Virology tidbits

Virology tidbits

Tuesday, 27 May 2014

Are broad spectrum antivirals for Coronavirus infections are just around the corner?

The emergence of a new highly pathogenic virus in animal as well as human populations presents a unique challenge for both veterinarians and physicians alike since vaccines more often than not are not readily available, leaving antiviral treatments the only option to contain an outbreak. Pharmaceuticals however take time to be developed and tested thus the only option available is to identify the antiviral pathways targeted by viral proteins in the hope that existing drugs are available and effective in activating these pathways and thus suppress viral replication. In the meantime, clinicians can only offer supportive care and use serum from convalescent patients - often a scarce commodity and not readily available. As an alternative however pharmaceuticals used and approved for the treatment of other viral diseases or indeed for other diseases might be repurposed.

The recent emergence of both the SARS-CoV in 2002 and MERS-CoV in 2012 have lead to substantial increase in Coronaviruses as a potential human pathogen. Following the emergence of MERS-CoV, the International Respiratory and Emerging Infection Consortium (ISARIC) compiled a list of pharmaceuticals available to physicians based on the experience gained during the SARS-CoV epidemic in 2002/2003, with the most promising drugs being Interferon and Ribavirin, which had been used in combination as well as separate to treat SARS-CoV and pandemic Influenza A/2009 patients. Indeed, both drugs are effective to prevent MERS-CoV replication in a rhesus macaque model but failed to be effective in patients with a severe infection. A screen of chemical library of 1280 pharmaceuticals known to be effective against Influenza A was also assessed for their ability to reduce viral yield and prevent the cytopathic effect following the infection of cells with MERS-CoV confirmed that at least under laboratory conditions MERS-CoV is sensitive to Interferon as well as to two antiretroviral drugs, nefinavir and lopinavir. At first it may seem surprising that two antiretroviral drugs can prevent the replication of a Coronavirus. Both drugs were developed to prevent the replication of HIV by targeting the HIV protease. As discussed before however, the Coronavirus genome encodes for a protease, 3CLpro which is required for the processing of the orf1ab polyprotein and has been shown sensitive to nefinavir and lopinavir due to the inhibition of the viral 3CLpro    protease.  Both drugs are non-specific for MERS-CoV and also effective in treating SARS-CoV related infections.
Other targets of antiviral therapy most certainly include preventing viral entry. As discussed in a previous post, monoclonal antibodies against the viral S protein and small molecules binding to the receptor-binding site of the S protein have been shown to be effective to neutralize viral particles. Another possibility is to target the release of the viral genome into the cytoplasm of the cell, which is dependent on a low pH within the endosome. The application of a lysosomotropic agent such as Chloroquine/Hydroxychloroquine (the protonated form of Chloroquine) (an antimalarial drug) or NH4Cl might therefore prevent the fusion of the virus with the endosome by raising the pH. Indeed the application of low doses of Chloroquine to cells infected with SARS-CoV or MERS-CoV as well as Influenza A have shown to prevent viral replication. In addition to prevent the fusion of the viral particle with the endosome, Chloroquine might also prevent the glycosylation of ACE2, the receptor for SARS-CoV and thus prevent binding of the SARS-CoV S1 subunit to its receptor (it remains to be seen if this is the case with DPP4, the receptor for MERS-CoV). The glycosylation of proteins is targeted by inhibiting glycosyltransferases, namely quinone reductase 2, which is involved in the biosynthesis of sialic acid, a component of cellular receptors. Sialic acid moieties are also present within the glycoproteins of HIV-1 glycoproteins, the SARS-CoV receptor ACE2, the MERS-CoV receptor DPP4/CD26, Coronavirus S proteins as well in the receptors for Influenza A thus explaining the broad spectrum activity of Chloroquine. Quinone reductase 2 inhibitors therefore reduce the glycosylation of SARS S proteins although it seems that the reduction has no effect on viral infectivity (or only a marginal effect).
So far however its effectiveness has not been demonstrated in the animal model of MERS and studies with Influenza A have shown that Chloroquine -although effective in cell lines- is not effective in humans thus adding some caution. Apart from being a potential pharmaceutical against a variety of human Coronaviruses, Chloroquine is well tolerated and better known in treating in Malaria patients at therapeutic doses in micro molar concentrations.  Pharmaceuticals effective specifically against MERS-CoV, two pharmaceuticals emerged recently, mycophenolic acid (MPA) and IFN-β, both of which have been discussed previously.      

As outlined previously, the polyprotein 1ab is processed further by auto proteolysis that generates a number of nonstructural proteins varying among the Coronaviridae, which includes not the RNA dependent RNA Polymerase (RdRp) but also an NTPase/Helicase known as nsp 12 and 13 respectively.  In simian Vero E6 cells infected with SARS-CoV these are located within perinuclear double membrane bound vesicles representing replication-transcription complexes containing nascent viral subgenomic RNAs, RdRp as well as viral positive strand RNA and dsRNA intermediates which are resolved by the viral Helicase. Although the precise mechanism and specific function of the Coronavirus Helicase is not known, the replication of SARS-CoV, MERS-CoV, and the murine MHV can effectively inhibited by a small compound, SSYA10-001, targeting the Helicase at amino acid residues K508, R507, and Y277 respectively, thus offering a potential broad spectrum inhibitor of Coronavirus mediated infections and highlighting the importance of the Coronavirus Helicase for viral replication since inhibition of the SARS-CoV Helicase by Bismuth has been shown to inhibit SARS-CoV replication in the past. In addition to its wide spectrum of antiviral activity, SSYA10-001 exhibits only minimal cytotoxicity if applied to cells. The viral RdRp itself can be inhibited by combination of Ribavirin and 5-Flourouracil, the latter being mutagenic and thus sensitizing infected cells to Ribavirin treatment (Ribavirin itself being ineffective). 
Overview of potential and existing antiviral strategies to treat Coronavirus infections

In conclusion, whilst future outbreaks of novel respiratory viruses cannot prevented, pharmaceuticals which are already available might be used in the treatment during a pandemic or an epidemic whilst bioinformatics in conjunction with the identification of ways that viral proteins interact with the host cell might identify effective broad spectrum inhibitors which target highly conserved proteins. A recent screen of potential antiviral pharmaceuticals revealed that even antipsychotic drugs can have an antiviral effect against MERS-CoV, revealing the hidden potential of many drugs already approved.

Further reading

Dyall J, Coleman CM, Hart BJ, Venkataraman T, Holbrook MR, Kindrachuk J, Johnson RF, Olinger GG Jr, Jahrling PB, Laidlaw M, Johansen LM, Lear CM, Glass PJ, Hensley LE, & Frieman MB (2014). Repurposing of clinically developed drugs for treatment of Middle East Respiratory Coronavirus Infection. Antimicrobial agents and chemotherapy PMID: 24841273

Falzarano D, de Wit E, Rasmussen AL, Feldmann F, Okumura A, Scott DP, Brining D, Bushmaker T, Martellaro C, Baseler L, Benecke AG, Katze MG, Munster VJ, & Feldmann H (2013). Treatment with interferon-α2b and ribavirin improves outcome in MERS-CoV-infected rhesus macaques. Nature medicine, 19 (10), 1313-7 PMID: 24013700

Falzarano D, de Wit E, Martellaro C, Callison J, Munster VJ, & Feldmann H (2013). Inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin. Scientific reports, 3 PMID: 23594967 

Chan, J., Chan, K., Kao, R., To, K., Zheng, B., Li, C., Li, P., Dai, J., Mok, F., Chen, H., Hayden, F., & Yuen, K. (2013). Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus Journal of Infection, 67 (6), 606-616 DOI: 10.1016/j.jinf.2013.09.029

Kilianski A, & Baker SC (2014). Cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors. Antiviral research, 101, 105-12 PMID: 24269477

Al-Tawfiq JA, Momattin H, Dib J, & Memish ZA (2014). Ribavirin and interferon therapy in patients infected with the Middle East respiratory syndrome coronavirus: an observational study. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 20, 42-6 PMID: 24406736

Smith EC, Blanc H, Vignuzzi M, & Denison MR (2013). Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics. PLoS pathogens, 9 (8) PMID: 23966862 

Hart BJ, Dyall J, Postnikova E, Zhou H, Kindrachuk J, Johnson RF, Olinger GG Jr, Frieman MB, Holbrook MR, Jahrling PB, & Hensley L (2014). Interferon-β and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell-based assays. The Journal of general virology, 95 (Pt 3), 571-7 PMID: 24323636

Coleman CM, Liu YV, Mu H, Taylor JK, Massare M, Flyer DC, Glenn GM, Smith GE, & Frieman MB (2014). Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice. Vaccine, 32 (26), 3169-74 PMID: 24736006

Keyaerts E, Li S, Vijgen L, Rysman E, Verbeeck J, Van Ranst M, & Maes P (2009). Antiviral activity of chloroquine against human coronavirus OC43 infection in newborn mice. Antimicrobial agents and chemotherapy, 53 (8), 3416-21 PMID: 19506054

Savarino A, Di Trani L, Donatelli I, Cauda R, & Cassone A (2006). New insights into the antiviral effects of chloroquine. The Lancet infectious diseases, 6 (2), 67-9 PMID: 16439323

Vincent MJ, Bergeron E, Benjannet S, Erickson BR, Rollin PE, Ksiazek TG, Seidah NG, & Nichol ST (2005). Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virology journal, 2 PMID: 16115318

van Hemert, M., van den Worm, S., Knoops, K., Mommaas, A., Gorbalenya, A., & Snijder, E. (2008). SARS-Coronavirus Replication/Transcription Complexes Are Membrane-Protected and Need a Host Factor for Activity In Vitro PLoS Pathogens, 4 (5) DOI: 10.1371/journal.ppat.1000054

Adedeji AO, Singh K, Kassim A, Coleman CM, Elliott R, Weiss SR, Frieman MB, & Sarafianos SG (2014). Evaluation of SSYA10-001 as a Replication Inhibitor of SARS, MHV and MERS Coronaviruses. Antimicrobial agents and chemotherapy PMID: 24841268 

Yang N, Tanner JA, Wang Z, Huang JD, Zheng BJ, Zhu N, & Sun H (2007). Inhibition of SARS coronavirus helicase by bismuth complexes. Chemical communications (Cambridge, England) (42), 4413-5 PMID: 17957304

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