European Review for Medical and Pharmacological Sciences 2021; 25: 556-566 Quinacrine as a potential treatment for COVID-19 virus

B. PINEDA1, V. PÉREZ DE LA CRUZ2, R. HERNÁNDEZ PANDO3, J. SOTELO1

1Laboratorio de Neuroinmunología y Neurooncología, Instituto Nacional de Neurología y Neurocirugía, Ciudad de México, México 2Laboratorio de Neurobioquímica y Conducta, Instituto Nacional de Neurología y Neurocirugía, Ciudad de México, México 3Unidad de Patología Experimental, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Ciudad de México, México

Abstract. – A novel coronavirus named se- been reported that high-risk individuals such as vere acute respiratory syndrome coronavirus-2 elderly subjects or those affected by multimorbid- (SARS-CoV-2) is a current outbreak of infection ities such as hypertension, diabetes mellitus, and termed Coronavirus Disease 2019 (COVID-19) by cardiovascular disorders, in which the virus pro- the World Health Organization (WHO). COVID-19 duces acute respiratory failure and consequently is currently a global that may cause 9 close to half a billion deaths around the world. it can reach to a high death rate . The world faces Until now, there is no effective treatment for its worst public health crisis in the modern era, COVID-19. Quinacrine (Qx) has been used since and the disease continues to spread without any the 1930s as preventive antimalarial compound. effective treatment. Consequently, there is an It is a recognized small molecule inhibitor of urgent necessity to find effective treatments to RNA virus replication, with known anti- ac- reduce its dissemination until successful preven- tivity, and identified as a potent Ebola virus in- hibitor both in vitro and in vivo. Recently, Qx has tion method is developed through vaccination. showed anti-SARS-CoV-2 activity. Herein, we re- Currently, there is no existence of any effective view the potential mechanisms associated with treatment against COVID-19 and clinical man- quinacrine as an antiviral compound. agement is only symptomatic10. Actually, the current pharmacological therapy for Key Words: Quinacrine, SARS-CoV-2, COVID-19, Antiviral. COVID-19 is focused on drugs with antiviral activ- ity (ribavirin, lopinavir, remdesivir, plasma/immu- noglobulin, /), im- munomodulators (interferons and corticosteroids), Introduction and supplements (, alpha lipoid acid)11-14. Herein, we proposed the research of quinacrine, Around the world, the scientific community is an antimalarial drug with potential antiviral effects facing a pandemic provoked by a virus from the along with immunomodulatory properties. family Coronaviridae, order Nidovirales and de- nominated SARS-CoV-2 that is the causal agent Quinacrine Background of COVID-19 pandemic1,2. SARS-CoV-2 has a Quinacrine is an aminoacridine, discovered positive-sense single-stranded RNA transmitted in the 1920s, that has been used for the treatment mainly from human to human through respira- of malaria since 1931. It is worth mentioning tory droplets3-5. The envelope spike protein of that its use was empirical during the first years the SARS-CoV-2 can attach the human angio- of World War II. The suppressive antimalarial tensin-converting enzyme II (ACE2) as an entry activity of quinacrine (also known as atabrine or receptor. ACE2 is expressed in several human ) was examined in both Plasmodium tissues including lungs, small intestine, kidneys, vivax and heart, thyroid, and adipose tissue6. induced by blood inoculation. Quinacrine was The SARS-CoV-2 infection mainly exhibits 200 times more active than , thus, the flu-like symptoms such as fever, cough, and as- oral dose of quinacrine was around one-third of thenia, similar to other coronaviruses7,8. It has that required of quinine15. Quinacrine was also

556 Corresponding Author: Benjamin Pineda, MD; e-mail: [email protected] Quinacrine as a potential treatment for COVID-19 virus infection effective to reduce around 97% of the tissue Nevertheless, quinacrine use for malaria, giar- damage induced by amebic hepatitis16. Quina- diasis, and tapeworm infections was supplanted crine inhibited the growth of Histoplasma cap- by other drugs. In 1955, the introduction of hy- sulatum in vitro and contributed to decreasing droxychloroquine as antimalarial resulted in a the mortality rates of infected mice17. In 1937, decrease in quinacrine research. However, quina- quinacrine was also used to treat , and crine was used for other purposes, such as a treat- it was found that doses similar to those given ment of erythematosus and intrauterine orally for malaria also eliminated giardiasis in sterilization23-35. In this context, it was showed children and adults18; similar satisfactory results that quinacrine is a safe method for the treatment were seen in Oriental sore19. of HIV-infected women36. Moreover, its high Regarding its clinical use in patients, there effectiveness against patients with nitroimidaz- are numerous studies about its pharmacological ole-refractory giardiasis was demonstrated37. characteristics. It has been known that the main Despite the favourable effects and adequate route of quinacrine administration is through oral use of quinacrine profile with its pharmacokinet- intake, but it can be also administrated by intral- ic properties and established record of safety31, esionally/paralesionally, intramuscularly, rectally, its clinical use has been abandoned. Howev- intravenously, transcervically, and interstitially. er, recent experimental studies show quinacrine Quinacrine is rapidly absorbed from the gastro- as a potential therapy, not only as antimalarial intestinal tract and also rapidly absorbed after and anthelmintic38,39, but also as anticancer, an- intrapleural and intrauterine administration20. ti-prion, and antiviral agent40,41. Recently, quin- Plasma levels increase after 2-4 h after its intake acrine has been tested in different trials of can- and reaching a peak between 8-12 h. Quinacrine cer (ClinicalTrials.gov identifiers: NCT01839955, is widely distributed in tissue but it is liberated NCT00417274, NCT01844076) and prion disease and eliminated slowly from the subject’s body21. (NCT00183092, NCT00104663). The administration of quinacrine produces some adverse reactions, most of which are minor or Mechanisms of Quinacrine for reversible (Table I). When quinacrine was admin- Antiviral Effects istrated daily for long periods, it was shown that Quinacrine is an acridine compound with an- quinacrine was well tolerated, with few adverse tiviral properties. The first studies that demon- effects (dermatitis, corneal edema and occasional strated its antiviral effects were against equine anaemia for 1 in 20,000 samples)22. encephalitis virus and louping-ill producing a

Table I. Adverse effects induced by quinacrine. Quinacrine Adverse effects Reference

Children 2 mg/kg three times daily × 5-7 days 28 % of study participants present bitter Wolfe23 taste, nausea, and vomiting Craft et al24 4-5% of the study participants showed yellow/orange discolouration of the skin, sclerae, and had urine effects (started after a week of treatment) Nausea, vomiting, dizziness, and headache Adults Doses with adverse effects Occasionally toxic psychosis (0.4% of Farthing et al25 incidence, reversible effect) Gardner et al26 100 mg three times daily, 5-7 days After prolonged use: Yellow discolouration Evans et al27 of the skin, sclerae and urine, chronic Lindenmayer et al28 dermatoses, and aplastic anemia (0.003%) Engel29 100 mg twice daily for 2 weeks Exacerbate psoriasis and glucose-6-phosphate Ward et al30 dehydrogenase-deficient individuals Ehsanian et al31 200-1200 mg per day for 10 days Cortical stimulatory effects, hyperirritability, insomnia, and nightmares Pregnant women Quinacrine ingestion during Renal agenesis and hydrocephalus in Humphreys et al32 the first 4 weeks of gestation an infant

557 B. Pineda, V. Pérez de la Cruz, R. Hernández Pando, J. Sotelo reduction in the amount of blood circulating virus of Hepatitis B virus52, inducing an Interferon-like during the early systemic phase of the infection. substance 44 and it is a strong inhibitor of somatic A protective effect was also observed when the mutations53,54. virus was injected intracerebrally. Additionally, A recent report shows that quinacrine inhibits when 1-10 lethal doses (LD100) were used, a the initiation of translation of encephalomyo- single dose of quinacrine reduced the mortality carditis virus (EMCV), hepatitis C virus, and from one to two-thirds as compared with mice poliovirus internal ribosome entry sites (IRESs) without treatment42,43. After these studies, the in a cell-free translation assay and it has also antiviral effects of quinacrine have been studied been demonstrated that quinacrine binds the vi- under several conditions (Table II) to determine ral IRES by a competition dialysis system test. the mechanisms by which quinacrine possesses Additional experiments showed that production antiviral properties. Divergent results explained of EMCV and poliovirus capsid proteins, as well by diverse factors, such as the severity of viral as replication of virus RNAs, was suppressed by infection, site of the infection or the virus strain, quinacrine in HeLa cells infected reducing virus have been reported43,44. generation without disrupting the internal cellular Among the mechanisms by which this acridine IRES55. The same effects on RNA intercalation, compound possess antiviral properties are: quin- inhibition of RNA transcription, and protein syn- acrine binds strongly to DNA with the associa- thesis without disrupting internal cellular IRES tion constant of 1.2 × 106 M-1 and stoichiometry was observed with Enterovirus 7156. Addition- of 1 quinacrine molecule/~ 4 nucleotides, being ally, it has been shown that quinacrine inhibits the 9-imino group of quinacrine involved in this RNA accumulation of RNA tomato bushy stunt intercalation binding45. Studies with polarized virus (TBSV) in plants and protoplasts57. In vi- fluorescence showed that the quinacrine bound to tro studies have shown that quinacrine inhibits flowing DNA, being the plane of the quinacrine the binding of protein p33, which is essential as ring nearer to the nucleotide bases than that of replication co-factor of the viral RNA template58; the helical axis of DNA46. Additionally, quina- quinacrine reduces the minus-strand synthesis crine restricts the conformational flexibility of by the tombusvirus replicase. Additionally, the the DNA and thus strengthening the ability of translation of TBSV genomic RNA was also in- topoisomerase II to form a ‘cleavable complex’ in hibited by quinacrine59. which DNA is covalently linked to the enzyme47. Similarly, it has been reported that quinacrine Spectroscopic and isothermal titration calo- inhibits (PLA2) without affect- rimetry studies showed that quinacrine binding ing phospholipase C60,61 and reduces the cytotox- is preferred to the HL-form of the RNA than to icity of gp120 protein of human immunodeficien- the A-form; while thermodynamic parameters cy virus due to inhibition of phospholipase A262. showed that the binding of quinacrine to RNA The inhibition of phospholipase by quinacrine reveals entropy-driven intercalation with no dif- also leads to a drop in the amount of choline ferentiation to A-form or HL-form of RNA48. liberated into the medium by poliovirus-infected Hence, quinacrine is a DNA and RNA strong cells63. Quinacrine inhibits the cytolytic activity intercalating substance preventing structural per- of TNF-α through inhibition of phospholipase A2 turbation of DNA which renders it as a poor activity64. substrate for endonucleolytic cleavage interfer- Quinacrine is a lysosomotropic agent, its dual ing with the process of viral replication49. In protonation at low pH induces cellular trapping this context, quinacrine and chloroquine, both that increases the endosomal pH. This proper- antimalarial drugs inhibit the Tat-mediated long ty of quinacrine seems related to its inhibition terminal repeat-drive gene expression necessary of the acidification organelles of as lysosome, for replication of human immunodeficiency virus endosome, and secretory granules of exocrine type 1 (HIV-1). It has been reported that quina- cells, necessary for degradation of cell debris, crine has a higher antiproliferative effect as com- signalling, and cell homoeostasis65. In this con- pared to chloroquine50. Also, quinacrine binds text, it has been proposed that quinacrine could to heparin and affects the interaction between interfere with the cellular mechanisms involved heparin interacting peptide from Tat (which acts in the process of HIV infection66. Additionally, it as a potent transactivator and it is crucial for viral was demonstrated that quinacrine reduced den- replication) and heparin51. Moreover, it has been gue virus (DENV2) titers and viral RNA copy shown that quinacrine inhibits DNA polymerase numbers in infected cells. In the same study,

558 Quinacrine as a potential treatment for COVID-19 virus infection

Table II. Quinacrine documented use as a treatment of experimental viral models.

Doses/ Toxic effects concentration induced by Model quinacrine Effects quinacrine Reference

Equine Mice Single-dose 10 mg/18 g Increased the survival of Not reported Hurst et al42 encephalomyelitis fatal cases and reduced the mortality by more than 72%. Louping-ill Mice Single-dose 10 mg/18 g Increased the survival of Not reported Hurst et al42 fatal cases and reduced the mortality Western equine Mice Single-dose (10 mg/20 g) Increased the survival of fatal Not reported Hurst et al43 encephalomyelitis 24 h before virus lethal cases and reduced the challenge mortality by around 91% as compared to the group of mice infected without treatment. St. Louis Mice Single-dose (10 mg/20 g) Reduced mortality by 40%. Not reported Hurst et al43 encephalitis 24 h before virus lethal Decreased virus titer. challenge Rabies-virus-fixe Mice Single-dose (10 mg/20 g) No changes were found as Not reported Hurst et al43 24 h before virus lethal compared to animals infected challenge without treatment Rift Valley fever Mice Single-dose (10 mg/20 g) Increased survival periods Not reported Hurst et al43 24 h before virus lethal of fatal cases. challenge Decreased virus titer Neurotropic Mice Single-dose (10 mg/20 g) Increased the survival period influenza – 24 h before virus lethal and a reduction of around 50% W.S. strain challenge in the deaths was reported as compared to untreated infected mice Not reported Hurst et al43 Lymphocytic Mice Single-dose (10 mg/20 g) Increased the survival period Not reported Hurst et al43 choriomeningitis 24 h before virus lethal of fatal cases and decreased the challenge number of deaths. Decreased virus titer Herpes febrilis Mice Single-dose (10 mg/20 g) Increased the survival period Not reported Hurst et al43 24 h before virus lethal of fatal cases with a decrement challenge in the mortality. Herpes A Mice Single-dose (10 mg/20 g) Increased the survival period Not reported Hurst et al43 24 h before virus lethal of fatal cases with a decrement challenge in the mortality. Herpes B Mice Single-dose (10 mg/20 g) Increased the survival period Not reported Hurst et al43 24 h before virus lethal of fatal cases with a decrement challenge in the mortality Vaccinia virus Mice 200 mg/kg, 22 h before The decreased quantity of Not evaluated Glaz et al44 virus infection plaque-forming units as compared to a case with no treatment considered on mice Herpes simplex Ganglia 1×10-4M Inhibited the viral reactivation No cytotoxic Kurane et al72 Virus 1 culture in the explanted ganglia, was observed no virus titer was found effect for the used concentration. Hepatitis B virus Preparation 100-800 µg/ml Inhibited hepatitis B Not evaluated Hirschman 52 of hepatitis DNA polymerase (IC50 et al B antigen 334 µg/ml). gp120 of Rat primary Pretreatment (30 min Decreased gp120 cytotoxicity. Not evaluated Ushijima HIV-1 Neuronal before infection) Increased the percentage of et al62 Cell culture 3-50 µM viable cells (3 µM of Qx increased 28%) HIV-1 Transfected 2-10 µM for 12 h Inhibited Tat-induced Not evaluated Jiang et al50 cells transactivation and showed antiproliferative effects

Continued

559 B. Pineda, V. Pérez de la Cruz, R. Hernández Pando, J. Sotelo

Table II (Continued). Quinacrine documented use as a treatment of experimental viral models.

Doses/ Toxic effects concentration induced by Model quinacrine Effects quinacrine Reference

Tomato bushy Nicotiana 5-20 µM Inhibited TBSV accumulation Not evaluated Sasvari et al59 stunt virus benthamiana around 50% when it was. (TBSV) protoplasts applied before electroporation Nicotiana 1600 µM Inhibited TBSV RNA benthamiana (via accumulation by 99%. leaves infiltration) Inhibited TBSV replication In vitro 20 µM (90-90%). replicase Inhibited minus-strand synthesis assembly by the tombusvirus replicase assay Inhibited the binding of p33 replication protein to the viral (+)RNA. Encephalomy- HeLa cells 0-20 µM Suppressed production of viral Not evaluated Gasparian55 ocarditis virus infected capsid proteins, replication of (EMCV) with EMCV viral RNAs, and virus titer. Cell-free s 25 µM Inhibited 80% of translation ystem from the EMCV Internal ribosome entry sites (IRESs). Poliovirus HeLa cells 0-20 µM Inhibited the synthesis of infected viral capsid proteins, production with of viral RNAs, and virus poliovirus replication in a dose-dependent Cell-free 0-25 µM manner. system Inhibited around 50-60% of Not evaluated Panavas et al58 translation from poliovirus IRES. Enterovirus 71 Rhabdomy- 1-20 µM Inhibited viral RNA replication, Not evaluated Wang et al56 (EV71) osarcoma viral capsid protein expression, cells and virus production. Protected cells from EV71. Coxsackieviruses Rhabdomy- 1-25 µM Inhibited viral replication. Not evaluated Wang et al56 (CoxA10, CoxA1 osarcoma Suppressed viral titer. and CoxB5) cells Echovirus Rhabdomy- 1-25 µM Inhibited viral replication Not evaluated Wang et al56 (Echo25) osarcoma (suppressed viral titer) cells White spot Pacific white 5 μg/g Reduced the mortality rate and No mortality Kang et al73 syndrome virus shrimp viral copy number. was found Litopenaeus with quinacrine vannamei treatment. Sf9 cells 25-30 µM Suppressed icp53 IRES activity. transfected Increased cell viability. with the bicistronic plasmid containing the icp35 IRES element

Continued the authors demonstrated that quinacrine inhibits (EBOV); decreasing the EBOV infection in vitro autophagy increasing pH and, as consequence, and in vivo protecting 70% of mice from a lethal inhibits infection by dengue virus67. Also, quin- challenge with mouse-adapted EBOV suggesting acrine inhibits cathepsin B which is involved in that this protective effect was induced by quina- LC3-II proteolysis. Quinacrine has recently been crine raising the pH of acidic cytoplasmic organ- proposed as an active drug against Ebola virus elles inhibiting the enzymatic activity of viral

560 Quinacrine as a potential treatment for COVID-19 virus infection

Table II (Continued). Quinacrine documented use as a treatment of experimental viral models.

Doses/ Toxic effects concentration induced by Model quinacrine Effects quinacrine Reference

74 Ebola virus HeLa cells 0.12-250 µM Decreased Ebola infection CC50 6.2 µM Ekins et al (EBOV) treated with (EC50 350 nM). infection mix containing Ebola-GFP virus

EBOV- EC50 0.5-0.98 µM Decreased EBOV activity. CC50 ̴7.94 µM Anantpadma infected et al69 HeLa cells Mouse- 25 mg/kg Increased survival rate by 70%. 50 mg/kg of Lane et al68 adapted quinacrine EBOV decreases by 50% of the effect observed with 25 mg/kg in survival. Dengue virus HEK293 5 nM and 10 μM Inhibited 94% of infection, Shum et al75

cells CI50 0.53 ± 0.04 µM. noculated with DENV-2

BHK-21 cell 0.78-200 µM Reduced DENV2 titers CC50 44.48 ± Balasubramanian line stably (CI50 7.09 ± 1.67 µM) 16.45 µM et al67 expressing and DENV2 viral RNA DENV2 RNA copy numbers.

Zika virus Vero cells 0-200 µM Inhibited Zika virus infectivity CC50 18.59 ± Balasubramanian transfected (EC50 2.27 ± 0.14 µM) 3.15 µM et al67 with Zika Inhibited Zika replication. virus (MR766) SARS-CoV-2 Lung and Inhibited viral replication and CC50 6.2-22 µM Han et al71

Colonic virus entry with EC50= Ianevski Organoids 0.84-3.01 µM through blocking et al76 Vero cells ACE2 receptor. cell entry68-70. Recently, it has been described that neous lupus erythematosus, due to its ability to Qx inhibited SARS-CoV-2 viral replication and inhibit the secretion of proinflammatory cyto- viral entry by the Qx binding to angiotensin-con- kines, such as tumour necrosis factor-α (TNF-α) verting enzyme 2 (ACE2) receptor71. from peripheral blood mononuclear cells (PB- MCs)82,83. Coincidentally, TNF-α is a relevant Mechanisms Associated with Quinacrine cytokine participating in the cytokine storm that Immune Modulator Effects some COVID-19 patients with clinical compli- Since the 1950s, quinacrine has been used as cations develop. an immunomodulator in asthma72-78. Afterwards, Quinacrine has shown inhibition of intracel- it was demonstrated that quinacrine had anti-ag- lular TLRs 7 and 9, two ligands involved in gregate activity by reduction of platelet-acti- the innate immune response by recognition of vating factor79. However, the most documented single-chain RNA and CpG motifs, inhibiting characteristic of quinacrine as an anti-inflam- the secretion of proinflammatory cytokines and matory factor is through PLA2 inhibition80. Its antigen presentation by peripheral blood mono- anti-asthmatic activity was due to improved nuclear cells (PBMCs)84,85. Recently, Zhou et al86 clinical features, as well as Th1/Th2 unbalanced elaborated a network-based drug for therapeu- response, simultaneously decreasing PLA2 ac- tic trials against novel coronavirus 2019-nCoV/ tivity and cysteinyl leukotrienes levels81. More SARS-CoV-2, in that list, quinacrine appears recently, quinacrine has also been used as an at the top of 16 network-predicted repurposable immunomodulator in dermatomyositis and cuta- drugs.

561 B. Pineda, V. Pérez de la Cruz, R. Hernández Pando, J. Sotelo

Quinacrine as a Potential Anti-SARS- Moreover, quinacrine inhibits intracellular TLRs CoV-2 Agent 7 and 9, ligands of single-chain RNA involved Quinacrine has several documented possible in the activation of the innate immune response targets for SARS-CoV-2 replication inhibition. against SARS-CoV-2 infection, which decrease As the authors have mentioned before, SARS- the secretion of proinflammatory cytokines by pe- CoV-2 is a positive single-stranded RNA virus ripheral blood mononuclear cells (PBMCs) with- with nucleocapsid. Therefore, quinacrine could out affecting the production of Type I interferons intercalate within the SARS-CoV-2 RNA, in- (IFN-α and IFN-β). These properties would allow hibiting both, RNA transcription and protein quinacrine to limit the cytokine storm-generated synthesis, without disrupting the internal cellu- in some COVID-19 patients without affecting the lar IRES. Additionally, after SARS-CoV-2 cell antiviral immune response (Figure 2). entry via endosomes quinacrine, as a lysosomo- tropic agent, might increase the pH in the endo- somes and lysosomes, thus, inhibiting lysosomal Conclusions proteases and the fusion of autophagosome and lysosome preventing the release of viral materi- In summary, the vast information about the med- al into the endosomes and autophagolysosomes ical uses of quinacrine by millions of patients and (Figure 1). its experimental efficacy inhibiting virus replication In addition to these antiviral properties, quin- supports the idea that quinacrine may be useful as acrine has several characteristics that suggest adjuvant antiviral compound against SARS-COV-2 immune modulator effects. Quinacrine acts as virus. Additionally, its effects on the innate immune anti-inflammatory through PLA2 inhibition in- response and on reducing the cytokine storm make hibiting the formation of arachidonic acid, COX quinacrine as a reliable candidate for the clinical activation and cysteinyl leukotrienes formation. test to repurposing it for therapy of SARS-COV-2

Figure 1. Potential anti-SARS-CoV-2 mechanisms of quinacrine.

562 Quinacrine as a potential treatment for COVID-19 virus infection

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