inhibitor of In Vitro Zika Virus Replication and Delays Disease Progression in a Robust Mouse Infection Model. PLoS Negl Trop Dis. 2016; 10 (5):e0004695. Zoulim F, Durantel D. Antiviral therapies and prospects for a cure of chronic hepatitis B. Cold Spring Harb Perspect Med. 2015; 5 (4).

5. APÊNDICE 5.1. ARTIGOS

5.1.1. DITERPENE FROM MARINE BROWN ALGAE Dictyota friabilis AS A POTENTIAL MICROBICIDE AGAINST HIV-1 IN TISSUE EXPLANTS

J Appl Phycol DOI 10.1007/s10811-016-0925-1

V REDEALGAS WORKSHOP (RIO DE JANEIRO, BRAZIL)

Diterpene from marine brown alga Dictyota friabilis as a potential microbicide against HIV-1 in tissue explants

Paulo Roberto Soares Stephens1,2 & Claudio Cesar Cirne-Santos2,3 & Caroline de Souza Barros2 & Valéria Laneuville Teixeira4 & Leila Abboud Dias Carneiro3 & Leonardo dos Santos Corrêa Amorim1,2 & Jurandy Susana Patrícia Ocampo1,5 & Luíz Roberto Ribeiro Castello-Branco6 & Izabel Christina Nunes de Palmer Paixão2

Received: 28 January 2016 /Revised and accepted: 3 August 2016 # Springer Science+Business Media Dordrecht 2016

Abstract Due to the great genetic diversity of HIV,producing to 90 % in peripheral blood cells (PBMC) and macrophages an effective vaccine is extremely difficult. The combined an- infected with HIV-1, respectively. Following the studies, we tiretroviral therapy, although not leading to a cure, is capable found that the dolabelladienetriol after being subjected to the of preventing opportunistic infections, since it greatly reduces experimental model of ex vivo cervical mucosa did not show HIV viral load in the blood. Considering a large number of toxicities in concentrations that were used for the experiments. HIV infections occur through sexual intercourse, the use of Subsequently, we evaluate the protective effect of topical microbicides could be very useful tools to reduce in- dolabelladienetriol in explant model in uterine cervix, show- fection rates. Studies by our group showed that the ing a curve of dose-dependent inhibition in treatment with dolabelladienetriol, isolated from the brown seaweed different concentrations of the compound in the presence of Dictyota friabilis, had a clear effect on replication by HIV-1 with 20 to 99 % in concentrations of 0.15 and 14.4 μM, inhibiting HIV-1 replication in cell culture. In this study, we respectively. These studies demonstrate the important effect of have demonstrated that pretreatment with this compound for dolabelladienetriol that can be recognized as a microbicide. 2 h or 1 to 5 days, showed an inhibitory effect ranging from 60 Keywords Phaeophyceae . Dictyota . Dolabelladienetriol . Microbicide . HIV-1 . Ex vivo explant * Valéria Laneuville Teixeira [email protected] * Izabel Christina Nunes de Palmer Paixão Introduction [email protected] The pandemic of infection with human immunodeficiency 1 Laboratório de Inovação em Terapias, Ensino e Bioprodutos virus type 1 (HIV-1), the etiologic agent of acquired immuno- (LITEB), Instituto Oswaldo Cruz/FIOCRUZ, Av. Brasil 4365, deficiency syndrome (AIDS), remains a serious public health Manguinhos, Pavilhão Cardoso Fontes, Sala 64, Rio de Janeiro, RJ 21045-900, Brazil problem worldwide, despite advances in understanding the pathogenesis of infection, prevention measures, and advances 2 Laboratório de Virologia Molecular e Biotecnologia Marinha, Departamento de Biologia Celular e Molecular, Instituto de Biologia, in treatment (Montagnier 2010;ArtsandHazuda2012). Universidade Federal Fluminense, Niterói, RJ 24020-141, Brazil According to the latest United Nations global report (UN) 3 Laboratório de Imunologia Clínica, Instituto Oswaldo Cruz / The United Nations Joint Programme on HIV (UNAIDS) in FIOCRUZ, Av. Brasil 4365, Manguinhos, Pavilhão Leonidas Deane 2014, there are 35.3 million people infected with the human / 409, Rio de Janeiro, RJ 21045-900, Brazil immunodeficiency virus (HIV) in the world, 2.1 million new 4 Laboratório Algamar, Departamento de Biologia Marinha, Instituto infections in 2013 and 240,000 new infections in children. It is de Biologia, Universidade Federal Fluminense, PO Box 100.644, estimated that in 2013, 1.5 million people died of AIDS, a Niterói, RJ 24010-970, Brazil decrease of 11.8 % compared to 1.7 million deaths in 2012. 5 Laboratório de Patologia Geral, Universidade Federal do Estado do In Brazil, it is estimated that 718,000 people living with HIV/ Rio de Janeiro, Rio de Janeiro, RJ, Brazil AIDS, and only in 2012 were reported 11,896 deaths 6 Fundação Ataulpho de Paiva, Rio de Janeiro, RJ, Brazil (UNAIDS 2014). J Appl Phycol

The current antiretroviral therapy is considered very effec- infected individuals is relatively lower than in several nearby tive in suppressing viral replication and subsequent reduction countries (Teas and Irhimeh 2012). of morbidity and mortality from HIV-1 (Arts and Hazuda Several algal lectins are considered as potential microbi- 2012). However, current drugs do not totally eliminate the cide candidates to prevent the sexual transmission of HIV viral population of infected tissues. Prolonged use may pro- through topical applications (Huskens and Schols 2012). mote metabolic disorders and toxicities, encouraging the The candidate microbicide Carraguard, a carrageenan derived emergence of resistant virus, besides being complex to admin- from seaweed, did not show efficacy in prevention of vaginal istrate (Sleiman et al. 2012; Sinha and Rubens 2014). transmission of HIV in phase 3 trials (Skoler-Karpoff et al. The actual treatment is based on combination of 2008), but acceptability findings regarding Carraguard indi- antiretrovirals, called BHighly Active Antiretroviral cate that it may be useful for next-generation candidate Therapy^ (HAART), an association of reverse transcriptase microbicides (Whitehead et al. 2011). inhibitors and protease, has proven effective in reducing viral Our group has been studying molecules isolated from sea- load and significantly contributes to immune reconstitution. weed with antiviral potential in order to develop microbicides The BHAART^ decreased mortality of HIV-1 and improved (Veazey et al. 2012) that are preventive and topical for the quality of life of patients who adhere to treatment genital mucosa. The use of explant technology was highly (Magiorkinis et al. 2002; Rodríguez-Arenas et al. 2006;Arts relevant to our analysis, principally because it facilitated the and Hazuda 2012). At the same time, the wide use of HAART study of human mucosal tissue. Thus, we were able to analyze of HIV infection leads to HIV drug resistance. The simulta- the action of the drug, the viral action in the tissue of the neous formation of the resistance to different groups of anti- substance under study, and the kinetics of the respective tissue viral drugs compromises the efficacy of HAART. Thus, the pathophysiology (Collins et al. 2000; Greenhead et al. 2000; problem of the drug resistance becomes of an enormous signif- Hu et al. 2004; Maher et al. 2005; Kelly and Shattock 2011; icance. We agree that the most important form of control this Shattock and Rosenberg 2012; Roan and Münch 2015). infection is the microbicide development that acting as a pre- Explant culture technology of human uterine cervix fulfills exposure prophylactic (PrEP) to prevent HIV transmission can the requirements for phase 1 of the preclinical study of significantly reduce the chain of transmission and decreasing the microbicides and should lead to the selection of those with number of new annual cases (Kelly and Shattock 2011;Morin the best potential to follow in clinical trials (phases 2, 3, and et al., 2012; Shattock and Rosenberg 2012). 4). It should be noted that this work involves the biotechno- Although several of these microbicide candidates are al- logical development of new preventive substances of great ready in advanced stages of clinical studies, there are many applicability against the background of the absence of HIV criteria to be considered for an ideal product. Among these vaccines and high toxicity present in numerous therapies properties, low toxicity is extremely important in order that (Cummins et al. 2007; Gengiah and Karim 2012). These structural integrity is preserved in mucosa. In this sense, many microbicides are unique alternative drugs that present low studies are still necessary in order to minimize this effect, toxicity, are topical, and can be developed at low cost (Kelly which is common in most of the products described in the and Shattock 2011; Shattock and Rosenberg 2012; Morin literature (Shattock and Rosenberg 2012). et al., 2012; Roan and Münch 2015). The development of drug candidates as microbicides for In our recent studies, we showed that compound extracted topical use requires deeper knowledge about the initial events from seaweed D. friabilis (as Dictyota pfaffii) called of infection through the genital mucosa. Therefore, the use of dolabelladienetriol inhibits HIV-1 replication in a dose- study models, such as the explants of the uterine cervix, has dependent manner. In this work, we determined the effect of been of great importance. This model consists of the in vitro pretreatment with dolabelladienetriol in inhibition of HIV-1 cultivation of small fragments of the uterine cervix, subjected replication and the protective effect of this compound in to HIV infection. Through this model, we can evaluate the ex vivo explant model. interactions of the virus with the immune cells present in such tissues, mimicking natural infection (Morin et al., 2012). Marine brown algae have been reported to produce mole- Experimental section cules with promising antiretroviral properties. We showed that dolabellane diterpene isolated from Dictyota friabilis (as Sample collection and obtaining of the diterpenes Dictyota pfaffii) inhibited the reverse transcriptase enzyme of HIV-1 (Cirne-Santos et al. 2006) and Trinchero et al. ThemarinebrownalgaD. friabilis was collected in (2009) showed that sulfated polysaccharides (fucoidans) from July 2009 at a depth of 6–9 m, by SCUBA divers at the Atol Adenocystis utricularis, interfere with early events of HIV-1 das Rocas reef, a biological marine reserve in Rio Grande do replication. Interestingly, in some tribal groups in Chad Norte State, lat. 03°51′03″S, long. 33°40′29″W, Brazil. The (Africa), that consume seaweeds regularly, the rate of HIV- legal authorization for sample collecting was obtained from J Appl Phycol

VLT (SISBIO/IBAMA Brazil) (number 17,352). The sea- Tissue explants weeds were collected and identified by Dr. Roberto Villaça (Departamento de Biologia Marinha, Instituto de Biologia, Cervical tissues were obtained from premenopausal women un- UFF). A voucher specimen (HRJ 9117) was deposited in the dergoing planned therapeutic hysterectomy at Hospital Federal herbarium of the Universidade do Estado do Rio de Janeiro de Bonsucesso and Hospital Municipal da Mulher Fernando (UERJ). The air-dried material of D. friabilis (65 g) was ex- Magalhães, Rio de Janeiro, Brazil. The tissues of the cervix were tracted three times with 500 mL of CH2Cl2, each time for 24 h. obtained using the following criteria: (a) absence of any ecto and The extracts were combined, filtered, and further dried under endocervical pathology; (b) pertaining to the patient’s clinical vacuum at 40 °C. The dried crude extract (3.5 g) was subject- condition, i.e., those of premenopausal women; (c) the indica- ed to vacuum column chromatography (CC) using CH2Cl2/ tion for hysterectomy was therapeutic; and (d) the patient had EtOAc (from 100 % CH2Cl2−9:1–8:2–7:3–6:4–5:5–3:7 to not been submitted to prior therapeutic hormonal stimulation. 100 % EtOAc) to obtain 90 fractions (F1 to F90). Fractions Cervical explants, comprising of both epithelial and stromal

60–78 (CH2Cl2/EtOAc, 9:1) yielded white crystals tissue, were produced by 3 mm diameter biopsy puncture on (1,000 mg) of majority compound, the 10,18-diacetoxy- ectocervix tissue, as described below. Intact explants were indi- dolabelladienetriol. Although it is a natural product isolated vidually cultured on squares of stainless-steel mesh in 24-well from D. friabilis (Pardo-Vargas et al. 2014), the flat-bottomed plates, such that a meniscus of culture medium dolabelladienetriol (1R,2E,4R,6E,8S,10S,11S,12R)-8,10,18- (Eagle’s minimal essential medium supplemented with peni- trihydroxy-2,6-dolabelladiene (Fig. 1) was obtained by reduc- cillin, streptomycin, L-glutamine, and HEPES buffer tion of the majority diterpene, in accordance with Barbosa [EMEM], Sigma) was in contact with the under surface of et al. (2004). the grid, as previously described by (Palacio et al. 1994). The explants were incubated at 37 °C in a humidified atmosphere

Cells and virus containing 5 % CO2, and two-thirds of the medium was changed every 2 to 3 days, taking care not to disturb any Peripheral blood cells (PBMCs) from normal individuals were migratory cells within the culture wells. The explants were obtained by density gradient on Ficoll-Hypaque and resus- cultured in 96-well flat-bottomed tissue culture plates in pended in culture medium RPMI 1640 with 10 % fetal calf RPMI with 10 % FBS. The biopsies were removed from the serum. PBMCs were stimulated with the mitogen phytohe- culture, fixed in phosphate-buffered formalin, and stained with magglutinin (PHA) for 2 to 3 days, and activated cells were hematoxylin and eosin. maintained in culture medium supplemented with Interleukin- 2 (IL-2), for further testing of viral infection. To obtain Determination of compound toxicity on explants tissue monocyte-derived macrophages, the PBMCs were distributed in culture plates of 48 wells (2 × 106 cells/well/500 μL) and For assessing the viability of the explants, we use the meth- cultured in Dulbecco (DMEM, Hyclone) with 10 % human odological principle of MTT dye reduction. The compounds serum (Sigma-Aldrich.) for 5 to 6 days. Then the nonadherent were added and incubated overnight, and subsequently the cells were removed, and complete medium was re-added. We − tissues were exposed to MTT (0.5 mg mL 1)for3hat used the HIV-1 isolates X4-tropic (CXCR4) and R5- tropic 37 °C. The formazan product was then released by incubation (CCR5) donated by Dr. Eva Maria Fenyo, the University of overnight in methanol (1 mL). Methanol-formazan absor- Lund, Sweden. bance was then determined at 570 nm. All data are expressed as the percent viability (normalized to the tissue weight) of Effect of pretreatment with dolabelladienetriol, inhibition compound treated wells compared to that of control wells of HIV-1 replication on PBMCs and macrophages untreated, and 50 % cytotoxic concentration (CC50)isdefined as the drug concentration at which the viability of the tissue To assess whether the treatment of target cells with was reduced to 50 % of free drug control. dolabelladienetriol prior to HIV-1 can inhibit viral replication, PBMCs were exposed to dolabelladienetriol for 2 h, 1 or 5 days, washed to remove excess (25 μ Mof Infectivity and inhibition assays on explants tissue dolabelladienetriol and then infected with HIV-1 (X4-tropic and R5-tropic) and maintained in culture. Macrophages were For executing of assays a standardized amount of virus culture treated with dolabelladienetriol for 1 or 5 days, washed, in- supernatant was normalized for infectivity. Serial dilutions of fected with HIV-1 (X4-tropic), and maintained in culture. compounds (between 14.4 and 0.15 μM) were realized and Viral replication was assessed by detection of p24 antigen in PM-1 cells were incubated for 1 h at 37 °C, and then virus was culture supernatants after 7 to 14 days for PBMC and added to cells and left for the time of the experiment. The cells macrophages. were washed with PBS and lysed and luciferase expression J Appl Phycol was assessed after addition of substrate (Luciferase Assay System, Promega) as previously described (Wei et al., 2002). The extent of virus replication was determined by measuring the p24 antigen concentration in supernatants (HIV-1 p24 enzyme-linked immunosorbent assay [ELISA] for all tissue explant models and migratory/PM-1 cells co-cultures; AIDS Vaccine Program, National Cancer Institute, Frederick, MD, USA) (Buffa et al. 2009). For all compounds tested, cell and tissue viabilities were determined by measuring tetrazolium salt [3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)], cleavage into a blue product (formazan) by viable cells (Wang et al. 2004) as described previously (Crucitti et al. 2012). Each experiment was performed three times with a different donor for each of the triplicates. The explants were incubated with 200 μL complete RPMI- MTT (0.5 mg mL−1) at 37 °C for 3 h. The formazan salts were solubilized by addition of 100—l20 % sodium dodecyl sulfate in 1:1 H2O–N,N-dimethylformamide (cells) or 1 mL methanol (explants), and viability was determined by measuring the optical density at 570 nm (reference, 690 nm) in a Synergy- HT plate reader. For tissue studies, this value was corrected for Fig. 2 Effect of pre and posttreatment with 25 μM of dolabelladienetriol on HIV-1 replication. Error bars indicate the standard deviation and explant dry weight. experiments were performed in triplicate. a: Pretreatment and posttreatment of PBMCs with Dolabelladienetriol. b: Pretreatment and posttreatment of macrophages with dolabelladienetriol. Pretreatment for 2 h; Pretreatment for 1 day; Pretreatment Results and discussion for 5 days; Posttreatment; R5-tropic HIV-1; X4-tropic HIV-1

We found that pretreatment of PBMCs with dolabelladienetriol it is one of the aspects related to the effect of this substance that (Fig. 1) resulted in strong inhibitory activity on HIV-1 replica- draws attention to different possibilities in the treatment of indi- tion (Fig. 2a), inhibition ranging between 60 and 80 % for both viduals living with HIV. HIV-1 isolates X4-tropic (CXCR4) and R5- tropic (CCR5). In Our data about explant viability in normal culture condi- macrophages, the pretreatment with dolabelladienetriol for 1 or tions demonstrated that the culture of explants decreased strat- 5 days resulted in a strong block in the replication of HIV-1 ified squamous tissue (ectocervix), with loss of cellularity (Fig. 2b). The same result was found when the within the first 72 h. From this point onward, the reduction dolabelladienetriol was added after cell infection. The results was slow and gradual until the end of the culture in a period of show that this compound, dolabelladienetriol, which is not re- 13 days (Fig. 3). In contrast, the loss of connective tissue added after infection, has a strong suppressive effect on the structures was slow up to the end of this period. We observed replication of HIV-1. According to Barros et al. (2016), diter- the presence of a small number of microvasculature and stro- penes was isolated from Canistrocarpus cervicornis,and mal cellularity compared with the initial phase. This histolog- inhibited HIV-1 replication in MT2 cells at the same concentra- ical pattern resembled that previously reported by Cummins tion (25 μM). Given these results, it is important to analyze if the et al. (2007). dolabelladienetriol exerts pharmacological effects on cells, for The use of explant cultures of the uterine cervix and example, decreased expression of receptors for HIV-1 or chang- the human penis glans meets the requirements of the es in the synthesis of macromolecules. However, we found that stages of preclinical studies for the development of microbicides. Considering a scenario of no existence of vaccines and drugs that cause severe side effects, the development of microbicides is a very important strategy for the prevention of disease (Cummins et al. 2007). Microbicides are alternatives that, because of their low toxicity and low cost of development, can be providential use for the prevention of HIV infection Fig. 1 Molecular structure of dolabelladienetriol (Shattock and Rosenberg 2012;Morinetal.2012; J Appl Phycol

effect by inhibiting viral replication without a loss in viability of the tissue in a dose-dependent manner by varying the inhibitions of 21 % ± 2.08 to 95 % ± 1.31 for the concentrations mentioned above, respectively. This compound, therefore, has a great potential as a possible microbicide. The red algal protein griffithsin (GRFT) and cyanovirin-N (CV-N), a protein originally purified from the cyanobacterium Nostoc ellipsosporum, were also tested in ex vivo cervical explant models. The GRFT protein showed potential activity in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes (O’Keefe et al. 2009), and CV-N was able to inhibit HIV-1 infection in cervical explants, with an

EC90 value of 1 μM(Buffaetal.2009). Microbicides are products that can be applied in the Fig. 3 Explant culture of normal human cervical mucosa. Tissue vagina or rectal mucosa to prevent or reduce transmis- viability was maintained for 13 days in culture. Predominant changes sion of sexually transmitted diseases including HIV-1, were present in ectocervical and connective tissue. The histological thus representing a potential for self-protection strategy, sections of each step (best viewed alongside) show these changes, especially women (Shattock and Rosenberg 2012). particularly the sharp decline in stratified squamous tissue (ectocervix) from day 0 to day 3, and the gradually reduction from day 3 to day 13 Studies of compounds with microbicidal activity are ad- (hematoxylin and eosin stain) vancing every day, but until now there is no microbicide available for use. Thus, our studies go for further Opoku-Anane et al. 2012). Our studies with the com- studies seeking confirmation as a microbicide. pound extracted from D. friabilis the dolabelladienetriol showed clear dose-dependent inhibition of HIV-1 replication. Our group has determined the effect and protec- Conclusions tion time caused by the compound, in addition to standard maintenance of the explant culture, confirming the The pretreatment of PBMCs and macrophages with importance of this model to study some aspects related dolabelladienotriol showed a strong inhibition of HIV-1 repli- to HIV-1 (Shattock and Rosenberg 2012). cation and the explant model confirm the potential of micro- After infection of the uterine cervix with HIV and bicidal activity of dolabelladienotriol at different concentra- incubating sections in 96 wells with 200 μLofculture tions. Besides that, the explant model has proven to be useful medium plates RPMI1640, the candidate microbicide for studying the infectivity of HIV and cytotoxicity of the was tested at different concentrations ranging from compound. However, further studies are needed to analyze 0.15 to 14.4 μM. As can be seen in Fig.4,the the mechanisms involved in the inhibition of HIV-1 replica- dolabelladienetriol was able to produce a protective tion by dolabelladienotriol.

Acknowledgments The authors are grateful to FIOCRUZ/IOC, UNESCO, CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support and this last for Productivity Fellowships to ICNPP (303368/2013-6) and VLT (301420/ 2010-6). ICNPP (E-26/103.024/2011) and VLT (E-26/103.176/2011) also thank the FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro) for the Cientista do Nosso Estado Fellowship. CSB thanks FAPERJ for the DSc fellowship (E-26/100.770/2012). CCCS thanks FAPERJ for the PHD fellowship (E-26/101.919/2009). Special thanks to Dr. Robin J. Shattock of the Department of Medicine, St Mary’s Campus, Imperial College London, London, United Kingdom.

Compliance with ethical standards

Fig. 4 Microbicidal activity of dolabelladienotriol at different Ethics approval The study was the study approved and registered with concentrations applied over the explant tissue. Error bars indicate the National Committee for Research Ethics, Brazil (CONEP) under protocol standard deviation and experiments were performed in triplicate number 15.958. J Appl Phycol

References safety and efficacy as a topical microbicide component. Proc Natl Acad Sci 106:6099–6104 Opoku-Anane J, Diouf K, Nour NM (2012) New success with microbicides and pre-exposure prophylaxis for human immunode- Arts E, Hazuda D (2012) HIV-1 antiretroviral drug therapy. Cold Spring ficiency virus (HIV): is female-controlled prevention the answer to Harb Perspect Med 2:a007161 the HIV epidemic? Rev Obstet Gynecol 5(1):50 Barbosa JP, Pereira RC, Abrantes J, Dos Santos CC, Rebello MA, de Palacio J, Souberbielle B, Shattock R, Robinson G, Manyonda I, Griffin Palmer Paixao Frugulhetti I, Teixeira V (2004) In vitro antiviral G (1994) In vitro HIV1 infection of human cervical tissue. Res Virol diterpenes from the Brazilian brown alga Dictyota pfaffii. Planta 145:155–161 Med 70:856–860 Barros CS, Cirne-Santos CC, Garrido V, Barcelos I, Stephens PRS, Pardo-Vargas A, Oliveira IB, Stephens PRS, Cirne-Santos CC, Paixão Giongo V,Teixeira VL, Paixão ICNP (2016). Anti-HIV-1 activity ICNP, Ramos FA, Jiménez C, Rodríguez J, Resende JALC, of compounds derived from marine alga Canistrocarpus Teixeira VL, Castellanos L (2014) Dolabelladienenols A-C, new cervicornis. J Appl Phycol 28:2523–2527 diterpenes isolated from Brazilian brown alga Dictyota pfaffii. Mar – Buffa V, Stieh D, Mamhood N, Hu Q, Fletcher P, Shattock RJ (2009) Drugs 12:4247 4259 Cyanovirin-N potently inhibits human immunodeficiency virus type Roan NR, Münch J (2015) Improving preclinical models of HIV micro- – 1infection in cellular and cervical explant models. J Gen Virol 90: bicide efficacy. Trends Microbiol 23:445 447 234–243 Rodríguez-Arenas MÁ, Jarrín I, Amo JD, Iribarren JA, Moreno S, Cirne-Santos CC, Teixeira VL, Castello-Branco LR, Frugulhetti IC, Bou- Viciana P, Peña A, Sirvent JLG, Vidal F, Lacruz J (2006) Delay in Habib DC (2006) Inhibition of HIV-1 replication in human primary the initiation of HAART, poorer virological response, and higher cells by a dolabellane diterpene isolated from the marine algae mortality among HIV-infected injecting drug users in Spain. AIDS Dictyota pfaffii. Planta Med 72:295–299 Res Hum Retrovir 22:715–723 Collins KB, Patterson BK, Naus GJ, Landers DV, Gupta P (2000) Shattock RJ, Rosenberg Z (2012) Microbicides: topical prevention Development of an in vitro organ culture model to study transmis- against HIV. Cold Spring Harb Perspect Med 2(2):a007385 sion of HIV-1 in the female genital tract. Nat Med 6:475–479 Sinha B, Rubens M (2014) Systemic immune activation in HIV and Crucitti G, Botta M, Di Santo R (2012) Will integrase inhibitors be used potential therapeutic options. Immunopharm Immunot 36:89–95 as microbicides? Cur HIV Res 10:36–41 Skoler-Karpoff S, Ramjee G, Ahmed K, Altini L, Plagianos MG, Cummins JE, Guarner J, Flowers L, Guenthner PC, Bartlett J, Morken T, FriedlandB,GovenderS,DeKockA,CassimN,PalaneeT Grohskopf LA, Paxton L, Dezzutti CS (2007) Preclinical testing of (2008) Efficacy of Carraguard for prevention of HIV infection in candidate topical microbicides for anti-human immunodeficiency women in South Africa: a randomised, double-blind, placebo- virus type 1 activity and tissue toxicity in a human cervical explant controlled trial. Lancet 372:1977–1987 – culture. Antimicrob Agents Chemother 51:1770 1779 Sleiman D, Goldschmidt V, Barraud P, Marquet R, Paillart J-C, Tisné C Gengiah TN, Karim QA (2012) Implementing microbicides in low- (2012) Initiation of HIV-1 reverse transcription and functional role – income countries. Best Pract Res Clin Obstet Gynaecol 26:495 501 of nucleocapsid-mediated tRNA/viral genome interactions. Virus Greenhead P, Hayes P, Watts PS, Laing KG, Griffin GE, Shattock RJ (2000) Res 169:324–339 Parameters of human immunodeficiency virus infection of human cer- Teas J, Irhimeh M (2012) Dietary algae and HIV/AIDS: proof of concept – vical tissue and inhibition by vaginal virucides. J Virol 74:5577 5586 clinical data. J Appl Phycol 24:575–582 Hu Q, Frank I, Williams V,Santos JJ, Watts P, Griffin GE, Moore JP,Pope Trinchero J, Ponce N, Córdoba OL, Flores ML, Pampuro S, Stortz CA, M, Shattock RJ (2004) Blockade of attachment and fusion receptors Salomón H, Turk G (2009) Antiretroviral activity of fucoidans ex- inhibits HIV-1 infection of human cervical tissue. J Exp Med 199: tracted from the brown seaweed Adenocystis utricularis.Phytother 1065–1075 Res 23:707–712 Huskens D, Schols D (2012) Algal lectins as potential HIV microbicide UNAIDS U, World Health Organization (2014) Global AIDS response candidates. Mar drugs 10:1476–1497 progress reporting 2014: construction of core indicators for moni- Kelly C, Shattock R (2011) Specific microbicides in the prevention of toring the 2011 United Nations political declaration on HIV and HIV infection. J Intern Med 270:509–519 AIDS. UNAIDS, Geneva Magiorkinis E, Paraskevis D, Magiorkinis G, Chryssou S, Chini M, Lazanas M, Paparizos V, Saroglou G, Antoniadou A, Giamarellou Veazey RS, Shattock RJ, Klasse PJ, Moore JP (2012) Animal models for – E (2002) Mutations associated with genotypic resistance to antire- microbicide studies. Curr HIV Res 10:79 87 troviral therapy in treatment naıve HIV-1 infected patients in Greece. Wang Q, Ding Z-H, Liu J-K, Zheng Y-T (2004) Xanthohumol, a novel Virus Res 85:109–115 anti-HIV-1 agent purified from hops Humulus lupulus. Antivir Res Maher D, Wu X, Schacker T, Horbul J, Southern P (2005) HIV binding, 64:189–194 penetration, and primary infection in human cervicovaginal tissue. Wei X, Decker JM, Liu H, Zhang Z, Arani RB, Kilby JM, Saag MS, Wu Proc Natl Acad Sci U S A 102:11504–11509 X, Shaw GM, Kappes JC (2002) Emergence of resistant human Montagnier L (2010) 25 years after HIV discovery: prospects for cure and immunodeficiency virus type 1 in patients receiving fusion inhibitor vaccine. Virol 397:248–254 (T-20) monotherapy. Antimicrob Agents Chemother 46:1896–1905 Morin SF, Yamey G, Rutherford GW (2012) HIV pre-exposure prophy- Whitehead SJ, McLean C, Chaikummao S, Braunstein S, Utaivoravit W, laxis. BMJ 345:e5412. doi:10.1136/bmj.e5412 van de Wijgert JH, Mock PA, Siraprapasiri T, Friedland BA, O’Keefe BR, Vojdani F, Buffa V, Shattock RJ, Montefiori DC, Bakke J, Kilmarx PH (2011) Acceptability of Carraguard vaginal microbicide Mirsalis J, d’Andrea A-L, Hume SD, Bratcher B (2009) Scaleable gel among HIV-infected women in Chiang Rai, Thailand. PLoS One manufacture of HIV-1 entry inhibitor griffithsin and validation of its 6(9):e14831 5.1.2. ANTIVIRAL EFFECT OF THE SEAWEED Osmundaria obtusiloba AGAINST THE ZIKA VIRUS

Vol. 12(25), pp. 387-395, 10 October, 2018 DOI: 10.5897/JMPR2018.6624 Article Number: B76A50558615 ISSN: 1996-0875 Copyright ©2018 Author(s) retain the copyright of this article Journal of Medicinal Plants Research http://www.academicjournals.org/JMPR

Full Length Research Paper

Antiviral effect of the seaweed Osmundaria obtusiloba against the Zika virus

Claudio Cesar Cirne-Santos1, Caroline de Souza Barros1,2 , Caio Cesar Richter Nogueira1,2, Leonardo dos Santos Corrêa Amorim1, Renata de Mendonça Campos3, Norman Arthur Ratcliffe4, Valeria Laneuville Teixeira2, Davis Fernandes Ferreira3 and Izabel Christina Nunes de Palmer Paixão1*

1Laboratório de Virologia Molecular e Biotecnologia Marinha, Programa de Pós-graduação em Ciências e Biotecnologia, Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense 24020-141, RJ, Brasil. 2Laboratório Algamar, Departamento de Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense 24020- 141, RJ, Brasil. 3Instituto de Microbiologia, Departamento de Virologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941- 902, Brazil. 4Department of Biociences, College of Science Swansea University, SA2 8PP. UK.

Received 13 June 2018; Accepted 30 July 2018

Zika virus (ZIKV), a mosquito-borne member of the family Flaviviridae, is a human pathogen of global significance. Recently, ZIKV, has become a public health problem with increases in numbers of cases and a strong association between ZIKV outbreaks and the spread of cases of Guillain-Barré Syndrome and microcephaly. In this study, the extracts of the seaweed Osmundaria obtusiloba (O. obtusiloba) (native to the Brazilian coast) against ZIKV using Vero cells was evaluated. The seaweed extract tested inhibited ZIKV replication in a dose-dependent manner at low concentrations with EC50 values of 1.82 μg/mL and a selective index (SI) of 288. Other results showed that this extract had significant virucidal effects. In addition, when the extract and Ribavirin were used concomitantly there was a significant synergistic effect. Our promising results suggest that extracts of O. obtusiloba are excellent candidates for further studies, and that marine algae are potentially important sources for the development of novel anti-ZIKV agents.

Key words: ZIKA, seaweeds, antiviral activity, marine algae, Osmundaria obtusiloba.

INTRODUCTION

The antiviral potential of marine macroalgae is widely number of species of algae against HIV-1 (Cirne-Santos recorded. Several studies have demonstrated activity of a et al., 2008; Barros et al., 2016), HSV-1 (Macedo et al.,

*Corresponding author. E-mail: [email protected]

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

388 J. Med. Plants Res.

2012; Mendes et al., 2012; Soares et al., 2012; Barros et large outbreak in Yap Island Federal Sate of Micronesia al., 2015), HSV-2 (Mendes et al., 2012; Soares et al., (Faye et al., 2014; Hamel et al., 2016). In October of 2013, 2012) and dengue type 2 strains (Talarico et al., 2007). a large outbreak occurred in French Polynesia where 396 Thus, marine algae provide a potentially rich source for laboratory confirmed cases were reported. Up to now, two the discovery of antiviral drugs. In this study, the native main distinct ZIKV geographic lineages have been algal extract of Osmundaria obtusiloba (C.Agardh) described (African and Asian) (Musso et al., 2014; R.E.Norris, from the Brazilian coast, was tested for Kucharski et al., 2016). inhibiting the replication of the Zika virus (ZIKV) (Hayashi A substantial proportion of ZIKV infections are et al., 2007). subclinical, but when clinical symptoms occur, the disease Arthropod-borne viruses, commonly called arboviruses, produced was considered moderate and self-limiting. normally circulate in nature through biological Recent studies, however, have described a strong transmission between susceptible vertebrate hosts and association between ZIKV outbreak and an increased blood-feeding arthropods, such as mosquitoes. Studies number of cases of Guillain-Barré Syndrome (GBS) in show that the most important mosquitoes in this French Polynesia, indicating a first complication resulting transmission are A. aegypti, although there is also strong from a ZIKV infection (Cao-Lormeau et al., 2016; Teixeira evidence for the role of A. albopictus in this process too et al., 2016). There is also strong evidence for the (Kraemer et al., 2015; Calvez et al., 2016). incidence of cases of microcephaly following ZIKV The main arboviruses causing disease in humans infection of women during pregnancy. These observations include the alphaviruses (Togaviridae: Alphavirus), have been supported by evidence accrued during different flaviviruses (Flaviviridae: Flaviviruses), Bunyaviruses outbreaks and resulted in great fear in pregnant women (Bunyaviridae) and some members of other virus families (Mlakar et al., 2016; Rodrigues, 2016). Other factors (Rhabdoviridae and Reoviridae). Currently, of the 534 associated with ZIKV infection, such as hyperglycemia, viruses listed in the International Catalogue of among other malignancies are clear demonstrations of Arboviruses, 214 are known to be, or are probably the a potent morbidity of this virus (Nielsen and Bygbjerg, associated with arthropods, 287 viruses are reported to be 2016). possible arboviruses, and 33 are probably or definitely not In May 15, 2015, the Ministry of Health of Brazil arboviruses (Gubler, 2001; Iranpour et al., 2016). In total, confirmed ZIKV circulation in the country after ZIKV 134 of the 534 arboviruses have been reported to cause identification in 16 samples (eight from Bahia and eight disease in humans and have a global distribution with the from Rio Grande do Norte) by the National Reference majority circulating in tropical areas, where climatic Laboratory. The symptoms that were described as are the conditions favor transmission throughout the year most common include, arthralgia, edema of the (Gyawali et al., 2016; Tabachnick, 2016). extremities, slight fever, headache, retro-orbital pain, Zika virus (ZIKV) is a mosquito-borne and from the conjunctival hyperemia and maculopapular rashes, often genus Flavivirus, family Flaviviridae and clusters with the spreading down the face to the limbs and often itchy, Spondweni serocomplex (Vorou, 2016). Flaviviruses have dizziness, myalgia and digestive disorders.(Junior et al., a positive sense single-strand RNA genome of 2015; Heukelbach et al., 2016). approximately 11,000 nucleotides in length. The genome There is no vaccine or specific antiviral therapy for the contains a long open-reading frame (ORF) that encodes prevention or treatment of infections by ZIKV (Barrows et three structural proteins (capsid, precursor membrane and al., 2016). A study identified the viral polymerase inhibitor, envelope) that form the viral particles and seven non- 7DMA, as an inhibitor of in vitro ZIKV replication, and, in structural proteins (NS1, NS2A, NS2B, NS3, NS4A, virus-infected mice significantly reduced viremia and NS4B, and NS5). The non-structural proteins participate in delayed virus-induced morbidity and mortality (Zmurko et viral replication, virion assembly, and evasion of the host al., 2016). Deng et al. (2016) also showed that an immune response (Lindenbach et al., 2013). The infection adenosine analog has in vitro and in vivo activity against in humans produces a self-limiting acute febrile illness ZIKV. with fever, headache, myalgia and rash, very similar to Previous studies have shown different biological other arboviruses like Dengue Virus and Chikungunya. activities for O. obtusiloba extract such as: In vivo tests Therefore, in regions where more than one arbovirus is using BALB/c mice infected with L. amazonenses in the detected, ZIKV could be circulating but not be notified control of the dissemination of this parasite (Lira et al., causing misleading and low incidences recorded (Tappe 2016); acute toxicity tests have demonstrated that O. et al., 2015; Vorou, 2016). obtusiloba extract does not produce significant toxic After the ZIKV was first detected in 1947, during a effects in BALB/c mice (de Souza Barros et al., 2018); and yellow fever surveillance program in Uganda, few reports studies have shown that compounds derived from O. of the disease were recorded until 2007 when there was a obtusiloba showed potent antiviral activity against HSV-1

Cirne-Santos et al. 389

and HSV-2 and had low toxicity to cell cultures (de Souza Plaque reduction assay et al., 2012). In this way, these studies reinforced our perspectives for the accomplishment of this work. VERO cells were cultured in DMEM medium and after confluence were incubated with the ZIKV (MOI of 0.1) for 2 h. Subsequently, the Characterization of the antiviral activity of the crude extract cells were washed with PBS to remove the residual virus and a 2% of the algae O. obtusiloba against ZIKV was done in this (w/v) mixture of CMC (Sigma Aldrich) was added with DMEM work. It is shown that the extract inhibited ZIKV replication supplemented with 5% FBS, 5 mmol/L L-glutamine and 0.20% of and thus our findings broaden the antiviral scope. sodium bicarbonate. Different concentrations of the seaweed extracts were added and incubated at 37°C in a 5% CO2 atmosphere and analyzed daily for plaque formation. Subsequently, the cells MATERIALS AND METHODS were fixed with 10% formaldehyde, then stained with 1% crystal violet and the plates were examined and plaque formation Seaweed material and extraction quantified. The assay was used for evaluation of antiviral activity and for viral titration. The infectious virus titer (PFU/mL) was determined The seaweed O. obtusiloba, is a native species of Brazil and was using the following formula: plate count × dilution factor × (1 / collected by snorkeling at a depth 1-3 m in various coastal sites in inoculation volume). Rasa Beach, Armação de Búzios, Rio de Janeiro State (lat. 22° 45’40”, long. 41° 54’ 32”). The seaweed was separated from sediments, epiphytes, and other associated organisms, washed with Antiviral assay sea water and air-dried (approximate ca. temperature 28-30°C for 7- 10 days) until the total evaporation of any water. Air-dried seaweed Antiviral activity was evaluated using a virus plaque reduction assay. (approximately 100 g) was powdered and exhaustively extracted Vero cells were grown in 24-well plates under conditions described three times using ethanol for 72 h in the approximate temperature of above and subsequently infected with ZIKV (MOI of 0.1) in the 28 to 30ºC. The extract was evaporated under reduced pressure, absence or presence of different concentrations of the crude yielding crude extract (15 to 20 mg), of which 2 to 5 mg was used in seaweed extracts ranging from 1.25, 2.5, 5, 10, 15, 20, 25 or 50 tests against the ZIKV. The ethanolic extract was chosen due to the μg/mL, respectively. After 1 h of adsorption at 37°C, residual efficiency of ethanol in extracting the phenolic compounds from O. inoculum was replaced by medium containing 2% methyl-cellulose obtusiloba (Carvalho et al., 2006) and also the low toxicity in vivo of and the corresponding dose of each extract. Plates were evaluated this extract (de Souza Barros et al., 2018). daily and counted between 5 to 10 days of incubation at 37°C in 5% CO2. The 50% inhibitory concentration (EC50) was calculated as the extract concentration required reducing the virus plaques by 50%. All Cells and virus experiments were performed twice and each in triplicate.

Vero cells (African green monkey kidney) were grown in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no 11960) Viral kinetics and time-of-drug addition studies supplemented with 5% Fetal Bovine Serum (FBS; Invitrogen), 2 mmol/L L-glutamine (Invitrogen, cat. no. 25030). Antibiotics were Vero cells were cultured in 24 well plates, as above and after 90% added at a final concentrations of 50 units/ml penicillin and confluence, was treated differentially. In some wells, the cells were streptomycin (Invitrogen, cat. no. 15070). ZIKV (ATCC® VR-1839™) pretreated with the crude extract at 5 μg/mL, from O. obtusiloba for was amplified in C6/36 mosquito cells line from A. albopictus, 1, 2 or 3 h prior to infection. Subsequently, these cells were adapted to grow at 28°C, was cultured in L-15 Medium (Leibovitz) incubated with ZIKV (MOI of 0.1) while in other wells cells were supplemented with 0.5% tryptose phosphate broth, 0.03% incubated at time 0 (immediately after infection) or at 1, 2 and 3 h glutamine, 1% MEM non-essential amino acids solution and 5% post-infection with the extract concentration of 5 μg/mL. Cells were FBS. then maintained under the conditions for the plaque assay production at 37°C in 5% in CO2. Inhibition of viral replication was evaluated in relation to the control cells, incubated without extract in Cellular cytotoxicity assays 3 independent experiments in triplicate.

To evaluate the cytotoxic effect of the seaweed extract, VERO cells were cultured in 96-well plates to 90% confluence. The cells were Virucidal effect then treated with increasing concentrations (25, 50, 100, 200, 400 and 800 μg/ml) of the crude extracts of algae and incubated for 2 to A suspension of ZIKV, containing the relative concentration which in 3 days in DMEM culture medium with 5% FBS at 37°C in a 5% CO2 atmosphere. For assessment of cell viability, the MTT method was culture corresponds to an MOI of 0.1, was incubated with the same used as previously described by Mosmann (1983). In the 96-well volume of algae extract from O. obtusiloba at concentrations of 2.5, plate previously treated with the extracts, the MTT reagent [3-(4,5- 5 or 10 μg/mL and incubated in microtiter plates for 2 h at 37°C. The dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] Sigma- pre-incubated samples were then added to Vero cells in 24-well Aldrich) was added at a concentration of 5 mg/ml and incubated for plates for 2 h, washed and incubated under plate-assay conditions. 3 h at 37°C. The MTT medium was then removed and 100 μM The virucidal effect was defined by the ability of the compound to dimethylsulfoxide (DMSO) was added to the plate and incubated for inactivate the particles not allowing infection and without generating 15 min to dissolve the formazan crystals (Mosmann, 1983).. The cytopathic effect as observed in the virus-only control. plate was read in an ELISA reader at 550 nm absorbance. The percentage of metabolically active cells was compared to the control of extract untreated cells, to determine the cytotoxicity of the Synergistic effect test O. obtusiloba plus Ribavirin compounds. These assays were performed three times independently, each in triplicate. For this analysis, Vero cells were cultured in 24-well plates

390 J. Med. Plants Res.

Table 1. Cytotoxicity (CC50), anti-ZIKV profile (EC50) and selectivity index (SI) of the O. obtusiloba extract compared with the Ribavirin control.

a b c Crude extracts CC50 (µg/mL) EC50 (µg/mL) SI O. obtusiloba 525 ± 3.11 1.82 ± 0.49 288 Ribavirin 297 ± 4.25 3.95 ± 0.95 75.2

The mean values ± standard deviations are representative of three independent experiments. aConcentration that reduced 50% cytotoxic concentration when compared to untreated controls. bConcentration that reduced 50% of ZIKV replication when compared to infected controls. c Selectivity index was defined as the ratio between CC50 and EC50 and represents the safety for in vitro assays.

subsequently infected with ZIKV MOI 0.1. Subsequently, the infected respectively, were better and gave significances and times cells were treated with a high dose of 10 μg / ml extract and 10 μM significantly better than those obtained by ribavirin (EC50= Ribavirin capable of inhibiting 90% of viral replication. The inhibition 3.95 µg/mL; SI=75.2) used as a control (Table 1). with a minimum dose of both 0.5 μg / mL extract and 0.5 μM Ribavirin which are concentrations that inhibit replication below 20% was also evaluated. For the synergism evaluation, we combined the concentrations of the compounds and added the infected cells. After Virucidal effect 72 h, inhibition of cytopathic effect was observed by inhibition of viral plaque formation. The virucidal activity of O. obtusiloba extract was

evaluated against ZIKV. The viral suspension was Statistical analysis maintained with different concentrations of the extracts (2.5, 5 and 10 µg/mL) for 2 h and then added to Vero cell cultures. The results showed that O. obtusiloba had a The data were analyzed by one-way analysis of variance (ANOVA) good inactivation capacity of the virus (virucidal effect). followed by a Tukey test using the GraphPad Instat version 3 Figure 2 shows that the O. obtusiloba extract significantly program. A p value of <0.05 was considered statistically significant. The values of p<0.05, p<0.01 and p<0.001 are shown in the figures. inhibited ZIKV infectivity at higher levels than Ribavirin at all concentrations tested and at 10 μg/mL of this extract inhibited about 80% of ZIKV replication. RESULTS

Cytotoxicity and effect of the extract on the ZIKV Time of drug addition replication in Vero cells To identify the step at which viral replication might be inhibited, time of addition experiments were performed The cytotoxicity (CC50) of the extract from the algae was assessed by MTT (Sigma-Aldrich), as previously with the compounds administered 5.0 µg/mL at 3, 2, 1 h described (Mosmann, 1983) with some modifications. The before infection. Subsequently, the virus was added at results in Table 1 show that the O. obtusiloba extract time 0, 1, 2, and 3 h after infection and Ribavirin was used as a control (5 µM). At time 0, O. obtusiloba extract produced the best CC50 with a value of 525 µg/mL. Subsequently, the antiviral activities of the extract were inhibited over 80% of viral replication. O. obtusiloba and evaluated. For these analyzes, different concentrations of Ribavirin maintained an inhibitory effect at the other times the extract was tested starting with a concentration having but with a decline recorded but even at 3 h after virus an inhibitory potential of 20 μg/mL and reducing the infection at least 60% inhibition occurred in viral replication concentration progressively. The extract inhibited the (Figure 3). replication of ZIKV in a dose-dependent manner (Figure 1). The results demonstrate that O. obtusiloba extract inhibited above 90% of ZIKV replication in the highest Synergism between the extract of Osmundaria concentrations (20 μg/mL) with low EC50 values of 1.82 obtusiloba and Ribavirin μg/mL. Based on these data, the Selectivity Index (SI), representing the degree of reliability of the extracts for The results obtained clearly show the inhibitory efficiency possible future use, was derived from the relationship of the O. obtusiloba extract on ZIKA was greatly increased between the CC50 and EC50 levels. The values of EC50 and with the addition of Ribavirin and can be attributed to a SI presented by O. obtusiloba extract, 1.82 µg/mL and 288 synergistic effect. As shown in Figure 4, the addition of

Cirne-Santos et al. 391

Figure 1. Inhibition of ZIKV replication by O. obtusiloba extract. Vero cells were infected with ZIKV (MOI 0.1) and treated at concentrations of 1.25, 2.5, 5, 10 or 20 µg / mL and varying concentrations of Ribavirin as a control. The results were evaluated from three independent experiments in triplicate. Data are presented as percentage of virus titer, when compared to control cells and are expressed as the mean of three experiments ± standard error. Statistical analysis was performed using Tukey test in comparison of O. obtusiloba with Ribavirin in each concentration: ***p<0.001.

Figure 2. Virucidal effect. Viral suspension (ZIKV) was incubated with O. obtusiloba extract at the concentrations of 2.5, 5 or 10 µg/ mL for 2 hours and then added to Vero cells. Data are presented as percentage of virus titer, when compared to Ribavarin control cells and are expressed as the mean of three experiments ± standard error. Statistical analysis was performed using Tukey test in comparison of O. obtusiloba with Ribavirin in each concentration: **p<0.01; ***p<0.001.

subdoses of O. obtusiloba extract (0.5 μg / mL) resulted in of Ribavirin recorded ca. 15% inhibition. However, when only ca. 20% virus inhibition, and, similarly, 0.5 μM doses the seaweed extract and Ribavirin were given together

392 J. Med. Plants Res.

Figure 3. Effect of the addition of O. obtusiloba extract on replication over time. Monolayers of Vero cells were infected with ZIKV at an MOI of 0.1 at time zero. At the times indicated, extract or ribavirin was added to a final concentration of 10 µg /mL for extract or 10µM / mL, respectively. Data are presented as percentage of virus titer, when compared to control cells and are expressed as the mean of three experiments ± standard error. Statistical analysis was performed using Tukey test in comparison of O. obtusiloba with Ribavirin in each time: **p<0.01; ***p<0.001.

and associated with the lowest concentrations, the Previously seaweed extracts have been reported with little inhibitory effect was potentiated and was inhibiting almost cytotoxicity (Alencar et al., 2014) but with considerable 3 times more than the effect of both added at 0.5 μM antiviral activity, for example, in studies with HIV alone, generating an inhibition of the replication of the (Nogueira et al., 2016) and against Herpes (de Souza ZIKV of approximately 90%, and thus showing Barros et al., 2017). characterizing a strong synergistic effect. In the present work, initially the seaweed extract was evaluated for virucidals activity and shown to inactivate the ZIKA particles. The studies demonstrated that the DISCUSSION extract of O. obtusiloba inactivated the viral particles up to 80% in concentrations of up to 10 μg/mL. Thus, additional In this study, the results demonstrate that O. obtusiloba studies of this virucidal compound are necessary to extract inhibited viral replication significantly when cells develop new strategies for the preparation of preventive were treated with various concentrations of the extract and measures. that inhibition was dose-dependent generating an EC50 of Looking for specific characteristics of the mechanism of 1.82 μg/mL. In addition, there was a low cytotoxicity of the action of the extract, studies such as the time of addition extract on Vero cells resulting in a CC50 of 525 μg/mL. of the extracts (Time of Addition Experiment) showed that Interestingly, the extract showed a selectivity index (SI) of the O. obsutiloba used at different times both pre- and 288 which, as demonstrated in the literature, is described post-infection has great potential to inhibit the replication as good for SI compounds with values greater than 100 of ZIKV, around 60% after treating up to 3 h prior to (Silva et al., 2011; Zandi et al., 2011). infection. At time 0, however, the addition of the extract The literature records a considerable number of studies was concomitant with the infection of the cells, and of antivirals for Dengue that has very significant results resulted in 90% inhibition of the virus replication. Even if (Zandi et al., 2012). However, few studies have been the extract was added up to 3 h after infection, there was undertaken for other arboviruses that have results as still inhibition of around 80% of the virus. As far as significant as those on Dengue. ZIKA was focused on, infections are concerned, the signs and symptoms can which has been associated with severe syndromes initially be confusing and the follow-up may be delayed but (Alvarado-Socarras et al., 2018; Barbi et al., 2018). a drug capable of treating late infections would be an

Cirne-Santos et al. 393

Figure 4. Evaluation of the synergistic effect in combination of the extract of O. obtusiloba and Ribavirin. Monolayers of Vero cells were infected with ZIKV at an MOI of 0.1 and subsequently treated with the extract and Ribavirin subdoses (0.5µg/mL and 0.5µM, respectively) and concentrations of 10µg/mL and 10µM. In addition, combinations of the extract and Ribavirin were also tested at both concentrations for assessment of synergism. Evaluation of the synergistic antiviral effect was determined by the inhibition of cytopathic effects by plaque assay. Data are presented as percentage of virus titer, when compared to control cells and are expressed as the mean of three experiments ± standard error. Statistical analysis was performed using Tukey test: * p<0.05; **p<0.01; **p<0.001.

important additional strategy. These data suggest the There is much interest in searching for combinations of potential of O. obtusiloba as an algal extract candidate for drugs for the inhibition of virus replication as described future development. Similarly, Zmurko et al. (2016) for Dengue (Yeo et al., 2015), HSV-1 (Mancini et al., performed time of drug addition studies against ZIKV with 2009), and also for Chikungunya (Mishra et al., 2016). the 7 DMA viral polymerase inhibitor, but without These analyzes are performed in order to reduce the pretreatment and with posttreatment at different times up concentration of substances used and to optimize the to 24 h. They showed that the addition of the compound treatments making them more effective and less toxic. to the infected cells could be delayed up to ~10 h after The results herein demonstrated an important synergistic infection without much loss of antiviral potency (Zmurko effect by the combination of Ribavirin and the O. et al., 2016). obtusiloba extract (Figure 4), since the use of both

394 J. Med. Plants Res.

combined in subdoses were able to inhibit viral replication Barbi L, Coelho AVC, Alencar LCA , Crovella S (2018). Prevalence three times more than both evaluated separately. of guillain-barre syndrome among zika virus infected cases: A systematic review and meta-analysis. The Brazilian Journal of Infectious Diseases 22(2):137-141. Barros CdS, Cirne-Santos CC, Garrido V, Barcelos I, Stephens PRS, Conclusions Giongo V, Teixeira VL ,de Palmer Paixão ICN (2016). Anti-hiv-1 activity of compounds derived from marine alga canistrocarpus cervicornis. Journal of Applied Phycology 28(4):2523-2527. The findings showed that the crude O. obtusiloba Barros CS, Teixeira VL, Paixão IC (2015). Seaweeds with anti-herpes seaweed extract tested had activity against ZIKV, simplex virus type 1 activity. Journal of Applied Phycology demonstrating that marine algae are an interesting 27(4):1623-1637. source for drug discovery and the development of novel Barrows NJ, Campos RK, Powell ST, Prasanth KR, Schott-Lerner G, Soto-Acosta R, Galarza-Muñoz G, McGrath EL, Urrabaz-Garza R, anti-ZIKV agents. Extracts of O. obtusiloba all gave very Gao J (2016). A screen of fda-approved drugs for inhibitors of zika promising results, and are candidates for further studies virus infection. Cell Host and Microbe 20(2):259-270. to isolate their active factors and better elucidate their Calvez E, Guillaumot L, Millet L, Marie J, Bossin H, Rama V, Faamoe A, mechanisms of action. In summary, O. obtusiloba extract Kilama S, Teurlai M, Mathieu-Daudé F (2016). Genetic diversity and phylogeny of aedes aegypti, the main arbovirus vector in the pacific. has anti-ZIKV with a particularly significant virucidal PLoS Neglected Tropical Diseases 10(1):e0004374. effects and synergistic effect in combination with Cao-Lormeau V-M, Blake A, Mons S, Lastère S, Roche C, Ribavirin. Currently, there are no vaccines or specific Vanhomwegen J, Dub T, Baudouin L, Teissier A, Larre P (2016). drugs for prevention and treatment of ZIKV infection. The Guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: A case-control study. The Lancet results demonstrate the importance of the marine 387(10027):1531-1539. environment in the search for antivirals drugs with activity Carvalho LRD, Guimarães SM, Roque NF (2006). Sulfated against ZIKV. bromophenols from osmundaria obtusiloba (c. Agardh) re norris (rhodophyta, ceramiales). Brazilian Journal of Botany 29(3):453-459. Cirne-Santos CC, Souza TML, Teixeira VL, Fontes CFL, Rebello MA, Castello-Branco LRR, Abreu CM, Tanuri A, Frugulhetti IC, Bou-Habib CONFLICT OF INTERESTS DC (2008). The dolabellane diterpene dolabelladienetriol is a typical noncompetitive inhibitor of hiv-1 reverse transcriptase enzyme. The authors have not declared any conflict of interests. Antiviral Research 77(1):64-71. de Souza Barros C, Garrido V, Melchiades V, Gomes R, Gomes MWL, Teixeira VL, de Palmer Paixão ICN (2017). Therapeutic efficacy in BALB/C mice of extract from marine alga canistrocarpus cervicornis ACKNOWLEDGMENTS (phaeophyceae) against herpes simplex virus type 1. Journal of Applied Phycology 29(2):769-773. The authors are grateful to Conselho Nacional de de Souza Barros C, Gomes MWL, Gomes RdSP, Melchiades V, Nogueira CCR, Cirne-Santos CC, esar, Garrido V, Pinto CEC, Desenvolvimento Científico e Tecnológico (CNPq) for the Teixeira VL, de Palmer Paixao ICN (2018). Acute toxicity evaluation financial support and for Productivity Fellowships to of ethanol extract of red algae, osmundaria obtusiloba, in balb/c mice. ICNPP and VLT (443930/2014-7 and 304070/2014-9). Journal of Medicinal Plants Research 12(17):217-221. ICNPP and VLT (E-26/201.442/2014) also thank de Souza LM, Sassaki GL, Romanos MTV, Barreto-Bergter E (2012). Structural characterization and anti-hsv-1 and hsv-2 activity of Fundação de Amparo à Pesquisa do Estado do Rio de glycolipids from the marine algae osmundaria obtusiloba isolated Janeiro (FAPERJ) for the Cientista do Nosso Estado from southeastern brazilian coast. Marine Drugs 10(4):918-931. Fellowship. CCCS thanks Coordenação de Deng YQ, Zhang NN, Li CF, Tian M, Hao JN, Xie XP, Shi PY, Qin CF Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (2016). Adenosine analog nitd008 is a potent inhibitor of zika virus. In Open forum infectious diseases. Oxford University Press 3(4). for the Postdoc fellowship and CSB thanks FAPERJ for Faye O, Freire CC, Iamarino A, Faye O, de Oliveira JVC, Diallo M, the Postdoc fellowship (E-26/201.344/2016) in the Zanotto PM (2014). Molecular evolution of zika virus during its postgraduate program in sciences and biotechnology of emergence in the 20 th century. PLoS Neglected Tropical Diseases UFF (PPBI-UFF). 8(1):e2636. Gubler DJ (2001). Human arbovirus infections worldwide. Annals of the New York Academy of Sciences 951: 13-24. Gyawali N, Bradbury RS, Taylor-Robinson AW (2016). Do neglected REFERENCES australian arboviruses pose a global epidemic threat? Australian and New Zealand Journal of Public Health 40(6):596-596. Alencar DB, Silva SR, Pires-Cavalcante KM, Lima RL, Pereira Junior Hamel R, Liégeois F, Wichit S, Pompon J, Diop F, Talignani L, Thomas FN, Sousa MB, Viana FA, Nagano CS, Nascimento KS, Cavada BS, F, Desprès P, Yssel H, Missé D (2016). Zika virus: Epidemiology, Sampaio AH ,Saker-Sampaio S (2014). Antioxidant potential and clinical features and host-virus interactions. Microbes and Infection cytotoxic activity of two red seaweed species, amansia multifida and 18(7-8):441-449. meristiella echinocarpa, from the coast of northeastern brazil. Anais Hayashi L, de Paula EJ, Chow F (2007). Growth rate and carrageenan da Academia Brasileira de Ciencias 86(1):251-263. analyses in four strains of kappaphycus alvarezii (rhodophyta, Alvarado-Socarras JL, Idrovo AJ, Contreras-Garcia GA, Rodriguez- gigartinales) farmed in the subtropical waters of são paulo state, Morales AJ, Audcent TA, Mogollon-Mendoza AC, Paniz-Mondolfi A brazil. Journal of Applied Phycology 19(5): 393-399. (2018). Congenital microcephaly: A diagnostic challenge during zika Heukelbach J, Alencar CH, Kelvin AA, de Oliveira WK, de Góes epidemics. Travel medicine and infectious disease. Cavalcanti LP (2016). Zika virus outbreak in brazil. The Journal of

Cirne-Santos et al. 395

Infection in Developing Countries 10(02):116-120. Soares AR, Robaina M, Mendes GS, Silva TS, Gestinari L, Pamplona Iranpour M, Moghadam AR, Yazdi M, Ande SR, Alizadeh J, Wiechec E, OS, Yoneshigue-Valentin Y, Kaiser CR, Romanos MTV (2012). Lindsay R, Drebot M, Coombs KM, Ghavami S (2016). Apoptosis, Antiviral activity of extracts from brazilian seaweeds against herpes autophagy and unfolded protein response pathways in arbovirus simplex virus. Revista Brasileira de Farmacognosia 22(4):714-723. replication and pathogenesis. Expert Reviews in Molecular Medicine Tabachnick WJ (2016). Climate change and the arboviruses: Lessons 18 p. from the evolution of the dengue and yellow fever viruses. Annual Junior VLP, Luz K, Parreira R, Ferrinho P (2015). Zika virus: A review to Review of Virology 3:125-145. clinicians. Acta Medica Portuguesa 28(6):760-765. Talarico LB, Duarte ME, Zibetti RG, Noseda MD, Damonte EB (2007). Kraemer MU, Sinka ME, Duda KA, Mylne AQ, Shearer FM, Barker CM, An algal-derived dl-galactan hybrid is an efficient preventing agent for Moore CG, Carvalho RG, Coelho GE , Van Bortel W (2015). The in vitro dengue virus infection. Planta Medica 73(14):1464-1468. global distribution of the arbovirus vectors aedes aegypti and Ae. Tappe D, Nachtigall S, Kapaun A, Schnitzler P, Gunther S ,Schmidt- Albopictus. Elife 4:e08347. Chanasit J, (2015). Acute zika virus infection after travel to malaysian Kucharski AJ, Funk S, Eggo RM, Mallet H-P, Edmunds WJ, Nilles EJ borneo, september 2014. Emerging infectious diseases 21(5):911- (2016). Transmission dynamics of zika virus in island populations: A 913. modelling analysis of the 2013–14 french polynesia outbreak. PLoS Teixeira MG, da Conceição N. Costa M, de Oliveira WK, Nunes ML, Neglected Tropical Diseases 10(5):e0004726. Rodrigues LC (2016). The epidemic of zika virus–related Lindenbach B, Murray C, Thiel H, Rice C (2013). Flaviviridae. Fields microcephaly in brazil: Detection, control, etiology, and future Virology 1:712-746. scenarios. American Journal of Public Health 106(4):601-605. Lira M-LF, Lopes R, Gomes AP, Barcellos G, Verícimo M, Osako K, Vorou R (2016). Zika virus, vectors, reservoirs, amplifying hosts, and Ortiz-Ramirez FA, Ramos CJB, Cavalcanti DN, Teixeira VL (2016). their potential to spread worldwide: What we know and what we Anti-leishmanial activity of brazilian green, brown, and red algae. should investigate urgently. International Journal of Infectious Journal of Applied Phycology 28(1):591-598. Diseases 48:85-90. Macedo NRPV, Ribeiro MS, Villaça RC, Ferreira W, Pinto AM, Teixeira Yeo KL, Chen Y-L, Xu HY, Dong H, Wang Q-Y, Yokokawa F, Shi P-Y VL, Cirne-Santos C, Paixão IC, Giongo V (2012). Caulerpin as a (2015). Synergistic suppression of dengue virus replication using a potential antiviral drug against herpes simplex virus type 1. Revista combination of nucleoside analogs and nucleoside synthesis Brasileira de Farmacognosia 22(4):861-867. inhibitors. Antimicrobial Agents and Chemotherapy 59(4):2086-2093. Mancini DAP, Torres RP, Pinto JR, Mancini-Filho J (2009). Inhibition of Zandi K, Teoh BT, Sam SS, Wong PF, Mustafa MR ,Abubakar S (2012). DNA virus: Herpes-1 (hsv-1) in cellular culture replication, through an Novel antiviral activity of baicalein against dengue virus. BMC antioxidant treatment extracted from rosemary spice. Brazilian Complementary and Alternative Medicine 12(1):214. Journal of Pharmaceutical Sciences 45(1):127-133. Zandi K, Teoh BT, Sam SS, Wong PF, Mustafa MR, Abubakar S (2011). Mendes GdS, Bravin IC, Yoneshigue-Valentin Y, Yokoya NS, Romanos Antiviral activity of four types of bioflavonoid against dengue virus MTV (2012). Anti-hsv activity of hypnea musciformis cultured with type-2. Virology Journal 8(1):560. different phytohormones. Revista Brasileira de Farmacognosia Zmurko J, Marques RE, Schols D, Verbeken E, Kaptein SJ, Neyts J 22(4):789-794. (2016). The viral polymerase inhibitor 7-deaza-2’-c-methyladenosine Mishra P, Kumar A, Mamidi P, Kumar S, Basantray I, Saswat T, Das I, is a potent inhibitor of in vitro zika virus replication and delays Nayak TK, Chattopadhyay S, Subudhi BB (2016). Inhibition of disease progression in a robust mouse infection model. PLoS chikungunya virus replication by 1-[(2-methylbenzimidazol-1-yl) Neglected Tropical Diseases 10(5):e0004695. methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (mbzm-n-ibt). Scientific Reports 6:20122. Mlakar J, Korva M, Tul N, Popović M, Poljšak-Prijatelj M, Mraz J, Kolenc M, Resman Rus K, Vesnaver Vipotnik T, Fabjan Vodušek V (2016). Zika virus associated with microcephaly. New England Journal of Medicine 374(10):951-958. Mosmann T (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65(1-2):55-63. Musso D, Nilles E, Cao-Lormeau V-M (2014). Rapid spread of emerging zika virus in the pacific area. Clinical Microbiology and Infection 20(10):O595-O596. Nielsen KK, Bygbjerg IC (2016). Zika virus and hyperglycaemia in pregnancy. The Lancet 387(10030):1812. Nogueira CCR, de Palmer Paixão ICN, Cirne-Santos CC, Stephens PRS, Villaça RC, de Souza Pereira H, Teixeira V, Laneuville E (2016). Anti-hiv-1 activity in human primary cells and anti-hiv-1 rt inhibitory activity of extracts from the red seaweed acanthophora spicifera. Journal of Medicinal Plants Research 10(35):621-625. Rodrigues LC (2016). Microcephaly and zika virus infection. The Lancet 387(10033):2070-2072. Silva A, Morais S, Marques M, Lima D, Santos S, Almeida R, Vieira I, Guedes M (2011). Antiviral activities of extracts and phenolic components of two spondias species against dengue virus. Journal of Venomous Animals and Toxins Including Tropical Diseases 17(4):406-413.

5.1.3. AMINOMETHYLNAPHTHOQUINONES AND HSV-1: IN VIVO AND IN SILICO EVALUATIONS OF POTENTIAL ANTIVIRALS

Antiviral Therapy 2016; 21:507–515 (doi: 10.3851/IMP3039)

Original article Aminomethylnaphthoquinones and HSV-1: in vitro and in silico evaluations of potential antivirals

Camilly P Pires de Mello1, Nathália S Sardoux1, Luciana Terra1, Leonardo C Amorim1,2, Maria D Vargas3, Gustavo B da Silva3, Helena C Castro1, Viveca A Giongo1, Lucianne F Madeira1, Izabel CNP Paixão1*

1PPBI, Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Campus do Valonguinho, Niterói, Brazil 2Laboratório de Inovações em Terapias, Ensino e Bioprodutos (LITEB), Instituto Oswaldo Cruz (IOC), FIOCRUZ, Rio de Janeiro, Brazil 3Departamento de Química Inorgânica, Instituto de Química, Universidade Federal Fluminense, Campus do Valonguinho, Niterói, Brazil

*Corresponding author e-mail: [email protected]

Background: Herpes simplex viruses (HSV) are leading causes activity. Our in vitro results showed that these compounds of human infections which result in severe manifestations, have significant anti-HSV-1 activity comparable to acy- especially in neonates, immunocompromised and/or trans- clovir, the antiviral currently used clinically. Importantly planted individuals. Current HSV type-1 (HSV-1) resistance two of them showed a lower cytotoxicity profile against to standard antiviral agents is a therapeutic challenge, espe- Vero cells than acyclovir. The inhibitory mechanism analy- cially for treating immunocompromised patients. sis using a time-of-addition assay revealed that all com- Methods: Herein we describe the promising antiviral profile pounds inhibit the late phase of lytic replication. Finally, of three 2-aminomethyl-3-hydroxy-1,4-naphthoquinones the highest selectivity index of the first tested compound against HSV-1 using Vero cells. was almost twice as high as that of acyclovir. Results: The in silico theoretical analysis indicated that Conclusions: Since resistance is still a problem for treat- the lowest unoccupied molecular orbital (LUMO) and the ing HSV infections, these compounds present a promis- conformational features of these molecules are impor- ing profile toward the development of new strategies for tant structural features for modulating their biological anti-HSV-1 therapy.

Introduction

Herpes simplex viruses (HSV) are important human new anti-HSV-1 agents with distinct mechanisms of pathogens that belong to the Herpesviridae family. action could offer a new strategy against drug-resistant These viruses cause different diseases ranging from viruses [5]. mucocutaneous infections (for example, herpes labia- The development of new drugs is based on several lis and herpes genitalis) to life-threatening diseases (for parameters including the understanding of their mech- example, herpes encephalitis and neonatal herpes). HSV anism of action and of their toxicological potential [6]. infections are caused by type-1 (HSV-1) and type-2 Natural (for example, lapachol and b-lapachone) and (HSV-2) viruses which are double-stranded DNA envel- synthetic naphthoquinones have been widely stud- oped viruses and can establish a life-long latent infec- ied due to their wide spectrum of biological activities tion within their hosts [1]. including fungicide, antileishmanial, antimalarial, anti- Currently the most common drugs used for the Schistosoma mansoni and anti-Trypanosoma cruzi treatment of HSV-1 and HSV-2 infections are syn- [7–10]. The presence of a nitrogen atom (for exam- thetic guanine nucleoside analogues such as acyclovir ple, of an alkyl or arylamino group) in quinone-type (ACV) [2]. However, the wide use of these molecules compounds is associated with a number of biological has been associated with the rise of drug-resistant activities such as antitumor, antimalarial and mollus- HSV strains [3,4]. cicide effects [11–13]. Several inhibitory mechanism In order to overcome this issue many non-nucleoside studies revealed the effect of aminonaphthoquinones inhibitors have been proposed as candidate drugs for on the topoisomerase-DNA with significant inhibition the treatment of herpes. Importantly, the discovery of of topoisomerase II-a [14].

AVT-15-OA-3675_Pires_de_Mello.indd 507 27/10/2016 14:52:52 CP Pires de Mello et al.

Recently we described the inhibition of the in vitro molecular orbital (HOMO) and lowest unoccupied replication of bovine herpesvirus 5 by the 2-amino- molecular orbital (LUMO), electrostatic potential map 3-hydroxy-methylnaphthoquinones derived from law- (MEP) and maps of the HOMO and LUMO coefficient sone [15] shown in Figure 1. Of the 12 derivatives con- distributions, and density maps of each compound also taining different substituents on the nitrogen atom and compared with ACV, the control drug. on the phenyl group, these compounds were the most The theoretical in silico analysis of the toxicologi- potent and selective inhibitors, with concentration that cal potential and the pharmacokinetic behaviour of

kills 50% of cellular population (CC50) and concentra- these compounds were obtained using the Osiris ® tion that inhibits 50% of viral plaque formation (EC50) Property Explorer program (Actelion Pharmaceuti- values of 1,867 µM ±8.3 and 3.8 µM ±1.2, respectively cals, Rio de Janeiro, Brazil). The toxicity (mutagenic, (ACV: 989 µM ±2 and 1.66 µM ±2, respectively). tumourigenic, irritant and effects on the reproduc- In the present work we investigated the potential of tive system), ‘druglikeness’ (similarities with market three compounds (Figure 1) as antiviral agents against drugs) and ‘drugscore’ (potential as a drug candidate) HSV-1. In addition, we also explored their antiviral were calculated. ‘Drugscore’ combines the values of mechanisms and performed in vitro and in silico evalu- ‘druglikeness’, cLogP (lipophilicity), logS (solubil- ations to analyse their toxicity and potential profile as ity), molecular weight and toxicity risks in one value a new drug prototype. The in vivo toxicity profile and to assess whether the compound has the potential in vivo anti-HSV-1 activity of these compounds were to become a drug. The risk of toxicity is based on also investigated and they confirmed ourin vitro results the database of chemicals that present toxic effect showing low toxicity and promising antiviral activity of (Registry of Toxic Effects of Chemical Substances the compounds (data not shown). [RTECS]) and validated with a database containing drugs commercially available and widely used in the Methods market. According to the literature, the data obtained with the Osiris Property Explorer® program may be In silico analysis of the potential profile of compounds used as parameters to evaluate pharmacokinetics The stereoelectronic properties were calculated using and toxicology with good reliability. The theoretical the Spartan'10 program (Wavefunction Inc., Irvine, CA, data obtained herein is for orientation more than for USA). The conformational analysis was performed using excluding any molecules and biological assays should molecular mechanics and MMFF94 force field. The com- not be simply replaced by in silico evaluation. pounds were submitted to geometry optimization using the RM1 semi-empirical method and the stereoelec- Cell culture, virus and compounds tronic properties were calculated with the Hartree-Fock Vero cells (ATCC) were cultured in Dulbecco’s modi- method using the 6-311G* basis set available in Spar- fied Eagle’s medium (DMEM) supplemented with 5% tan'10. The calculated parameters include: area, molecu- fetal bovine serum (FBS; HyClone, Logan, UT, USA), lar weight, dipole moment, energy of highest occupied 100 U/ml penicillin, 100 µg/ml streptomycin and

Figure 1. The series of 2-aminomethyl-3-hydroxy-1,4-naphthoquinones 1–3

O O O

OH OH OH

H H H N N N

O Cl O Cl O 2 1 5 3 2 3 4

Cl Cl NO2

AVT-15-OA-3675_Pires_de_Mello.indd 508 27/10/2016 14:52:53 Potential activity of aminomethylnaphthoquinones against HSV-1

amphotericin B (25 mg/ml; Cultilab, São Paulo, Brazil), suspension was diluted in DMEN medium in the pres-

at 37°C in a humidified 5% CO2 atmosphere. ence or absence of the compounds (50 µM) and incu- HSV-1 strain KOS (ATCC) was used for all experi- bated at 4ºC for 1, 2, 3 or 4 h [20]. Then, Vero cells ments [16] and was routinely grown. VERO cells and maintained in 24-multiwell plates (2×105 cell/well) virus stock cultures were prepared from supernatants were infected with the incubated virus for 1 h at 37ºC. of infected cells and stored at -80°C until use. Hereafter, the monolayer was covered with DMEN The three 2-aminomethyl-3-hydroxy-1,4-naphthoqui- 2X, 5% of fetal calf serum and 2% of methylcellulose nones 1–3 (Figure 1) were synthesized as described else- (Fluka) for 72 h. The viral titre was determined accord- where [11,17]. Their identity was confirmed by 1H NMR ing to the number of viral plaque units per ml (PFU/ml). (Varian VNMRS 300 MHz spectrometer) and their purity, by elemental analysis (Perkin-Elmer CHN 2400 Time-of-addition assay micro analyzer at Central Analítica IQ-USP, São Paulo, In order to evaluate the effect of the derivatives against Brazil) and melting point measurement (Digital Melt- the HSV-1 lytic replication process, a time-of-addition ing Point IA9100, ThermoFischer Scientific, Waltham, assay was performed, followed by a virus yield reduction MA, USA). They were dissolved in dimethylsulfoxide assay. Briefly, monolayers of Vero cells were inoculated (DMSO) 100% sterile and stored at -20°C. This stock with HSV-1 at an MOI of 1 and incubated for 60 min at solution (50 mM) was diluted in DMEM for the tests. 37ºC, after which the viral inoculum was removed, and the cells were maintained at 37ºC and treated with com- Cytotoxicity assay pounds 1, 2 or 3 (50 µM) for 0–3, 3–6 or 6–24 h post- Vero cells cultivated in 96-multiwell plates (1×104 infection (hpi). After 24 h, viral particles were titred as cells/well) were treated with the derivatives in different described in Plaque reduction assay. The percentage of concentrations (50, 250, 500 and 1,000 µM) for 72 h. viral inhibition of each treated sample was calculated Then, 50 µl of a 1 mg/ml solution of 3-(4,5-dimeth- by comparing it with virus titres of untreated controls. ylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma, São Paulo, Brazil) was added to evalu- Western blot ate cell viability according to the procedure described Monolayers of Vero cells were inoculated with HSV-1

elsewhere [18,19]. The CC50 was calculated by linear KOS at an MOI of 1 and the plates were incubated for regression analysis of the dose–response curves. 60 min at 37ºC. Infected cells were treated with compounds 1, 2 or 3 (50 µM) for 6–24 hpi, and then incu- Plaque reduction assay bated for 24 h. After that the cells were lysed with the Vero cells maintained in 24-multiwell plates (2×105 extraction buffer (10 mM Tris-Hcl pH 7.5, 100 mM cells/well) were infected with HSV-1 KOS strain (multi- NaCl, 1% NP-40, 1% Triton X-100, 1% sodium deox- plicity of infection [MOI] of 1) for 1 h at 37ºC and 5% ycholate, 0.1% SDS, 5 mM EDTA, 10 µg/ml aprotinin,

CO2 atmosphere. Then the viral inoculum was removed 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 µg/ml sodium and cells were treated with different concentrations orthovanadate and 0.5 mM sodium fluoride). Each (0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 µM) of the sample was analysed separately on a 12% SDS–PAGE compounds for 24 h. gel and electroblotted onto nitrocellulose membranes. In order to determine the viral titre, Vero cells were After blocking, membranes were incubated overnight maintained in 24-multiwell plates (2×105 cells/well) with anti-gD (1:5,000; Santa Cruz Biotechnology, Dal- and infected with various dilutions (1:10) of the HSV-1 las, TX, USA) and then with the corresponding second- from cells cultivated in 24-multiwell plates, for 1 h at ary antibodies (1:4,000) conjugated to horseradish

37ºC and 5% CO2 atmosphere. Hereafter, the mon- peroxidase. Protein bands were revealed by chemilu- olayers were covered with DMEN 2X, 5% of fetal calf minescence on ChemiDocTM XRS (Bio-Rad) using ECL serum and 2% of methylcellulose (Fluka) for 72 h. The substrate according to the manufacturer’s protocol viral titre was determined according to the number (Santa Cruz Biotechnology). The anti-tubulin antibody

of viral plaque units per ml (PFU/ml) and EC50 value, was used as a control for total protein loading and the which shows the concentration that inhibits 50% of the graphic was made according to results generated by the viral plaque formation. This was determined by linear software ImageJ Fiji. regression compared with an infected untreated control and infected treated with DMSO, to ensure that DMSO Statistical analysis is not interfering with inhibition of viral plaques. All assays were performed at least three times in triplicate and the statistical analysis was made using the GraphPad Virucidal assay Prism 4.0 program (GraphPad Software Inc., La Jolla, The assay was carried out in a cell-free system to evalu- CA, USA). The analysis of variance test was used, fol- ate the virucidal activity of compounds 1–3. The viral lowed by multiple comparisons using the Kruskal–Wallis

Antiviral Therapy 21.6 509

AVT-15-OA-3675_Pires_de_Mello.indd 509 27/10/2016 14:52:53 CP Pires de Mello et al.

test. Differences were considered statistically significant high antiviral activity in a dose-dependent manner

when P<0.05. (Table 1). Compound 2 showed the lowest EC50 (0.83 ±0.4 µM) which was close to compounds 1 and 3, and

Results also presented EC50 values (1.74 ±0.22 µM and 2.13

±0.11 µM, respectively) comparable to ACV (EC50=1.09 In silico analysis ±0.25 µM; Table 1). The following stereoelectronic and physico-chemical Since the selectivity index represents the degree of properties of compounds 1–3 were evaluated using a safety for using a compound, it was calculated (SI

molecular modelling approach: clogP, molecular weight = CC50/EC50) for each naphthoquinone (Table 1). (MW), molecular volume (MV), topological polar sur- Our results reinforce their promising profile with face area (TPSA), number of hydrogen bond acceptors both compounds 1 and 2 presenting SI values (SI = (nHBA) and donors (nHBD), HOMO and LUMO ener- 1,525.29 and 1,161.45, respectively) higher than gies, dipole moment and number of rotatable bonds ACV (SI = 880.73) and the compound 3 value was (nrotb). Among these, only the HOMO and LUMO similar to it (SI = 808.45). energies, dipole moment and number of hydrogen bond acceptors (nOHNH) seem to be directly or indirectly Antiviral mechanism evaluation related to the observed antiviral activity (Figure 2). Virucidal profile The distribution of HOMO and LUMO orbital In order to identify the naphthoquinones as potential coefficients was also analysed. Interestingly, the microbicides their ability to deactivate directly the viral LUMO distribution data presented singular results particles was investigated. Accordingly, the HSV-1 sus-

for compound 2, which exhibits the lowest EC50 and pensions were pretreated with these compounds for 1–4 LUMO distribution on the naphthoquinone ring. h, and then Vero cells were infected with the suspensions

Accordingly, compound 3 showed the highest EC50 for analysis by testing viral plaque formation. The results in accord to the highest LUMO distribution on the are presented as a percentage of virus control and are same ring (Figure 2). The conformational analysis also the mean values from three independent experiments. reinforced these data, since 3 presented a different ori- Importantly, our results showed that compounds 1–3 entation of the benzyl ring, which may be associated don’t have a time-dependent pattern and they have a low with its lower antivirus activity compared to 1 and 2 virucidal activity at 50 µM (less than 50%; Figure 3). (Figure 2). The analysis of the theoretical potential of these compounds as new drugs revealed that 1 and 2 Time-of-addition assay display the highest drugscore and druglikeness values According to the literature, HSV-1 replicates with a compared with ACV. Once more 3 showed the lowest predictable time dynamic in a replicative cycle of 18 to values, thus revealing the lowest theoretical potential 20 h, and spike proteins at times 0–3 h (a or immedi- to become a future drug (Figure 2). Finally in silico ate early [IE]-phase), 3–6 (b or early [E]-phase) and toxicological features were calculated using the Osiris 6–20 h (g or late [L]-phase) [21]. In order to identify an Property Explorer on-line system, including irritant, HSV target for the active compounds among the virus mutagenic, tumourigenic and reproduction effects. replication phases, Vero cells were infected for 1 h and The theoretical calculations showed all compounds as then treated with the compounds (50 µM) during time safe as or better than ACV with small theoretical risk points of the replicative cycle (0–3 h, 3–6 h, 6–20 h). of mutagenicity, tumourigenicity, reproductive and The virus of each sample was titred and the results irritant effects (Figure 2). were calculated as percentage of inhibition relative to infected untreated control (virus control [VC]). The Cytotoxic and antiviral activity evaluation of results showed inhibition of the L phase (6–24 h) by all compounds 1–3 compounds. In addition, compound 1 and compound

The in vitro cytotoxicity (CC50) of all compounds 2 also inhibited the IE and E phases although to differ- towards Vero cells was also investigated. All compounds ent degrees (compound 1: IE = 56.77%, E = 74.86%;

presented similar (3 = 964 ±56.04 µM) or higher CC50 compound 2: IE = 98.55%, E = 100%; Figure 4). (1 = 2,654 ±135.76 µM and 2 = 1,722 ±175.9 µM) than that of the reference drug (ACV = 960 µM; Table 1). Viral protein expression The antiviral activity of the 2-aminomethyl-3-hy- Since all compounds inhibited the L-phase of the HSV-1 droxy-1,4-naphthoquinones 1–3 was evaluated by incu- replicative cycle, we investigated the expression level of bating HSV-1 infected Vero cells (titre 1×108 PFU/ml) the gD protein, an L-phase protein, by western blot analy- with different concentrations of the compounds (0.78, sis. We also tested the expression of the ICP27 protein, an 1.56, 3.125, 6.25, 12.5, 25 and 50 µM) or ACV used as IE-phase protein, but the compounds had no effect on it reference. Interestingly, all three compounds exhibited (data not shown). The results showed that compounds 1

AVT-15-OA-3675_Pires_de_Mello.indd 510 27/10/2016 14:52:53 Potential activity of aminomethylnaphthoquinones against HSV-1

Figure 2. In vitro (EC50) and in silico analysis of compounds 1–3 and ACV

Parameters 1 2 3 ACV

Anti-HSV-1 EC50 (µM) 1.74 ±0.22 0.83 ±0.4 2.13 ±0.11 1.09 ±0.25

EHOMO -8.89 -8.87 -9.10 -8.19

ELUMO 0.20 0.42 0.06 3.08

Dipole moment (µ) 4.28 4.76 8.99 6.08

nOHNH 4 4 7 5

1 2 3 ACV

Druglikeness Drugscore 3 4 0.35 Mutagenic 1 Tumourigenic 2 Irritant -2 0.3 Reproductive effect -5 1 0.25 -8

-11 0.2 0 1 2 3 ACV 1 2 3 ACV 1 2 3 ACV

The in silico evaluation included the analysis of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies, dipole moment and number of hydrogen bond acceptors (nOHNH), the distribution and spatial conformation of the LUMO and druglikeness, drugscore and theoretical toxicity profiles

related to irritant, mutagenic, tumourigenic and reproductive effects. ACV, acyclovir; EC50, concentration that inhibits 50% of viral plaque formation; HSV-1, herpes simplex virus type-1.

and 2 decreased the gD protein expression in accordance Discussion with the time-of-addition assay results. Compound 1 also influenced the molecular weight of gD indicating a possi- Currently, the main challenge associated with the ble glycosylation inhibition. In contrast, compound 3 did development of new antivirals is the design of mol- not affect this L-phase protein expression, which points ecules that are both active against virus and exhibit to a different L-phase protein as a possible target for this low cell and systemic toxicities [22,23]. In this work compound (Figure 5). the evaluation of the anti-HSV-1 activity of three

Antiviral Therapy 21.6 511

AVT-15-OA-3675_Pires_de_Mello.indd 511 27/10/2016 14:52:53 CP Pires de Mello et al.

Table 1. CC50, EC50 and selectivity index (CC50/EC50) of aminonaphthoquinones (1–3) showed high antiviral compounds 1–3 and ACV activity in a dose-dependent manner. Interestingly, com-

Compounds CC , µM EC , µM SI, CC /EC pound 2 exhibited the lowest EC50 (EC50 = 0.83 µM) 50 50 50 50 compared not only with compounds 1 and 3 but also 1 2,654 ±135.76 1.74 ±0.22 1,525.29 with ACV and other compounds, for example, PMEO- 2 964 ±56.04 0.83 ±0.4 1,161.45 DAPym (6-phosphonylmethoxyethoxy-2,4-diaminopy- 3 1,722 ±175.9 2.13 ±0.11 808.45 rimidine), an acyclic nucleoside phosphonate analogue ACV 960 ±156 1.09 ±0.25 880.73 recently described in the literature, whose anti-HSV activity is lower than that of ACV [24]. sd Data are mean ± . ACV, acyclovir; CC50, concentration that kills 50% of cellular

population; EC50, concentration that inhibits 50% of viral plaque formation; SI, An initial structure–activity relationship (SAR) anal- selectivity index. ysis showed that structural modifications on compound

2 (EC50 = 0.83) involving substitution of the benzyl group on the nitrogen atom for an n-butyl group (com- Figure 3. Virucidal profile of compounds1 –3 after 1, 2, 3 and pound 1, EC = 1.74 µM) or of the two chlorine atoms 4 h incubation 50 on the phenyl ring for a nitro group (compound 3,

45 EC50 = 2.13 µM) resulted in lower activity. These data 40 therefore point to both the benzyl substituent on the nitrogen atom and the two chlorine substituents on the 35 phenyl group as important structural features for the 30 positive modulation of the aminonaphthoquinone anti- 25 viral activity. 20 The in vitro and in silico toxicity of these aminonaphthoquinones were also evaluated. The aminonaphtho- 15

relative to control relative quinones were analysed theoretically using a molecular 10 modelling approach. Among the different stereoelec- 5 tronic and physico-chemical properties, we explored the theoretical chemical reactivity of these aminonaphtho- Percentage of viral particle inactivation Percentage 0 1a 2a 3b quinones by analysing both the energy and distribution of their HOMO and/or LUMO. Thus, the higher the dif- 1 h 2 h 3 h 4 h ference between the HOMO and LUMO energy levels

The number of viral plaque units per ml (PFU/ml) and the percentage of viral the greater was the activity profile of this small series. particle inactivation was calculated (aP>0.05; bP<0.05; n=3). Even though no recognizable relationship between the

Figure 4. Antiviral mechanism analysis of compounds 1–3 through inhibition of the replicative cycle

A B

Time-of-addition 120

100 1 80 0–3 h

60 3–6 h 6–24 h 40 2 20 relative to control, % to control, relative Inhibition of viral plaque 0 1 2 3 Compounds 3 0–3 h 3–6 h 6–24 h VC

(A) Graphical representation and (B) viral plaques of Vero cells treated with each compound (50 mM) or not (virus control [VC]) at time points of the replicative cycle: 0–3 h (a or immediate early phase), 3–6 h (b or early phase) and 6–24 h (g or late phase).

AVT-15-OA-3675_Pires_de_Mello.indd 512 27/10/2016 14:52:54 Potential activity of aminomethylnaphthoquinones against HSV-1

Figure 5. Western blot analyses of gD protein expression (a late phase protein) of infected cells treated with compounds 1–3 or not (VC)

A B 70

1 2 3 CC VC 60 1 50 gD 2 40 2 30

20 Tubulin 10 Reduction % of gD relative to Reduction % of gD relative control normalized with tubulin control

0 1 2 3 gD 66.57% 60.79% 0%

Uninfected and untreated cells were used as cellular control (CC) whereas tubulin antibody was used for detecting tubulin as a control for total protein loading. (A) Western blot protein bands. Arrows: 1= glycosylated gD; 2= non-glycosylated gD. (B) Percentage of reduction of gD relative to control normalized with tubulin values. VC, virus control.

HOMO distribution and the activity was encountered, chain length of the substituent on the nitrogen atom

the lower the LUMO distribution on the naphthoqui- (C4H9

be improved for further investigation [24]. Once know- culated by using the CC50 and EC50 ratio. The higher ing the true targets (viral proteins or host cell proteins) the SI value, the more promising is the substance for of these compounds, the quantitative structure–activity further in vivo and in vitro studies. Compounds 1 and relationship (QSAR) will be meaningful to establish the 2 presented SI values (1,525.29 and 1,161.45, respec- direct relationship of these molecules tively) higher than ACV (SI = 880.73), which reinforced The in vitro concentration that inhibits 50% of their potential profile for further studies. These aminon-

the cellular mitochondrial activity (CC50) ranged aphthoquinones have a higher antiviral potential than from 964 to 2,654 µM for these compounds, which some molecules described in the literature, including

is higher than for ACV. The highest CC50 value found natural and synthetic products such as flavones from

for compound 1 (CC50 = 2,654 µM) suggests that it Ficus benjamina (SI = 100–666) [26] and the 4′-phe- is a promising antiviral prototype. Apparently the in nylflavone (SI = 213.35) [27], the sulfonoquinovosyl- vitro cytotoxicity profile of these molecules depends diacylglyceride from Azadirachta indica (SI = 12.4) [28] on the nature of the substituent on the phenyl group and the tricyclic analogue of ACV and ganciclovir car- and on the nitrogen atom. These results are in accord rying the 3,9-dihydro-9-oxo-5H-imidazo[1,2-a]purine with our previous cytoxicity studies of other analo- system (SI = 1,000) [29]. gous Mannich bases derived from lawsone, which To start to understand the mechanism of action of showed increased cytoxicity when varying the carbon these aminonaphthoquinones, firstly, we evaluated

Antiviral Therapy 21.6 513

AVT-15-OA-3675_Pires_de_Mello.indd 513 27/10/2016 14:52:54 CP Pires de Mello et al.

their virucidal profile, which shows if the compounds LUMO distribution and energy profiles seem to modu- can inhibit directly the viral particle. According to late the antiviral activity of these compounds. Based Schuhmacher et al. [20], compounds that present not on these results we conclude that this series of 3-ami- only a dose-dependent pattern but also a time-depend- nomethylnaphthoquinones, which are structurally and ent one show significant virucidal activity. Our results functionally different from ACV, have a promising pro- showed that, despite the dose-dependent pattern of the file for further in vitro and in vivo studies and future compounds, aminonaphthoquinones 1–3 don’t have a development of new derivatives with antiviral action. time-dependent profile, and all of them presented a low virucidal activity at 50 µM (less than 40% of viral par- Acknowledgements ticle inhibition). Most of the marketed drugs for the treatment of We thank Conselho Nacional de Desenvolvimento HSV infections (nucleoside analogues) target the viral Científico e Tecnológico (CNPq), Coordenação de DNA polymerase, which increases the selection pres- Aperfeiçoamento de Pessoal Docente (CAPES) and sure risk of resistant strains [22]. Hence, the search Fundação de Amparo à Pesquisa do Estado do Rio de for new antiviral agents with different mechanisms of Janeiro (FAPERJ) for the financial support and fellow- action is a key goal for future treatment of resistant- ships. The technical assistance of Samara Nascimento, strain-caused infections. Among new feasible targets Hania Rosado and Sandro Portela (Universidade Fed- of HSV replicative cycle stages are: proteins that eral Fluminense, Niterói, Brazil) is acknowledged. regulate viral replication – IE proteins (for example, ICP27); proteins that synthesize and package DNA – E Disclosure statement proteins (for example, UL42); and other virion proteins – L proteins (for example, gD). Molecules that The authors declare no competing interests. target any of these proteins may stop HSV replication and present a potential profile as antiviral drugs. Inter- References estingly, all aminonaphthoquinones tested herein were 1. Jones CA, Isaacs D. Management of herpes simplex virus able to inhibit the L-phase of the HSV-1 replicative infections. Curr Paediatr 2004; 14:131–136. cycle. In fact compounds 1 and 2 also inhibited the 2. Jerome KR. The road to new antiviral therapies. Clin Appl other two phases, primarily compound 2 with almost Immunol Rev 2005; 5:65–76. 100% effectiveness. Importantly, further mechanism 3. Morfin F, Thouvenot D. Herpes simplex virus resistance to assays revealed that only aminonaphthoquinones 1 antiviral drugs. J Clin Virol 2003; 26:29–37. 4. James SH, Prichard MN. Current and future therapies for and 2 were able to inhibit gD protein expression. It herpes simplex virus infections: mechanism of action and is a possibility that this effect on gD expression may drug resistance. Curr Opin Virol 2014; 8:54–61. be caused by an early inhibition on HSV-1 replication 5. Pires de Mello CP, Bloom DC, Paixão ICNP. Herpes cycle but not for compound 3 since the time-of-addi- simplex virus type-1: replication, latency, reactivation and its antiviral targets. Antivir Ther 2016; doi: 10.3851/ tion assay for this compound only showed inhibition IMP3018. on late phase. Furthermore, compound 1 also influ- 6. Buxton ILO. Pharmacokinetics and pharmacodynamics: enced the molecular weight of gD indicating a possible the dynamics of drug absorption, distribution, action and elimination: introduction. In Brunton LL (editor). glycosylation inhibition. Thus, substitution of the two Goodman & Gilman’s the pharmacological basis of chlorine atoms on the phenyl ring of compounds 1 therapeutics. 11th ed. New York: McGraw–Hill 2006; pp. 1–39. and 2 for a nitro group in compound 3, seems to have 7. Bourguignon SC, Cavalcanti DF, De Souza AM, et al. affected its target orientation. Overall these biological Trypanosoma cruzi: insights into naphthoquinone effects results reinforced the promising profile of these com- on growth and proteinase activity. Exp Parasitol 2011; pounds for further in vitro and in vivo analysis and as 127:160–166. 8. Gafner S, Wolfender J-L, Nianga M, Storckli‑Evans H, potential new antivirus drugs. Hostettman K. Antifungal and antibacterial In conclusion, we have identified a small series of naphthoquinones from Newbouldia Laevis roots. 3-aminomethylnaphthoquinones active against HSV-1 Phytochemistry 1996; 42:1315–1320. with low in vitro and in silico toxicity profile. In spite 9. Dalgliesh CE. Naphthoquinone antimalarials. Mannich bases derived from lawsone. J Am Chem Soc 1949; of the highest antiviral activity against HSV-1 of com- 71:1697–1702. pound 2, the most promising molecule is probably 10. Leffler MT, Hathaway RJ. Naphthoquinone compound 1 due to its greater SI value. All compounds antimalarials. XIII. 2-hydroxy-3-substituted- aminomethyl derivatives by Mannich reaction. J Am inhibited L-phase of lytic replication. Compounds 1 and Chem Soc 1948; 70:3222–3223. 2 also inhibited IE and E phases to different degrees and 11. Neves AP, Pereira MXG, Peterson E, et al. Exploring they affect gD protein expression (L-phase). Structural the DNA binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3-(aminomethyl)- features, such the nature of the substituent on the nitro- 1,4-naphthoquinone Mannich bases and their platinum(II) gen atom (benzyl versus n-butyl), the conformation and complexes. J Inorg Biochem 2013; 119:54–64.

AVT-15-OA-3675_Pires_de_Mello.indd 514 27/10/2016 14:52:54 Potential activity of aminomethylnaphthoquinones against HSV-1

12. Francisco AI, Casellato A, Neves AP, et al. Novel 21. Roizman B, Sears AE. Herpes simplex viruses and their 2-(R-phenyl)amino-3-(2-methylpropenyl)-[[[1,4]]]- replication. In: Fields Virology. 3rd edition. Philadelphia: naphthoquinones: synthesis, characterization, Lippincott-Raven 1996; pp. 2231–2295. electrochemical behavior and antitumor activity. J Braz 22. De Clercq E. The clinical potential of the acyclic (and cyclic) Chem Soc 2010; 21:169–178. nucleoside phosphonates: the magic of the phosphonate 13. Silva TMS, Camara CA, Barbosa TP, et al. Molluscicidal bond. Biochem Pharmacol 2011; 82:99–109. activity of synthetic lapachol amino and hydrogenated 23. De Clercq E. Ten paths to the discovery of antivirally derivatives. Bioorg Med Chem 2005; 13:193–196. active nucleoside and nucleotide analogues. Nucleosides 14. Cunha AS, Lima ELS, Pinto AC, et al. Syntheses of novel Nucleotides Nucleic Acids 2012; 31:339–352. naphthoquinone-spermidine conjugates and their effects on a 24. Balzarini J, Andrei G, Balestra E, et al. A multi-targeted drug DNA-topoisomerases I and II- . J Braz Chem Soc 2006; candidate with dual anti-HIV and anti-HSV activity. PLoS 17:439–442. Pathog 2013; 9: e1003456. 15. Pinto AMV, Paixão ICNP, Leite JPG, Neves AP, Silva GB, Vargas MD. Synthetic aminomethylnaphthoquinones inhibit 25. van de Waterbeemd H, Gifford E. ADMET in silico the in vitro replication of bovine herpesvirus -5. Arch Virol modeling: towards prediction paradise? Nat Rev Drug 2014; 159:1827–1833. Discov 2003; 2:192–204. 16. Smith KO. Relationship between the envelope and the 26. Yarmolinsky L, Huleihel M, Zaccai M, Ben-Shabat S. Potent infectivity of herpes simplex virus. Proc Soc Exp Biol Med antiviral flavones glycosides from Ficus benjamina leaves. 1964; 115:814–816. Fitoterapia 2012; 83:362–367. 27. Hayashi K, Iinuma M, Sasaki K, Hayashi T. In vitro 17. Neves AP, Barbosa CC, Greco SJ, et al. Novel and in vivo evaluation of a novel antiherpetic flavonoid, aminonaphthoquinone Mannich bases derived from ′ lawsone and their copper(II) complexes: synthesis, 4 -phenylflavone, and its synergistic actions with acyclovir. characterization and antibacterial activity. J Braz Chem Soc Arch Virol 2012; 157:1489–1498. 2009; 20:712–727. 28. Bharitkar YP, Bathini S, Ojha D, et al. Antibacterial and 18. Vega-Avila E, Pugsley MK. An overview of colorimetric antiviral evaluation of Sulfonoquinovosyldiacylglyceride assay methods used to assess survival or proliferation (SQDG): a glycolipid isolated from Azadirachta indica of mammalian cells. Proc West Pharmacol Soc 2011; leaves. Lett Appl Microbiol 2014; 58:184–189. 54:10–14. 29. Goslinski T, Wenska G, Golankiewicz B, Balzarini J, De Clercq E. Synthesis and fluorescent properties of 19. Mosmann T. Rapid colorimetric assay for cellular growth 6-(4-biphenylyl)-3,9-dihydro-9-oxo-5H-imidazo[1,2-a] and survival: application to proliferation and cytotoxicity purine analogues of acyclovir and ganciclovir. Nucleosides assays. J Immunol Methods 1983; 65:55–63. Nucleotides Nucleic Acids 2003; 22:911–914. 20. Schuhmacher A, Reichling J, Schnitzler P. Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro. Phytomedicine 2003; 10:504–510. Accepted 23 February 2016; published online 25 February 2016

Antiviral Therapy 21.6 515

AVT-15-OA-3675_Pires_de_Mello.indd 515 27/10/2016 14:52:54 5.1.4. SYNTHESIS OF 4-OXOQUINOLINE ACYCLONUCLEOSIDE PHOSPHONATE ANALOGS AND ANTI-MAYV EVALUATION

Synthesis of 4-oxoquinoline acyclonucleoside phosphonate analogs and anti-MAYV evaluation.

Leonardo dos Santos Corrêa Amorim1, LetíciaVillafranca Faro2, Andrew Molção Meireles1, Vitor Won-Held Rabelo1, Paula Alvarez Abreu3, Claudio Cesar Cirne dos Santos1, Caroline de Souza Barros1, Maria Cecilia Bastos Vieira de Souza2, Izabel Christina Nunes de Palmer Paixão1

2Pós-graduação em QuímicaOrgânica, Instituto de Química, Universidade Federal Fluminense,; Campus do Valonguinho - Centro - Niterói – RJ, CEP: 24020-141,Brazil.

3Instituto de Biodiversidade e Sustentabilidade, Universidade Federal do Rio de Janeiro, Macaé, RJ, CEP 27965-045, Brazil.

Abstract The Mayaro virus (MAYV) is an RNA virus, has a viral envelope and is classified in the Togaviridae family, genus Alfavirus. Mayaro fever is caused by MAYV, and it has overlapping symptoms of other arbovirus diseases like Chikungunya and Dengue fevers. This disease is mainly characterized by acute febrile illness and last-longing severe arthralgia. To date, there are no antiviral drugs or vaccines against infection with MAYV and resources for the prevention or treatment of other alphaviruses are very limited. In this study we evaluated the antiviral potential of 4-oxoquinoline acyclonucleoside phosphonate analogs as anti-MAYV agents. The compound 7a was able to reduce viral production effectively at concentration that was non-toxic for Vero cells. Compound 7a showed more potent and safer in vitroprofile (CC50= 956.72, EC50= 0.83 and SI= 1152.67) than the commercial drug chloroquine. Regarding its mechanism of action, 7a was shown to inhibit early events in MAYV replication.Docking studies suggested that the compound binds tôThe envelope proteíns of MAYV and might impairs vírus entry or fusion. In addition, this compound showed a satisfactory theoretical pharmacokinetics and toxicological profile. Altogether, our results reinforce The potential of compound 7a as a drug candidate against MAYV and support further in vivo investigations.

Keywords:Mayaro, oxoquinoline, antivirals, arbovirus,

Introduction

Mayaro virus (MAYV) is a positive-sense single-stranded RNA virus that belongs to the Togaviridae family and Alphavirus genus(Mackay and Arden, 2016). Its RNA is about ~11.4 kb in size and encodes for four non-structural proteins (nsP1, nsP2, nsP3, and nsP4) and five structural proteins, namely capsid protein (C), envelope protein 1, 2 and 3 (E1, E2, and E3) and the peptide 6K(Mackay and Arden, 2016; Muñoz and Navarro, 2012). The viral capsid is icosahedral and surrounded by a lipid envelope where trimers comprised of three heterodimers of E1 and E2 are found (Napoleão-Pego et al., 2014). Mayaro fever is caused by MAYV, and it has overlapping symptoms of other arbovirus diseases like Chikungunya and Dengue fevers. This disease is mainly characterized by acute febrile illness and last-longing severe arthralgia (Agarwal et al., 2017; Mavian et al., 2017). This virus was first isolated in 1954 from blood samples of rural workers with febrile illness in Trinidad and Tobago.Recently, MAYV is responsible for small-scale outbreaks, especially in the Pan-Amazon region (Aguilar-Luis et al., 2020; Mavian et al., 2017), though imported cases have been notifiedin other areas like North America and Europe(Acosta-Ampudia et al., 2018; Thevarayan et al., 2010).In 2015, the largest outbreak occurred in Venezuela, where 77 rural workers tested positive for this virus (Hotez and Murray, 2017). MAYV is an arthropod-borne virus (arbovirus), and Haemagogus mosquitoes are the primary vectors(Mota et al., 2015). However, further studies revealed that other mosquitoes like Aedes spp. and Anopheles spp. could act as vectors as well (Brustolin et al., 2018; Kantor et al., 2019; Wiggins et al., 2018). These species are distributed in countries from different continents, which raises concerns about the spread of this virus across the world,such as other arboviruses like Chikungunya (CHIKV) and Zika (Mota et al., 2015). In fact, MAYV infections were reported in countries from Central America and the Caribbean like Mexico and Panama, suggesting that its geographic range is already increasing (Pezzi et al., 2019). In Brazil, surveillance has sporadically identified cases of Mayaro fever throughout the years, indicating that this is one of the most affected countries by this virus(Acosta-Ampudia et al., 2018). Besides, the cases of Mayaro fever are probably underestimated because of the lack of efficient diagnostic methods (Esposito and Fonseca, 2017). More recently, a mathematical model suggested that MAYV is likely to cause an epidemic in Rio de Janeiro city as CHIKV (Dodero- Rojas et al., 2020). Taken together, these data show that this virus has the potential to become a significant threat to public health soon. Nonetheless, there are no approved vaccines or antiviral agents available to treat this disease to date (de Mello et al., 2020). Consequently, our group has been putting efforts into finding novel compounds with anti-MAYV potential (Amorim et al., 2017). 4-oxoquinoline derivatives have been drawing the attention of the scientific community for decades and have been associated with a wide range of biological activities.In the past years, we have uncovered the antiviral activity of 4-oxoquinoline derivatives against several viruses such as HIV (Faro et al., 2012; Matta et al., 1999), HSV-1 and HSV-2 (Abreu et al., 2011; Canuto et al., 2007; Souza et al., 2008), and BoHV-5 (Pinto et al., 2019).Considering the experience of our research group involving the synthesis of potentially antiviral 4-oxoquinoline derivatives, we described here the synthesis of a series of 4-oxoquinoline acyclonucleoside phosphonate analogs containing an acylhydrazide or acylhydrazone unit in its structure and their antiviral evaluation against MAYV.

Material and Methods Synthesis 1.1.1. General Methods

Melting points were obtained on a Fischer-Johns apparatus (Fischer-Scientific, Pittsburgh, PA, USA), and are uncorrected. 1D and 2D NMR spectra were recorded on a Varian Unity Plus VXR300 MHz spectrometer (Varian Inc., Palo Alto, CA, USA) at 300.13, 121.42 and 75.47 1 31 13 MHz, for H, P and C, respectively. DMSO-d6 was used as solvent, tetramethylsilane as internal reference or 85% H3PO4 as external reference. The chemical shifts are expressed in  (ppm), coupling constants (J) are reported in Hertz, and refer to apparent peak multiplicities. Proton and carbon NMR spectra were typically obtained at room temperature. The two- dimensional experiments were acquired using standard Varian Associates automated programs for data acquisition and processing. High resolution mass spectra (HRMS) were recorded on Maxis Impact HD Mass Spectrometer (Bruker Daltonics) using ESI-qTOF (Electrospray Ionization Time of Flight).

1.1.2. Synthesis of 1-[(diisopropoxyphosphoryl)methyl]-4-oxo-1,4-dihydroquinoline-3- carbohydrazides (7a-f) A solution of the appropriate acyclonucleosides phosphonates 6a-m(3.70 mmol) and 3.7 mL of 80% hydrazine monohydrate in 10 mL of ethanol, was stirred at reflux for 2 h. The reaction mixture was then concentrated under reduced pressure giving a solid which was collected by filtration, washed with cold water and dried under vacuum, leading to the desired acyclonucleosides phosphonates acylhydrazides 7a-f.

1-[(diisopropoxyphosphoryl)methyl]-6-methyl-4-oxo-1,4-dihydroquinoline-3- carbohydrazide (7a-ANPH08): White solid, m.p.: 189-190 °C, Yield: 35%;1H NMR (300.13 MHz, DMSO-d6):  10.64 (s, 1H, NHNH2), 8.81 (s, 1H, H-2), 8.16 (d, 1H, J = 2.3 Hz, H-5), 7.90 (d, 1H, J = 8.8, H-8), 7.68 (dd, 1H, J = 9.0 and 2.4 Hz, H-7), 5.06 (d, 2H, J = 11.4 Hz, CH2P), 4.64 (dsep, 2H, J = 7.3 and 6.2 Hz, CH(CH3)2), 2.50 (s, 3H, CH3), 1.24 (d, 6H, J = 6.1 13 Hz, CH(CH3)2), 1.15 (d, 6H, J = 6.2 Hz, CH(CH3)2). C NMR (75.47 MHz, DMSO-d6):  175,4 (C-4), 164.1 (C=ONH), 148.7 (C-2), 137.9 (C-8a), 135.2 (C-6), 134.2 (C-7), 127.3 (C-4a), 125.6 (C-5), 118.9 (C-8), 111.1 (C-3), 72.1 (d, JCP = 7.0 Hz,CH(CH3)2), 48.7 (d, JCP = 153.6 Hz, CH2P), 24.1 (d, JCP = 3.7 Hz, CH(CH3)2), 23.9 (d, JCP = 5.0 Hz, CH(CH3)2), 20.9 (CH3).HRMS + (ESI) m/zcalcd for C18H26N3O5P [M+H] 396.1683, found 396.1682.

1-[(diisopropoxyphosphoryl)methyl]-7-methyl-4-oxo-1,4-dihydroquinoline-3- carbohydrazide (7b-ANPH09): White solid, m.p.: 196-197 °C, Yield: 42%; 1H NMR (300.13 MHz, DMSO-d6):  10.58 (s, 1H, NHNH2), 8.79 (s, 1H, H-2), 8.23 (d, 1H, J = 9.2 Hz, H-5), 7.79 (s, 1H, H-8), 7.35 (d, 1H, J = 8.8 Hz, H-6), 5.05 (d, 2H, J = 11.4 Hz, CH2P), 4.62 (dsep, 2H, J = 7.0 and 6.5 Hz, CH(CH3)2), 2.51 (s, 3H, CH3), 1.20 (d, 6H, J = 6.1 Hz, CH(CH3)2), 1.13 13 (d, 6H, J = 6.2 Hz, CH(CH3)2). C NMR (75.47 MHz, DMSO-d6):  174.9 (C-4), 163.4 (C=ONH), 148.4 (C-2), 143.0 (C-8a), 139.3 (C-7), 126.5 (C-5), 125.7 (C-6), 124.7 (C-4a), 124.7 (C-4a), 117.9 (C-8), 110.6 (C-3), 71.5 (d, JCP = 4.3 Hz,CH(CH3)2), 48.0 (d, JCP = 153.7 Hz, CH2P), 23.5 (d, JCP = 3.7 Hz, CH(CH3)2), 23.3 (d, JCP = 5.0 Hz, CH(CH3)2), 21.4 (CH3).HRMS + (ESI) m/zcalcd for C18H26N3O5P [M+H] 396.1683, found 396.1682.

1-[(diisopropoxyphosphoryl)methyl]-6-nitro-4-oxo-1,4-dihydroquinoline-3-carbohydrazide 1 (7c-ANPH10): White solid, m.p.: 306-308 °C, Yield: 40%; H NMR (300.13 MHz, DMSO-d6):  10.31 (s, 1H, NHNH2), 9.04 (d, 1H, J = 2.8 Hz, H-5), 8.93 (s, 1H, H-2), 8,23 (d, 1H, J = 9.6, H- 8), 7.56 (dd, 1H, J = 9.4 and 2.8 Hz, H-7), 5.18 (d, 2H, J = 11.4 Hz, CH2P), 4.64 (dsep, 2H, J = 7.5 and 6.0 Hz, CH(CH3)2),1.21 (d, 6H, J = 6.2 Hz, CH(CH3)2), 1.15 (d, 6H, J = 6.1 Hz, 13 CH(CH3)2). C NMR (75.47 MHz, DMSO-d6):  175.0 (C-4), 162.7 (C=ONH), 150.7 (C-2), 144.5 (C-6), 143.3 (C-8a), 127.0 (C-4a), 126.6 (C-7), 122.4 (C-5), 121.4 (C-8), 112.9 (C-3), 72.3 (d, JCP = 7.1 Hz,CH(CH3)2), 48.1 (d, JCP = 152.8 Hz, CH2P), 24.2 (d, JCP = 3.7 Hz, CH(CH3)2), + 24.0 (d, JCP = 5.0 Hz, CH(CH3)2). HRMS (ESI) m/zcalcd for C17H23N4O7P [M+H] 427.1377, found 427.1376.

1-[(diisopropoxyphosphoryl)methyl]-7-nitro-4-oxo-1,4-dihydroquinoline-3-carbohydrazide 1 (7d-ANPH11): White solid, m.p.: 310-312 °C, Yield: 29%; H NMR (300.13 MHz, DMSO-d6):  10.75 (d, 1H, J = 0.5 Hz, NHNH2), 8.56 (s, 1H, H-2), 7.99 (d, 1H, J = 9.3 Hz, H-5), 6.76-6.74 (m, 2H, H-8 and H-6), 6.02 (d, 2H, J = 0.5 Hz, NHNH2), 4.73 (d, 2H, J = 11.4 Hz, CH2P), 4.62 (dsep, 2H, J = 7.0 and 6.5 Hz, CH(CH3)2), 1.23 (d, 6H, J = 6.2 Hz, CH(CH3)2), 1.15 (d, 6H, J = 13 6.1 Hz, CH(CH3)2). C NMR (75.47 MHz, DMSO-d6):  174.8 (C-4), 164.5 (C=ONH), 153.5 (C-7), 147.8 (C-2), 142.1 (C-8a), 127.9 (C-5), 117.6 (C-4a), 114.3 (C-6), 110.3 (C-3), 98.3 (C-8), 72.0 (d, JCP = 7.3 Hz,CH(CH3)2), 48.3 (d, JCP = 154.8 Hz, CH2P), 24.2 (d, JCP = 3.6 Hz, + CH(CH3)2), 23.9 (d, JCP = 5.0 Hz, CH(CH3)2). HRMS (ESI) m/zcalcd for C17H23N4O7P [M+H] 427.1377, found 427.1379.

1-[(diisopropoxyphosphoryl)methyl]-6-methoxy-4-oxo-1,4-dihydroquinoline-3- carbohydrazide (7e-ANPH12): White solid, m.p.: 187-189 °C, Yield: 79%;1H NMR (300.13 MHz, DMSO-d6):  10.62 (s, 1H, NHNH2), 8.67 (s, 1H, H-2), 7.94 (d, 1H, J = 9.4 Hz, H-8), 7.75 (d, 1H, J = 3.1, H-5), 7.44 (dd, 1H, J = 9.4 and 3.1 Hz, H-7), 5.06 (d, 2H, J = 11.4 Hz, CH2P), 4.61 (dsep, 2H, J =7.0and6.5 Hz, CH(CH3)2), 3.90 (s, 3H, OCH3), 1.21 (d, 6H, J = 6.1 13 Hz, CH(CH3)2), 1.12 (d, 6H, J = 6.2 Hz, CH(CH3)2). C NMR(75.47 MHz, DMSO-d6):  174.8 (C-4), 164.1 (C=ONH), 157.3 (C-6), 148.0 (C-2), 134.4 (C-8a), 128.4 (C-4a), 122.4 (C-5), 121.0 (C-8), 110.5 (C-3), 106.5 (C-7), 72.0 (d, JCP = 6.9 Hz,CH(CH3)2), 56.1 (OCH3), 48.8 (d, JCP = 153.4 Hz, CH2P), 24.1 (d, JCP = 3.7 Hz, CH(CH3)2), 23.9 (d, JCP = 4.9 Hz, CH(CH3)2).HRMS + (ESI) m/zcalcd for C18H26N3O6P [M+H] 412.1632, found 412.1630.

1-[(diisopropoxyphosphoryl)methyl]-7-methoxy-4-oxo-1,4-dihydroquinoline-3- carbohydrazide (7f-ANPH13): White solid, m.p.: 198-200 °C, Yield: 68%; 1H NMR (300.13 MHz, DMSO-d6):  10.60 (s, 1H, NHNH2), 8.79 (s, 1H, H-2), 8.25 (d, 1H, J = 9.0 Hz, H-5), 7.35 (d, , J = 2.2 Hz, 1H, H-8), 7.13 (dd, 1H, J = 9.0 and 2.2 Hz, Hz, H-6), 5.06 (d, 2H, J = 11.4 Hz, CH2P), 4.62 (dsep, 2H, J = 7.0 and 6.5 Hz, CH(CH3)2), 3.96 (s, 3H, OCH3), 1.22 (d, 6H, J = 13 6.2 Hz, CH(CH3)2), 1.13 (d, 6H, J = 6.2 Hz, CH(CH3)2). C NMR (75.47 MHz, DMSO-d6):  175.0 (C-4), 165.8 (C=ONH), 163. 2 (C-7), 149.3 (C-2), 141.7 (C-8a), 128.2 (C-5), 121.3 (C-4a), 114.8 (C-6), 110.9 (C-3), 101.7 (C-8), 72.0 (d, JCP = 7.0 Hz,CH(CH3)2), 56.4 (OCH3), 48.9 (d, JCP = 153.3 Hz, CH2P), 24.1 (d, JCP = 3.8 Hz, CH(CH3)2), 23.9 (d, JCP = 4.5 Hz, CH(CH3)2). + HRMS (ESI) m/zcalcd for C18H26N3O6P [M+H] 412.1632, found 412.1630.

Cells, virus propagation and compounds Vero cells obtained from American Type Culture Collection (ATCC) were maintained in Dulbecco's modified Eagle’s Medium (DMEM) medium (Gibco) supplemented with 4.5 g/L glucose, 2.0 g/L sodium bicarbonate, 2 mM L-glutamine, 100 U/mL penicillin (Gibco), 100 µg/mL streptomycin (Gibco), 2.5 µg/mL amphotericin B (Sigma), and 10% inactivated fetal bovine serum (FBS, Gibco) at 37 °C in a humidified atmosphere with 5% CO2 unless otherwise stated. MAYV (ATCC VR 66, strain TR 4675) was kindly provided by Dr. Davis Fernandes Ferreira. Viral stocks of MAYV were propagated in Vero cells. The cell monolayer was infected at a multiplicity of infection (MOI) of 0.1 and, 48 hours post-infection (hpi), cells were lysed by three cycles of freezing and thawing. Cell lysates were centrifuged at 400g for 20 minutes at 4ºC, and the supernatant was tittered and stored at -80 °C until further use. Compounds were dissolved in sterile 100% dimethyl sulfoxide (DMSO) at 50mM and stored at -20 ºC until use.

Cytotoxicity assay

The cytotoxicity of the 4-oxoquinoline acyclonucleoside phosphonate analogs on Vero cells was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described elsewhere (Pires Mello et al., 2019) with a few modifications. Briefly, Vero cells were seeded into a 96-well plate at a cell density of 1.0 x 104 cells per well and incubated overnight at 37 °C in a humidified atmosphere with 5% CO2. On the next day, cells were treated with compounds at different concentrations (50, 100, 200, 400, and 800μM) for 72 h at 37 °C in a humidified 5% CO2 atmosphere. Then, media containing compounds was discarded, and cells were incubated with MTT dye solution (5 mg/mL) for 4 h at 37 °C in 5% CO2 atmosphere. MTT solution was discarded, and cells were incubated with DMSO under rocking at room temperature for 20 minutes. Finally, absorbance was measured on a microplate reader at 540nm. The cell viability of treated cells was calculated relative to the untreated control. The experiment was carried out in triplicate and repeated three times. The compound concentration required to reduce cell viability by 50% (CC50) was estimated using linear regression.

Antiviral screening and plaque formation assay

Vero cells (3x105 cells/well) were seeded in 24-well plates and incubated for 24h at 37°C in 5% CO2 atmosphere. Confluent cells were then infected with MAYV at a MOI of 1.0 for 1.0 h at 37 °C in 5% CO2 atmosphere. Following this, the virus inoculum was replaced by fresh media containing 10% FBS and compounds at a final concentration of 50 µM. Chloroquine (50 µM; Sigma) was used as positive control, while untreated cells were used as MAYV infection control. At 24 hours post-infection, cells were lysed by three cycles of freezing and thawing, lysates were harvested and stored at -80 °C until titration. The virus titer was determined by standard plaque-forming assay (PFA). Confluent Vero cells grown in 24-well plates were infected with 10-fold dilutions of the MAYV lysates collected previously and incubated for 1.0 h at 37 °C, 5% CO2. The virus inoculum was discarded, cells were covered with DMEM containing 5% FBS and 1.5% methylcellulose and incubated for 72 h at 37 °C in 5% CO2atmosphere. Afterward, the overlay media was removed, and cells were fixed and stained with 10% formaldehyde and 0.2% crystal violet solution. The viral titers were determined according to the number of plaque-forming units per milliliter (PFU/mL), and inhibition of viral plaque formation by the compounds was calculated relative to the untreated control.

EC50 determination

The determination of the concentration of the compound 7a required to reduce the number of PFU by 50% (EC50) was carried out for compounds that inhibited over 50% of viral plaque formation at 50 µM. Vero cells were grown in 24-well plates at a cell density of 3x105 cells per well at 37 °C, 5% CO2for 24h before the experiment. Confluent monolayers were infected at a MOI of 1.0 for 1.0 h at 37 °C, 5% CO2; then,the virus inoculum was discarded. Media containing compounds at increasing concentrations (1.56, 3.125, 6.25, 12.5, 25µM) were added, and cells were further incubated for 24 h at 37 °C in 5% CO2 atmosphere. Finally, cells were lysed by three cycles of freezing and thawing, and cell lysates were collected and tittered by PFA. Chloroquine was used as a positive control, whereas infected and untreated cells were taken as infection control. The inhibition of viral plaque formation by the compounds was calculated relative to the untreated control. This assay was performed in triplicate in three independent experiments. EC50 was calculated by linear regression from dose-response curves.

Time-of-drug-addition

Vero cells (3x105cells/well) were cultured in 24-well plates at 37 °C in a humidified atmosphere with 5% CO2for 24h prior to the assay. To evaluate at which stage of virus replication the compounds might act, cells were treated with compound 7a (100 µM) at different time points. For instance, compounds were added to the cellsat 1 or 2h before viral infection (-1 and -2 hpi; pre-treatment conditions), compounds were added simultaneously with virus inoculum (0 hpi; simultaneous treatment), and compounds were added after viral infection (1, 2 and 3 hpi; post-treatment conditions). Cells were infected at a MOI of 1.0, and attachment occurred for 1.0 h at 37 °C, 5% CO2. Then, virus inoculum was removed, and media containing compounds or not according to the treatment scheme were added. Cells were incubated for further 24 h at 37 °C, 5% CO2. Next, cells were lysed by freezing and thawing, and lysates were collected and tittered by PFA. Chloroquine was used as a positive control, while infected and untreated cells were used as a negative control. Inhibition of viral plaque formation by compounds at different times was calculated relative to the infected and untreated cells. The experiment was carried out in triplicate in three different assays.

Molecular modeling

Homology modeling

The amino acid sequence of the envelope protein 1, 2, and 3 (E1, E2, and E3, respectively) complex from MAYV (ATCC VR-66, strain TR 4675) was retrieved from GenBank under the code AZM66146.1. A template search within the Protein Data Bank (PDB) was carried out using the Basic Local Alignment Search Tool (Blast) server. The X-ray structure of the mature envelope protein complex (E1-E2-E3) from the Chikungunya virus (PDB code 3N42) was used as the template for the construction of the homology model of MAYV envelope proteins. The crystal structure of the Chikungunya virus envelope protein has a resolution of 3.0 Å and shares 57.49% of sequence identity with MAYV envelope proteins considering all together. The three-dimensional (3D) model of MAYV E1-E2-E3 was constructed using the Modeller v. 9.22 software(Šali and Blundell, 1993). Further, the model was submitted to energy minimization cycles using the GROMOS96 force field and Swiss PDB Viewer 4.1 program.The model quality was assessed by a stereochemical analysis of amino acid residues using the Ramachandran plot generated with Procheck (Laskowski et al., 1993); a residue environment analysis using the 3D-1D score calculated with Verify-3D (Lüthy et al., 1992); an overall energy analysis using the Z-score calculated in ProSA-web server (Wiederstein and Sippl, 2007). The protonation state of the charged residues at pH 7.4 was predicted using Propka 3.0 available at the PDB2PQR server(Dolinsky et al., 2004).

Binding site prediction and molecular docking

The 3D structure of 7a compound was constructed using the Spartan`10 program (Wavefunction Inc. Irvine, CA, USA). First, the structure was submitted to a conformational analysis using molecular mechanics and the MMFF force field. Then, the selected conformer was subjected to a geometry optimization step using the RM1 semi-empirical method, followed by the single point calculation using the density functional theory (DFT) method with B3LYP/6- 311G* basis set. Molecular docking studies were carried out using Autodock Tools 1.5.7 (ADT) and Autodock 4.2.6 software. Gasteiger charges and hydrogens were added to the protein structure obtained by homology modeling, while polar hydrogen atoms were added to the ligand`s structure, using ADT. The protein structure was treated as rigid, whereas the ligand was kept flexible. Its rotatable bonds were defined automatically using ADT.Initially, a blind docking approach was undertaken to identify potential binding sites for 7a within the MAYV envelope complex. For this purpose, a grid box encompassing the whole protein was centered on the protein complex and had dimensions of 100 x 126 x 98 points with a grid spacing of 1.0 Å. The Lamarckian genetic algorithm was employed as the search engine, and a total of 100 poses were calculated. Next, focused docking was performed on three potential binding sites to explore the interaction of 7a with them in-depth. The selection of the three binding sites relied on favorable binding energy (≤ 0.0 Kcal/mol) and structural features reported in the literature regarding binding sites within envelope proteins of alphaviruses and mechanisms of inhibition of early steps of the virus replication cycle. The grid space used for each site is described as follows: for site 1, a grid with dimensions of 60 x 60 x 60 points centered on H353 from E2; for site 2, a grid with dimensions of 70 x 80 x 86 points centered on E989 from E1; for site 3, a grid with dimensions of 70 x 70 x 60 points centered on V434 from E2. For the focused docking, 0.375 Å grid spacing was used. The Lamarckian genetic algorithm was also employed, but a total of 50 poses was obtained for each site. Other searching parameters were kept with default values in both docking approaches. Finally, molecular interaction analysis was performed using Pymol version 1.2r2 (The PyMOL Molecular Graphics System, Version 1.2r2 Schrödinger, LLC).

Prediction of pharmacokinetic and toxicological properties

To evaluate the drug-likeness profile of 7a, we calculated some stereoelectronic features of this compound such as octanol-water partition coefficient (LogP), molecular weight (MW), number of hydrogen bond donor and acceptor groups (HBD and HBA, respectively), number of rotatable bonds (nRot) and the topological polar surface area (tPSA) using the FAF-Drugs4 server(Lagorce et al., 2017) while the polar surface area (PSA) was calculated using the Spartan`10 software as above mentioned. Further, we applied different rules developed by pharmaceutical companies like Lipinski “rule of five” (Lipinski et al., 2001), Veber criteria(Veber et al., 2002), GlaxoSmithKline (GSK) 4/400(Gleeson, 2008), and Pfizer 3/75(Hughes et al., 2008). These rules can guide the selection of compounds with the most promising pharmacokinetics and toxicological properties. Moreover, we predicted some pharmacokinetic and toxicological properties. For instance, human intestinal absorption (HIA) and carcinogenic risk were predicted using the admetSAR 2 server(Yang et al., 2019) whereas the genotoxicity (based on Ames test), hepatotoxicity and cardiotoxicity (based on hERG I or II inhibition) were predicted using the pkCSM server(Pires et al., 2015). For comparison purposes, we also evaluated the same properties for the marketed drugs chloroquine and ribavirin, which exhibits antiviral activity against MAYV.

Results Chemistry The compounds tested in the present study (7a-f) were previously synthesized through the Gould-Jacobs methodology (Gould and Jacobs, 1939; Riegel et al., 1946; Snyder et al., 1947; Stern et al., 2006), using a three-step procedure that involved the condensation of anilines 2 with diethyl ethoxymethylenemalonate (EMME) in refluxing ethanol followed by thermal cyclization of the aniline acrylate intermediates 3a-m. The N-alkylation reaction between oxoquinolines4a- m and the tosyl methoxy phosphonatediisopropyl ester 5 (Schultze et al., 1998) in N,N- dimethylphormamide, afforded the respective acyclonucleosides phosphonates 4-oxoquinolines- 3-carboxylates 6a-maccording to our previous work(Faro et al., 2012). Acyclonucleosides phosphonates 6a-mwere submitted tonucleophilic substitution reaction with hydrazine monohydrate(80%) (He et al., 2005; Santos et al., 2009) in refluxing ethanol affording acyclonucleosides phosphonates acylhydrazides7a-f, in 81-14% yields. The structures of the new compounds (7a-f) were assigned on the basis of their 1H and 13C 1D and 2D-NMR spectra and their molecular formulas were confirmed by HRMS. Regarding the 1H NMR spectra of the compounds 7a-f, it is important to highlight the following: The singlet between 8.90 and 8.56 ppm refers to H-2 oxoquinolinic hydrogen; the singlet at 10.75-10.31 ppm was attributed to hydrogen NHNH2 of the acylhydrazide moiety; the signals of oxoquinolinic hydrogens between 9.04 and 6.74 ppm; the methylenichydrogens CH2P as a doublet at 6.02-5.05 ppm; the methynic hydrogen CH(CH3)2 as the double septet at 4.73-

4.60 ppm; and the methyl hydrogens CH(CH3)2 as doublets at 1.30-1.10 ppm.

Scheme 1.Synthesis of 1-[(diisopropoxyphosphoryl)methyl]-4-oxo-1,4-dihydroquinoline-3- carbohydrazides (7a-m). Reagents and conditions: (i) EtOH, diethyl ethoxymethylenemalonate, reflux; (ii) diphenylether, 275 ºC; (iii) dry DMF/potassium carbonate, 80 ºC; (iv) hydrazine monohydrate(80%), ethanol, reflux.

Cytotoxicity and antiviral activity of 4-oxoquinoline acyclonucleoside phosphonate analogs against MAYV

Initially, we evaluated the toxicity of the 4-oxoquinoline acyclonucleoside phosphonate analogs on Vero cells. These compounds presented low toxicity on these cells with CC50 values ranging from 659.48 µM with 7d to 2,815.83 µM with 7f. However, most of them did not inhibit MAYV replication at 50 µM (Table 1). By contrast, compound 1ashowed potent activity against this virus and presented an EC50 of 0.83 µM. Interestingly, this compound was shown to be less cytotoxic and more potent against MAYV than the drug chloroquine, which had a CC50 of

470.20 µM and EC50 of 25.83 µM. Consequently, we observed that 1a has a significantly higher selectivity index (1152.67) compared to chloroquine (18.19), indicating that this compound is safer than amarketed drug with antiviral activity.

Table1. Cytotoxicity and anti-MAYV profile and selectivity index (SI) ofthe4-oxoquinoline derivatives and chloroquine. ND: Notdetermined.

a b c d Compound CC50 (µM) Inhibition (%) EC50 (µM) SI 7a 956.72 0.83 1152.67 7b 1500.00 <50 >50 ND 7c 813.59 <50 >50 ND 7d 659.48 <50 >50 ND 7e 1284.10 <50 >50 ND 7f 2815.83 <50 >50 ND Chloroquine 470.20 25.83 18.19 a CC50: Compound concentration at which compound decreases cell viability by 50%. bInhibition percentage of PFU formation by the tested compound at 50 µM in comparison to the untreated control. c EC50: Effective concentration that the compound exhibits 50% activity normalized against infection. d SI: Selectivity index values are based on the ratio of CC50 to EC50 values.

Compound 1a acts at early steps of MAYV replication

To get more insights about the mechanism of antiviral action of compound 7a, we added it at different time points throughout viral replication and compared it with chloroquine (Figure 1). Chloroquine showed more significant inhibition potential when added before virus infection, especially at 1.0 h prior to the infection, but also inhibited virus replication at a similar extent when added 3.0 h post-infection. For compound 7a, the highest inhibition of MAYV replication was reached when the compound was added simultaneously to the virus inoculum (~55% inhibition). Pre-treatment of cells with compound 7a also exhibited considerable inhibition of MAYV replication, suggesting that this compound act on early events in the MAYV life cycle.

Figure 1. Effect of the addition of the 7a or chloroquine on MAYV replication over time. Monolayers of Vero cells were infected with MAYV at MOI of 1.0 at time zero. At the times indicated, compounds or chloroquine was added at a final concentration of 100 µM. Data are presented as percentage of PFU inhibition relative to the infected and untreated control, and are expressed as the mean of three experiments ± standard deviation

Binding mode of 7a with the potential target, the envelope protein complex from MAYV

The highest antiviral activity of 7a was observed when infected cells were treated before or during infection according to the time-of-drug-addition assay. Also, this compound did not inhibit CHIKV replication (data not shown?), suggesting that it acts directly on the MAYV structure and not on host cells. Thereby, we employed molecular modeling tools to investigate whether this compound can bind to the MAYV envelope protein complex. Since the 3D structure of the envelope proteins of MAYV was not solved experimentally, we constructed a homology model based on the CHIKV envelope protein complex. The final model showed good stereochemical quality, presenting 85.5% and 1.4% of the residues in favorable and disallowed regions of the Ramachandran plot, respectively, similar to the ones observed for the template (85.0% and 0.9%, respectively). Besides, 77.30% of the model residues exhibited 3D-1D score values ≥ 0.2, which indicates good compatibility between the amino acid sequence and its 3D structure. Likewise, the template showed 88.92% of residues with a 3D-1D score of ≥ 0.2. Finally, the Z-score of the model was -6.61, whereas the template showed a value of -9.34 (Figure S1). Although these values are not similar, both model and template presented values within the range of other proteins whose 3D structure has been solved experimentally. These results indicated the reliability of the constructed model, which allows its use for docking studies. Initially, blind docking was carried out to find potential binding sites of 7a within the MAYV envelope complex model. We evaluated the poses and their binding sites with a favorable binding affinity (binding energy ≤ 0.0 Kcal/mol). Among them, three potential sites were identified and could be related to virucide or entry inhibitor profile of the compound (Figure 2). Site 1 is a solvent-exposed binding site and is comprised of residues from domain A of E2 and domain II of E1. This site also encompasses residues of the fusion loop, an essential structure of E1 that triggers the fusion and uncoating steps of MAYV replication cycle (Fields and Kielian, 2013; Kielian et al., 2010). Site 2 is also located at the complex surface and has been identified to be the heparan sulfate binding region in the CHIKV complex (Sahoo and Chowdary, 2019), which is a host cell receptor for virus entry. Site 3 is a cleft found between the E1 (domain II) and E2 (β-ribbon region), and it was also identified in the CHIKV envelope protein as a potential drug binding site (Nguyen et al., 2018). Following this, we performed focused docking of 7a at each site to improve the accuracy in the binding site prediction by increasing the conformational sampling(Ghersi and Sanchez, 2009). The compound showed higher theoretical affinity with site 3 (binding energy = -2.41 Kcal/mol), followed by site 1 (-1.84 Kcal/mol) and site 2 (-1.31 Kcal/mol). Consequently, we further explored the binding mode and interaction network of the compound bound in site 3 and site 1 in-depth (Figure 2). At site 1, we observed that the hydrazide group could establish hydrogen bonds with M894 and W895, and van der Waals contacts with G896 and G897, residues comprising the fusion loop of E1. Meanwhile, the 4-oxoquinoline was hydrogen-bonded with H353 of E2 and interacted with F893 of the fusion loop by van der Waals interaction. Also, the 6-methyl group allowed the van der Waals interactions with G1033 and N1034 of E2. Of note, the phosphonate substituents were positioned towards outside the binding site. For site 3, the hydrazide group interacted with G988, N1058, and I1067 by hydrogen bonds, while van der Waals contacts were established with Q1068. The 4-oxoquinoline ring was stacked with E989 via anion-π interaction, and, due to the introduction of 6-methyl, it also interacted with K987 through van der Waals interaction. The phosphonate group was hydrogen-bonded with R462 of E2 while the i-propyl group was involved in van der Waals contacts with P1056.

Figure 2. 3D structure of the MAYV envelope protein complex and docking results with 7a. A) The overall structure of the MAYV envelope protein complex model (E1, green; E2, cyan; E3, yellow). (B) Lowest energy conformer of 7a bound to the three potential binding sites explored in the focused docking strategy (Site 1, orange; Site 2, red; Site 3, purple). Binding mode of ANPH08 within (C) site 1 and, (D) site 3. Residues are colored according to the protein color scheme, while the black and orange dashed lines represent hydrogen bonds and anion-π interactions, respectively.

Pharmacokinetic and toxicological profile of 7a.

The pharmacokinetics and toxicological properties of 7a were analyzed and compared with marketed drugs with anti-MAYV activity to assess its drug-likeness (Table 2). Like chloroquine and ribavirin, 7a was predicted to be absorbed in the human gastrointestinal system. According to its stereoelectronic features, this compound fulfills all requirements of Lipinski “rule of five” (Log ≤ 5; MW ≤ 500 Da; HBA ≤ 10; HBD ≤ 5)(Lipinski et al., 2001) and Veber criteria (nRot ≤ 10 and PSA ≤ 140 Å2)(Veber et al., 2002), which suggest a good oral bioavailability and reinforces its potential as a drug to be orally delivered.

Table 2.Comparison of stereoelectronic descriptors, pharmacokinetics, and toxicological properties and classification of 7a and the marketed drugs chloroquine and ribavirin regarding pharmaceutic industry rules.MW: molecular weight (Da), LogP: octanol-water partition coefficient; HBA and HBD: hydrogen- bond acceptor and donor groups, respectively; nRot: number of rotatable bonds; tPSA: topological polar surface area (Å2); PSA: polar surface area (Å2); HIA: Human intestinal absorption. Compounds 7a Chloroquine Ribavirin MW (Da) 395.39 319.87 244.20 LogP 2.30 4.63 -1.85 HBA 8 3 9 HBD 3 1 5 nRot 7 8 3 tPSA (Å2) 122.46 29.36 143.72 PSA (Å2) 95.64 23.06 110.50 HIAa Yes (0.95) Yes (1.00) Yes (0.87) Lipinski “ruleoffive” Approved Approved Approved Vebercriteria Approved Approved Approved GSK 4/400 rule Approved Approved Approved Pfizer 3/75 rule Approved Rejected Approved Carcinogenicitya No (0.81) No (0.83) No (0.99) Genotoxicity No Yes No Hepatotoxicity Yes Yes No Cardiotoxicityb No Yes No aProbability of classification accuracy by the predictive model is shown in parentheses.bCardiotoxicity was predicted based on the inhibition of hERG I or hERG II potassium channels located in the cardiac muscle.

Additionally, this compound was approved according to both GSK 4/400 and Pfizer 3/75 rules. The first one defines that structures with Log P < 4.0 and MW < 400 Da are most likely to present satisfactory pharmacokinetics and toxicological properties(Gleeson, 2008), whereas the last one states that compounds with LogP< 3 and tPSA> 75 Å2 are less likely to exhibit preclinical toxicity(Hughes et al., 2008). Unlike 7a and ribavirin, chloroquine was rejected according to Pfizer 3/75 rule due to its low tPSA. Furthermore, other toxicity risks were evaluated, like carcinogenicity, genotoxicity, hepatotoxicity, and cardiotoxicity. Ribavirin exhibited low toxicity for all risks evaluated. Likewise, 7a showed a low risk for all effects predicted, except for hepatotoxicity, which was associated with the phosphonate group. Chloroquineexhibitedgenotoxicity, hepatotoxicityandalsocardiotoxicityrisks.

Discussion

The results of this study have shown that 7a compound has a potent anti-Mayaro activity, demonstrating a potent effect on viral replication with an EC50 at a concentration of 0.83 µM, and an SI selectivity index above 1000, which seems to be considered an excellent value for an antiviral. In addition, as shown in Table 1, this seems to be an exclusive characteristic for that compound since we do not observe anti-MAYV effects in the other tested compounds. Recently, several studies have shown compounds with effects on Mayaro replication, however the EC50 values of these substances were higher than demonstrated in our study(Amorim et al., 2017; Ferraz et al., 2019). In general, what makes an antiviral differentiated is its mechanism of action, therefore, the search and understanding of the possible effects that this compound may cause in the phases of the replicative cycle must be carried out strategically and carefully evaluated, that is why in our study we started by performing the time-of-addition. Our data suggest that in the tested concentration of the compound 7a, we observed that its effects seem to be in very early events of viral replication, since in a pre-treatmentof 2 hours it was already possible to observe an effect greater than 40% inhibition in viral replication. Andwith a greater effect on the concentration used when we carry out the simultaneous treatment of the infection, leading to na inhibition of approximately 60% (Fig 1), demonstrating to be a different behavior from other studies presented for the Mayaro virusor for different viruses(Amorim et al., 2017; Kaur et al., 2013; Wintachai et al., 2015). Interestingly, thesepercentagesofinhibitionslightlydecrease in thetreatmentfromthefirst hour afterinfection, demonstrating a lastingeffectthatalthough it isless, butismaintaineduntilthethird hour. In this context, we employed computational tools to investigate the putative antiviral target of 7a accordingly to the experimental data. We observed that 7a can bind theoretically to the envelope protein complex from MAYV, especially at site 3. Importantly, the binding of 7a between the E1 and E2 proteins could prevent conformational changes of the envelope proteins spikes, which, in turn, could lead to the virucide effect of this compound as well as the inhibition of early events in virus replication (Rashad and Keller, 2013). Additionally, this structural effect could also impact post-entry steps like virus assembly and budding(Byrd and Kielian, 2017; Chen et al., 2018), which may explain the maintenance of the antiviral activity of 7a when added few hours post-infection (though a lower activity was observed). Pharmacokinetic and toxicological issues are major concernsin the drug development pipeline and one main reason for drug failure in clinical trials (Dong et al., 2018; Wang et al., 2015). Consequently, we employed in silico tools to evaluate the drug-like profile of compound 7a. Interestingly, this compound showed promising theoretical pharmacokinetics and toxicological properties. For instance, this compound is compatible with oral administration because it showed good oral bioavailability and intestinal absorption as well as low probability to present pharmacokinetic and pre-clinical toxicity according to pharmaceutical industry rules. The predicted toxicity risks suggested a safety profile for ribavirin comparable to the one observed for 7a. On the other hand, a hepatotoxicity warning was raised for this compound, probably associated with the phosphonate group. It is important to note that chloroquine exhibited hepatotoxicity risk as well as cardiotoxicity and genotoxicity risks.Indeed, many of these toxic effects have been related to chloroquine experimentally(Fang et al., 2013; Rossmann-Ringdahl and Olsson, 2007; Thanacoody, 2016; Traebert et al., 2004; Wielgo-Polanin et al., 2005) and, yet, it reached the market. Therefore, our results reinforce the potential of 7a as an anti-MAYV drug candidate which deserves future investigations.

Conclusions Herein, we evaluated the antiviral potential of 4-oxoquinoline acyclonucleoside phosphonate analogs as anti-MAYV agents. Compound 7a showed more potent and safer in vitroprofile than the commercial drug chloroquine. Regarding its mechanism of action, 7a was shown to inhibit early events in MAYV replication. Docking studies suggested that this compound binds to the E1-E2 complex and might impair conformational changes important for interactions with host cell or fusion processes. In addition, this compound showed a satisfactory theoretical pharmacokinetics and toxicological profiles comparable to drugs available in market with antiviral properties.Therefore, 7a could serve as a start point to develop novel antivirals against MAYV.

Acknowledgments This work was supported by the Brazilian agencies CAPES (Finance Code 001).

References

Abreu, P.A., Da Silva, V.A.G.G., Santos, F.C., Castro, H.C., Riscado, C.S., De Souza, M.T., Ribeiro, C.P., Barbosa, J.E., Dos Santos, C.C.C., Rodrigues, C.R., Lione, V., Correa, B.A.M., Cunha, A.C., Ferreira, V.F., De Souza, M.C.B.V., Paixão, I.C.N.P., 2011. Oxoquinoline derivatives: Identification and structure-activity relationship (SAR) analysis of new anti-HSV-1 agents. Curr. Microbiol. 62, 1349–1354. https://doi.org/10.1007/s00284- 010-9860-6 Acosta-Ampudia, Y., Monsalve, D.M., Rodríguez, Y., Pacheco, Y., Anaya, J.M., Ramírez- Santana, C., 2018. Mayaro: an emerging viral threat? Emerg. Microbes Infect. https://doi.org/10.1038/s41426-018-0163-5 Agarwal, A., Parida, M., Dash, P.K., 2017. Impact of transmission cycles and vector competence on global expansion and emergence of arboviruses. Rev. Med. Virol. https://doi.org/10.1002/rmv.1941 Aguilar-Luis, M.A., del Valle-Mendoza, J., Silva-Caso, W., Gil-Ramirez, T., Levy-Blitchtein, S., Bazán-Mayra, J., Zavaleta-Gavidia, V., Cornejo-Pacherres, D., Palomares-Reyes, C., del Valle, L.J., 2020. An emerging public health threat: Mayaro virus increases its distribution in Peru. Int. J. Infect. Dis. 92, 253–258. https://doi.org/10.1016/j.ijid.2020.01.024 Amorim, R., de Meneses, M.D.F., Borges, J.C., da Silva Pinheiro, L.C., Caldas, L.A., Cirne- Santos, C.C., de Mello, M.V.P., de Souza, A.M.T., Castro, H.C., de Palmer Paixão, I.C.N., Campos, R. de M., Bergmann, I.E., Malirat, V., Bernardino, A.M.R., Rebello, M.A., Ferreira, D.F., 2017. Thieno[2,3-b]pyridine derivatives: a new class of antiviral drugs against Mayaro virus. Arch. Virol. 162, 1577–1587. https://doi.org/10.1007/s00705-017- 3261-0 Brustolin, M., Pujhari, S., Henderson, C.A., Rasgon, J.L., 2018. Anopheles mosquitoes may drive invasion and transmission of Mayaro virus across geographically diverse regions. PLoS Negl. Trop. Dis. 12. https://doi.org/10.1371/journal.pntd.0006895 Byrd, E.A., Kielian, M., 2017. An alphavirus E2 membrane-proximal domain promotes envelope protein lateral interactions and virus budding. MBio 8. https://doi.org/10.1128/mBio.01564- 17 Canuto, C.V.B. dos S., Gomes, C.R.B., Marques, I.P., Faro, L. V., Frugulhetti, I.C. de P.P., Souza, T.M.L., Cunha, A.C., Romeiro, G.A., Ferreira, V.F., Souza, M.C.B.V., 2007. Synthesis and Anti-HSV-1 Activity of 1,4-dihydro-4-oxoquinoline Ribonucleosides. Lett. Drug Des. Discov. 4, 404–409. https://doi.org/10.2174/157018007781387818 Chen, L., Wang, M., Zhu, D., Sun, Z., Ma, J., Wang, Jinglin, Kong, L., Wang, S., Liu, Z., Wei, L., He, Y., Wang, Jingfei, Zhang, X., 2018. Implication for alphavirus host-cell entry and assembly indicated by a 3.5Å resolution cryo-EM structure. Nat. Commun. 9. https://doi.org/10.1038/s41467-018-07704-x de Mello, M.V., Domingos, T.F., Ferreira, D., Ribeiro, M., Ribeiro, T., Souza, A., Rodrigues, C., 2020. Antiviral drug discovery and development for Mayaro Fever – What do we have so far? Mini-Reviews Med. Chem. 20. https://doi.org/10.2174/1389557520666200316160425 Dodero-Rojas, E., Ferreira, L.G., Leite, V.B.P., Onuchic, J.N., Contessoto, V.G., 2020. Modeling Chikungunya control strategies and Mayaro potential outbreak in the city of Rio de Janeiro. PLoS One 15. https://doi.org/10.1371/journal.pone.0222900 Dolinsky, T.J., Nielsen, J.E., McCammon, J.A., Baker, N.A., 2004. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 32, W665–W667. https://doi.org/10.1093/nar/gkh381 Dong, J., Wang, N.N., Yao, Z.J., Zhang, L., Cheng, Y., Ouyang, D., Lu, A.P., Cao, D.S., 2018. Admetlab: A platform for systematic ADMET evaluation based on a comprehensively collected ADMET database. J. Cheminform. 10. https://doi.org/10.1186/s13321-018-0283-x Esposito, D.L.A., Fonseca, B.A.L. da, 2017. Will Mayaro virus be responsible for the next outbreak of an arthropod-borne virus in Brazil? Brazilian J. Infect. Dis. https://doi.org/10.1016/j.bjid.2017.06.002 Fang, H., Liu, A., Dahmen, U., Dirsch, O., 2013. Dual role of chloroquine in liver ischemia reperfusion injury: reduction of liver damage in early phase, but aggravation in late phase. Cell Death Dis. 4, e694. https://doi.org/10.1038/cddis.2013.225 Faro, L. V., De Almeida, J.M., Cirne-Santos, C.C., Giongo, V.A., Castello-Branco, L.R., Oliveira, I.D.B., Barbosa, J.E.F., Cunha, A.C., Ferreira, V.F., De Souza, M.C., Paixão, I.C.N.P., De Souza, M.C.B.V., 2012. Oxoquinoline acyclonucleoside phosphonate analogues as a new class of specific inhibitors of human immunodeficiency virus type 1. Bioorganic Med. Chem. Lett. 22, 5055–5058. https://doi.org/10.1016/j.bmcl.2012.06.020 Ferraz, A.C., Moraes, T. de F.S., Nizer, W.S. da C., Santos, M. dos, Tótola, A.H., Ferreira, J.M.S., Vieira-Filho, S.A., Rodrigues, V.G., Duarte, L.P., de Brito Magalhães, C.L., de Magalhães, J.C., 2019. Virucidal activity of proanthocyanidin against Mayaro virus. Antiviral Res. 168, 76–81. https://doi.org/10.1016/j.antiviral.2019.05.008 Fields, W., Kielian, M., 2013. A Key Interaction between the Alphavirus Envelope Proteins Responsible for Initial Dimer Dissociation during Fusion. J. Virol. 87, 3774–3781. https://doi.org/10.1128/jvi.03310-12 Ghersi, D., Sanchez, R., 2009. Improving accuracy and efficiency of blind protein-ligand docking by focusing on predicted binding sites. Proteins Struct. Funct. Bioinforma. 74, 417–424. https://doi.org/10.1002/prot.22154 Gleeson, M.P., 2008. Generation of a set of simple, interpretable ADMET rules of thumb. J. Med. Chem. 51, 817–834. https://doi.org/10.1021/jm701122q Gould, R.G., Jacobs, W.A., 1939. The Synthesis of Certain Substituted Quinolines and 5,6- Benzoquinolines. J. Am. Chem. Soc. 61, 2890–2895. https://doi.org/10.1021/ja01265a088 He, J.F., Yun, L.H., Yang, R.F., Xiao, Z.Y., Cheng, J.P., Zhou, W.X., Zhang, Y.X., 2005. Design, synthesis, and biological evaluation of novel 4-hydro-quinoline-3- carboxamide derivatives as an immunomodulator. Bioorganic Med. Chem. Lett. 15, 2980–2985. https://doi.org/10.1016/j.bmcl.2005.04.040 Hotez, P.J., Murray, K.O., 2017. Dengue, West Nile virus, chikungunya, Zika—and now Mayaro? PLoS Negl. Trop. Dis. https://doi.org/10.1371/journal.pntd.0005462 Hughes, J.D., Blagg, J., Price, D.A., Bailey, S., DeCrescenzo, G.A., Devraj, R. V., Ellsworth, E., Fobian, Y.M., Gibbs, M.E., Gilles, R.W., Greene, N., Huang, E., Krieger-Burke, T., Loesel, J., Wager, T., Whiteley, L., Zhang, Y., 2008. Physiochemical drug properties associated with in vivo toxicological outcomes. Bioorganic Med. Chem. Lett. 18, 4872–4875. https://doi.org/10.1016/j.bmcl.2008.07.071 Kantor, A.M., Lin, J., Wang, A., Thompson, D.C., Franz, A.W.E., 2019. Infection Pattern of Mayaro Virus in Aedes aegypti (Diptera: Culicidae) and transmission potential of the virus in mixed infections with chikungunya virus. J. Med. Entomol. 56, 832–843. https://doi.org/10.1093/jme/tjy241 Kaur, P., Thiruchelvan, M., Lee, R.C.H., Chen, H., Chen, K.C., Ng, M.L., Chu, J.J.H., 2013. Inhibition of Chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression. Antimicrob. Agents Chemother. 57, 155–167. https://doi.org/10.1128/AAC.01467-12 Kielian, M., Chanel-Vos, C., Liao, M., 2010. Alphavirus entry and membrane fusion. Viruses. https://doi.org/10.3390/v2040796 Lagorce, D., Bouslama, L., Becot, J., Miteva, M.A., Villoutreix, B.O., 2017. FAF-Drugs4: free ADME-tox filtering computations for chemical biology and early stages drug discovery. Bioinformatics 33, 3658–3660. https://doi.org/10.1093/bioinformatics/btx491 Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., 1993. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291. https://doi.org/10.1107/S0021889892009944 Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J., 2001. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3–26. Lüthy, R., Bowie, J.U., Eisenberg, D., 1992. Assessment of protein models with three- dimensional profiles. Nature 356, 83–85. https://doi.org/10.1038/356083a0 Mackay, I.M., Arden, K.E., 2016. Mayaro virus: a forest virus primed for a trip to the city? Microbes Infect. https://doi.org/10.1016/j.micinf.2016.10.007 Matta, A.D. da, Santos, C.V.B. dos, Pereira, H. de S., Frugulhetti, I.C. de P.P., Oliveira, M.R.P. de, Souza, M.C.B.V. de, Moussatché, N., Ferreira, V.F., 1999. Synthesis of novel nucleosides of 4‐oxoquinoline‐3‐carboxylic acid analogues. Heteroat. Chem. 10, 197–202. https://doi.org/10.1002/(SICI)1098-1071(1999)10:3<197::AID-HC4>3.0.CO;2-Z Mavian, C., Rife, B.D., Dollar, J.J., Cella, E., Ciccozzi, M., Prosperi, M.C.F., Lednicky, J., Morris, J.G., Capua, I., Salemi, M., 2017. Emergence of recombinant Mayaro virus strains from the Amazon basin. Sci. Rep. 7. https://doi.org/10.1038/s41598-017-07152-5 Mota, M.T.D.O., Ribeiro, M.R., Vedovello, D., Nogueira, M.L., 2015. Mayaro virus: A neglected arbovirus of the Americas. Future Virol. 10, 1109–1122. https://doi.org/10.2217/fvl.15.76 Muñoz, M., Navarro, J.C., 2012. Mayaro: a re-emerging Arbovirus in Venezuela and Latin America. Biomedica 32, 286–302. https://doi.org/10.1590/S0120-41572012000300017 Napoleão-Pego, P., Gomes, L.P., Provance-Júnior, D.W., De-Simone, S.G., 2014. Mayaro Virus Disease. J. Hum. Virol. Retrovirology 1, 00018. https://doi.org/10.15406/jhvrv.2014.01.00018 Nguyen, P.T.V., Yu, H., Keller, P.A., 2018. Molecular Docking Studies to Explore Potential Binding Pockets and Inhibitors for Chikungunya Virus Envelope Glycoproteins. Interdiscip. Sci. Comput. Life Sci. 10, 515–524. https://doi.org/10.1007/s12539-016-0209-0 Pezzi, L., Rodriguez-Morales, A.J., Reusken, C.B., Ribeiro, G.S., LaBeaud, A.D., Lourenço-de- Oliveira, R., Brasil, P., Lecuit, M., Failloux, A.B., Gallian, P., Jaenisch, T., Simon, F., Siqueira, A.M., Rosa-Freitas, M.G., Vega Rua, A., Weaver, S.C., Drexler, J.F., Vasilakis, N., de Lamballerie X, Boyer, S., Busch, M., Diallo, M., Diamond, M.S., Drebot, M.A., Kohl, A., Neyts, J., Ng, L.F.P., Rios, M., Sall, A., Simmons, G., 2019. GloPID-R report on chikungunya, o’nyong-nyong and Mayaro virus, part 3: Epidemiological distribution of Mayaro virus. Antiviral Res. https://doi.org/10.1016/j.antiviral.2019.104610 Pinto, A.M. V, Leite, J.P.G., Marinho, R.S., Forezi, L. da S., Batalha, P.N., Boechat, F. da C., Cunha, A.C., Silva, D.O., Gama, I.L., Faro, L. V, de Souza, M.C., Paixão, I.C.P., 2019. Antiviral activity of 4-oxoquinoline-3-carboxamide derivatives against bovine herpesvirus type 5. Antivir. Ther. https://doi.org/10.3851/imp3329 Pires Mello, C., Quirico-Santos, T., Amorim, L.F., Silva, V.G., Fragel, L.M., Bloom, D.C., Palmer Paixão, I., 2019. Perillyl alcohol and perillic acid exert efficient action upon HSV-1 maturation and release of infective virus. Antivir. Ther. https://doi.org/10.3851/imp3315 Pires, D.E. V., Blundell, T.L., Ascher, D.B., 2015. pkCSM: Predicting Small-Molecule Pharmacokinetic and Toxicity Properties Using Graph-Based Signatures. J. Med. Chem. 58, 4066–4072. https://doi.org/10.1021/acs.jmedchem.5b00104 Rashad, A.A., Keller, P.A., 2013. Structure based design towards the identification of novel binding sites and inhibitors for the chikungunya virus envelope proteins. J. Mol. Graph. Model. 44, 241–252. https://doi.org/10.1016/j.jmgm.2013.07.001 Riegel, B., Lappin, G.R., Adelson, B.H., Jackson, R.I., Albisetti, C.J., Dodson, R.M., Baker, R.H., 1946. The Synthesis of Some 4-Quinolinols and 4-Chloroquinolines by the Ethoxymethylenemalonic Ester Method. J. Am. Chem. Soc. 68, 1264–1266. https://doi.org/10.1021/ja01211a038 Rossmann-Ringdahl, I., Olsson, R., 2007. Porphyria Cutanea Tarda: Effects and Risk Factors for Hepatotoxicity from High-dose Chloroquine Treatment. Acta Derm. Venereol. 87, 401–405. https://doi.org/10.2340/00015555-0260 Sahoo, B., Chowdary, T.K., 2019. Conformational changes in Chikungunya virus E2 protein upon heparan sulfate receptor binding explain mechanism of E2–E1 dissociation during viral entry. Biosci. Rep. 39. https://doi.org/10.1042/BSR20191077 Šali, A., Blundell, T.L., 1993. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815. https://doi.org/10.1006/jmbi.1993.1626 Santos, F.D.C., Abreu, P., Castro, H.C., Paixão, I.C.P.P.P.P., Cirne-Santos, C.C., Giongo, V., Barbosa, J.E., Simonetti, B.R., Garrido, V., Bou-Habib, D.C., Silva, D.D.O., Batalha, P.N., Temerozo, J.R., Souza, T.M., Nogueira, C.M., Cunha, A.C., Rodrigues, C.R., Ferreira, V.F., de Souza, M.C.B.V.B.. V., 2009. Synthesis, antiviral activity and molecular modeling of oxoquinoline derivatives. Bioorg. Med. Chem. 17, 5476–5481. https://doi.org/10.1016/j.bmc.2009.06.037 Schultze, L.M., Chapman, H.H., Dubree, N.J.P., Jones, R.J., Kent, K.M., Lee, T.T., Louie, M.S., Postich, M.J., Prisbe, E.J., Rohloff, J.C., Yu, R.H., 1998. Practical synthesis of the anti-HIV drug, PMPA. Tetrahedron Lett. 39, 1853–1856. https://doi.org/10.1016/S0040- 4039(98)00131-2 Snyder, H.R., Freier, H.E., Kovacic, P., Van Heyningen, E.M., 1947. Synthesis of 4- Hydroxyquinolines. VIII. Some Halogen Containing 4-Aminoquinoline Derivatives. J. Am. Chem. Soc. 69, 371–374. https://doi.org/10.1021/ja01194a061 Souza, T.M.L., De Souza, M.C.B. V., Ferreira, V.F., Santos Canuto, C.V.B., Marques, I.P., Fontes, C.F.L., Frugulhetti, I.C.P.P., 2008. Inhibition of HSV-1 replication and HSV DNA polymerase by the chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(β-d- ribofuranosyl) quinoline-3-carboxylic acid and its aglycone. Antiviral Res. 77, 20–27. https://doi.org/10.1016/j.antiviral.2007.08.011 Stern, E., Muccioli, G.G., Millet, R., Goossens, J.F., Farce, A., Chavatte, P., Poupaert, J.H., Lambert, D.M., Depreux, P., Hénichart, J.P., 2006. Novel 4-oxo-1,4-dihydroquinoline-3- carboxamide derivatives as new CB 2 cannabinoid receptors agonists: Synthesis, pharmacological properties and molecular modeling. J. Med. Chem. 49, 70–79. https://doi.org/10.1021/jm050467q Thanacoody, R., 2016. Quinine and chloroquine. Medicine (Baltimore). 44, 197–198. https://doi.org/10.1016/J.MPMED.2015.12.003 Thevarayan, S., Tolou, H., Doornum, G. van, Genderen, P.J. van, Hassing, R.J., Leparc-Goffart, I., Blank, S.N., Thevarayan, S., Tolou, H., van Doornum, G., van Genderen, P.J., 2010. Imported Mayaro virus infection in the Netherlands. J. Infect. 61, 343–345. Traebert, M., Dumotier, B., Meister, L., Hoffmann, P., Dominguez-Estevez, M., Suter, W., 2004. Inhibition of hERG K+ currents by antimalarial drugs in stably transfected HEK293 cells. Eur. J. Pharmacol. 484, 41–8. Veber, D.F., Johnson, S.R., Cheng, H.-Y., Smith, B.R., Ward, K.W., Kopple, K.D., 2002. Molecular Properties That Influence the Oral Bioavailability of Drug Candidates. J. Med. Chem. 45, 2615–2623. https://doi.org/10.1021/jm020017n Wang, Y., Xing, J., Xu, Y., Zhou, N., Peng, J., Xiong, Z., Liu, X., Luo, X., Luo, C., Chen, K., Zheng, M., Jiang, H., 2015. In silico ADME/T modelling for rational drug design. Q. Rev. Biophys. 48, 488–515. https://doi.org/10.1017/S0033583515000190 Wiederstein, M., Sippl, M.J., 2007. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res. 35, W407–W410. https://doi.org/10.1093/nar/gkm290 Wielgo-Polanin, R., Lagarce, L., Gautron, E., Diquet, B., Lainé-Cessac, P., 2005. Hepatotoxicity associated with the use of a fixed combination of chloroquine and proguanil. Int. J. Antimicrob. Agents 26, 176–178. https://doi.org/10.1016/J.IJANTIMICAG.2005.04.019 Wiggins, K., Eastmond, B., Alto, B.W., 2018. Transmission potential of Mayaro virus in Florida Aedes aegypti and Aedes albopictus mosquitoes. Med. Vet. Entomol. 32, 436–442. https://doi.org/10.1111/mve.12322 Wintachai, P., Kaur, P., Lee, R.C.H., Ramphan, S., Kuadkitkan, A., Wikan, N., Ubol, S., Roytrakul, S., Chu, J.J.H., Smith, D.R., 2015. Activity of andrographolide against chikungunya virus infection. Sci. Rep. 5, 1–14. https://doi.org/10.1038/srep14179 Yang, H., Lou, C., Sun, L., Li, J., Cai, Y., Wang, Z., Li, W., Liu, G., Tang, Y., 2019. admetSAR 2.0: web-service for prediction and optimization of chemical ADMET properties. Bioinformatics 35, 1067–1069. https://doi.org/10.1093/bioinformatics/bty707

Supplementary material

Figure S1. Validation results obtained for the MAYV envelope protein complex model and its template, the CHIKV envelope protein complex (PDB code 3N42). (A) Ramachandran plots of the MAYV (left) and CHIKV (right) complexes; (B) Z-score plot of the MAYV (left) and CHIKV (right) complexes; (C) 3D-1D score for each residue of the MAYV (gray) and CHIKV (black) complexes.

5.1.5. HIV EPIDEMIC - THE IMPORTANCE OF SEXUAL EDUCATION AND AIDING PROGRAMS

116

ISSN: 2687-8100 DOI: 10.33552/ABEB.2020.03.000571 Archives in Biomedical Engineering & Biotechnology

Correa-Amorim LS1,2*, Moncao-Meireles, AB1, Cine-Santos CC1, Barros, CS1, Oliveira MC 1,2 and Paixeo ICNP1,2 1Marine Biotechnology and Molecular Virology Laboratory, Department of Cellular and Molecular Biology, Biology Institute, Brazil 2Department of Biology Institute, Brazil

*Corresponding author: Correa-Amorim LS, Department of Cellular and Received Date: December 31, 2019 Molecular Biology, Biology Institute, Fluminense Federal University - Niterói - Rio de Janeiro, Brazil. Published Date: January 30, 2020

Abstract

Despite all global efforts against the HIV/AIDS virus, it is alarming the high number of new infections episodes and unaware people regarding their serology, putting at risk one of the main goals of the WHO 90-90-90 program on the epidemic’s control and eradication. In spite of the elevated number of countries that present systems of supporting and drawing seropositive individuals, one of the most significant concerns nowadays is, still, the transmission of HIV. PrEP, Pre-Exposure Prophylaxis is helping, considerably, in decreasing the cases of new infections. However, it also warns about the resurgence and growing of other Sexually Transmitted Infections (STI’s).Which only makes us recognize sexual education as the central pillar, on raising population awareness about safe sexual practices, as well as predictors and risk factors, and acknowledge that the lack of a solid educationalKeywords: basis facilitates the practice of unsafe sex and increases the chances of exposing the person to HIV throughout life.

HIV; Sexual education; Sexual risk factors; PrEP

Introduction Retrovirus, nowadays recognized as HIV or Human decade. Due to the incomes generated by GPA and the significant number of donating countries, many underdeveloped nations Immunodeficiency Virus, was isolated only in 1983.Even ifthe were able to establish national controlling programs of HIV-AIDS Acquired Immune Deficiency Syndrome (AIDS) was originally considering many countries were successful reverting epidemics or infection. Their programs proved to be very efficient at that time, acknowledged in 1981 when the number of opportunistic infections increased among young homosexuals. Since its first identification, simply avoiding the worsening of the disease. However, the efforts HIV has been thoroughly studied. But it was only in 1985, that it were not enough, culminating in the closing of GPA’s initiative and was possible to develop licensed serology tests capable of detecting its substitution for the United Nations program UNAIDS [4,5]. the virus infection. It is estimated that in over thirty years of HIV pandemic, the virus has been responsible for the infection of In 1996 the Highly Active Antiretroviral Therapy (HAART) was approximately 60 million people, and more than 25 million deaths, introduced in the treatment of HIV. This therapy consists of the around the world [1-3]. blending of three antiretroviral that focuses on at least two different molecular targets. This therapy suppresses viral replication Although the World Health Organization (WHO) had drastically and also reduces its quantity, to levels lower than the underestimated the pandemic in the past, since 1987, the detected limits established in the most sensible laboratory essays. organization has released a special program considering HIV Resulting in significant reconstitution of the immunologic system named Global AIDS Plan (GAP). Hence, it has been observed a [6]. In 2003 WHO conceded the compulsory licensing of drugs in considerable advance in the treatment and control of the disease. national emergencies, which enabled the importing of generic The Nucleoside Reverse Transcriptase Inhibitors (NRTIs), as AZT medicines by underdeveloped countries, and in some cases, as in and DDI were the first drugs available to treat the HIV infection. Brazil, their production [5,7]. The efforts to contain the HIV pandemic continued after the 1990’s

This work is licensed under Creative Commons Attribution 4.0 License ABEB.MS.ID.000571. Page 1 of 5 Archives in Biomedical Engineering & Biotechnology Volume 3-Issue 5

Since the isolation and presentation of HIV, many studies Regions demonstrated a considerable reduction in the number focused in its morphology. After it became clear that AIDS was of cases, while in other regions the increase was not expressive, caused by two distinct types of HIV, later nominated HIV-1 and except for the northern region where there was an increase from HIV-2. Numerous HIV-1 strains are globally disseminated making 7.0 to 20.6 cases [23]. clear the heterogeneity exhibited among them. Those two viral Since 1980 when AIDS pandemic started until December 2017, types present similarities in their morphology and tropism due it has been reported by the responsible organs in Brazil, more than to their lymphocytes T CD4+, nevertheless, they are genetically 327 thousand deaths having as primary cause the HIV/AIDS. The Epidemiologydistinguished [2]. higher percentage of deaths occurred in the Southeast Region, 58.9%, followed by South and Northeast Regions both with 17.7% The HIV epidemic is responsible for approximately 1.5 million and 13.3% respectively, leaving the last positions to Central West deaths around the world in 2017 [8], HIV-1 is considered the Predictorsand North Regions of Sexual with 5.2% Risk and 4.9% respectively [23]. main causative agent of AIDS. UNAIDS published data of 2018, demonstrates that in the year 2017, 36 million people [30,8 – Sexual risk predictors and converging strategies are important 42,9 million] were infected with HIV. Two types of HIV have been to recognize behavioral targets that may be susceptible to the identified, type 1 and type 2, (HIV-1 and HIV-2) classified in groups risk of HIV infection. Those risk predictors are important for and subtypes according to their distinct geographic distribution prevention programs, besides from helping on the recognition of and origins [9]. HIV-1 isolated in 1983 [10], is widely distributed infected individuals that may be at a moderate level of infection. worldwide. While HIV-2 isolated in 1986 [11], can be found in HIV is mainly transmitted through sexual intercourse worldwide. Western Africa and some regions of Europe [12]. The transmission rate of new HIV cases among youngsters in the The strains of HIV-1 are organized in four different groups United States unrelated to sexual intercourse is lower than 5% [24]. based on the viral genome, named: M (main), O (outlier), N (nom M Therefore, for an effective prevention against HIV, it is necessary to or O) and P (putative) [13,14]. The “M” group exhibits a significant have clear communication on the implications of the disease and the number on a global scale responsible for 95% of infections by HIV- risk behaviors that should be avoided [25]. The most common via 1 [15]. The predominance of the other groups is extremely inferior, of HIV transmission is through sexual relations, representing 99% limited to Western Central Africa [16,17]. of new occurrences in some Latin America countries. Behavioral monitoring can help to better evaluate the HIV prevalence as well Divided into 9 subtypes, A, B, C, D, F, G, H, J and K, the M group as other Sexually Transmitted Infections (STI’s), contributing to the has been evenly disseminated around the world. One example of development of effective prevention initiatives [26]. that extensive dispersion is the subgroup B, the most prevalent form present in the American Continent and Europe yet considered At the beginning of the HIV epidemic, individuals that rare in Africa [18]. Nowadays, the most common subtype of HIV- demonstrated higher levels of schooling, higher available income, 1 in the world is subtype C portraying the most prominent rate better possibilities of locomotion, and more favorable chances of of infections (50%), followed by subtype A (12%) and subtype B establishing a wide sexual network, including sex professionals, were (10%) [19,20]. more susceptive to contract the disease. Deeper conscientization, safer sexual practices and access to precautionary health services Today it is estimated that 40 million people in the world are produced an alteration in the scene of the correlation between the living with HIV [21], experiencing social, personal and interpersonal formal years of education and HIV [27]. Studies have demonstrated traumas [22]. The annual global index of deaths regarding people that youngsters who abandon formal education showcase a larger who contracted HIV, according to UNAIDS, demonstrated a decline chance of having a higher number of sexual partners throughout of 1.9 million deaths [1,4 – 2,7 million] in 2004 to a peak of 940 their lives. Hence, increasing the frequency of sexual relations thousand deaths [670 thousand – 1,3 million] in 2017. Since 2010 and also the incidence of unsafe sex when compared to those who the mortality related to AIDS cases presented a decay of 34% continued in school [28]. [8]. The number of cases of HIV infection in the world continued to decline until 2017. Estimates indicate that there has been a Those statistics only confirm the fact that education is significant decline in the number of new infections, with a decrease extremely important. It shapes sexual behavior with the aid of from 3.4 million [2,6 – 4,4 million] of new cases in 1996 to 1.8 several mechanisms, and helps transform the dominant values of million [1,4 – 2,4 million] in 2017 [8]. human behavior, such as knowledge, mindset, and social media action, consequently generating improvement in the socioeconomic In Brazil since the manifestation of HIV in 1980 until 2011, level [29]. Information about sexual risk predictors should be it was recorded 608.203 registered occurrences of the disease used to provide the population with a better understanding manifestation. In 2010 alone were notified 34.218 new cases through transparent, specific and scientifically updated messages. elevating the illness rate of incidence to 17.9 casesper 100.000 Enlightening people about the risks of transmission and infection habitants. According to statistics in the last ten years there were by HIV and also its prevention strategies [25]. changes on the sickness rates, where the South and the Southeast Citation: Page 2 of 5 . Correa-Amorim LS, Moncao-Meireles, AB, Cine-Santos CC, Barros, CS, Oliveira MC, Paixeo ICNP. HIV Epidemic - The Importance of Sexual Education and Aiding Programs. Arch Biomed Eng & Biotechnol. 3(5): 2020. ABEB.MS.ID.000571. DOI:10.33552/ABEB.2020.03.000571 Archives in Biomedical Engineering & Biotechnology Volume 3-Issue 5

HIV Intervention/Prevention Programmes shielding effect produced after correcting daily administration, was higher than 90% [36,37]. UNAIDS’s goal is to reduce the number Public health strategies to prevent HIV infection include of new infections to less than 500.000 cases per year until 2020, educational campaigns to promote safe sexual practices, to combining access to different prevention methods, including PrEP expand and facilitate HIV tests and serotyping, to ease access to [38]. men circumcision and promote the antiretroviral prescription to seropositive individuals to diminish the circulating viral load, thus The motivation to use PrEP antiretroviral as an agent of minimizing the risks of transmission [30]. At the beginning of the preventing transmission of HIV infection was initially based on 2000s, the World Health Organization (WHO) created an initiative researches using test animals and case studies with humans that so people from poor countries living with HIV, could have total use post-exposure prophylaxis [30]. access to the antiretroviral treatment until the end of 2005 [31]. Depending on the country, PrEP can be prescribed like other The efforts to expand the treatment were based on scientific prophylaxis drugs (one pill a day) or as “on-demand” prophylaxis evidence which confirmed that the early treatment could prevent medicine, before and after the person presents unsafe sexual the disease and, consequently, the deaths it caused. It also behavior (two pills before the sexual activity, and one pill 24 and 48 presented a decrease on virus transmission and helped to reduce hours after the first medicine) [38]. health expenses [31]. The United States was the first country to authorize the use of The increasing global necessity and its compromise in solving PrEP as a continuous prophylaxis drug in 2012. Nevertheless, only in the AIDS epidemic brought the UN Programme on HIV/AIDS 2016, France permitted the usage of PrEP as continuous prophylaxis (UNAIDS) to establish in 2014, the 90-90-90 goals (32). Those medicine “on-demand”. France was also the first country to offer purposes provide a vital infrastructure to guide the response a full refund to users of PrEP, which was later adopted by other to HIV and to continue monitoring the progress of the epidemic European countries [38]. The World Health Organization (WHO) termination [33]. advises the prescription to PrEP to all individuals at high risk of HIV infection, for example, any person who belongs to a population The UNAIDS 90-90-90 program consists of reaching 90% of group with an HIV incidence rate superior to 3/100 people a year people living with HIV (PVHIV) to acknowledge their infection, [39]. Those recommendations include men that make oral sex with 90% of them taking antiretroviral therapy (TARV), and 90% on other men (HSH), transsexual women, heterosexual men and of those getting their viral load suppressed [33]. UNAIDS predicts women that maintain contact with unknown serology partners or that, if we reach those goals until 2020, we will be able to end the untreated HIV positive people [38]. AIDS epidemic until 2030 [32]. The identification of suitable candidates to the requirements of The 90-90-90 program focus on enhancing and maintaining PrEP needs a complete and periodic review of the patients’ drug constant care from the diagnosis to the viral suppression on the use and sexual practices standards, so the higher-risk patients HIV infected individuals. The proportion of the specification among may be identified and beneficiated by PrEP [30]. One of the factors diagnosis, treatment, and viral suppression is the central pillar relevance of developing an adequate treatment to everyone in that discourage PrEP prescription is mostly the concern about an in response to HIV. The program’s goal explicitly emphasizes the increase of unsafe sexual behavior; incentive to unprotected sex practice that consequentially, increases the ISTs incidence due to need and implicitly attests the requirement for monitoring systems supposed protection provided by preventive treatment [38]. to measure its progress [31]. The collected information about continued care is restricted now and describing the local, national According to some studies, PrEP volunteers exhibited and global continuous care has presented difficulties. Considering seropositivity on the initial screening to syphilis, HHV-2, gonorrhea that there are no standardized approaches, or they neglected to (oral, rectal and urethral) and chlamydia. The high-level presence detail how estimations had been determined [31]. of ISTs before and after PrEP treatment had started, illustrates the importance of a screening routine for ISTs amid those patients [30]. Despite all efforts to reach that goal, we are still affected by PrEP completely changed the situation of HIV infection prevention, 1.8 million HIV new infections every year, which demonstrates especially to HSH. It is clear that combined with ISTs screening and that prevention strategies are imperative. Clinical trials have their immediate treatment, PrEP will considerably contribute to a been demonstrating that the use of medicines, like Pre-Exposure large-scale incidents reduction, in countries where the treatment is Prophylaxis (PrEP), can help decrease the risk of HIV transmission available [38]. Strategies on HIV prevention are applied worldwide when used with high adherence [34]. and widely divulged. Among those strategies, prevention through PrEP is composed of daily therapeutics, employed through oral education continues to be a key factor in intervention policies, on co-formulation of Tenofovir Disoproxil Fumarate combined with Conclusionnational and global levels [39,40]. Emtricitabine (TDF/FTC). This combination has demonstrated to be safe and effective in the prevention of HIV infection in many population groups [35]. Validating those efficiency estimates, the HIV infection still produces a substantial impact on public health due to its alarming epidemiological numbers. Despite all Citation: Page 3 of 5 . Correa-Amorim LS, Moncao-Meireles, AB, Cine-Santos CC, Barros, CS, Oliveira MC, Paixeo ICNP. HIV Epidemic - The Importance of Sexual Education and Aiding Programs. Arch Biomed Eng & Biotechnol. 3(5): 2020. ABEB.MS.ID.000571. DOI:10.33552/ABEB.2020.03.000571 Archives in Biomedical Engineering & Biotechnology Volume 3-Issue 5

WHO efforts, in association with the treatment centers scattered 12. Kanki PJ, Travers KU, S MB, Hsieh CC, Marlink RG, Gueye NA, et al. (1994) Slower heterosexual spread of HIV-2 than HIV-1. Lancet (London, around the world, the infection by HIV still reachs thousands of England) 343(8903): 943-946. people, which makes even harder to achieve the goals established 13. Plantier JC, Leoz M, Dickerson JE, De Oliveira F, Cordonnier F, et al. by WHO until 2020.When 90% of the infected population will have (2009) A new human immunodeficiency virus derived from gorillas. consciousness of their serology and 90% of that group, will be under Nature medicine 15(8): 871-872. antiretroviral treatment, and 90% of those treated individuals, will 14. Simon F, Mauclere P, Roques P, Loussert-Ajaka I, Muller-Trutwin MC, et al. (1998) Identification of a new human immunodeficiency virus type 1 be viral-suppressed. 15. distinct from group M and group O. Nature medicine 4(9): 1032-1037. One of the strategies to be used is the PrEP, which has been Sharp PM, Hahn BH (2008) AIDS: prehistory of HIV-1. Nature 455(7213): proving to be a highly effective tactic against HIV transmission. Many 605-606. countries, including Brazil, employ that tool as a neutralization 16. Peeters M, Gueye A, Mboup S, Bibollet-Ruche F, Ekaza E, et al. (1997) Geographical distribution of HIV-1 group O viruses in Africa. AIDS mechanism of new infections, although, it is disturbing the 17. (London, England) 11(4): 493-798. emergence of strains resistant to the medicines, once PrEP is often Vallari A, Holzmayer V, Harris B, Yamaguchi J, Ngansop C, et al. (2011) constituted of drugs that compose the cocktail. Confirmation of putative HIV-1 group P in Cameroon. Journal of virology 85(3): 1403-1407. With the usage of PrEP, many individuals feel protected, which 18. Gilbert MT, Rambaut A, Wlasiuk G, Spira TJ, Pitchenik AE, et al. (2007) makes them vulnerable to ISTs. Therefore, the use of PrEP only shall The emergence of HIV/AIDS in the Americas and beyond. Proceedings not be enough to reduce the number of new infections worldwide. of the National Academy of Sciences of the United States of America Coupled to this tool, sexual education, as well as formal schooling, 104(47): 18566-18570. are vital pillars to control the HIV infection. Researches indicate 19. Hemelaar J, Gouws E, Ghys PD, Osmanov S (2011) Global trends in molecular epidemiology of HIV-1 during 2000-2007. AIDS (London, that the lack of knowledge regarding the virus, just as the failings England) 25(5): 679-689. on people’s education, seems to be key factors on the fight against 20. Taylor BS, Sobieszczyk ME, McCutchan FE, Hammer SM (2008) The Acknowledgmentone of the greatest epidemics of all time. challenge of HIV-1 subtype diversity. The New England journal of medicine 358(15): 1590-1602. 21. Jaspal R, Kennedy L, Tariq S (2018) Human Immunodeficiency Virus and The authors thank the Brazilian funding agencies CNPq, CAPES, Trans Women: A Literature Review. Transgender health 3(1): 239-250. Conflictsand FAPERJ. of Interest 22. Dulin AJ, Dale SK, Earnshaw VA, Fava JL, Mugavero MJ, et al. (2018) Resilience and HIV: a review of the definition and study of resilience. AIDS Care 2019: 1-12. ó ReferencesNo conflicts of interest. 23. Boletim E (2018) Boletim epidemiol gico HIV/Aids 2018. 49(HIV / 1. AIDS): 72. 24. Mustanski BS, Newcomb ME, Du Bois SN, Garcia SC, Grov C (2011) HIV Del Rio C (2017) The global HIV epidemic: What the pathologist needs to in young men who have sex with men: a review of epidemiology, risk know. Seminars in diagnostic pathology 34(4): 314-317. and protective factors, and interventions. Journal of sex research 48(2- 2. Sauter D, Kirchhoff F (2019) Key Viral Adaptations Preceding the AIDS 3): 218-53. Pandemic. Cell host & microbe 25(1): 27-38. 25. Hess KL, Hu X, Lansky A, Mermin J, Hall HI (2017) Lifetime risk of a 3. Sharp PM, Hahn BH (2011) Origins of HIV and the AIDS pandemic. Cold diagnosis of HIV infection in the United States. Annals ofepidemiology Spring Harbor perspectives in medicine 1(1): a006841. 27(4): 238-243. 4. Merson MH, O Malley J, Serwadda D, Apisuk C (2008) The history and 26. Stuardo Avila V, Fuentes Alburquenque M, Munoz R, Bustamante Lobos challenge of HIV prevention. Lancet (London, England). 372(9637): L, Faba A, et al. (2019) Prevalence and Risk Factors for HIV Infection in a 5. 475-88. Population of Homosexual, Bisexual, and Other Men Who Have Sex with Men in the Metropolitan Region of Chile: A Re-emerging Health Problem. Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, et al. (2006) AIDS and behavior. Disease Control Priorities in Developing Countries. Second Edition (eds) Oxford University Press and The World Bank, p. 1401. 27. Bunyasi EW, Coetzee DJ (2017) Relationship between socioeconomic status and HIV infection: findings from a survey in the Free State and 6. Arts EJ, Hazuda DJ (2012) HIV-1 antiretroviral drug therapy. Cold Spring Western Cape Provinces of South Africa. BMJ open 7(11): e016232. 7. Harbor perspectives in medicine. 2(4): a007161. 28. Hargreaves JR, Morison LA, Kim JC, Bonell CP, Porter JD, et al. (2008) Brito AM, Castilho EA, Szwarcwald CL (2001) AIDS and HIV infection The association between school attendance, HIV infection and sexual in Brazil: a multifaceted epidemic. Revista da Sociedade Brasileira de behaviour among young people in rural South Africa. Journal of Medicina Tropical 34(2): 207-217. epidemiology and community health. 62(2): 113-119. 8. UNAIDS. Global, AIDS update 2018 / UNAIDS. Report 2018. 29. Jukes M, Simmons S, Bundy D (2008) Education and vulnerability: 9. Essex M (1999) Human immunodeficiency viruses in the developing the role of schools in protecting young women and girls from HIV in 10. world. Advances in virus research 53: 71-88. southern Africa. AIDS (London, England). 22(Suppl 4): S41-S56. Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, et al. 30. Riddell JT, Amico KR, Mayer KH (2018) HIV Preexposure Prophylaxis: A (1983) Isolation of a T-lymphotropic retrovirus from a patient at risk for Review. Jama 319(12):1261-1268. acquired immune deficiency syndrome (AIDS). Science (New York, NY) 31. Granich R, Gupta S, Hall I, Aberle-Grasse J, Hader S, et al. (2017) Status 11. 220(4599): 868-871. and methodology of publicly available national HIV care continua and Clavel F, Guyader M, Guetard D, Salle M, Montagnier L, et al. (1986) 90-90-90 targets: A systematic review. PLoS medicine 14(4): e1002253. Molecular cloning and polymorphism of the human immune deficiency 32. Staveteig S, Croft TN, Kampa KT, Head SK (2017) Reaching the ‘first virus type 2. Nature 324(6098): 691-695. 90’: Gaps in coverage of HIV testing among people living with HIV in 16 African countries. PloS one 12(10): e0186316. Citation: Page 4 of 5 . Correa-Amorim LS, Moncao-Meireles, AB, Cine-Santos CC, Barros, CS, Oliveira MC, Paixeo ICNP. HIV Epidemic - The Importance of Sexual Education and Aiding Programs. Arch Biomed Eng & Biotechnol. 3(5): 2020. ABEB.MS.ID.000571. DOI:10.33552/ABEB.2020.03.000571 Archives in Biomedical Engineering & Biotechnology Volume 3-Issue 5

33. Hakim AJ, MacDonald V, Hladik W, Zhao J, Burnett J, et al. (2018) Gaps and 38. Siguier M, Molina JM (2018) HIV preexposure prophylaxis: An essential, opportunities: measuring the key population cascade through surveys safe and effective prevention tool for sexual health. Medecineet maladies and services to guide the HIV response. Journal of the International AIDS infectieuses 48(5): 318-326. Society. 21(Suppl 5): e25119. 39. WHO Guidelines Approved by the Guidelines Review Committee. 34. Gibas KM, Van den Berg P, Powell VE, Krakower DS (2019) Drug Guidelines for The Diagnosis, Prevention and Management of Resistance During HIV Pre-Exposure Prophylaxis. Drugs 79(6): 609-619. Cryptococcal Disease in HIV-Infected Adults, Adolescents and Children: Supplement to the 2016 Consolidated Guidelines on the Use of 35. Fonner VA, Dalglish SL, Kennedy CE, Baggaley R, O Reilly KR, et al. (2016) Antiretroviral Drugs for Treating and Preventing HIV Infection. Geneva: Effectiveness and safety of oral HIV preexposure prophylaxis for all World Health Organization populations. AIDS (London, England). 30(12): 1973-1983. 40. (2018) World Health Organization 2018. 36. Anderson PL, Glidden DV, Liu A, Buchbinder S, Lama JR, et al. (2012) Emtricitabine-tenofovir concentrations and pre-exposure prophylaxis 41. Adelekan M (2017) A critical review of the effectiveness of educational efficacy in men who have sex with men. Science translational medicine interventions applied in HIV/AIDS prevention. Patient education and 4(151): 151ra25. counseling 100(Suppl 1): S11-S16. 37. Donnell D, Baeten JM, Bumpus NN, Brantley J, Bangsberg DR, et al. (2014) HIV protective efficacy and correlates of tenofovir blood concentrations in a clinical trial of PrEP for HIV prevention. Journal of acquired immune deficiency syndromes (1999). 66(3): 340-348.

Citation: Page 5 of 5 . Correa-Amorim LS, Moncao-Meireles, AB, Cine-Santos CC, Barros, CS, Oliveira MC, Paixeo ICNP. HIV Epidemic - The Importance of Sexual Education and Aiding Programs. Arch Biomed Eng & Biotechnol. 3(5): 2020. ABEB.MS.ID.000571. DOI:10.33552/ABEB.2020.03.000571 5.2. PATENTES

122

5.2.1. USO DE DERIVADOS QUILÔNICOS NA PREVENÇÃO DO HIV-1 E SUA AÇÃO VIRUCIDA IN VITRO.

123

870180168756 28/12/2018 13:59

29409161812933605 Pedido nacional de Invenção, Modelo de Utilidade, Certificado de Adição de Invenção e entrada na fase nacional do PCT

Número do Processo: BR 10 2018 077412 3

Dados do Depositante (71)

Depositante 1 de 1

Nome ou Razão Social: UNIVERSIDADE FEDERAL FLUMINENSE Tipo de Pessoa: Pessoa Jurídica CPF/CNPJ: 28523215000106 Nacionalidade: Brasileira

Qualificação Jurídica: Instituição de Ensino e Pesquisa Endereço: Rua Miguel de Frias, 9/3o andar - Icaraí Cidade: Niterói Estado: RJ CEP: 24220-900 País: Brasil Telefone: (21) 26295946 Fax: Email: [email protected]

Dados do Pedido

Natureza Patente: 10 - Patente de Invenção (PI) Título da Invenção ou Modelo de USO DE DERIVADOS QUINOLÔNICOS NA PREVENÇÃO DO HIV- Utilidade (54): 1 E SUA AÇÃO VIRUCIDA IN VITRO Resumo: A presente invenção descreve o uso de derivados quinolônicos para o preparo de medicamentos que atuam na prevenção e tratamento da infecção pelo HIV-1 e também descreve um método virucida in vitro. A presente invenção situa-se nos campos da Medicina e da Farmácia.

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 1/67 Dados do Inventor (72) Inventor 1 de 16

Nome: IZABEL CHRISTINA NUNES DE PALMER PAIXÃO CPF: 46322264753 Nacionalidade: Brasileira Qualificação Física: Professor do ensino superior Endereço: Av. Prefeito Dulcídio Cardoso nº 2500, bloco 4 apto 1004, Barra da Tijuca Cidade: Rio de Janeiro Estado: RJ CEP: 22631-051 País: BRASIL Telefone: (21) 999 737508 Fax: Email: [email protected]

Inventor 2 de 16

Nome: MARIA CECÍLIA BASTOS VIEIRA DE SOUZA CPF: 44470134791 Nacionalidade: Brasileira Qualificação Física: Professor do ensino superior Endereço: Av. Ary Parreiras 691 apto 1501, bl 2, Icaraí Cidade: Niterói Estado: RJ CEP: 24230-321 País: BRASIL Telefone: (21) 998 037155 Fax: Email: [email protected]

Inventor 3 de 16

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 2/67 Nome: ANNA CLAUDIA CUNHA CPF: 91618983768 Nacionalidade: Brasileira Qualificação Física: Professor do ensino superior Endereço: Wilson Nogueira, 332 – Bairro Nova Cidade, Nilópolis Cidade: Rio de Janeiro Estado: RJ CEP: 26535-150 País: BRASIL Telefone: (21) 984 107428 Fax: Email: [email protected]

Inventor 4 de 16

Nome: FERNANDA DA COSTA SANTOS BOECHAT CPF: 09181266731 Nacionalidade: Brasileira Qualificação Física: Professor do ensino superior Endereço: Rua dos Girassóis, Qd5 Lt11 Inoã Cidade: Maricá Estado: RJ CEP: 24942-360 País: BRASIL Telefone: (21) 987 312871 Fax: Email: [email protected]

Inventor 5 de 16

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 3/67 Nome: PAULO ROBERTO SOARES STEPHENS CPF: 93196644715 Nacionalidade: Brasileira Qualificação Física: Doutorando Endereço: Jornalista Alberto Francisco Torres, 463/Bl. A/Apto. 202 – Icaraí Cidade: Niterói Estado: RJ CEP: 24230-008 País: BRASIL Telefone: (21) 982 533299 Fax: Email: [email protected]

Inventor 6 de 16

Nome: CLAUDIO CESAR CIRNE SANTOS CPF: 03202612719 Nacionalidade: Brasileira Qualificação Física: Estudante de Pós Graduação Endereço: Rua Toni Moraes nº 70 casa 94, Arsenal Cidade: São Gonçalo Estado: RJ CEP: 24755-425 País: BRASIL Telefone: (21) 995 715707 Fax: Email: [email protected]

Inventor 7 de 16

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 4/67 Nome: CAROLINE DE SOUZA BARROS CPF: 09909535726 Nacionalidade: Brasileira Qualificação Física: Estudante de Pós Graduação Endereço: Rua Des. João Claudino de Oliveira e Cruz 50 Ap 2007, Barra da Tijuca Cidade: Rio de Janeiro Estado: RJ CEP: 22793-920 País: BRASIL Telefone: (21) 986 182775 Fax: Email: [email protected]

Inventor 8 de 16

Nome: JURANDY SUSANA PATRICIA MORALES OCAMPO CPF: 54856655787 Nacionalidade: Brasileira Qualificação Física: Professor do ensino superior Endereço: Rua José Higino 388 ap 402, Tijuca Cidade: Rio de Janeiro Estado: RJ CEP: 20510-412 País: BRASIL Telefone: (21) 988 980393 Fax: Email: [email protected]

Inventor 9 de 16

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 5/67 Nome: LEONARDO DOS SANTOS CORRÊA AMORIM CPF: 11344133789 Nacionalidade: Brasileira Qualificação Física: Doutorando Endereço: Rua Professor Miguel Couto - 344 - Bloco B - Apartamento 102 - Icaraí Cidade: Niterói Estado: RJ CEP: 24230-240 País: BRASIL Telefone: (21) 992 404533 Fax: Email: [email protected]

Inventor 10 de 16

Nome: PEDRO NETTO BATALHA CPF: 11647606705 Nacionalidade: Brasileira Qualificação Física: Estudante de Pós Graduação Endereço: Rua Nossa Senhora das Mercês, 113, Apartamento 303, Fonseca Cidade: Niterói Estado: RJ CEP: 24130-050 País: BRASIL Telefone: (21) 975 120115 Fax: Email: [email protected]

Inventor 11 de 16

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 6/67 Nome: NATHALIA MOTTA DE CARVALHO TOLENTINO CPF: 12952941769 Nacionalidade: Brasileira Qualificação Física: Doutorando Endereço: Rua Geminiano Góis, 151, bl 2 apt 306, Freguesia - Jacarepaguá Cidade: Rio de Janeiro Estado: RJ CEP: 22743-670 País: BRASIL Telefone: (21) 980 587642 Fax: Email: [email protected]

Inventor 12 de 16

Nome: THIAGO MOTA DO VALE CPF: 13625765730 Nacionalidade: Brasileira Qualificação Física: Estudante de Graduação Endereço: Rua Laurentina Alves Pereira, 16A, Badu Cidade: Niterói Estado: RJ CEP: 24320-070 País: BRASIL Telefone: (21) 980 200935 Fax: Email: [email protected]

Inventor 13 de 16

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 7/67 Nome: FERNANDA SAVACINI SAGRILLO CPF: 11173250751 Nacionalidade: Brasileira Qualificação Física: Doutorando Endereço: Rua Geraldo Martins, nº 23, apto 803, Icaraí Cidade: Niterói Estado: RJ CEP: 24220-380 País: BRASIL Telefone: (21) 986 068197 Fax: Email: [email protected]

Inventor 14 de 16

Nome: LETÍCIA VILLAFRANCA FARO CPF: 09544441751 Nacionalidade: Brasileira Qualificação Física: Estudante de Pós Graduação Endereço: Rua Lopes da Cunha 145, Bloco 6 - Apto. 604 Cidade: Niterói Estado: RJ CEP: 24120-095 País: BRASIL Telefone: (21) 991 688168 Fax: Email: [email protected]

Inventor 15 de 16

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 8/67 Nome: LUANA DA SILVA MAGALHÃES FOREZI CPF: 07303352678 Nacionalidade: Brasileira Qualificação Física: Estudante de Pós Graduação Endereço: Rua Quinze de Novembro, 102 Centro Cidade: Niterói Estado: RJ CEP: 22631-051 País: BRASIL Telefone: (21) 992 372325 Fax: Email: [email protected]

Inventor 16 de 16

Nome: VANESSA DA GAMA OLIVEIRA CPF: 09977187738 Nacionalidade: Brasileira Qualificação Física: Doutorando Endereço: Rua Riodades, 145, blo3/ap. 804, Fonseca Cidade: Niterói Estado: RJ CEP: 24130-247 País: BRASIL Telefone: (21) 988 237904 Fax: Email: [email protected]

Documentos anexados

Tipo Anexo Nome

Comprovante de pagamento de GRU 200 GRU_Nosso Numero_29409161812933605.pdf

Relatório Descritivo RELATÓRIO DESCRITIVO.pdf

Reivindicação REIVINDICAÇÕES.pdf

Resumo RESUMO.pdf

Procuração Procuração Co-Titularidade UNIRIO.pdf

Comprovantes de Vínculo Declarações de Vínculo Inventores.pdf

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 9/67 Acesso ao Patrimônio Genético

Declaração Negativa de Acesso - Declaro que o objeto do presente pedido de patente de invenção não foi obtido em decorrência de acesso à amostra de componente do Patrimônio Genético Brasileiro, o acesso foi realizado antes de 30 de junho de 2000, ou não se aplica.

Declaração de veracidade

Declaro, sob as penas da lei, que todas as informações acima prestadas são completas e verdadeiras.

Esta solicitação foi enviada pelo sistema Peticionamento Eletrônico em 28/12/2018 às 13:59, Petição 870180168756

Petição 870180168756, de 28/12/2018, pág. 10/67 Petição 870180168756, de 28/12/2018, pág. 11/67