Perspectives for repurposing drugs for the coronavirus disease 2019

Main protease (Mpro)/ 3CLpro inhibitors - Lopinavir and/or lopinavir-ritonavir, cinanserin, herbacetin, rhoifolin and pectolinarin

The Mpro is a promising viral target for the design of drugs against SARS/MERS, as the polyprotein cleavage by the Mpro facilitates the formation of the RNA-dependent RNA polymerase (RdRp) and the helicase which are the major proteins of viral replication811122223. Various classes of protease inhibitors, such as halomethylketones, phthalhydrazide ketones, α, β-epoxyketones, glutamic acid and glutamine peptides with a trifluoromethylketone group, zinc or mercury conjugates, C2-symmetric diols, peptidomimetic-α, β-unsaturated esters, aldehydes, anilides, nitriles, pyrimidinone and pyrazole analogues, benzotriazole, N-phenyl-2-acetamide and biphenyl sulphone, are reported to inhibit the SARS-CoV-1 Mpro/ 3CLpro2425 (Fig. 2). Of these prospective Mpro inhibitors, the common FDA-approved ones are well-known HIV-1 protease inhibitors26. Among these, lopinavir and/or a ritonavir-boosted form of lopinavir has been reported to have anti-CoV activity in vitro and also has shown improved outcomes in non-human primates infected with MERS-CoV and in non-randomized trials with SARS patients27. Both lopinavir and ritanovir are under phase II/III clinical trials for MERS-CoV (NCT02845843)28. These are also reported to have activity against HCoV-229E, HCoV-NL63 and animal CoVs29.

Cinanserin (SQ 10,643) a serotonin antagonist, demonstrated antiviral activity against SARS-CoV-1, and the inhibition of replication was probably by blocking the activity of Mpro14. Flavonoids, herbacetin, rhoifolin and pectolinarin that are known to possess antioxidant effects associated with diseases such as cancer, Alzheimer's disease and atherosclerosis were also noted to efficiently inhibit SARS-CoV-1 Mpro15.

Papain-like protease (PLpro) inhibitor - Disulfiram

Disulfiram, which is an approved drug for the treatment of alcohol dependence, demonstrated in vitro inhibition of the PL2pro enzyme of SARS and MERS30. The study also provided future directions for the development of fragment-linked inhibitors for improving its potency31.

RNA-dependent RNA polymerase (RdRp) inhibitors - Ribavirin, immucillin-A/ galidesivir, remdesivir and acyclovir

The RdRp which is critical for CoV transcription and replication is involved in producing the genomic and subgenomic RNAs. Nucleoside analogues such as favipiravir, ribavirin, penciclovir, remdesivir and galidesivir are well-known RdRp inhibitors. A guanosine analogue, ribavirin, showed broad-spectrum antiviral activity against several viruses including respiratory syncytial virus, hepatitis C and E viruses (HCV, HEV), chikungunya and viral haemorrhagic fevers3233. Though the mechanism of action is not fully understood, it is hypothesized that the drug may be involved in the inhibition of mRNA capping or viral RNA synthesis. The in vitro antiviralactivityofribavirin was demonstrated against SARS-CoV-1 and MERS-CoV34 and in rhesus monkeys infected with MERS-CoV35. The drug has been used in the treatment of SARS and MERS patients, though the benefits are ambiguous. Further, in severely infected CoV patients, there could be side effects associated with high doses36.

Immucillin-A (galidesivir), an adenosine analogue, has been shown recently as a broad-spectrum RdRp inhibitor against several RNA viruses, such as paramyxoviruses, flaviviruses, togaviruses, bunyaviruses, arenaviruses, picornaviruses, filoviruses and also against SARS/MERS-CoVs37. Though it has been reported as a treatment option during the 2014-2016 West Africa Ebola virus epidemic, no data for animal/human were reported for CoVs until recently for the SARS-CoV-216.

Sheahan et al38 showed that another nucleoside analogue, remdesivir (GS-5734), presently under clinical trials for the Ebola virus, demonstrated inhibition of the replication of SARS-CoV-1 and MERS-CoV inprimary human airway epithelial cells. They also demonstrated broad-spectrum anti-CoV activity against bat-CoVs and human CoVs in primary human lung cells1738. In another recent study, remdesivir was shown to possess better in vitro antiviral efficacy against MERS-CoV in comparison to lopinavir and ritanovir1739. In mice, remdesivir improved pulmonary function with lower viral loads in the lungs both as a prophylactic and as a therapeutic1740.

Another nucleoside analogue, acyclovir that was modified by incorporating fleximers to increase its binding affinity has been reported to be effective in vitro against MERS-CoV and HCoV-NL633941, though to the best of our knowledge, no animal or human data are available.

Inhibitors of spike glycoprotein - Griffithsin

CoVs possess a surface structural spike glycoprotein (S) which is vital for interaction with the host cell receptor and subsequent virus entry into the cell. The S protein constitutes two subunits, the S1 (receptor-binding) and the S2 (membrane fusion) domains40. Griffithsin, a lectin extract red algae, has been reported to bind to oligosaccharides on the surface of various viral glycoproteins, including HIV glycoprotein 120 and SARS-CoV glycoproteins41.

Other inhibitors with unknown site of action - Resveratrol, amodiaquine, mefloquine, loperamide

Resveratrol, a natural compound from grape, which is in a clinical phase for heart and other diseases, was also reported to effectively inhibit MERS-CoV in vitro by downregulation of the apoptosis induced by the virus42. The possible site of action was suggested to be the nucleocapsid protein. Amodiaquine and mefloquine, antimalarial drugs, were also found to be effective against MERS-CoV43. Loperamide, an antidiarrhoeal agent that was identified by the screening of an FDA-approved compound library, showed in vitro antiviral activity against MERS44.

Inhibitors of viral nucleic acids - Mycophenolic acid

Viral nucleic acids are mainly composed of nucleosides and nucleotides. The drugs that target these have mycophenolic acid (MPA) as the active compound and inhibit inosine monophosphate dehydrogenase and guanine monophosphate synthesis45. Broad-spectrum activity has been reported by MPA against a broad range of viruses including orthohepadnaviruses (hepatitis B), flaviviruses (HCV), arboviruses and CoVs. MPA possessed anti-MERS-CoV activity in vitro, though it was shown to result in a worsened outcome in the marmoset primate model26. Treatment of renal transplant recipients with MPA resulted in severe MERS46. Combination therapy with interferon beta-1b (IFN-β-1b) was, however, reported to be synergistic in vitro47, implying that monotherapy with the drug might not be useful for treating CoVs.

Inhibitors targeting endocytosis or cell entry - Chlorpromazine, ouabain, bufalin, chloroquine

Chlorpromazine, an antipsychotic/tranquilizer drug, is also known to affect the assembly of clathrin-coated pits at the plasma membrane44. It showed broad-spectrum in vitro activity against viruses such as HCV, alphaviruses, SARS-CoV-1 and MERS-CoV. Ouabain and bufalin, examples of a class of steroids which bind sodium- or potassium-transporting ATPase subunit α1, also inhibited the endocytosis of MERS-CoV mediated by clathrin56. However, very high EC₅₀/C_max (half-maximal effective concentration value/peak serum concentration level) ratios at the typical dosages or toxicity, limit the clinical use of these endocytosis inhibitors. Acidification of the endosome can also affect endocytosis. Chloroquine, an antimalarial drug, can increase the intracellular pH by directing protons into the lysosomes57. It possesses broad-spectrum in vitro antiviral activities against flaviviruses, HIV, Ebola, Nipah and numerous CoVs58. However, it did not show activity in SARS-CoV-infected mice59. The anti-CoV activity of different endocytosis inhibitors thus need further in vivo evaluation.

Inhibitors of host receptor mediated viral entry - N-(2-aminoethyl)-1-aziridine-ethanamine (NAAE), peptides, mAb YS110

Specific peptide inhibitors and monoclonal or polyclonal antibodies can be used to target the host receptor48. N-(2-aminoethyl)-1-aziridine-ethanamine, a small-molecule inhibitor and synthetic ACE2-derived peptides showed inhibition of ACE2 activity and cell fusion via the S protein of SARS-CoV-1 in vitro6061. However, these inhibitors have not been tested in CoV patients. Monoclonal antibodies (mAbs) such as anti-dipeptidyl peptidase 4 (DPP-4) have also been reported to block cell entry of MERS-CoV in vitro62. YS110, an anti-DPP4 recombinant humanized IgG1 mAb, used in a phase I clinical trial, was found to be well tolerated in patients with advanced malignancies19. However, considering that host cell receptor usage differs in different CoVs, the anti-CoV activity of these agents may be narrow-spectrum. Further, based on the vital biological functions of these receptors, the risks of immunopathology such as blood pressure regulation, glucose metabolism etc., would need assessment52.

Inhibitors of host proteases used for viral entry - Camostat mesylate, nafamostat

Camostat mesylate, a synthetic serine protease inhibitor, that is used to treat patients with chronic pancreatitis, works against the serine protease TMPRSS26364. It has shown broad-spectrum activity against enveloped RNA viruses such as CoVs and paramyxoviruses. Camostat mesylate is reported to inhibit SARS and MERS in ex vivo studies and improves the survival of mice infected with SARS6465. Nafamostat, another serine protease inhibitor used to treat disseminated intravascular coagulation and pancreatitis, blocked MERS-CoV infection by inhibiting TMPRSS2 in human airway epithelial Calu-3 cells6566.

Enhancers of host innate immune response - Interferons, polyinosinic: polycytidylic acid [poly(I:C)] and nitazoxanide

Though on viral infection suppression of the IFN response is an integral part for immune evasion, several viruses and CoVs are noted to be susceptible to IFN treatment. The effectiveness of recombinant IFN-β over IFN-α has been demonstrated by in vitro studies against both SARS and MERS67.IFN-α mediated reduction of viral titres was observed in SARS-CoV-infected in vivo models3559, while IFN-β administration via different routes was found to be effective in MERS-CoV in vivo models26. Combinations of IFN-α/β, ribavirin and lopinavir/ritonavir-boosted lopinavir for treatment of SARS/MERS patients, demonstrated varying benefits333668.

Another type I IFN enhancer, polyinosinic: polycytidylic acid [poly(I:C)], a dsRNA synthetic analogue, demonstrated reduction in viral load in MERS-CoV-infected BALB/c mice69. In phase II clinical trials, poly (I:C) was shown to be beneficial for patients suffering from malignant gliomas70.

Nitazoxanide, a synthetic derivative of nitrothiazolyl-salicylamide which is used as a treatment for parasitic infections, is an effective type I IFN inducer71. It has been shown to exhibit antiviral activities against several viral families and canine CoVs. Nitazoxanide was found to be safe in phase II and III clinical trials against HCV and influenza72.

Inhibitors of signaling pathways involved in viral replication - Cyclosporine, trametinib and others

Drugs interfering with the viral replication signaling pathways are noted to have broad spectrum activity against several viruses such as HCV, HIV, vesicular stomatitis virus, human papilloma virus, vaccinia virus and CoVs55. Cyclosporine, a calcineurin pathway inhibitor, inhibited a broad range of CoVs in vitro by interacting with the nsp1 protein and modulating immune response mediated by T cells73. The clinical application of this drug is, however, restricted due to immune-suppressive effects and a higher EC₅₀/C_max ratio at standard dose levels. Other calcineurin inhibitors such as alisporivir, have demonstrated activity against HCoV-NL6320.

The extracellular signal-regulated kinase (ERK) pathway mediates intracellular signals from membrane-associated Ras to the cytoplasmic kinase cascade Raf, Mek and Erk74. The kinase signaling pathway inhibitors, such as trametinib (Mek inhibitor), selumetinib (Erk inhibitor), everolimus, rapamycin, dasatinib and imatinib have also demonstrated anti-CoV effects through inhibition of early viral entry or post-entry events75. However, their toxicities may be a concern in severe infections.

Targeting viral translation - Silvestrol

Initiation of translation in many viruses happens through the usage of the host eukaryotic initiation factors (eIFs)76. The helicase eIF4A unwinds 5′-untranslated region of the mRNA, facilitating assembly of the translation pre-initiation complexes. A natural compound, silvestrol, being an inhibitor of eIF4A and reported to show anti-cancer activity77, demonstrated inhibition of MERS-CoV and HCoV-229E translation and replication in MRC-5 lung fibroblast cells78.