The Discovery of Antiviral Compounds Targeting Adenovirus and Herpes Simplex Virus

Mårten Strand The Discovery of Antiviral Compounds Targeting Adenovirus and Herpes Simplex Virus

The Discovery of Antiviral Compounds Targeting Adenovirus and Herpes Simplex Virus Assessment of Synthetic Compounds and Natural Products

Mårten Strand

Umeå University

Department of Clinical Microbiology, Virology ISBN: 978-91-7601-043-3 Department of Clinical Microbiology, Virology Umeå University, SE-901 87 Umeå, Sweden ISSN: 0346-6612 Umeå University 2014 Serie nr: 1647 Umeå University Medical Dissertations, New Series No 1647

The Discovery of Antiviral Compounds Targeting Adenovirus and Herpes Simplex Virus

Assessment of Synthetic Compounds and Natural Products

Mårten Strand

Akademisk avhandling som med vederbörligt tillstånd av Rektor vid Umeå universitet för avläggande av filosofie doktorsexamen framläggs till offentligt försvar i Betula, byggnad 6M, fredagen den 16:e Maj, kl. 09:00. Avhandlingen kommer att försvaras på engelska.

Fakultetsopponent: Professor, Lieve Naesens, Rega Institute, Leuven, Belgien.

Department of Clinical Microbiology, Virology Umeå University Umeå 2014

Organization Document type Date of publication Umeå University Doctoral thesis 25 April 2014 Clinical Microbiology, Virology

Author Mårten Strand

Title The Discovery of Antiviral Compounds Targeting Adenovirus and Herpes Simplex Virus Assessment of Synthetic Compounds and Natural Products

Abstract There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most often associated with respiratory disease, diarrhea and infections of the eye. These infections can be severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus infections are associated with morbidity and high mortality rates. These patients are mainly stem cell or bone marrow transplantation recipients, however solid organ transplantation recipients or AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated disease. Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One compound, designated Benzavir-1, was identified with activity against representative types of all adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR), the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2. To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV types. To expand our search for compounds with antiviral activity, we turned to the natural products. An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active compounds could be isolated. However, several compounds had previously been characterized with antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this compound was identified as a butenolide. Additional butenolide analogues were found and we proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was characterized and substantial differences in their toxic potential were observed. One of the most potent butenolide analogues had minimal toxicity and is an attractive starting point for further optimization of the anti-adenoviral activity. This thesis describes the discovery of novel antiviral compounds that targets adenovirus and HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead to the development of new antiviral drugs for clinical use.

Keywords Adenovirus, Herpes Simplex Virus, Antiviral, Natural Products, Screening, Drug discovery.

Language ISBN ISSN Number of pages English 978-91-7601-043-3 0346-6612-1647 104 + 4 papers

The Discovery of Antiviral Compounds Targeting Adenovirus and Herpes Simplex Virus Assessment of Synthetic Compounds and Natural Products

Mårten Strand

Department of Clinical Microbiology Umeå 2014

Responsible publisher under swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7459-368-6 ISSN: 0346-6612 Cover art: Front: An adenovirus virion surrounded by small molecules. Back: R/V Jan Mayen on the hunt for marine actinobacteria. Elektronisk version tillgänglig på http://umu.diva-portal.org/ Tryck/Printed by: Print&Media Umeå, Sverige 2014 Table of Contents

Table of Contents i! Abstract iii! Summary in Swedish – Populärvetenskaplig sammanfattning på svenska v! List of papers vii! Abbreviations viii! Introduction 1! 1. General introduction 1! 2. Adenovirus 2! 2.1. History 2! 2.2. Taxonomy and classification 3! 2.3. Virion structure 4! 2.4. Genome organization and gene products 6! 2.5. Viral life cycle 9! 2.6. Human adenovirus spread and infections 16! 2.7. Antiviral drugs used in the clinic 20! 3. Herpes Simplex Virus 23! 3.1. History 23! 3.2. Taxonomy and classification 23! 3.3. Virion structure 24! 3.4. Genome organization and gene products 25! 3.5. Viral life cycle 26! 3.6. Latency and reactivation 29! 3.7. HSV spread and infections 30! 3.8. Antiviral drugs used in the clinic 33! 4. Antiviral strategies and drug modes-of-action 35! 4.1. Binding inhibitors 36! 4.2. Inhibitors of DNA replication – Nucleoside/Nucleotide analogues 37! 4.3. Inhibitors of viral maturation - Protease inhibitors 40! 4.4. Antiviral prospects 40! 5. Drug discovery and development 42! 5.1. Targets and assays 42! 5.2. Compound Libraries 43! 5.3. Screening and lead identification 43! 5.4. Lead optimization 44! 5.5. Drug development 45! 5.6. Orphan drugs 45! 5.7. Natural products in drug discovery 47! Aims 49! Methods 50! Results and Discussion 53!

i Paper I 53! Paper II 55! Paper III 56! Paper IV 58! Concluding remarks and future prospects 59! References 63! Acknowledgements 104!

ii Abstract

There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most often associated with respiratory disease, diarrhea and infections of the eye. These infections can be severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus infections are associated with morbidity and high mortality rates. These patients are mainly stem cell or bone marrow transplantation recipients, however solid organ transplantation recipients or AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated disease. Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One compound, designated Benzavir-1, was identified with activity against representative types of all adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR), the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2. To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV types. To expand our search for compounds with antiviral activity, we turned to the natural products. An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active compounds could be isolated. However, several compounds had previously been characterized with antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this compound was identified as a butenolide. Additional butenolide analogues were found and we proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was characterized and substantial differences in their toxic potential were observed. One of the most potent

iii butenolide analogues had minimal toxicity and is an attractive starting point for further optimization of the anti-adenoviral activity. This thesis describes the discovery of novel antiviral compounds that targets adenovirus and HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead to the development of new antiviral drugs for clinical use.

iv Summary in Swedish – Populärvetenskaplig sammanfattning på svenska

Det finns ett behov av fler läkemedel för behandling av virussjukdomar. De antivirala läkemedel som finns idag kan inte effektivt behandla svåra sjukdomar orsakade av vissa typer av virus. En sådan virustyp är adenovirus. Hos friska personer orsakar adenovirus oftast luftvägsinfektioner eller infektioner i ögat. Hos barn är diarrésjukdomar orsakade av adenovirus vanliga. Dessa infektioner brukar kroppens immunförsvar kunna ta hand om och de leder sällan till allvarliga komplikationer. Men hos individer med nedsatt immunförsvar kan en adenovirusinfektion vara direkt dödlig. Dessa individer är främst patienter som har genomgått en benmärgs- transplantation eller en organtransplantation. Man har medvetet försvagat deras immunförsvar för att undvika att transplantatet ska stötas bort. Detta innebär att om patienten blir infekterad med adenovirus kan infektionen spridas obehindrat i kroppen och vara livshotande. Det är främst barn som drabbas av svåra och livshotande adenovirusinfektioner. Idag finns det inget godkänt antiviralt läkemedel som kan behandla adenovirusinfektioner, trots att det finns ett behov. Vi har utvecklat en screeningmetod som möjliggör att man snabbt kan identifiera substanser med antiviral aktivitet. Denna metod bygger på ett modifierat adenovirus som uttrycker ett protein som lyser grönt när viruset förökar sig i cellen. Med denna metod har vi screenat 9800 syntetiska substanser. Bland dessa substanser identifierade vi ett flertal med antiviral aktivitet utan att påverka värdcellen. Vi valde ut en substans, som vi kallar Benzavir-1, och analyserade dess aktivitet. Benzavir-1 visade kraftfull antiviral aktivitet mot alla adenovirustyper vi testade och hade låg påverkan på värdcellen. Effekten av Benzavir-1 var t.o.m. bättre än cidofovir, som är det antivirala läkemedel som används idag mot adenovirusinfektioner. Genom att modifiera den kemiska strukturen av Benzavir-1 kunde vi förbättra den antivirala aktiviteten. Den förbättrade substansen kallar vi Benzavir-2. För att utreda om den antivirala aktiviteten var specifik för adenovirus, undersökte vi om Benzavir-1 och -2 kunde hämma en annan typ av virus, nämligen herpes simplex virus (HSV). Benzavir-1 och -2 var aktiva mot både HSV typ 1 och typ 2 samt så visade den förbättrade substansen, Benzavir-2, bättre effekt än Benzavir-1. Benzavir-2 var lika effektiv som acyclovir, som är det läkemedel som används mot herpes virus idag, och var dessutom aktiv mot acyclovir- resistenta kliniska HSV typ 1 och typ 2 isolat.

v Baserat på den potenta aktiviteten av Benzavir-2, är vår avsikt att utreda om Benzavir-2 kan vara en möjlig läkemedelskandidat för behandling av adenovirusinfektioner hos patienter med nedsatt immunförsvar, där det idag finns ett stort behov. Parallellt med karaktäriseringen av Benzavir-2, utvidgade vi sökandet av substanser med antiviral aktivitet mot naturprodukter. Från havsbotten utanför Norges kust togs sedimentprover. Från dessa sedimentprover isolerades aktinobakterier, som är kända för sin förmåga att producera substanser med biologisk aktivitet. Genom att växa aktinobakterier i diverse stressframkallande lösningar, framtvingas produktion och frisättning av substanser från bakterierna. Dessa substansrika extrakt, screenades för antiviral aktivitet med vår screeningmetod. Ett flertal extrakt visade antiviral aktivitet och genom att fraktionera dessa extrakt kunde vi identifiera de aktiva substanserna. Några substansers antivirala aktivitet var redan känd men vi hittade en substans som tidigare inte hade visat antiviral aktivitet. Denna substans visade sig vara en butenolide. När vi försökte optimera produktionen av butenoliden upptäckte vi ett flertal analoger till butenoliden som vi lyckades isolera. Vi kunde även visa i vilken ordning, i relation till varandra, som analogerna producerades. När vi analyserade den biologiska aktiviteten av butenoliderna, upptäckte vi stora skillnader. Två butenolider hade likvärdig antiviral aktivitet men beroende på strukturen av butenolidens sidokedja, skiljde de sig åt i avseende den toxiska effekten på värdcellen. Genom att modifiera butenolidernas struktur, kunde vi även identifiera den del av strukturen som gav upphov till den antivirala aktiviteten. Sammanfattningsvis beskriver den här avhandlingen upptäckten av antivirala substanser som kan leda till att nya antivirala läkemedel kommer till kliniskt bruk.

List of papers

Small-molecule screening using a whole-cell viral replication reporter gene assay identifies 2-[2-(benzoylamino)benzoylamino] benzoic acid as a novel antiadenoviral compound. Andersson EK, Strand M, Edlund K, Lindman K, Enquist PA, Spjut S, Allard A, Elofsson M, Mei YF, Wadell G. Antimicrob. Agents Chemother., 2010, 54 (9): 3871-3877.

Synthesis, biological evaluation, and structure-activity relationships of 2-[2-(benzoylamino)benzoylamino]benzoic acid analogues as inhibitors of adenovirus replication. Öberg CT, Strand M, Andersson EK, Edlund K, Tran NP, Mei YF, Wadell G, Elofsson M. J. Med. Chem., 2012, 55 (7): 3170-3181.

III

2-[4,5-Difluoro-2-(2-fluorobenzoylamino)-benzoylamino]benzoic acid, an antiviral compound with activity against acyclovir- resistant isolates of herpes simplex virus types 1 and 2. Strand M, Islam K, Edlund K, Öberg CT, Allard A, Bergström T, Mei YF, Elofsson M, Wadell G. Antimicrob. Agents Chemother., 2012, 56 (11): 5735-5743.

Isolation and characterization of anti-adenoviral secondary metabolites from marine actinobacteria. Strand M*, Carlsson M*, Uvell H, Islam K, Edlund K, Cullman I, Altermark B, Mei YF, Elofsson M, Willassen NP, Wadell G, Almqvist F. Mar. Drugs, 2014, 12(2): 799-821. (*equally contributing authors).

vii

Abbreviations

Adpol Adenovirus DNA polymerase AIDS Aquired immunodeficiency syndrome ADP Adenovirus death protein BMT Bone marrow transplant CAR Coxsackie and adenovirus receptor CDK Cyclin-dependent kinase

CC50 Half maximal cytotoxic concentration CMV Cytomegalovirus DBP DNA binding protein DSG-2 Desmoglein-2 EBV Epstein-Barr virus

EC50 Half maximal effective concentration EMA European medicines agency EKC Epidemic keratoconjunctivitis FDA Food and drug administration GFP Green fluorescent protein HCS High content screening HSCT Hematopoetic stem cell transplantation HSV Herpes simplex virus HTS High throughput screening PK Pharmacokinetics pTP Pre-terminal protein qPCR Quantitative polymerase chain reaction (PCR) RCAD11GFP Replication competent GFP-expressing adenovirus 11 SAR Structure activity relationship SCT Stem cell transplant TP Terminal protein TK Thymidine kinase

viii

Introduction

1. General introduction

The word virus is Latin for poison or toxin. A virus is an intracellular molecular parasite that is unable to replicate outside the cell. Viruses can infect all forms of life and many viruses have co-evolved with their host organisms. The origin of viruses is not clear, however three main hypotheses are defined. The first hypothesis states that virus arose from mobile cellular genetic elements that gained the ability to exit one cell and enter another. The second hypothesis states that virus originates from cellular organisms that have lost genetic information over time and have adopted a parasitic replication approach. The third and last hypothesis postulates that virus was the first self-replicating unit that was evolved to form cells [1]. The first infectious agent that was designated as virus, was the tobacco mosaic virus. In 1898, Martinus Beijerinck was convinced that the filtrated solution from infected tobacco plants contained a new form of agent, distinct from bacteria. He believed that the infectious agent was a liquid, thus with the development of the plaque assay in 1917, and when the first electron micrographs of tobacco mosaic virus in 1939, it became more obvious that viruses were particles [2]. Virus may have beneficial functions in the evolution of organisms. It is estimated that about 5 % of the human genome originates from retroviruses [3]. Hence, viruses may have a role in the evolution of man. However, viruses are most commonly associated with a variety of diseases and have been described as “a piece of bad news wrapped in a protein coat” by Peter Medawar, who is regarded as the father of transplantation [4]. This description could have arisen from the fact that viral infections are more difficult to treat compared to e.g. bacterial infections. This is mainly due to that it is challenging to target the intracellular replication of viruses without negative effects on the cell and the host organism. Thus, the limited number of antiviral drugs today generates a need for development of new antiviral drugs.

2. Adenovirus

2.1. History

In 1953, Wallace Rowe and Robert Huebner cultured human tonsils and adenoids in tissue culture with the aim of identifying the agent causing “common cold”. Rowe noted that cells derived from adenoids changed their morphology and degenerated over time [5]. The causative agent for this was considered to be a virus. Almost coincidently in 1954, Hilleman and Werner, isolated an agent that presumably also was a virus, but not an influenza virus, that caused acute respiratory disease in military recruits [6]. Subsequently, it was shown that these viruses were related [7]. In 1956, it was proposed that this group of viruses should be commonly named as “adenoviruses” based on the cell type where it was first isolated [8]. This was the onset of an era in virology research that has led to great achievements and impacts in understanding both fundamental virological events such as: viral structure and viral replication as well as understanding cellular events and the interplay between virus and cell and ultimately the effect on the whole organism. For example, in 1977, Phillip Sharp and Richard Roberts discovered that genes were not merely parts of a long continuous segment of DNA, as previously believed, but could consist of several small segments of DNA [9, 10]. This was discovered using adenovirus as a model system, since its genes display important similarities to higher organisms, including man. The phenomenon that genes could originate from discontinuous segments of DNA is called splicing, and is a fundamental principle in modern molecular biology. Sharp and Roberts were jointly awarded the 1993 Nobel Prize in Physiology/Medicine for their discovery. Another important event in the field of adenovirus is the usage of adenovirus in gene therapy, which has both scientific implication and great clinical impact. Gene therapy is when a functional gene (DNA) is introduced to the organism to replace a dysfunctional gene. Most often is the gene packaged into a virus (vector) that delivers the gene to target cells. Adenoviruses are today the most commonly used gene therapy vectors, followed by retrovirus vectors, and different types of cancers are by far the most common indication [11]. There are several reasons why adenoviruses are so commonly used as gene delivery vectors; i) adenovirus can give a high gene expression. ii) adenovirus can infect both dividing and non-dividing cells without incorporation of viral DNA into the host genome. iii) the overall viral biology is well understood. However, despite successful clinical therapies with adenovirus vectors, a major drawback was in 1999, when 18- year-old Jesse Gelsinger, died after a large dose of an adenovirus vector that was administrated into arteria hepatica [12]. Gelsinger died of multi-organ

failure after that substantial amounts of the vector for unknown reasons had spread to the circulation and triggered a massive inflammatory response. Although Gelsinger was exposed to a replication incompetent vector, it is important to be able to suppress and control the infection when performing clinical gene therapy trials. An effective antiviral drug is obviously also needed in the field of gene therapy.

2.2. Taxonomy and classification

The International Committee on Taxonomy of Viruses (ICTV) has classified adenoviruses to the family Adenoviridae that is divided into five genera; Atadenovirus (contains A and T-rich genomes, capable of infecting a broad range of hosts), Aviadenovirus (infecting birds), Ichtadenovirus (currently only one species isolated from sturgeon), Siadenovirus (infecting frogs and some birds) and Mastadenovirus (infecting mammals). Human adenovirus is within the genus of Mastadenovirus and consists of over 54 types, divided in seven species, A to G (Table 1). The classification of the different types is originally based on serology, their hemagglutination patterns and their oncogenicity in rodents among other biological attributes [13, 14]. However, it is currently debated whether the types should be classified according to whole-genome sequencing instead [15-17]. Depending on the method of classification, the number of adenovirus types varies. According to the whole-genome sequencing technique, over 60 isolated adenovirus types have been characterized so far [18]. As seen in Table 1, there is a correlation between the species and their tropism, e.g. the tropism of species F and G is only enteric and species D does not infect respiratory tissue, reasons for these speificities are discussed in section 2.5.1.

Table 1. Categorization of adenovirus types within species and their respective tropisms.

Species Type Tropism A 12, 18, 31 Enteric and respiratory B:1 3, 7, 16, 21, 50 Respiratory and ocular B:2 14, 11, 34, 35 Renal, respiratory and ocular C 1, 2, 5, 6 Respiratory, ocular, hepatic and lymphoid D 8-10, 13, 15, 17, 19, 20, 22-30, Ocular and enteric 32, 33, 36-39, 42-49, 51, 53, 54 E 4 Respiratory and ocular F 40, 41 Enteric G 52 Enteric

2.3. Virion structure

Adenovirus is between 70 – 100 nanometers in size and its outer structure is composed of a non-enveloped capsid with icosahedral architecture. The most abundant protein in the capsid is the hexon protein with a total of 240 hexon trimers distributed to 12 trimers at each of the side of the twenty- sided structure. At each of the 12 vertices of the capsid is a penton that consists of a penton base and a protruding fiber (Fig. 1). The fiber is a trimeric protein with a distal globular knob domain that functions as the cellular attachment site. The length of the fiber varies between adenovirus types, depending on the number of amino acid sequence repetitions in the shaft, from six repeats in Ad3 to 22 repeats in Ad2 and Ad5 [19, 20]. In addition, Ad40 and Ad41 have two different lengths of fibers and some avian adenovirus can have two different fibers attached on the same penton base [21-23]. The minor proteins, IIIa, VI, VIII and IX are capsid associated proteins and are important for virion assembly, the transient virion stability and also when the virion disassembles on infection [24]. For example, protein IIIa is involved in the viral DNA packaging and protein VI may aid the virion to escape from the intracellular endosome and facilitates the nuclear import of hexon proteins [25, 26]. Protein IX is located in the cavities between the hexon proteins and stabilizes the capsid formation [27]. Furthermore, IX has also been reported to be involved in transcriptional activation of late adenoviral genes [28].

Figure 1. The structure of the adenovirus virion. Reprinted from [29] with permission from the publisher.

Inside the core of the capsid is a double-stranded DNA genome with a covalently attached terminal protein at each 5´ end [30]. The linear genome is condensed by three basic DNA binding proteins, V, VII and µ [31]. Protein VII is a histone-like protein that in a sequence-independent fashion binds to the DNA and wraps it up in nucleosome-like structures. Protein V connects the DNA to the capsid via protein VII and the penton base [32, 33]. Protein µ is a small protein that is tightly associated with the DNA and facilitates the DNA condensation [34]. Moreover, the core protein IVa2 binds sequence- specific to motifs in the major late promoter-region of the adenovirus genome and is suggested to be a transcriptional activator and/or involved in the packaging of DNA in the virion core [35, 36]. The last, to date, known protein inside the core is the adenovirus protease. The adenovirus protease

(adpol) is a monomeric cysteine protease that is essential for the virion to be infective [37]. The protease is involved in both the entry of the adenovirus into cells and the assembly of new virion particles [38, 39]. The protease requires two cofactors to be active. One cofactor is the 11-amino acid C- terminal part of the precursor protein VI and the other cofactor is the viral DNA [40, 41]. The precursor protein VI has been suggested as a “molecular sled” that carries the protease along the viral DNA to efficiently locate its substrates [42] . The adenovirus protease is considered to be an attractive target for antiviral drug development [43] (discussed in section 4).

2.4. Genome organization and gene products

The adenovirus genome is a linear, double-stranded DNA molecule with two inverted terminal repeats (ITR) and two covalently bound terminal proteins at each end of the DNA. The size of the genome in the Mastadenovirus genus is between 34-36 kb, although the genome size can vary between 26-45 kb in the other genera in the Adenoviridae family [44]. The genome organization is well conserved among the presently known adenoviruses, except for the avian adenovirus CELO with some distinctive differences [45]. Henceforth, the genome of Mastadenovirus will be discussed. The size of the genome is critical in the biology of adenoviruses, since it has to fit into the virion capsid. It is quite remarkable that the ~36 kb genome can produce over 40 proteins. What makes this possible is the effective organization of the genome. There are 8 RNA polymerase II transcription units that are located at either strand of the genome. These units are divided in five early (E1A, E1B, E2, E3, E4), two intermediate (IX and IVa2) and one late (L1-L5). In addition, there are two virus-associated RNAs (VA RNAI and II) (Fig. 2).

Figure 2. Schematic genome organization of adenovirus type 5. Figure reprinted from [46] with permission from the publisher.

E1A is the first transcription unit to be expressed during infection. E1A products be detected in the cell as early as one hour post-infection [47]. The gene products are two major proteins (243R and 289R) that are transcriptional modulators and stimulate the cell to enter S-phase [46]. E1A is considered as an oncogene since it has potential to immortalize cells [48]. E1B is transcribed shortly after E1A, after approximately 1.5-2 hours post- infection is the mRNA in the cytoplasm [47]. The E1B encodes the 19K and 55K proteins that prevent the cell from apoptosis. The 55k protein binds to, and inhibits the function of the tumor suppressor protein p53 [49]. The 19K protein is a viral analog to the cellular Bcl-2 protein and can inhibit both p53 dependent and p53-independent apoptosis [50]. E2 is expressed from two regions (E2A and E2B) and yields three proteins by alternative splicing. The E2 gene products are directly involved in, and initiate the viral DNA replication. The proteins are the terminal binding protein (TP), the DNA polymerase (Adpol) and the DNA binding protein (DBP). TP is part of a quite unique feature in DNA replication. Adenovirus replicates by protein-priming mechanism instead of a RNA/DNA single stranded primer mechanism [51] (DNA replication is discussed in more detail in section 2.5.4). This feature is dependent on TP, which after cleavage of the adenovirus protease, is linked to each end of the viral DNA by a phospodiester bond from a serine to the terminal genomic dCMP by the

adenovirus DNA polymerase [52, 53]. In addition to functioning as a primer for the viral DNA, this protein linkage may also protect from exonucleases and also prevent that cellular DNA end-binding proteins bind to the viral DNA and inhibit the replication [54]. The second protein expressed from E2 is the 140-kD viral DNA polymerase. The polymerase has an intrinsic 3´- 5´ proofreading exonuclease activity and belongs to the Pol α family. The last expressed protein is the DBP, which is a 72-kDa protein with affinity to single-stranded DNA and RNA as well as double stranded DNA [55]. E3 encodes between 6-9 mRNAs, depending on adenovirus type [56]. All of these proteins function as immunomodulatory proteins to avoid the immune system of the host. Hence, the E3 region is not essential for virus growth in cell culture [57]. Due to the variation in the E3 region among adenovirus types it is believed that the E3 region is important in the species- specific characteristics. The E3 proteins are mostly transmembrane proteins, except 14.7 kD, 12.5 kD. The 11.6 kD is the adenovirus death protein (ADP) that induces cell lysis, maybe through interaction with the mitotic spindle assembly checkpoint protein, MAD2B [58]. ADP is also believed to be the switch between lytic and persistent adenovirus infection [59]. E4 is located at the 5´ end of the genome with a single promoter. This promoter generates at least 18 distinct mRNAs after alternative splicing and at least six proteins have been demonstrated to exist in infected cells named after their open reading frames: ORF1, ORF2, ORF3, ORF4, ORF6 and ORF6/7 [60]. These gene products are involved in DNA replication, viral transcription, apoptosis and regulation of cell cycle signaling [61, 62]. The interplay between the proteins seems to be complex since only the deletion of the entire E4 region impairs virus growth but individual mutations within the proteins have minimal or modest effect [63, 64]. The virus associated RNAs (VA RNAI and II) are small and highly structured low molecular weight regulatory RNAs found in infected cells. These are RNA polymerase III transcripts that are produced with different kinetics [65, 66]. However, some adenovirus types encode only one VA RNA and not all encoded VA RNAs contribute to the virus infectivity [67]. The functions of the VA RNAs are mostly studied in adenovirus type 2 and 5, where it has been shown that VA RNAI inhibits the interferon-induced PKR protein and thereby keeps the eIF-2α transcription factor and the general protein translation active [68]. Additionally, the VA RNAs can be processed to small RNAs (mivaRNA) by the Dicer complex, and be incorporated into the RNA-induced silencing complex (RISC). This results in inhibition of the host cell antiviral defense by suppression of the RNA interference (RNAi) machinery that can degrade viral mRNA [69]. The intermediate transcript gene products (IX and IVa2) are partly discussed in section 2.3. These are expressed prior to the late gene expression, and it has been shown that the IVa2 product contributes to the

late gene expression whereas the IX product serves not only as a capsid protein but also as a transcriptional activator [70, 71]. The major late transcriptional unit (MLTU) is expressed with the onset of viral DNA replication from the major late promoter (MLP). However, the MLP is also active in the early phase of the infection but to a lower transcriptional rate [72]. Coincidentally with the onset of viral DNA replication the transcription rate from MLP is increased dramatically [73]. The transcriptional units from MLTU are grouped into five families (L1-L5) and represent mostly structural proteins needed for progeny virus, e.g. penton base (L2), hexon (L3) and fiber (L5). Additionally, the adenovirus protease gene is in the L3 family.

2.5. Viral life cycle

2.5.1. Binding

The initial step of the virus life cycle is the recognition of the target cell. For adenovirus, this recognition is mediated by the fiber sand the fiberknobs. This initial contact is also the decisive factor for the adenovirus type-specific tissue tropism, since different types have different fibers that target specific cell surface proteins (receptors) (Fig. 3). Hence, receptor characterization is important, especially in gene therapy where specific cell types (most often cancerous) are targeted. Furthermore, with the techniques in molecular cloning that are available today, it is possible to retarget an adenovirus type to a certain receptor, and cell type, by modulation of the fiber and fiberknob. The first receptor that was characterized was for adenovirus type 2 and 5 in 1997. It was shown that the adenovirus and coxsackie virus shared the same receptor, this protein was then named coxsackie and adenovirus receptor (CAR) [74]. CAR is a transmembrane tight-junction protein involved in the cell-to-cell attachment and is abundantly expressed in a wide variety of tissues, including the intestine, prostate, heart, testis and pancreas. The fiber of adenovirus species A and C-F (not species B) have affinity for CAR but only species C have been found to use CAR as a functional receptor [75, 76]. However, since CAR is located in the tight-junction of the cells and is not accessible on the apical surface of the cells, the biological CAR-fiber interaction is debated [77]. It has been suggested that lesions in the epithelial layer would be needed prior infection or that adenovirus first infect non- polarized cells that spreads the infection to neighboring polarized cells [78]. Furthermore, the interaction with CAR has been considered to function as an exit mechanism instead of an entry mechanism in vivo. This is due to that during infection, an excess of fibers and pentons are produced that could

bind to CAR and thereby disrupt the cellular tight-junctions allowing virus escape [79].

Figure 3. Receptors used by human adenoviruses. Figure reprinted from [80] with permission from the publisher.

Several additional receptors have subsequently been identified. CD46, Desmoglein-2 (DSG-2) and the sialic acid-containing proteins are the most characterized. CD46 is expressed on all nucleated cells and is a regulator of the complement activation (RCA) family of proteins. Adenovirus types from species B, and species D types Ad37 and Ad49, have been demonstrated to use CD46 as a receptor [81-86]. Moreover, CD46 is used as receptor for several other pathogens as well, and is considered to be a pathogen magnet [87]. Interestingly, it has been shown that when the fiberknob of Ad11 binds to CD46, this alters the confirmation of CD46 from bent to a planar structure [88]. Quite recently, the cadherin protein Desmoglein-2 that is important in cell adhesion, was found to be the receptor of the species B types 3, 7, 11 and 14 [89]. As noted, Ad11 and other species B types can utilize more than one receptor depending on the affinity/avidity characteristics of the interaction [90, 91]. This implies also for type Ad37 from species D, which is reported to use CD46 and/or sialic acid proteins as cellular receptor(s) [84, 92]. However, the molecular interaction between the Ad37 fiberknob and ganglioside GD1a has been characterized, strongly suggesting that sialylated glycoproteins are functional receptors [93]. Ad37, Ad8, and Ad19, all from

species D, can infect the eye and cause epidemic keratoconjunctivitis. Their primary interactions with sialylated proteins are currently considered as a target for development of novel antiviral therapeutics for treatment of eye- infections caused by adenovirus [94].

2.5.2. Internalization

In general, after the initial binding of the virion to e.g. CAR, the virion needs to interact with an additional receptor to be internalized by the cell (Fig 4). These receptors are designated co-receptors and belong to the integrin family. Integrins are, similarly to CAR, involved in the cell-to-cell adhesion, although integrins are also engaged in adhesion to the extracellular matrix. They consists of two subunits, α and β, and today the αvβ3, αvβ5, αvβ1, α5β1 and α3β1 integrins have been shown to facilitate cell entry [95, 96]. The virion interaction with the integrins is mediated by the penton base, which possesses a tripeptide amino acid sequence, Arginine-Glycine-Aspartic acid (RGD), which can bind to integrins. This RGD amino acid sequence is conserved in all adenovirus types, except the typical enteric types 40 and 41 of species F, suggesting that the interaction with integrins is functioning independently from the initial receptor specificity [97]. The RGD sequence is part of a flexible loop in the penton base. However, the penton base is part of the capsid in a distal direction from the fiberknob, where the binding to CAR takes place. Hence, the length of the fibershaft would be expected to affect the physical interaction between the penton base and integrin. However, it has been suggested that the type 5 (and all species C) fibers contain a “hinge region” that facilitates the interaction [98]. The penton base–integrin interaction triggers phosphatidylinositol-3-OH kinase (PI3K), which is an intracellular lipid kinase [99]. The phospholipid products act as second messengers that lead to actin reorganization and promote endocytosis of the virion by clathrin-coated vesicles [100-102]. In less than 5 min after the initial receptor binding, adenovirus can be observed in endosomes (Fig. 4). Although, clathrin-mediated endocytosis of adenovirus is the most characterized entry pathway, macropinocytosis entry has been described as well. Macropinocytosis leads to vacuole formation at the cellular periphery and has been reported for both species C (type 2 and 5) and species B (type 3 and 35) adenovirus. For species C adenovirus, a simultaneous macropinocytosis and clathrin-mediated uptake was reported, that both were integrin-dependent [103]. For the species B adenovirus, it was reported that both CD46 and integrins was needed and macropinocytosis was suggested to be the major entry mechanism for type 3 and 35 [104, 105].

Figure 4. Illustration of adenovirus uptake and transport to nucleus. Figure adopted from [106] with permission.

When the endocytosis is initiated, a stepwise dismantling of the virion starts in parallel. The fiber proteins starts to detach from the penton base at the plasma membrane followed by a conformational change in the penton base protein that exposes hydrophobic parts and weakens the structure of the capsid [101, 107]. This is followed by release of internal proteins, such as IIIa, V, VIII and VI [108]. Protein VI plays an essential role in the early endosome escape [25]. The virion uncoating process and endosomal escape is very rapid, within 15 min after the initial extracellular binding, free virions can be detected in the cytosol [38]. Although, the rate of the endosomal escape seem to differ between adenovirus types [109]. The exact mechanism for the endosomal escape is not known, although it is known that an acidic pH is essential [110]. This acidification is believed to activate the adenovirus protease that cleaves protein VI, thus activating the membrane lytic capacity of VI [38].

2.5.3. Transportation to nucleus

After the endosomal escape to the cytoplasm, the virion is now partly disassembled and consists of the structural proteins hexon, a fraction of penton base and protein IX that secure the capsid formation. Inside the capsid is the DNA condensed with protein V and VII. The cytoplasm is a very crowded and viscous milieu, hampering a spontaneous diffusion to the nucleus. The virion overcomes this obstacle by active transportation to the nucleus along the microtubules. This transportation is facilitated by a motor protein, called dynein, which literally “walks” on the microtubule towards the microtubule-organizing center (MTOC) near the nucleus [111]. However, kinesin, which is a motor protein but is directed away from the nucleus, has been shown to compete for the intracellular transportation of species C adenovirus [112]. It has been demonstrated that the interaction between the capsid and dynein is mediated through the hexon protein [113]. The transport towards the nucleus is rapid, after approximately 60 min post- infection virions can be located around the nucleus [114]. When the capsid arrives at the MTOC, near the nucleus, the capsid cannot enter the nucleus since the nuclear pores are roughly half the size of the capsid, but need to dock to the nuclear pore [115]. This docking is facilitated by interaction between the capsid and the nuclear pore proteins CAN/Nup214 that are proteins of the cytoplasmic filaments of the nuclear pore (Fig. 4) [114]. This interaction allows nuclear histone H1 proteins to dock to hexon proteins followed by interaction to importin β proteins that tear the capsid apart. The viral DNA can then freely diffuse through the nuclear pore into the nucleus. Alternatively, chaperone protein Hsc70 or protein VII interaction with transportin mediate the translocation of the viral DNA into the nucleus [116, 117].

2.5.4. DNA replication

The replication of the viral DNA starts 6 hours post infection and is a highly efficient process that generates high amounts of progeny molecules. Within 40 hours after infection, one cell can produce approximately one million copies [118]. Prior to the onset of DNA replication the E1a proteins must have been transcribed and triggered the cell to enter S-phase. Also, the E2- gene products (TP, DBP and adpol) must have been expressed. The adenovirus DNA replication utilizes a protein-priming mechanism, in which the pre-terminal protein (pTP) acts as the protein primer. The pTP and the adenovirus DNA polymerase (adpol) are present in the infected cell as a heterodimer [119]. The onset of the DNA replication is when the adenovirus polymerase covalently attaches a dCMP nucleotide to the hydroxyl group of a serine residue of the pTP [54]. This facilitates binding of the pTP-adpol-

complex to specific DNA sequences in the origin of replication that is located at the initial 1-50 bp of either end of the viral genome (Fig 5A). Two cellular proteins enhance the initiation of replication vastly; nuclear factor-1 (NF1) and octomer-binding transcription factor-1 (Oct-1), which bind to the auxiliary region, close the pTP-adpol-complex [120, 121]. Subsequently, the unwinding of the DNA is needed. The mechanism for this is not fully understood, though the adenovirus DNA binding protein (DBP) has an essential role due to its high affinity to single-stranded DNA [122]. After DNA unwinding, the pTP-adpol complex forms a trinucletide CAT sequence that upon formation “jumps back” three basepairs to the 3´end of the genome [123] (Fig 5B). Sequentially, the adpol is released from the pTP and starts the chain elongation [124]. Later in infection the adenovirus protease cleaves pTP to TP, that is the covalently attached protein at each end of the viral DNA [125]. The newly synthesized double stranded DNA can serve as a template for subsequent replication rounds. In addition, the displaced single strand is protected by the DBP protein and can form pan-handles that could serve as replication templates [126, 127].

Figure 5. The initiation of adenovirus DNA replication. A, The origin of replication. B, The protein priming and “jumping back” mechanisms. Figure reprinted with permission from [118], copyright to Cold Spring Harbor Laboratory Press.

2.5.5. Assembly and release

The assembly of progeny virions takes place in the nucleus. However, the protein translation occurs in the cytoplasm, meaning that there is both an import and export trafficking over the nuclear pore complex. It has been shown that adenovirus proteins utilizes the cellular pathways for nuclear transportation by presenting nuclear localization signals (NLSs) and nuclear export signals (NESs), that are short stretches of basic amino acid residues. Although, no NLS has been found on the hexon protein, instead protein VI is suggested to shuttle the hexon proteins between the cytoplasm and nucleus [26]. Inside the nucleus the hexon proteins trimerize to form capsomeres that together with the other capsid proteins form the empty capsid structure. It is believed that the viral DNA is “injected” into the empty capsids, though it is also suggested that the capsid is formed around the DNA core [128, 129]. In the infected cell, a large amount of empty capsids and capsids with incomplete DNA are produced, in parallel to mature virions [130-132]. These incomplete virions have the potential to induce inflammation without any gene expression [133]. The complete virions need a maturations process to be infective progeny virions. This virion maturation is processed by the adpol which cleaves pIIIa, pVI, pVII, pVIII, pre-µ and pTP, possibly also the L1 52/55k polypeptide, to their functional forms [134]. The protease is also encapsidated and is essential for the progeny virus entry into new cells when released. When the nucleus is packed with virions, the cytoskeleton is disrupted and the cell rounds up to the typical cytophatic effect (CPE) that ends in cell lysis. It has been shown that adpol can cleave the cellular cytokeratin K18 in HeLa cells, which results in perturbation of the formation of cytoskeleton filaments, thus adpol is involved in the CPE and promotes cell lysis [135]. However, the precise mechanism of adenovirus-caused cell lysis is poorly understood. Though, the adenovirus death protein (ADP), also known as E3-11.6K protein, has a major role in this process. ADP is abundantly expressed late in infection and is, at least partly, involved in cell lysis since virus lacking ADP replicates as well as wild type virus, but the cell lysis phase is delayed [136]. This corresponds to that ADP levels have been suggested to be the switch between lytic and persistent infections [59]. Furthermore, both autophagy and apoptosis mechanisms have been reported to be involved in the adenovirus induced cell killing [137, 138]. In the end when the cell lyse, up to 104 progeny virions are produced per cell, along a vast excess of viral proteins and DNA that have not been encapsidated [139]

2.6. Human adenovirus spread and infections

Adenoviruses are easily spread from one person to another through aerosols when sneezing or coughing, or through the fecal-oral route. The virus is also very stable outside the host, on surfaces and in water. Most types are still infectious at 36 °C for a week and several weeks at room temperature [140]. In addition, the virus can be viable on dry surfaces for several months [141]. The adenoviruses are also resistant to lipid disinfectants (soap) but are sensitive to 70 % ethyl alcohol, heat, formaldehyde and chlorine [142, 143]. Due to the stability of the virus and that it is easily spread, large outbreaks of adenovirus infections have been described, especially in hospital settings, child-care centers and among military recruits [144-146]. However, community-wide outbreaks have also been reported [147].

2.6.1. Adenovirus infections in immunocompetent persons

In otherwise healthy, immunocomptetent persons, adenovirus infections can be severe but is most often self-limiting and can even be asymptomatic. Adenovirus infections are common in society and can occur throughout the year. Respiratory infections are often associated with species C adenovirus, and account for 5-10 % of all respiratory infections in children [143]. Fever, pharyngitis, and conjunctivitis are symptoms of pharyngoconjuctival fever (PCF) caused by adenovirus type 3, 7 and 14. PCF affects mostly children and can cause localized outbreaks in e.g. primary schools and swimming pools [148, 149]. Acute respiratory disease (ARD), caused predominately by species C and type 7, 14 from species B plus type 4 from species E, is associated with fever, rhinorrhea, cough and sore throat that last for 3-5 days, usually with tonsillitis and otitis media. In 1971 an oral vaccine was implemented to reduce ARD in military recruits where it was a major concern with occasional fatalities. The vaccine reduced adenovirus-related respiratory illnesses in recruits, by more than 95 %, however the vaccine production ceased in 1996 [150, 151]. Adenovirus can cause pneumonia with sporadic fatalities [152, 153]. Moreover, adenovirus type 40 and 41 infect the gastrointestinal tract and are responsible for 5-15 % acute diarrheal illness in children [143]. Adenovirus type 11 and 21 from species B can infect the urinary tract and cause haemorrhagic cystitis that occurs mostly in boys. It is characterized by gross hematuria and adenovirus accounts for 20 % of all reported hemorrhagic cystitis cases in US [154]. Myocarditis, hepatitis and meningoencephalitis are rarely reported manifestations of adenovirus infections [155-158]. More common is the highly contagious eye infection epidemic keratoconjunctivitis (EKC) that is mainly caused by type 8, 19 and

37 from species D. EKC cause significant morbidity for the patient, with photophobia, pain, red-eyes, tearing, and blurred sight. EKC can lead to prolonged impaired vision or blindness. Due to the infectiousness, outbreaks are common in densely populated areas. In Japan, between half a million to one million people acquire EKC every year [159].

Table 2. Predominant adenovirus types associated with adenovirus disease. Table adopted from [143] with permission from the publisher.

Diseases caused by adenoviruses Predominant types Infants: Pharyngitis, pneumonia 1, 2, 5 Otitis media 1, 2, 5 Diarrhea 40, 41

Children: Pharyngoconjunctivial fever 3, 7 Pneumonia 3, 7, 21 Diarrhea, mesenteric adenitis 40, 41, 2 Haemorrhagic cystitis 11, 21 Myocarditis 1, 2, 5

Young adults: Acute respiratory disease 4, 7, 14

Adults: Epidemic keratoconjuctivitis 8, 19, 37

Immunocompromised patients: Pneumonia 1, 2, 5 Gastroenteritis, hepatitis 5 Haemorrhagic cystitis, nephritis 11, 34, 35 Meningoencephalitis 1, 2, 5

As seen in Table 2, the age of the person, when infected, and the adenovirus type are closely correlated to the clinical manifestation of the infection. Although, most adults have acquired immunity to multiple adenovirus types due to infections in childhood [160]. Furthermore, adenoviruses can establish persistent infections in lymphatic tissues (tonsils and adenoids), the gastrointestinal tract (lymphocytes) and in the urinary tract [161-163]. These infections lead to shedding of the virus in the stool or urine, for a long time after the acute infection [164, 165]. In addition, since adenovirus infections can be asymptomatic, shedding of virus without prior clinical manifestation has been observed [166]. Adenovirus type 36 has been shown to be associated with obesity, both in animals and humans, however the medical implications of these findings are debated [167-169].

2.6.2. Adenovirus infections in immunocompromised patients

Opportunistic viral infections cause considerable morbidity and mortality in immunocompromised patients. Adenoviruses are increasingly recognized as pathogens causing severe infections and death, in these patients. The clinical manifestations of adenovirus infections in persons with insufficient immune system are more severe and prolonged with risk of disseminated disease and multi-organ failure. Adenovirus infections are associated with high morbidity and mortality rates in these patients [170]. The insufficient immune system could either be inborn deficiencies (congenital or primary immunodeficiency) or due to immunosuppressant medication when undergoing haematopoetic stem cell (HSCT), bone marrow (BMT) or solid organ transplantations (acquired or secondary immunodeficiency). In addition, the adenovirus infection can either be a primary infection or a reactivation of a persistant infection. However, due to the limited number of persons with severe congenital immunodeficiencies, reports of adenovirus disease within this group are limited. Several case reports describe fatal adenovirus infections in patients with severe combined immunodeficiency syndrome (SCID), Bruton´s X-linked agammaglobulinemia, DiGeorge syndrome [171-174]. Adenovirus infections in patients with acquired immunodeficiencies have been more characterized, though HSCT and BMT is also performed in patients with congenital immunodeficiences, together with hematological malignancies and other types of cancers. The incidence of adenovirus infections in HSCT and BMT patients varies depending the age of the patient, T-cell counts, HLA-match and more. In general, immunocompromised patients are a very heterologous group with expected variations in disease incidence, severity and outcome. A recent review of several reports concludes that adenovirus infections were reported in 5 - 47

% of the BMT and SCT patients [175]. Of these, 10 - 89 % had symptomatic adenovirus disease with diarrhea as the most common manifestation followed by respiratory disease and hemorrhagic cystitis. And ultimately, 6 - 70 % of the infected patients died due to the adenovirus infection [175]. In children, general mortality rates greater than 50 % are frequently reported [176]. Baldwin et al. reported 26 % adenovirus positive children and 9 % of the adults were positive, following BMT [177]. This is similar to Howard et al. that reported adenovirus in 23 % for children and 9 % of adults [178]. This illustrates that adenovirus infections are more common in pediatric than adult BMT and SCT patients, hence associated with higher mortality rates. However, the underlying reason for this is currently unclear, though it is believed that primary adenovirus infections are more frequent in children compared to adults. In addition, primary infections are associated with higher viral loads. Adenovirus infections are also a more pronounced problem in children than adult solid organ transplant recipients. In the solid organ transplant recipients, the adenovirus disease is usually related to the transplanted organ [179]. For liver transplant recipients, incidence of adenovirus infection is 6-11 % with symptoms as jaundice and hepatitis, and associated with 53 % mortality rates if hepatitis is developed [170]. Notably, it is mainly species C adenoviruses that are associated with hepatitis in the immunocompromised host, whereas in immunocompetent persons species C is associated with respiratory disease. Hence, reactivation or transmission from the donor liver has been demonstrated in serum negative recipients that have received donor liver from serum positive donor [180]. In renal transplant recipients, adenovirus incidence has been reported to 12 % leading to haemorrhagic cystitis and nephritis. Mortality rates of 18 % in these patients are reported, caused by species B adenovirus types [170]. In lung transplant recipients, adenoviruses have been reported in 50 % of the recipients and were associated with respiratory failure, graft loss and death [181]. Adenovirus infections in AIDS patients can be fatal, although since the introduction of effective antiretroviral therapy the mortality rates have decreased. Adenoviruses are most commonly isolated from the stool in AIDS patients and may be the cause of diarrhea that frequently is observed in AIDS patients [182, 183]. In addition, adenovirus-associated prostatitis, hepatitis, nephritis, parotitis and encephalitis, have also been reported in these patients [184-188]. To conclude, adenovirus infections in immunocompromised patients are associated with severe morbidity and mortality rates. However, it is difficult to assess how many patients that are affected by severe adenovirus infections worldwide. European Medicines Agency (EMA) reported 2001 in an orphan drug designation for ribavirin, that 0.6 – 3 people per 10,000 in EU are affected [189]. Converted to current number of citizens in EU, this

corresponds to 30,000 to 150,000 people in EU. In addition, EMA reported in 2014 that adenovirus infection in allogenic HSCT recipients affected less than 0.2 in 10,000 people in the EU [190]. Furthermore, considering the growing number of patients undergoing blood stem cell- and solid organ transplantations worldwide, severe and potentially fatal adenovirus infections will become an increasing threat without effective antiviral therapies.

2.7. Antiviral drugs used in the clinic

Today there are no approved specific antiviral drugs available for treatment of adenovirus infections. However, cidofovir and ribavirin are used off- license for adenovirus therapy, while no other alternatives are available (Fig. 6). Cidofovir is a nucleoside analog that suppresses the replication of viral DNA by inhibiting the viral DNA polymerase (mode-of-action for antiviral drugs is addressed in more detail in section 4). Cidofovir displays broad antiviral activity against several DNA viruses but is only regulatory approved for treatment of cytomegalovirus retinitis in AIDS patients [191]. Several reports describe the usage of cidofovir in immunocompromised patients with adenovirus infections, however both successes and failures are reported. In addition, cidofovir has low bioavailability when administrated orally, thus intravenous administration is needed. Intravenous administration of cidofovir is associated with nephrotoxicity due to intracellular accumulation of cidofovir to toxic levels, in renal tubule cells [192]. Consequently, frequent monitoring of renal function is required and potentially cidofovir needs to be co-administrated with probenicid to reduce nephrotoxicity [193]. Initial administration of cidofovir is recommended when adenovirus viremia is observed but cidofovir is not recommended to use prophylactically [194]. In general, monitoring adenovirus DNA load in blood is essential to be able to foresee dissemination of the infection. However, Lion et al. reported that adenovirus levels in stool could be observed 11 days prior viremia. Hence, early initiation of antiviral therapy is possible for preventing progression of the infection [195]. Ribavirin is also a nucleoside analog, with broad antiviral activity against several viruses, both RNA- and DNA viruses. The exact mode-of-action of ribavirin is not clearly understood and several mechanisms have been suggested (see section 4). Ribavirin is approved in the US for inhalation therapy of lower respiratory tract respiratory syncytial virus infections in children and for systemic treatment, in combination with interferon, of Hepatitis C infections, both in EU and US. As with cidofovir, several reports describe the treatment of adenovirus, though with both successes and failures [196-198]. The antiviral activity of ribavirin has also been suggested to differ between adenovirus types in vitro and differences were also

observed in clinical isolates [199, 200]. According to “European guidelines for treatment of adenovirus infections 2011”, by Matthes-Martin et al., ribavirin is not recommended for treatment of adenovirus infections [194].

Figure 6. Structures of cidofovir and ribavirin.

Other nucleoside analog drugs have been clinically used for treatment of adenovirus infections, including ganciclovir and vidarabine [201, 202] (Fig. 7). However, the reports are sporadic case reports and more clinical information is needed for conclusive data. Recent developments of lipid conjugates of existing antiviral drugs have resulted in Brincidofovir (CMX001) (Fig. 7). Brincidofovir, is an orally bioavailable lipid conjugate of cidofovir, that uses cellular plasma membrane proteins (flippases) for rapid entry into cells, that compared to cidofovir, results in higher intracellular concentrations [203]. Brincidofovir has shown promising clinical effect in a small number of patients and is currently under development by Chimerix [204, 205].

Figure 7. Structures of Ganciclovir, Vidarabine and Brincidofovir.

Furthermore, adoptive cell therapy has been implicated in treatment of adenovirus infections in immunocompomised patients. Infusion of adenovirus-specific T cells from donors to adenovirus infected immunocompromised patients has shown successful transfer of the adenovirus immunity, thus potentially inhibiting virus reactivation [206]. Cell Medica has developed Cytovir™ ADV, which is in a phase I/II study that will be completed in early 2015.

3. Herpes Simplex Virus

3.1. History

The first documented cases of Herpes Simplex Virus (HSV) infections can be traced back to the ancient Greeks. Hippocrates used the term “herpes” to describe lesions on the skin that appeared to creep or crawl [207]. In 1736, Jean Astruc discovered that the condition was sexually transmitted among French prostitutes [208]. In 1921, Gruter isolated HSV and demonstrated the infectious nature of the virus, by serial transmission of the infection from rabbit to rabbit [209]. In 1930, Andrews and Carmichael described that herpes could recur in infected persons and in 1939, Burnet and Williams defined the concept of latency for herpes simplex infection, i.e. once infected the infection is persistent throughout life [210, 211]. Moreover, the introduction of electron microscopy techniques in the 1930’s, was the onset of virus characterization that has yielded the knowledge we have today about HSV.

3.2. Taxonomy and classification

HSV is constituted of two types, HSV type 1 and 2 (HSV-1 and HSV-2) and they belong to the subfamily Alphaherpesviridae, in the family Herpesviridae. There are three subfamilies in the human Herpesviridae family, α, β, and γ subfamily, each with different characteristics (Table 3).

Table 3. Classification of human herpesviruses.

Subfamily and characteristics Members Diseases α - Short growth cycle, rapid spread. HSV-1 and HSV-2. Labial and genital Latent in sensory neurons. lesions. Varicella-zoster virus Chickenpox, zoster (shingles). β - Long growth cycle, slow spread. Cytomegalovirus Mononucleosis, Latent in secretory glands and hepatitis, fetal lymphoreticular cells. abnormalities. Human herpesvirus Roseola 6A, 6B and 7 γ - Growth primarily in epithelial Epstein-Barr virus Mononucleosis, cells and B or T lymphocytes. Latent Burkitt´s lymphoma, in lymphocytes. nasopharyngeal carcinoma. Human herpesvirus 8 Kaposi´s sarcoma !

HSV-1 and HSV-2 share homology in approximately 50 % of their genome sequences [212]. However, the site of infection differs between them. HSV-1 infects the oropharynx and the mucosa of the facial lips while HSV-2 infects the genital mucosa, even though HSV-1 and HSV-2 may infect at either of these sites. HSV-1 has been the prototype for all herpes viruses. Hence, the herpes simplex biology, described below, is exemplified by HSV-1.

3.3. Virion structure

The HSV virion is approximately 120-200 nm in diameter and a linear double-stranded DNA is located within the core (Fig. 8). The DNA is encapsidated in an icosahedral capsid (similar to adenovirus) that is approximately 100-110 nm in diameter. The capsid is composed of 162 capsomeres that mainly contain four proteins, VP5, VP26, VP23 and VP19c.

In addition, the capsid has a cylindrical portal, or tube, formed by the UL – 6 protein that mediates viral DNA entry and exit from the capsid [213]. The capsid is enclosed in a lipid envelope, in which at least 12 different glycoproteins are embedded (gB to gM) together with several nonglycosylated proteins. These envelope proteins are observed as spikes, with lengths of 10 to 25 nm, and about 600 – 750 spikes cover each virion [214]. The space between the capsid and the envelope is called tegument [2]. The tegument contains several virus-encoded proteins that are introduced in the cell upon infection and may play important functions in the viral replication [215].

Figure 8. The structure of the HSV virion. Figure reprinted with permission from the Swiss Institute of Bioinformatics.

3.4. Genome organization and gene products

The length of the HSV genome is estimated to be approximately 150 kb and is linear within the capsid, though immediately after release into the cellular nucleus, the genome circularizes [216]. The genome is generally divided in unique regions, long (UL) and short (US), according to their relative length, and is flanked by inverted repeats. The repeats for UL are designated a and b, whereas for US the repeats are called c (Fig. 9). In total, the genome contains at least 84 different genes. The gene name defines in what sequence (L or S) and in what order they are found (1-56 in L and 1-12 in S) in the genome. In addition, some genes are placed in the b and c segments. The initially described proteins were named virion protein (VP) or glycoprotein (gp) or, if found in infected cell extracts, ICP (Infected Cell Protein) [2].

Figure 9. The HSV genome map. Figure reprinted from [217] with permission from the publisher.

3.5. Viral life cycle

3.5.1. Binding and uptake

The glycoproteins, or spikes, of the envelope mediate the initial attachment to the cells (Fig. 10). The gC, gB and gD glycoproteins are necessary for attachment to primarily heparan sulfate and potentially also chondroitin sulfate on the cell surface [218]. Initial attachment is followed by the binding of gD to specific co-receptors that triggers fusion of the envelope to the plasma membrane. Four different co-receptors have been characterized: herpes virus entry mediator (HVEM), nectins (-1 and/or -2), specific sites on heparan sulfate generated by certain 3-O-sulfotransferases and paired immunoglobulin-like type 2 receptor alpha (PILRα) [219-221]. The glycoproteins gH/gL have been suggested to regulate the fusogenic properties of gB [222]. However, exactly how the virion fuses with the cellular membrane is not fully understood, although conformational changes in the gD protein seems to be involved [223]. The cytoplasmic entry of the virion is pH-independent (different from adenovirus), thus the tegument proteins that covers the capsid are released into the cytoplasm. As a consequence the host macromolecular metabolism and DNA synthesis ceases [2]. Subsequently, the capsid is transported along the microtubule, by the dynein protein, towards the nuclear pore. Upon arrival to the nucleus, the viral DNA is introduced in the nucleus where it immediately circularizes [224].

Figure 10. The receptors and ligands for HSV entry. Reprinted from [225] with permission from the publisher.

3.5.2. DNA replication

Once the viral DNA is in the nucleus, transcription of the viral genome occurs by the host cell RNA polymerase II. The viral genes can be classified into immediate early (α), early (β) and late genes (γ1 and γ2). The immediate early genes are regulatory proteins that, upon expression, activate the early and late γ1 genes. The γ2 genes are expressed after the onset of the DNA replication. The early gene products include enzymes (e.g. thymidine kinase and DNA polymerase) that are essential for virus DNA replication. After the expression of the required gene products, the HSV DNA replication starts in a theta replication but then switches to a rolling circle mechanism [226, 227]

(Fig. 11). The UL9 protein binds to the origin sequence, CGTTCGCACTT, and recruits ICP8, which is a single-stranded DNA binding protein. ICP8 can unwind the double-stranded DNA and thereby enable binding of both the helicase-primase complex and the DNA polymerase complex. The helicase- primase complex further unwinds the double-stranded DNA and consequently, allows the polymerase complex to replicate the viral DNA [228].

Figure 11. Illustration of the HSV DNA replication. Figure reprinted from [229] with permission from the publisher.

3.5.3. Assembly and release

The assembly of progeny virions occurs in the nucleus. The major capsid protein VP5, together with VP26 and several additional proteins form an immature capsid around a protein scaffold, containing VP21, VP22a and VP24 (Fig. 12). The viral DNA is then packed inside the capsid. Motor proteins, UL-6, UL-15 and UL-28, which recognize the a-sequence in the viral genome, mediate the packaging of the DNA. When they encounter the next a-sequence, the packaging is aborted. Subsequently, the scaffold proteins are cleaved by the protease and are ejected from the capsid [230, 231]. In this process, the DNA is enclosed and the capsid undergoes morphological changes to form a more robust capsid structure [2, 228].

The mechanism how the progeny viruses are released from the nucleus, and finally from the cell, is not clear. One theory is that the nuclear pores become enlarged thus allows the capsid to exit to the cytoplasm. Subsequently, Golgi membranes envelop the naked capsids in the cytoplasm [233]. Another theory is that the capsis are initially enveloped by the nuclear inner membrane but is de-enveloped upon release from the nuclear membrane and is the re-enveloped by Golgi membranes in the cytoplasm [234]. Furthermore, the third and original theory is that the capsids are enveloped by the nuclear inner membrane and is released from the nucleus in an enveloped form to the cytoplasm [235]. Common for all theories is that the enveloped capsids is in, or ends up in, secretory vesicles in the cytoplasm that later fuse with the plasma membrane to release the enveloped virions from the cell.

3.6. Latency and reactivation

All today known herpesviruses can establish life-long latent infections. The precise mechanism for this is however not known. Although, certain events have been characterized which are described here. Following infection at the primary site, the progeny virus is spread within the tissue and infects sensory neurons. This facilitates a retrograde transport of the capsids along the axons towards the cell bodies of dorsal root neurons that innervate the primary site of infection [236]. In general, HSV-1 resides in the trigeminal ganglia, whereas HSV-2 resides in the sacral ganglia. Dynein, the motor protein, transports the HSV capsids to the neuronal cell bodies [237]. The viral DNA enters the nucleus and is circularized and associates with nucleosomes in a chromatin structure [238]. This chromatin structure is believed to suppress most of the gene expression to keep the infection latent [239]. The only viral gene products abundantly expressed are specific RNAs, called latency-associated transcripts (LATs) [240]. The roles of LATs are controversial, however common themes are that they protect the neurons from death and that they are involved in suppression of the lytic genes [241, 242]. Upon local trauma to the innervated primary site of infection, or upon systemic stress, e.g. UV-light, hormonal imbalance and emotional stress, the latent virus can be reactivated. It has been shown that induction of reactivation causes decreased LAT levels and disruption of the chromatin structure [243, 244]. This triggers the replication of viral DNA and subsequently the production of progeny virus. The progeny capsids are transported in an anterograde direction along the axons towards the primary site of infection. Kinesins are the motor proteins that transport the capsids in anterograde direction [245]. Upon arrival at the primary site of infection, the

tissue is infected and the virus is shed. However, reactivated infections can shed virus asymptomatically, which have impact on the viral transmissions in the population [246].

3.7. HSV spread and infections

HSV is highly contagious. It is most commonly spread through direct contact of infected saliva or secretions from viral ulcerations. However, transmission from aerosol droplets has been reported [247]. The lipid envelope of the virion is sensitive to ethanol, heat, chlorine and dry atmosphere. HSV infections occur worldwide without any specific seasonal distribution. Geographical location, socioeconomic status and age, influence the seroprevalence of HSV among populations. The highest incidence of both HSV-1 and -2 are in developing countries, e.g., in African countries up to 82 % of the women are seropositive for HSV-2 [248]. In USA, 21 % of the women and 12 % of the men are infected with HSV-2, with higher incidence among Afro-Americans [249]. In general, women are more susceptible to HSV-2 infections, because HSV is sexually spread more easily from men to women than vice versa. Furthermore, 58 % of the Americans are positive for HSV-1 [250]. The incidence of HSV varies among European countries as well. The highest incidence of HSV-1 is reported in Bulgaria with 84 %, whereas in Finland the seroprevalence is 52 %. Moreover, the HSV-2 seroprevalence is 24 % in Bulgaria, compared to 13 % in Finland. The lowest incidence of HSV-2 is in England and Wales with 4 % [251]. Conclusively, both HSV-1 and HSV-2 infections are common in societies, albeit HSV-1 is more common than HSV-2. Socioeconomic status and geographical location affects the incidence among populations greatly.

3.7.1. HSV infections in immunocompetent persons

In otherwise healthy persons, both HSV-1 and HSV-2 infections are commonly associated with recurrent ulcerations and blisters. However, HSV infections have other clinical manifestations as well. Primary infections, if symptomatic, are generally more severe than recurrent infections and can cause a more widespread infection with respiratory disease, fever, swollen tonsils, rash, and more [252]. Most primary HSV-1 infections occur in children and the infections are most often asymptomatic [2]. However, if HSV-1 is acquired later in life, pharyngitis and upper respiratory tract infections are more common [253]. Reactivated HSV-1 is associated with mucosal ulcerations at the mucocutaneous junction of the lip (known as cold sores or fever blisters). In addition, HSV-1 can cause generalized

dermatologic disease such as primary herpes dermatitis, eczema herpeticum (primary HSV-1 infection of the skin) and traumatic herpes (infection in wounds due to burns or lesions). Herpetic whitlow is an occupational hazard among dentists, hospital personnel and wrestlers, where HSV-1 or HSV-2 infects skin abrasions of the fingers [254, 255]. Furthermore, HSV-1 can infect the eye and lead to stromal keratitis, which is one of the leading causes of blindness in the Western world [256]. Herpes simplex encephalitis is a rare infection of the central nervous system, caused mainly by HSV-1. It is a severe condition associated with very high mortality rates if untreated. Moreover, serious neurological sequelae, with memory impairments and personality disturbances are common in survivors of HSV encephalitis [257]. Primary genital HSV-2 infections are symptomatic in approximately 40 % of the cases [258]. Primary symptomatic infections are associated with more severe and prolonged duration of symptoms, compared to recurrent infection. Genital HSV-2 symptoms include vesicles on the genitals, that develops to painful ulcers which ultimately crusts. In addition, especially in females, primary genital HSV infection can be associated with systemic symptoms such as fever, headache, malaise and muscle pain. HSV-1 is increasingly recognized as a pathogen causing genital infections, probably due to alterations in sexual behavior in society. However, genital HSV-1 infections seem to be less severe and not as prone to cause recurrent infections, compared to HSV-2 [259]. Genital HSV infections and HIV-1 positivity is tightly linked together. One reason for this is that genital ulcerations can increase the transmission rate of HIV-1 [260]. The risk of HSV transmission between mother and baby is between 30-50 % if the mother acquires a primary genital HSV infection in the third trimester [261, 262]. Neonatal HSV infections are associated with high mortality rates and great morbidity. Hence, caesarian sections are recommended if the mother has symptomatic genital infection. Furthermore, there is a correlation between cervical HSV infections and miscarriage [263].

3.7.2. HSV infections in immunocompromised patients

Both HSV and adenovirus (described in section 2.6.2), are problematic opportunistic pathogens in patients with suppressed immune system, e.g. BMT-, SCT-, solid organ transplantation recipients. The symptoms of HSV infection, in these patients, are more severe and generalized. In addition, suppression of the immune system can potentially trigger the reactivation of an endogenous latent infection. However, in distinction to adenovirus infections in the immunocompromised patients, effective antiviral therapies can suppress HSV disease. The rate of HSV reactivation among SCT recipients has been reported to be up to 80 %, 4-6 weeks after

transplantation when the immunosuppression is the most pronounced [264]. The most common symptoms are mucocutaneous disease, more in the orofacial region than the genital tract [265]. Oropharyngeal infections and esophagitis are also frequently observed, with symptoms as, nausea, chest pain and vomiting [266]. In addition, HSV caused pneumonitis, hepatitis and meningitis have been reported in immunocompromised patients [267- 269]. The golden standard antiviral drug for HSV treatment today, is acyclovir (see section 3.8). Acyclovir is extensively used for treatment in the clinic and prophylactically used in immunocompromised patients to prevent onset of herpesvirus disease. However, in HSV (or other herpesvirus) seronegative patients, no prophylactic antiviral treatment is necessary due to that these patients are at low risk of acquiring primary infections. In seropositive patients, the current recommendation is that acyclovir should be given intravenously or orally, 3-5 weeks (or longer) after the transplantation or chemotherapy [270]. The extensive and preemptive administration of acyclovir, in mainly immunocompromised patients, is associated with development of acyclovir-resistant HSV that cause more progressive disease with a prolonged symptomatic phase when reactivated. Acyclovir-resistant HSV is most commonly isolated from patients with severe mucocutaneous disease [271, 272]. However, isolation from pneumonia-, esophagitis- and encephalitis affected patients is also reported [273-275]. The isolation rate of acyclovir-resistance is higher in immunocompromised patients than immunocompetent persons. The general prevalence of HSV with reduced susceptibility to acyclovir, in immunocompromised patients has been reported to be between 3,5 - 10 %, compared to 0.1 – 0.7 % in immunocompetent persons [276]. Thus, the risk of developing acyclovir-resistance is more than ten-fold higher in immunocompromised patients compared to immunocompetent persons. Contradictory and a bit surprisingly, a long duration (> 1 year) of acyclovir therapy, in immunocompromised patients, is associated with low incidence of acyclovir-resistance [277]. The reason for this is unclear, although based on my experience the acyclovir-resistant isolates display reduced “fitness” and have a potentially lower replication rate, compared to acyclovir-sensitive isolates. In addition, GlaxoSmithKline that markets acyclovir (Zovirax), has supported the authors of this report which could undermine the credibility [277]. However, when acyclovir-resistance is observed, alternative antiviral drugs with differences in their mode-of-action are used. These drugs are mainly foscarnet and cidofovir that are discussed in section 3.8.

3.8. Antiviral drugs used in the clinic

Acyclovir (Zovirax®, Fig. 13) is the golden standard antiviral drug for HSV treatment today. It is mainly used topically and intravenously and not orally administrated, due to its low oral absorption. The structure of acyclovir is based on the discovery of nucleosides from the Caribbean sponge Tethya crypta [278, 279]. These nucleoside structures led to the synthesis of Ara-A, or vidarabine, which was primarily aimed for anti-cancer use but was re- targeted towards antiviral use after the discovery of its antiviral activity [280, 281]. Vidarabine is still used in the clinic, mainly as an eye ointment for HSV keratitis. In addition, one of the first antivirals was trifluridine (Viroptic®), which also is still used in the clinic as eye drops. Vidarabine was previously used intravenously but was later replaced by acyclovir, which had higher selectivity and potency [282-284]. Elion, G., et al. published the discovery of acyclovir in 1977 and in 1979 a patent application was filed. Elion, G., was not included among the inventors but she received a shared Nobel prize in 1988 [285]. In the 1980’s acyclovir was introduced to the market and since then, several analogs to acyclovir have been developed (Fig. 13). Valacyclovir (Valtrex®), which is a L-valyl-ester prodrug of acyclovir, displays better bioavailability after oral administration than acyclovir and can thus be administrated orally for treatment of various herpesvirus diseases [286]. Penciclovir display antiviral potency similar to acyclovir but is, as acyclovir, poorly absorbed when given orally. This led to the development of famciclovir, which is an oral diacetylated prodrug of penciclovir [287]

Figure 13. Structures of Acyclovir and Penciclovir, with their respective prodrugs Valacyclovir and Famciclovir.

Since acyclovir and penciclovir have similar mechanisms of antiviral action (antiviral mode-of-actions are described below in section 4), acyclovir-resistant HSV isolates are also resistant to penciclovir [288]. Hence, alternative antiviral drugs with different mode-of-action are needed for treatment of acyclovir/penciclovir-resistant HSV. Foscarnet (Foscavir®) is the second in line drug used (Fig 14). Foscarnet is given intravenously for 7-21 days, or until complete healing is observed [270]. However, foscarnet usage can be associated with nephrotoxicity, hemoglobin disturbances and seizures [289]. If foscarnet therapy was unsuccessful, the third in line drug is cidofovir (Fig. 6). As in the case when used to treat severe adenovirus infections, Cidofovir is given intravenously in combination with probenicid to reduce potential nephrotoxicity.

Figure 14. Structure of Foscarnet.

A new class of anti-HSV drugs that are in the pipeline is the helicase- primase inhibitors, with Pritelivir (BAY571293, AIC316) in the lead (Fig. 15). Pritelivir is developed by AiCuris and has shown promising reduction of HSV-2 symptoms with low toxicity, both in animal models and in phase II clinical trials [290, 291]. Unfortunately, the food and drug administration (FDA) has recently halted the development of Pritelivir due to skin and haemotological toxicity in monkeys [292].

Figure 15. Structure of Pritelivir.

4. Antiviral strategies and drug modes-of- action

Today there are over 200 different virus species that are known to infect humans and three to four additional species are discovered each year [293]. Each one of these viruses has different ways to infect the eukaryotic cell and secure progeny spread. However, all human viruses share general steps in their infection cycle (Fig. 16). Consequently, antiviral drugs that target each one of these steps have been developed.

Figure 16. General steps and antiviral drug targets in viral infection cycle.

This section will cover the stages where antiviral drugs have been developed to combat adenovirus and HSV infection. Currently, the main antiviral group for adenovirus and HSV treatment is the nucelsoside analogs. In addition, recent focus on entry and protease (maturation) inhibitors has yielded interesting compounds. Hence, these groups are discussed here. Moreover, since both adenovirus and HSV are DNA viruses and share common molecular features, especially in the DNA replication machinery, an overlap of antiviral drugs inhibiting both adenovirus and HSV is expected.

Figure 17 displays a generalized infection cycle for both adenovirus and HSV. Steps in the infection cycle that have been targeted for antiviral therapy are marked red.

Figure 17. A generalized infection cycle for adenovirus and HSV. Currently, most antivirals against adenovirus and HSV target the DNA replication, binding and protease (maturation) steps, as marked in red.

4.1. Binding inhibitors

Inhibition of the initial step of the infection cycle is an effective approach to block viral replication and to inhibit viral spread within the tissue. Attachment of the virus to host cell receptors can be blocked by compounds that either bind to the virion or on the cellular receptor. Focus has been on development of soluble compounds that mimic part of the cellular receptors that interact with the virion. One example is sialylated compounds for treatment of adenovirus EKC [294]. By attachment of the cellular receptor sialic acid on carrier molecules, the viral spread is reduced [94]. Adenovir Pharma AB develops these sialyated compounds and is currently in a phase

II clinical study with an eye drop administration [295]. Separately, NovaBay Pharmaceuticals is currently conducting clinical trials for treatment of adenovirus-caused EKC with their NVC-422 drug, a N-chlorotaurine analogue. NVC-422 has broad antimicrobial activity, and acts as a virucidal agent for adenovirus type 5 by oxidizing amino acids in the hexon and fiber proteins [296]. In addition, virucidal activity of NVC-422 was observed on HSV-1, coxsackievirus A24, enterovirus 70, adenovirus types 8, 19, 37, which all are ocular pathogens [297]. Adenovirus type 5 manifests a strong liver tropism, which impairs the efficacy of type 5 as a vector in gene therapy. Hence, small molecules have been identified that reduce the strong liver tropism [298]. In the blood, factor X, a central component of the coagulation system, binds to the virion and mediates virus entry in, specifically, liver cells [299]. Consequently, inhibition of the specific liver tropism may aid the gene therapy vector to be distributed to other tissues in the patient. In the HSV field, sulfated polysaccharides that mimic the HSV receptor heparane sulfate, are under development [300]. In addition, several peptides and proteins have displayed antiviral activity by blocking the viral entry. Example of this is lactoferrin/lactoferricin that can block both HSV and adenovirus entry by either interacting with glycosaminoglycan receptors on the host cell or by direct binding to the virion [301-304].

4.2. Inhibitors of DNA replication – Nucleoside/Nucleotide analogues

The most successful approach for antiviral drug development has been the nucleoside and nucleotide analogues. However, the initial development of nucleoside analogues was aimed at treatment of cancer, and it was later discovered that some compounds were more suited to inhibit the viral replication. Today, nucleoside analogues are used as therapeutic drugs for the treatment of both cancer and viral diseases. Nucleosides and nucleotides are endogenous molecules and are the building blocks of DNA and RNA. They are inserted in the growing DNA strand by the DNA polymerase. Nucleotides have a phosphonate group attached to the sugar moiety of the nucleoside structure. However, the term nucleoside analogues may cover both nucleosides and nucleotides. Currently, there are over 25 approved antiviral nucleoside analogues [305]. The general mode-of-action features are similar among them. Hence, the mode-of-action for nucleoside/nucleotide analogues is exemplified here by cidofovir and acyclovir that are used against adenovirus and HSV. Cidofovir, (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine or HPMPC, is an acyclic nucleoside (cytidine) phosphonate with broad antiviral

activity against several DNA viruses (Fig. 6 and 18). The phosphonate mimicks a phosphate group but is protected against cleavage by cellular esterases [306]. Nucleotides need three phosphates to be incorporated in the DNA. Since the phosphonate group functions as a phosphate, cidofovir needs to be phosphorylated twice. Once inside the cell, cidofovir is phosphorylated by pyrimidine nucleoside monophosphate kinase. The second and last phosphorylation is performed by nucleoside diphosphate kinase, pyruvate kinase or creatine kinase [307] (Fig 17). Both these phosphorylation steps can occur in both infected and uninfected cells, though is more efficacious in infected cells [308]. When cidofovir is di-phosphorylated, at the phosphonate group, it is a more specific substrate (or competitive inhibitor) for viral DNA polymerases than cellular DNA polymerases, hence the antiviral specificity [309, 310]. Integration of the mono-phosphate cidofovir molecule into the growing DNA strand terminates further chain elongation (Fig. 18). However, two consecutive incorporations of cidofovir is needed for chain elongation termination, at least in cytomegalovirus inhibition [311].

Figure 18. Mode-of-action for cidofovir. Figure reprinted from [312] with permission.

Acyclovir, 9-[(2-hydroxyethoxy)methyl]guanine, is an acyclic nucleoside (guanosine) analogue specifically active against herpesviruses (Fig. 13 and 19). The herpesvirus specificity is due to the initial phosphorylation of acyclovir, conducted by the virus-encoded thymidine kinase (TK) [285, 313]. Acyclovir monophosphate is successively phosphorylated twice by cellular kinases [314] (Fig. 18). Similarly to cidofovir, the triphosphate structure of acyclovir is a substrate for the viral DNA polymerase, which incorporates the mono-phosphate structure in the DNA strand and further chain elongation is inhibited. Resistance to acyclovir is most commonly due to mutations in the viral TK gene that inhibits the initial phosphorylation of acyclovir [315]. However, mutations in the viral DNA polymerase, with or without TK mutations have been reported as well [274, 316].

Figure 19. Mode-of-action for acyclovir. Figure reprinted from [312] with permission.

Foscarnet, phosphonoformic acid (Fig. 14), is used on acyclovir-resistant HSV and is active against all herpesviruses. It inhibits the viral DNA polymerases in a different way compared to acyclovir. The structure of foscarnet mimics pyrophosphate, which is produced during DNA chain elongation [317]. Consequently, foscarnet binds to pyrophosphate sites on the viral DNA polymerase and inhibits the release of pyrophosphates formed when tri-phosphorylated nucleotides are incorporated in the DNA [276]. It has been stated that foscarnet is about 100-fold more selective for herpes

DNA polymerase than cellular DNA polymerases [318]. Foscarnet is not active against adenoviruses [319]. Ribavirin, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Fig. 6), has previously been used for treatment of adenovirus disease but is currently not recommended as antiviral alternative [194]. The mode-of-action is controversial since several contradictory observations have been reported. Ribavirin is similarly to cidofovir, tri-phosphorylated by cellular kinases and it has been observed that ribavirin depletes nucleotide pools, causes lethal mutagenesis of viral RNA genomes, potentiates interferon action, reduces viral transcription, and more [320-323].

4.3. Inhibitors of viral maturation - Protease inhibitors

Targeting viral proteases is a promising antiviral strategy since they are virus-encoded and most often have essential roles in the infection cycle [324]. Thus, selective inhibition of the viral protease is less likely to have cellular side effects. Development of protease inhibitors has been focused on HIV-1 and Hepatit C Virus (HCV). However, the adenovirus and HSV protease have recently been assessed as potential antiviral targets. Both the adenovirus protease and the HSV protease are essential for infective progeny virions and they are involved in the late virion maturation process (See section 2.5.5. and 3.5.3, respectively). Broad cysteine protease inhibitors have antiviral activity against adenovirus [325]. Additionally, by utilizing a virtual docking approach, several small-molecule inhibitors of the adenovirus protease have been identified [43]. Characterization of these compounds is needed to determine their in vivo potential. In the herpesvirus field, protease inhibitors have been identified although mainly focused on the CMV protease [326, 327]. More general cysteine protease inhibitors have been reported to have activity on HSV protease [328]. In addition, the clinically used HIV-1 protease inhibitor Nelfinavir, has been reported to inhibit the maturation and export of HSV-1 [329].

4.4. Antiviral prospects

A recent aspect in antiviral drug development is to target the host cell instead of the virus, since there are several events in the viral infection cycle that are dependent on cellular proteins. Advantages of this approach may be that virus is less prone to develop resistance to cellular proteins and that these antiviral drugs will be active against viral mutants that already have developed resistance to conventional antivirals. Antiviral effects mediated via cellular targets would also be expected to have a broad antiviral activity since several viruses require mutual cellular proteins. In addition, a combination therapy with conventional antiviral drugs and cellular antiviral

drugs would have beneficial effects. Unfortunately, targeting cellular proteins may be associated with more cytotoxicity and side effects, although several drugs on the market today targets cellular proteins without severe toxicity. Identification and inhibition of a cellular protein that is upregulated during viral infection would be a desirable target. Cyclin-dependent kinases (CDK) are essential for replication of several viruses and have been expoited as antiviral targets. CDK inhibitors such as, roscovitine with selectivity for CDK2, CDK7 and CDK9, and the ATP competitor olomoucine, with selectivity for CDK2/cyclin interaction, have displayed antiviral activity against HSV-1 and CMV, respectively [330, 331]. Since then, several purine containing CDK inhibitors have been reported to have antiviral activity against HSV-2, HIV, Varicella-zoster virus (VZV) and Epstein-Barr virus (EBV) [332-334]. Roscovitine has displayed both positive and negative results in mouse models. In SCID mice with skin VZV xenografts, viral reduction was observed, whereas in a HSV-1 keratitis model no significant reduction was seen with roscovitine treatment [335, 336]. Interestingly, cidofovir in combination with the CDK inhibitor olomoucine II have been reported to almost completely eliminate the spread of adenovirus type 4 spread from infected cells [337]. However, to truly address CDK inhibitors as antiviral agents, further developments in vivo are needed. Moreover, with the increasing acceptance of cellular targets as antiviral agent, several reviews with focus on cellular targets as antivirals have been published. These include influenza virus, HIV (entry inhibitors) and arenavirus [338-340]. Another evolving aspect in antivirals, that is not drug-related, is adoptive T-cell transfer. The first report of adoptive transfer of immunity dates back to 1955 [341]. However, with the recent expansion of molecular techniques and knowledge the idea is more and more clinically used. Briefly, in this case, adoptive T-cell transfer is collection of virus-specific T-cells that, after in vitro expansion, are transferred to the patient. The virus-specific T-cells are collected from seropositive donors and transferred to seronegative recipients. This approach has proven successful for a number of viral infections (as well as cancers). In immunocompromised patients, the depletion of virus specific T-cells is a prominent risk for disseminated infection of opportunistic viruses, such as adenovirus. Hence, preemptive treatment with adenovirus specific T-cells infusion has been reported to reduce the rate of primary infections and reactivations [206, 342]. Due to the effective therapy with acyclovir and its analogs, adoptive T-cell transfer is not emphasized for recurrent HSV infections. However, for other more latent species of herpesvirus, such as CMV and EBV that also may have major complications in the immunocompromised patient, adoptive T-cell transfer is implicated [343-345].

5. Drug discovery and development

The driving force for each drug discovery project is to find cures for diseases or conditions that lack satisfactory therapies. Knowledge of the underlying mechanisms for the disease is critical to identify possible drug targets and to develop assays that can distinguish active compounds (hits) from non-active. Basic research within the academic, is most often the foundation for the drug discovery process. The drug discovery process is divided in several phases and in general each phase must be completed before proceeding to the next (Fig. 20). However, based on the outcome, reassessments within the process are frequent.

Figure 20. The drug discovery and development process.

5.1. Targets and assays

Most often, the target is a protein essential for manifestation of the disease. Enzymes and receptors are attractive drug targets since their activity easily can be measured [346]. Target identification is an important, if not the most important, step in drug discovery since all of the succeeding steps are dependent on a good target. A good target has to fulfill many criteria such as, i) the activity of the target is specific for the disease; ii) it is safe to modify the function of the target; iii) the target is “druggable”. Druggable targets are prone to interact with compounds and upon compound interaction, the function of the target may be altered to benefit disease treatment. In addition, the function of the target can easily be measured in vitro and in vivo. When screening for compounds that directly affect the function of a target protein, the assay is called biochemical or target-based assay. However, when assessing the impact of compounds in whole cells, e.g. using a GFP-expressing viral vector, the assay is called cell-based or phenotypic. Prior to screening, the assay needs to be optimized and quality assured. It is fundamental to have a significant separation between positive and negative

signals to be able to select hit compounds. By calculating the Z´-factor of the assay, the separation and ultimately the quality of the assay can be determined [347].

5.2. Compound Libraries

Today there are numerous commercially available compound libraries. The size varies between a few hundred compounds to several hundred thousands compounds. Initially, the philosophy was the bigger the better, i.e. the aim was to screen as large library as possible. And still in the pharmaceutical industry, the compound collections can be one million compounds and beyond. However, more and more are now aiming for more focused libraries. These smaller libraries can be diverse and still represent the chemical space, although with less number of compounds, and more focused towards interaction with known target families, e.g. kinases, ion-channels, hormone receptors etc. In addition, NMR-guided fragment-based library screening is common. Fragment-based libraries consist of smaller compound entities that potentially can interact with different parts of the target. Hence, by combining the segments a larger chemical space can be covered compared to conventional compound libraries [348]. The compound source is mainly from synthetic chemistry, e.g. fragment-based, diverse and targeted libraries, however commercial natural product libraries are also available that include samples from e.g. plants, invertebrates and microorganisms. In addition, library collections with already developed drugs are available. These compounds may have been used in the clinic or have passed phase 1 clinical trials. Screening these drugs against new targets may lead to that approved drugs can be re-targeted for other indications.

5.3. Screening and lead identification

High-throughput screening (HTS) is widely used in the pharmaceutical industry and it enables large numbers of compounds to be screened for biological activity in a short period of time. Using fully integrated robots for liquid handling and pipetting, 100,000, or more, compounds can be assessed against a target each day [349]. In general, HTS is mainly used for target- based screening, whereas for cell-based screening, High-content screening (HCS) is mainly used. The end analysis for HTS is measurement of a proteins activity using, e.g. microplate readers, whereas for HCS, the cellular phenotype is analyzed using, e.g. automated microscopy. In an academic setting, screening may be directed against diseases but can also be directed to identify chemical probes that alter specific biological pathways. These chemical probes are used to understand basic biological processes. Additionally, academic screens usually analyze smaller sets of compound

libraries and are not fully automated but most often semi-automated, e.g. with pipetting robots that aid manual laboratory work. When the screening assay is developed and quality assured, the primary screen is usually performed at one concentration in a single experiment. Based on the Z´-factor and preferably the activity of positive control compounds, the criteria for hit selection are determined. Hit selection rate describes how many compounds of the compound library that fulfill the criteria. The activity of the hits needs to be confirmed, primarily by re-testing the compounds under the same conditions and generation of dose-response curves. In addition, the activity of the hit compound is validated, e.g. to ensure that the signal is truly due to interaction with the target and not general toxicity or assay interference. Subsequently, the hit compounds are tested in orthogonal assays, i.e. different assays that quantify the activity of the compounds. Both the efficacy and the toxicity of the compound are addressed. Based on the outcome, compounds are selected for medicinal chemistry programs to optimize the efficacy and cytotoxicity of the compounds. Structure-activity relationship (SAR) analysis assesses the chemical space around a compound with the intention to find structural analogues with improved activity. Most often the analogues are synthesized in generations and the activity of each generation guides the synthesis of the next. The SAR analysis narrows down the number of hits to a few compounds with improved activity. These optimized hits are called leads.

5.4. Lead optimization

Lead optimization involves both in vitro and in vivo assessment and includes a variety of parameters. The overall aim is to determine the pharmacological aspects and to ascertain that the lead compounds have potential to be used as drugs in man. Exploratory pharmacokinetic (PK) analysis of the leads is performed, both in cell culture and in animal models, which mainly are mouse models at this phase. PK parameters include the ADME steps (Absorption, Distribution, Metabolism and Excretion) that describe behavior of the compounds in an organism (here, most often in mouse). If a pharmaceutical formulation of the compound is needed, the compound liberation rate from the formulation, among other parameters, is analyzed as well. Thus, L may be added to the ADME parameters to form LADME. In addition, preclinical proof-of-concept studies in vivo are performed to establish the activity of the compound in an organism. With medicinal chemistry the compounds are optimized and modified if any unfavorable results are observed. Based on the outcome for each lead compound, one, or possibly two to three compounds are selected for further development.

5.5. Drug development

Drug development is the later stage when candidate drugs will be assessed with a clear aim to reach the market and the patients. Drug development includes thorough preclinical analysis in two or more species of rodents and non-rodent animal models. Here, a full ADME-tox analysis is performed to determine the absorbance rate of the drug to the blood, how the drug is distributed in the body, how the drug is metabolized, the complete toxicity of the drug with its metabolites and the mechanism when drug metabolites are excreted. The drug and its formulation, needs to be produced according to good manufacturing practice (GMP) to secure high quality. Once the preclinical process is complete, a regulatory approval is needed for candidate drugs to enter the phase I-III clinical assessment in humans. Briefly, in phase I trials the drug is administrated to a limited number of healthy volunteers to assure that the drug is tolerated without apparent side-effects. Phase II trials includes the efficacy of the drug against the target in a larger group of patients with the disease/condition. In addition, the intervals of the dosage of the drug are analyzed. Phase III trials includes more patients with the disease, which are closely clinically monitored to detect any potential side effects of the drug. When the drug is approved, by regulatory authorities, it can be marketed and become accessible for the patients. In summary, it takes between 10-15 years to develop a new drug, including the drug discovery phases. To develop a drug that reaches the market generally costs more than 1 billion US dollars. However, this number is increasing for each year and currently, big pharmas can spend up to 5 billion US dollar per drug [350].

5.6. Orphan drugs

Adenovirus infections in immunocompromised patients are associated with morbidity and high mortality rates, especially in children (see section 2.6.2). Fortunately, the number of patients affected by life-threatening adenovirus disease is low and the condition is considered to be rare. Rare conditions are seldom attractive for larger pharmaceutical companies, mainly due the massive costs when developing a drug contra the small market for rare conditions. However, it has been estimated that between 6 – 8 % of the total EU population suffer from rare diseases [351]. Hence, to stimulate development of drugs for rare conditions, the FDA implicated an orphan drug act in 1983 [352]. This legislation was successively adopted by Singapore in 1991, Japan in 1993, Australia in 1997 and in 2000 by Europe [353]. The orphan drug legislation includes stimulatory incentives if the development of a drug is intended for the treatment of a rare disease.

However, the disease needs to fulfill certain criteria to be classified as rare. These criteria differ between countries. In EU and US the number of persons affected by a rare disease should not exceed 5 in 10,000 citizens. In Japan, any disease with fewer than 50,000 prevalent cases (0.4 %) is defined as a rare disease. In Australia, if a drug used to treat a disease that affects fewer than 2,000 individuals, the disease is considered to be rare [354]. In addition, no satisfactory treatment for the diseases should be available. If the disease meets these criteria, the inventors, or sponsors, can apply to regulatory authorities for an orphan drug status. However, the European commission is the decisive body in consultation with the Committee for Orphan Drug Medicinal Products (COMP), which handle the applications. In the US, the FDA Office of Orphan Products Development (OOPD) is responsible for the decisions of orphan drug status. If the applicaton is approved, the drug is designated with an orphan drug status and the development of the drug may be stimulated under the orphan drug legislation. The legislation includes several incentives. Perhaps the most appealing incentive for pharmaceutical companies is the ten-year, or seven years in US, market exclusivity of the drug [355]. The additional incentives are reduced charge for scientific advice and reduced fees for regulatory activities, e.g. application inspections and support. In addition, in the EU, the marketing authorization is central and valid in all EU member countries. Importantly, other drugs intended for the same rare disease must be proven superior to the current orphan drug, for orphan drug status approval and access to the stimulatory incentives [356]. However, if the second applicant of the orphan drug status has the consent from the original orphan drug applicant, two orphan drug designations are possible for the same disease [355]. The European Medicines Agency (EMA) has classified adenovirus infection in the immunocompromised host, as a rare disease at least twice. In 2001 they stated that 0.6-3 in 10,000 persons in EU are affected and in 2014, that 0.2 in 10,000 persons in the EU were affected [189, 190]. Hence, orphan drug status could possible be achieved. To my knowledge, Cell Medica Ltd. is the only applicant with approved orphan drug status for treatment of adenovirus infection in allogeneic haematopoetic stem cell transplant recipient [357]. Cell Medica Ltd. develops immunotherapeutics and adenovirus specific T cell transfer (see section 4.5). The promising drug Brincidofovir (see section 2.7), is a broad-acting antiviral that is in phase III for treatment of severe adenovirus disease but is not (officially) designated as an orphan drug. However, Brincidofovir was recently approved for compassionate use in Josh Hardy, a 7-year old boy suffering from a dissiminated adenovirus disease. This case has recently been spread in social media worldwide [358, 359].

5.7. Natural products in drug discovery

Natural products include all compounds produced by living organisms. However, from a pharmaceutical perspective, natural products mainly include secondary metabolites that are produced by an organism in response to external stimuli. These stimuli can be nutritional changes, infection or competition between organisms. Hence, for the organism, secondary metabolites may function as differentiation effectors, competitive weapons, sexual hormones, spore formation stimulators, quorum sensing signals and more [360, 361]. In addition, secondary metabolites are not essential for the growth, development and reproduction of the producing organism. The production of secondary metabolites does not follow the growth phase (trophophase) of the organism but is produced in a subsequent stage (idiophase) [362]. Many organisms have the potential to produce secondary metabolites, including plants, microbes, fungi and animals. However, this section will mainly focus on secondary metabolites derived from microbes. Secondary metabolites have been a rich source for the identification of new compounds in drug discovery. The most well known drugs that originate from secondary metabolites may be the antibiotics, including the first discovered antibiotic penicillin. However, numerous secondary metabolite- derived drugs are currently used for treatment of a variety of disease areas, e.g. paclitaxel and doxorubicin (anti-cancer), rapamycin (immunosuppressant), amphotericin B (anti-fungal), avermectin (antihelmetic), acarbose (anti-diabetes) and more [363-367]. In addition, approximately 50 % of the approved new chemical entities between 1981- 2006 are secondary metabolites or derivates thereof [368]. However, with the introduction of HTS in the 1990´s, the focus on secondary metabolites as a source for new drug leads was shifted towards combinatorial chemistry and synthetic compound libraries [369]. The advantages of combinatorial chemistry are the production of enormous amounts of new compound structures, which biological activity easily can be assessed using HTS and future favorable intellectual property aspects. Nonetheless, the combinatorial chemistry approach has resulted in relatively few new approved drugs on the market. Consequently, the secondary metabolites have recently regained focus as a source for discovery of new drug leads, particularly in infection research. Secondary metabolites are generally more complex in their structure, compared to synthetic compounds. This complexity has been selected during evolution to interact with biological targets. Hence, a combinatorial approach, designated diversity-oriented synthesis (DOS), has been utilized to combine the complexity of secondary metabolite structures with synthetic chemistry for the production of more natural product-like compounds [369]. DOS, applied for natural products, assesses the chemical space around a privileged structure of a bioactive

secondary metabolite and considers secondary metabolites as starting points for further structural optimization. This may result in numerous analogues that may have more potent and more targeted activity than the original structure. In addition, by structural classification of scaffolds for the to-date known secondary metabolites, the chemical space explored by nature can be visualized [370]. Plants are the major producers of secondary metabolites followed by microbes. Among the microbes, actinomycetes are known to produce a variety of bioactive secondary metabolites and are responsible for about half of the to-date known microbial secondary metabolites [371]. Actinomycetes, or actinobacteria, are gram-positive bacteria abundantly found in soil where they play important roles in the decomposition of organic matter. In the phylum of actinobacteria, the genus Streptomycetes is the major producer of secondary metabolites that have been developed to clinically important drugs, mainly antibiotics, e.g. streptomycin, chloramphenicol and tetracycline [372]. The high productivity of secondary metabolites among actinobacteria is associated with their large genomes, which harbors several gene clusters for secondary metabolism. The genus Streptomyces has in general a genome, between 8-10 Mb, which contains over 20 secondary metabolic gene clusters for biosynthesis of polyketides, peptides, terpenoids, shikimate-derived metabolites and more [373]. Thus, actinobacteria, and especially the genus Streptomycetes, which both are easily accessible in soil, have been extensively studied for their capacity to produce bioactive compounds with drug development potential. Consequently, the isolation rate of new actinobacteria-derived secondary metabolites has declined while the rate of re-isolation for previously known compounds with already characterized bioactivity has increased [374]. With the emerging drug- resistance worldwide, it is eminently crucial to find new compounds that have drug development potential. This has lead to a quest to identify new sources and environments that may harbor uncharacterized actinobacteria, which may produce new types of secondary metabolites. The oceans cover more than 70 % of the surface of the earth and its microbial diversity is still relatively unexploited. It has recently been recognized that marine actinobacteria can produce secondary metabolites with unusual structures and properties [375, 376]. Hence, marine actinobacteria are an unexploited source for new secondary metabolites. Isolation and characterization of these compounds will hopefully lead to new therapeutic agents.

Aims

The overall aim of this thesis was to identify and characterize compounds with antiviral activity against adenovirus. The specific aim for each paper was:

I - To develop a whole cell-based screening assay utilizing a replication competent GFP-expressing adenovirus vector. Furthermore, to screen a diverse set of 9800 small molecules for anti-adenoviral activity and select potential hit compounds for further characterization.

II - To perform SAR-analysis of a hit compound class found in the screening, for evaluation of the chemical space around the structure and for structure optimization.

III - To determine the antiviral specificity for the optimized compound class, by evaluation of the antiviral activity against HSV and clinical acyclovir- resistant HSV isolates.

IV - To establish a library of secondary metabolites derived from marine actinobacteria and to screen the library for anti-adenoviral activity. Furthermore, to identify and characterize potential hit compounds.

Methods

Screening assay

This thesis is based on the screening of compounds for anti-adenoviral activity using a robust screening assay based on a unique replication- competent adenovirus type 11 GFP-expressing vector (RCAd11GFP). RCAd11GFP has the GFP-gene inserted upstream of the E1-region of the adenovirus genome without hampering the infectivity of the virus [377]. In addition, RCAd11GFP manifests a strong GFP expression that initially can be visualized 4 h post infection (unpublished data). However, 24 h post infection the GFP expression reaches its maximal plateu and the GFP signal can easily be observed in microscope or be quantified using a plate reader [378]. We used this vector as a tool for detection of compounds that reduced the GFP expression without killing the cells. In the first paper, we assessed a diverse collection of 9800 small drug-like molecules using suspensional human erythroleukemic K562 cells that are highly susceptible for adenovirus type 11 [379]. Compounds and RCAd11GFP were added simultaneously to the wells, to achieve 50 µM compound concentrations and 1 pg per cell of the vector. The GFP expression was measured in a plate reader, 24 h post infection. In parallel, the cytotoxicity of the compounds was quantified using an MTT-based cytotoxicity assay. This assay measures the potential of mitochondrial enzymes to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) to a coloured formazan, which is quantified [380]. Thus, the viability of the cells can be determined. We defined criteria for selecting hit compounds as, reduction of 80 % or more of the GFP-signal and 50 % or more of viable cells. These criteria yielded a hit rate of approximately 4 %. These compounds were re-tested in a dose- response analysis and the most potent compounds were selected for further hit evaluation. In the second screening campaign (paper 4) the parameters in the screening assay were slightly different. Here, we utilized a High Content Screening (HCS) automated microscope for measuring the RCAd11GFP derived GFP-expression. In addition, this automated microscope facilitated an initial evaluation of the cytotoxicity of the compounds since the morphology of the Hoschst-stained nuclei easily could be assessed. Adherent human lung carcinoma, A549, cells were used in the screening and we lowered the amount of RCAd11GFP to 0.025 pg per cell due to the high sensitivity of the HCS automated microscope. Our established extract library of secondary metabolites from marine actinobacteria contained unknown compounds at unknown concentrations. Hence, the compound concentrations in the wells were unknown. However, based on previous

biological assessments of the extracts (unpublished data), we diluted the extracts to 1:200 in the wells. As previously, RCAd11GFP and the compounds (extracts) were added simultaneously and the microplates were analyzed 24 h post infection. Hoeschst was added to the wells 20 min prior to analysis. After analysis in the HCS automated microscope, the cytotoxicity was measured using an XTT-based toxicity assay. This assay measures the potential of mitochondrial enzymes to reduce 2,3-bis-(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) to a coloured formazan, which is similar to the MTT assay, albeit with fewer steps and higher sensitivity [381]. Extracts that decreased the GFP-signal without apparent toxicity were selected for further dose-response analysis. Potent extracts were fractionalized using HPLC and each fraction was re-tested in the screening assay for identification of active fractions. The compounds in the active fractions were structurally determined. Database and literature searches revealed that three of our hit compounds were previously known with assessed antiviral activity (Pentalenolactone, Nonactin and Cycloheximide). However, the fourth compound was a known butenolide compound with previously uncharacterized antiviral activity. Due to the lack of effective antivirals that targets adenovirus infections, the Z´factor for the two screening campaigns could not be determined. The Z´factor requires both negative and positive controls to define the quality of the screening assay (see section 5.1).

Hit expansion

The synthesis of compounds followed conventional chemistry using typical methods as liquid chromatography-mass spectrometry (LC-MS), high- performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). The purity of the synthesized compounds was above 95 %.

Assays for hit evaluation

To characterize the antiviral activity of the hit compounds and their analogs, we have utilized several assays that each analyse different stages in the infection cycle. In chronological order; to assess if the compounds inhibited the binding of the virus to the cells we used a binding assay with 35S labelled adenovirus virions. This facilitates the quantification of the amount of virions that are bound to the cell surface at 4°C (used in paper I and IV). Furthermore, to analyse if the compounds inhibited the uptake and the intracellular transportation to the nucleus, we labelled adenovirus type 5

virions with the fluorphore Alexa fluor 555. Using confocal microscope and inverted fluorescence microscope, individual adenovirus particles could be traced intracellulary along the microtubules and to the nuclear pore (data not published). To assess the replication of viral DNA, during which most antiviral compounds have inhibitory effect, we performed quantitative PCR (qPCR). The number of newly synthezised viral genomes was determined using specific primers against the genes of structural proteins of both adenovirus and HSV [382-384]. In parallel, the number of analysed cells was determined by the use of primers for the housekeeping gene RNAseP. Hence, the number of viral genomes per cell can be expressed. The qPCR assay is a very sensitive assay that can distinguish small variations in the number of genomes. Consequently, the qPCR-assay was the most frequently used assay for assessment of the antiviral activity of the compounds. In addition, dose- response analysis and EC50 determination of the compounds were performed using qPCR. The later steps in the infection cycle was analysed using flow cytometry. Using a monoclonal antibody (mAB8052), directed against newly synthezised adenoviral hexon proteins, the potential antiviral effect on the viral protein translation could be evaluated. However, if the antiviral effect is due to inhibition of a prior step of the infection cycle, the antiviral effect will be observed in the later steps as well. Assessment of the toxicity of compounds was performed with several assays. The predominant assay used, was the XTT-assay (described in section screening assay above). Additionally, the DNA intercalating agent, propidium iodide (PI) was used to stain the DNA of dead cells, using flow cytometry (used in paper I). Moreover, to determine any cell membrane disruption, the extracellular release of lactose dehydrogenase (LDH) was analyzed using a LDH cytotoxicity kit (unpublished data).

Results and Discussion

The discussed figures and tables herein refer to each paper, unless otherwise stated.

Paper I

There is a need for new antiviral drugs, especially for the treatment of severe adenovirus infections. Today there are no approved anti-adenoviral drugs available, although adenovirus infections are associated with morbidity and high mortality rates in, particularly immunocompromised children. With this in mind, we have developed a whole-cell based screening assay using a unique replication competent GFP-expressing adenovirus vector (RCAd11GFP), to screen a diverse chemical library of 9800 small molecules. This library was selected to cover a large chemical space. The main advantages of cell-based screening assays are the discovery of potential novel targets and the knowledge that the compounds have potential to pass the cell membrane. In addition, in cell-based assays the preliminary cytotoxicity of the compounds can be assessed. In target-based assays, non-target interactions that may result in toxicity, is not observed. Additionally, potent compounds that interact with the intracellular target may not sufficiently pass the cell membrane. The major disadvantage of cell-based assays is that the study design will not provide any information on the mode-of-action of the compounds. However, when considering the need for novel antiviral compounds with, preferably, new modes-of-action, a cell-based assay is to prefer. The screening campaign resulted in 408 hit compounds that fulfilled our hit selection criteria (see previous section Methods – Screening assay). These 408 compounds were re-tested in dose-response analysis and we selected 24 of these compounds for further analysis. The antiviral activity of these 24 compounds was assessed in a number of assays, each reflecting a different step in adenovirus infection cycle (see previous section Methods – assays for hit evaluation). Based on the overall results, we selected 2-[2- (benzoylamino)benzoylamino]-benzoic acid. This compound was designated as A02 in paper I and as 1 in paper II. In paper III we designated the

compound class as Benzavir and 2-[2-(benzoylamino)benzoylamino]- benzoic acid as Benzavir-1. Hence, 2-[2-(benzoylamino)benzoylamino]- benzoic acid will hereinafter be designated as Benzavir-1. Benzavir-1 was active against representative adenovirus types from several species with a EC50 value between 2.4 - 4.7 µM. In addition, Benzavir-1 was about 5 times more potent than cidofovir (Table 2). Benzavir-1 did not display any inhibitory effect on the binding of the virus to the cells (Fig. 5a). However, moderate antiviral activity was observed when assessing the GFP-expression from the RCAd11GFP vector (Fig. 1). Surprisingly, when re-assessing Benzavir-1 in conditions similar to the original screening, the percentage inhibition does not reach 80 %, which was one of the hit selection criteria for the screening (Fig. 1a). However, when the GFP signal was measured in A549 cells and analyzed in flow cytometry, the percentage inhibition reached 80 % (Fig. 1b). Assessment of the effect of Benzavir-1 on the replication of adenovirus DNA revealed prominent inhibition, using the more sensitive qPCR assay (Fig. 4). In the step subsequent to DNA replication, the expression of structural hexon proteins, the antiviral potency is moderate (Fig. 5b). The reason for this is unknown but this could be due to the fact that transcription and expression of hexon proteins still can occur from a reduced number of viral DNA templates.

The toxicity of Benzavir-1 is low. The CC50 value was determined to 199 µM on A549 cells and approximately 1 % of the A549 cells were non-viable according to PI toxicity assay (Fig. 4 and Fig. 5c). To our surprise, the purchased batch of Benzavir-1 turned out to contain two additional compounds, one two-ring structure and one four-ring structure (Fig. 2). These compounds are most probable remnants during synthesis. Fortunately, the three-ring Benzavir-1 structure was determined to be the active structure (Fig. 3). However, this represented an initial structure-activity relationships (SAR) analysis of Benzavir that was expanded upon in paper II.

Paper II

To assess the chemical space around Benzavir-1 and to potentially discover analogues with improved potency, we performed SAR analysis of Benzavir-1. Paper I displayed that only the trimeric Benzavir-1 structure was active, hence, only trimeric analogues were analyzed. The SAR-analysis was performed in three generations of studies where the biological activity of the analogues in each generation guided the synthesis of the next. A total set of 42 compounds was analyzed. In brief, the aim of the first generation of analogues was to determine the importance of the ortho-ortho substituent pattern of Benzavir-1 (designated as 1 in paper II). The carboxylic acid on the right-hand ring and the substituents of the central ring was moved to meta and para positions. In addition, the importance of the carboxylic acid was determined. The general conclusion of generation 1, was that the ortho-ortho substituent pattern and the presence of the carboxylic acid in the ortho position was of importance for the antiviral activity (Table 1 and 2). The second generation focused on the N-terminus, left-hand side, of the structure. By modifications of the ring-structure and by substituent attachments at various positions, we concluded that the overall antiviral activity of these compounds were moderate. However, by attachment of an electron-withdrawing substituent, fluoride (F), in the ortho position in the left-hand ring the activity was slightly increased compared to the activity of Benzavir-1 (compound 17h, Table 3). The third and last generation of analogues was a continuation of compound 17h, where electron-donating substituents, methoxy (OMe) or electron-withdrawing substituents, F or chloride (Cl), was added to the central or right-hand ring of the structure (Table 4). Substituation of the middle ring was favorable, whereas on the right-hand ring substituation resulted in a decreased activity. Attachment of Cl and OMe on meta and para positions on the middle-ring was beneficial. However, two F on the middle-ring yielded the greatest antiviral activity (compound 35j in Table 4). Cl is larger and more lipophilic than F, thus increasing the size and lipophilicity of the molecule. The size of F is comparable with hydrogen (H), thus little change in the overall steric bulk of the molecule is expected. We excluded dual substituation with OMe and Cl on the middle-ring due to steric hinderence. Further evaluation of compound 35j displayed an improved EC50 value compared to Benzavir-1, from 3.7 µM

for Benzavir-1 to 0.58 µM. In addition, the toxicity at two fixed concentrations, 30 µM and 60 µM, was reduced (Table 5). We decided to assess the toxicity at two fixed concentrations below 100 µM to ascertain that no solubility issues would interfere with the toxicity read out. The improved analogue to Benzavir-1 is hereinafter designated as Benzavir-2. Benzavir-2 is produced in a five-step synthesis and is currently our lead candidate, however synthesis and biological assessments of additional Benzavir-2 analogues may result in further improved compounds.

Paper III

The discovery of Benzavir-1 as a novel anti-adenoviral compound, in paper I, was followed by paper II, in which the exploration and evaluation of Benzavir-1 analogues was described. Paper II yielded the improved compound Benzavir-2. Here, in paper III, the antiviral activity of both Benzavir-1 and Benzavir-2 were assessed on another virus of medical importance, namely HSV. In addition, the activity on clinical acyclovir- resistant isolates of HSV was determined. Since Benzavir-1 is active against representative types from all adenovirus species (Table 2 in paper I), the initial aim of this paper was to evaluate the antiviral specificity of this compound class. HSV and adenovirus are both DNA viruses, and they share general steps in their replication cycle. However, HSV and adenovirus utilizes and express different proteins, all of which each may serve as an antiviral target. Hence, to determine if the antiviral activity of Benzavir-1 and -2, is specific for adenovirus or if the antiviral activity would also include HSV, we assessed the number of HSV genomes by the qPCR assay and determined the antiviral activity on both types of HSV. For initial analysis we assessed the anti-HSV activity of both Benzavir-1 and -2, at two fixed concentrations, 15 and 5 µM (Fig. 2). Acyclovir was included as positive control and as observed, all three compounds almost completely inhibited the HSV infection at 15 µM. Additionally, at 5 µM, Benzavir-2 and acyclovir were the most potent compounds with approximately 80 %, or more, inhibition whereas Benzavir-1 displayed around 70 % inhibition. Further characterization concluded that the EC50 values of Benzavir-2 and acyclovir on both HSV types were similar, ranging from 1.5 to 1.6 µM (Fig. 3).

However, as concluded in Fig. 2, Benzavir-1 was less potent with an EC50 of 3 and 4.3 µM, respectively. In addition, the clinically used antiviral drug cidofovir was assessed (Fig. 3b). Cidofovir displayed an EC50 value of 11 µM on HSV type 1, which indicates that our improved compound, Benzavir-2, is approximately seven times more potent than cidofovir. The profile of the dose-response curves differs between our Benzavir compounds and, most notably, acyclovir. The reason for this is unclear, however acyclovir and cidofovir inhibits the viral DNA replication in a two-step process, with essential phosphorylation steps prior to insertion to the growing viral DNA strand. The initial phosphorylation step of acyclovir, by the viral TK, is a rate-limiting step. Hence, a 1:1 stoichiometry and a straighter dose-response slope can be expected for acyclovir. However, cidofovir does not require the initial phosphorylation, due to its phosphonate group, thus the dose- response slope is not as straight as for acyclovir. The mode-of-action for Benzavir-1 and -2 is currently not known, consequently the impact of the steep dose-response curve can only be speculated upon. As in paper II, we determined the toxicity of the compounds at two fixed concentrations, 30 and 60 µM. Benzavir-2 has reduced toxicity compared to Benzavir-1, however no toxicity was observed for acyclovir (Table 4). Nonetheless, acyclovir is developed to be active only in virus-infected cells that produce the viral TK protein. Thus, acyclovir in uninfected cells is considered to be inactive. In collaboration with professor Tomas Bergström at Sahlgrenska University Hospital in Gothenburg, we obtained five acyclovir-resistant clinical isolates of both HSV types. Four isolates were identified as HSV type 2 and one was HSV type 1. These isolates were collected at various lesion sites from patients aged 52 to 83 years (Table 1). In comparison to the age of the patients with acyclovir-sensitive HSV isolates, which were collected from a 22- and a 24-year-old patient, the age of the patients suffering from acyclovir-resistant HSV is substantially higher. In general, acyclovir- resistant HSV is more frequently isolated from elderly patients than younger patients. The reason for this is probably due to conditions that are associated with a compromised immune system, which is more common in elderly persons. As expected, acyclovir had low or moderate effect on these isolates. However, both Benzavir-1 and -2 was active in the initial antiviral assessment at 15 and 5 µM (Table 2). Although as previously, Benzavir-2 displayed improved potency compared to Benzavir-1. Thus, Benzavir-2 was selected for thorough dose-response analysis (Fig. 4). Here, Benzavir-2 displayed similar EC50 values ranging from 1.1 to 1.6 µM, against all acyclovir-resistant isolates. Consequently, Benzavir-2 is active against both acyclovir-sensitive and acyclovir-resistant HSV isolates of both types, with similar potency. Hence, it can be concluded that Benzavir-2 and acyclovir does not share exactly the same mode-of-action. In addition, Benzavir-2 is a

promising antiviral compound that deserves further characterization as a drug candidate.

Paper IV

The previous three papers originated from a screening campaign where 9800 small molecules were screened for anti-adenoviral activity, using a cell- based assay. In this paper, we expanded our screening-based approach and screen ethyl acetate extracts derived from marine actinobacteria that were isolated from sediments of the Arctic sea. Since actinobacteria is a major source for a variety of bioactive secondary metabolites, we wanted to assess the potential to discover compounds with antiviral activity. In addition, there are 1 million, or more, virus particles per ml of seawater [385]. Hence, organisms living in the sea must have evolved defense mechanisms against the persistent attack of viruses. This defense mechanism may have a broad antiviral activity and be active against other types of viruses, i.e. human adenovirus. The collaboration partners at University of Tromsö, Norway, performed the collection of sediment samples. The research vessel Jan Mayen (depicted on the back cover of this thesis), collected sediment samples from various depths outside the coast of Norway. From these sediment samples, actinobacteria were selected for their potential to produce diverse bioactive secondary metabolites (see section 5.7). In total, 57 actinobacterial strains were isolated. Furthermore, these actinobacteria were grown in different media compositions to form an ethyl acetate extract library containing 912 extracts. These extracts were screened using the RCAd11GFP vector and analyzed in an automated microscope (see previous section Methods – Screening assay). After bioactivity-guided fractionation of active compounds, one fractionation contained a diastereomeric mixture of butenolide compounds, previously known to the literature but with previously uncharacterized antiviral capacity (Fig 2). The bacterial strain that produced these compounds was isolated at GPS coordinates N67 52.11512 E16 22.97003 at a depth of 417 m. However, when optimizing the production of these butenolide compounds, three additional analogues were discovered (Fig. 2). We proposed biosynthetic pathway for these compounds based on their relative time of appearance during cultivation and their structure (Fig. 4). All isolated butenolide compounds were assessed in a dose-response

analysis, where potency differences among the compounds were observed (Fig. 5). In addition, the two most potent butenolide compounds, an analogue with keton side-chain (3) and an analogue with a non- functionalized side-chain (4), had similar EC50 values around 90 µM but displayed substantial differences in toxicity (Table 3 and Fig. 6). The toxicity was determined on two different cell types, human lung carcinoma (A549) cells and primary foreskin fibroblast (FSU) to assess any cell type specificity of the toxicity. However, the toxicity of compound 4 did not differ between the cell types. Hence, the most potent and non-toxic butenolide analogue 3 was selected for further characterization. Time-of-addition analysis revealed that cellular pre-treatment with compound 3, completely inhibited the adenovirus infection (Fig. 7A). However, no inhibitory effect of the viral binding was observed, or on the integrity of the viral particle (Fig. 7B and C). Interestingly, reduction of the 2-furanone moiety in the butenolide structure, eliminated the antiviral activity (Fig. 8B). In addition, the toxic potential of the analogue with a non-functionalized side-chain, 4, was slightly reduced (Fig. 8C). This suggests that both the side-chain and the 2-furanone moiety are involved in the bioactivity. It could be speculated that, the more lipophilic nature of 4 may increase the transport over the cell membrane, thus the intracellular concentration of 4 would be higher than 3. However, this is a bit contradictory since the antiviral potency for 3 and 4 is similar. Hence, the mode-of-action for these compounds needs to be determined to assess the importance of side-chain substituents. In sum, the butenolide compound 3 is an anti-adenoviral non-toxic compound that is small and amendable for further optimization of the anti- adenoviral activity.

Concluding remarks and future prospects

The overall aim of this thesis was to discover and characterize novel anti- adenoviral compounds that may meet the unmet clinical need among patients affected by severe adenovirus infections. Hence, the discoveries included in this thesis may lead to that new antiviral drugs will be evaluated for clinical use. Our most well characterized compound, Benzavir-2, has low toxicity and is active against adenovirus with an EC50 value below 1 µM. In addition, similar potency is displayed against HSV and clinical acyclovir- resistant HSV isolates. Thus, Benzavir-2 is a promising new antiviral compound. However, the mode-of-action for Benzavir-2 is currently not known. We intend to determine the mode-of-action for Benzavir-2. It is most likely that Benzavir-1 and -2 share the same mode-of-action. We have performed attempts to determine the mode-of-action, so far with inconclusive results. A biotinylated Benzavir-1 compound was synthesized

and was used as a bait to “fish-out” potential target proteins in infected and uninfected cell lysates. However, no specific target interactions could be determined. Thus, our next approach will be to develop Benzavir-2 - resistant adenovirus mutants. This approach has been used to identify mutations responsible for cidofovir-resistance in adenovirus [386]. Adenovirus will be propagated in the presence of increasing concentrations of Benzavir-2, which will be expected to trigger the development of escape mutants that are resistant to Benzavir-2. Subsequently, the mutant virus isolates will be sequenced and their genome composition analyzed for mutations. However, if Benzavir-2 interacts with a cellular protein(s) or a virus-induced cellular protein(s), the interpretation of the developed mutations will be a challenge. An alternative approach is to assess the antiviral activity on other types of viruses, including RNA-viruses, to expand assessment of antiviral specificity of Benzavir-2. Hopefully, we would identify a virus type that is not inhibited by Benzavir-2, which in comparison to other Benzavir-2 sensitive virus types, may aid the mode-of-action assessment by reducing the number of potential Benzavir-2 targets. Additionally, we can exclude steps in the adenovirus infection cycle that we know are not involved in the antiviral activity of Benzavir-1 and -2. We know that Benzavir-1 does not affect the integrity of the adenovirus virion (unpublished data) or inhibit the binding of the virion to the cell (Fig. 5a in paper I). In addition, we know that Benzavir-1 inhibits neither the uptake nor the intracellular transport of the viral particles to the nucleus, analyzed by fluorophore-labeled adenovirus virions (unpublished data). We also know that Benzavir-2 is not a general DNA intercalating agent (unpublished data). However, the major antiviral activity is observed in conjunction with the viral DNA replication. Hence, it is tempting to assume that Benzavir-2 affects the initiation or progress phase of the viral DNA replication. This hypothesis may also be supported by the requirement of Benzavir-2 presence either prior to, or during, the production and initiation of the viral DNA replication machinery that occurs 6-8 h post infection (unpublished data). If Benzavir-2 is added 8 h post infection no, or moderate, antiviral activity is observed. However, the molecular mechanisms that are involved in the antiviral activity for Benzavir-2 needs to be determined. So far, Benzavir-2 seems to be a promising antiviral compound in vitro. However, the in vivo situation may differ from the in vitro situation. Hence, we have initiated in vivo assessments of Benzavir-2 in mouse models. At present, Benzavir-2 is assessed in a vaginal HSV-2 mouse model in collaboration with professor Kristina Eriksson at Sahlgrenska University hospital in Gothenburg. In addition, we plan to develop an adenovirus mouse model, using a luciferase-expressing adenovirus vector and a non- invasive in vivo imagining platform, to quantify the potential anti-adenoviral activity of Benzavir-2 in the mouse.

To conclude paper I - III, we discovered Benzavir-1 as a novel anti- adenoviral compound, which potential was enhanced by the development of Benzavir-2. Both the antiviral activity and the toxic potential of Benzavir-2 have been thoroughly characterized. We aim to continue the characterization of Benzavir-2, to assess its potential as a new antiviral drug. Consequently, a Umeå Biotech Incubator (UBI) project was initiated that has resulted in a spin-off company, Eirium AB. Eirium AB has the intention to continue the characterization of Benzavir-2 towards the pharmaceutical market. Eirium AB has applied for patent that covers Benzavir-2 and additional chemical space, for both EU and US, and will perform PK analysis of Benzavir-2 both in vitro and in vivo. In addition, Eirium AB will assess the anti-adenoviral efficacy in vivo and the mode-of-action for Benzavir-2.

Paper IV was a new endeavor. Here, we established an extract library originated from actinobacteria isolated from the depths of Vestfjorden in Norway. We identified a butenolide compound class that inhibited the adenovirus infection and we proposed a biosynthetic pathway for the production of the isolated butenolide compounds. The two most potent butenolide compounds had similar potency but differed widely in their toxic potential. One of these butenolides, compound 3, had no prominent toxicity at 2 mM, which was the highest assessed concentration, and an EC50 value of 91 µM. This potency may not be that appealing, especially when comparing it to e.g. Benzavir-2, which has an EC50 value of 0.58 µM. However, when considering the low toxicity of 3, a favorable therapeutic window is expected for this compound. The concentration range between the efficacy and toxicity is always to be considered in drug discovery and especially in antiviral drug discovery, since the virus require a viable host cell. Thus, cellular toxicity may be interpreted as antiviral effect. The antiviral mode-of-action for these butenolide compounds is currently unknown. However, the time-of-addition assay may indicate that these compounds target a cellular mechanism, since pre-treatment of the cells had improved antiviral activity (Fig. 7A). Interestingly, if this is the case, targeting this cellular mechanism does not result in any prominent toxicity. The reduction of the 2-furanone moiety of the butenolide structure resulted in elimination of the antiviral activity. Hence, this structure is responsible for the inhibition of the adenovirus infection but is not solely responsible for the toxic effect (Fig. 8B and C). This enables the usage of the 2-furanone moiety as a bait to fish out target proteins. In addition, the fatty tail may be used as a linker that could be covalently attached to a surface, e.g. on magnetic beads. Subsequently, separation and characterization of 2- furanone moiety interacting proteins will potentially reveal the mode-of- action. Another future prospective is to identify the enzyme(s) responsible for the oxidation of compound 4, as proposed in the biosynthetic pathway

(Fig. 4). This unknown enzyme oxidizes compound 4 to form compound 1 and one of the two possible diastereomers of compound 2. However, the stereochemistry of compound 2 has not been determined. Hence, identification of the enzyme would enable the cloning and expression of the enzyme. Ultimately it would lead to the determination of the stereochemistry of the butenolide compounds and support our proposed biosynthetic pathway. To conclude paper IV, we have identified anti-adenoviral secondary metabolites derived from actinobacteria isolated from the sediment of Vestfjorden, Norway. The compounds display interesting differences in their bioactivity and further characterization may identify the antiviral target(s) of the active 2-furanone moiety. In addition, the interaction between the 2- furanone moiety and the target is not associated with prominent toxicity in our assays. Consequently, this moiety could serve as a starting point for further characterization and optimization of antiviral activity.

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Acknowledgements

So this was it. The acknowledgements should really be on the frontpage instead of the back, since there are a lot of people that have been involved in this thesis to whom I´m very grateful. However, I would like to acknowledge following people that have been of particular importance for the completion of this thesis and the included papers. My main supervisor, Göran Wadell, for guidance in the scientific world and for that you have shared your knowledge, enthusiasm and experience in the field of virology (and beyond) with me. Mikael Elofsson, my co- supervisor, for your inspiring drive and effectiveness. Also for your brilliant inputs and comments on various documents, including this thesis, and for the one and only (so far) Grit-session. Ya-fang Mei, my second co- supervisor, for constructing the RCAd11GFP-vector, without the vector this thesis would not be what it is today. Karin Edlund, for all your support over the years and for always taking time to help and answering questions (when you’re actually busy). Katrine Isaksson-Nyström for all your help with everything, and for fixing all the things that only you can fix. Kristina “Kickan” Lindman, for always being able to help with lab issues. You and Karin are, as many have said before me, the cornerstones of the virology lab. Koushikul Islam, for all your qPCR runs and screening assistance. Emma Andersson, for teaching me the fantastic qPCR-assay. Nitte and Marko for the hard-hitting and exhausting squash matches and sorry Lars that I broke your arm. The poker-gang for letting me win your money. Johan Skog for introducing me to virology during my degree work and thanks to all present and former lab members of the virology unit for creating a pleasant work atmosphere. Marcus Carlsson and Hanna Uvell, for your collaboration and positive attitude. Producing paper IV was fun with you two. Fredrik “Frippe” Almqvist for the trip to Tromsö and for teaching me basic organic chemistry when I was a student. Christopher Öberg and Michael Saleeb for your synthesis of Benzavir, among other compounds. The people at LCBU for help and assistance when screening. UBI for fruitful seminars and discussions and in particular, Pia Keyser, for your coaching and organization of meetings. With your support, let us now take Eirium AB to the next level. The collaboration partners at Sahlgrenska University hospital, Tomas Bergström, for the HSV isolates and Kristina Eriksson, for assessing Benzavir-2 in your in vivo model. Friends and family My beloved Jenny, for cheerful “you can do it” support when writing. Let´s now sail the Swedish archipelagos (when we have bought a boat).

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