Exploring molecular targets for inhibition of alpha- and enterovirus replication

Chikungunya virus (CHIKV) is a mosquito-transmitted virus belonging to the genus alphavirus of the Togaviridae family. The symptoms of an acute infection with CHIKV include fever and arthralgia which may evolve in some patients into persistent disabling polyarthritis that can last for weeks and even up to several years after the acute phase. Despite the worldwide re-emergence of CHIKV and the high morbidity rate associated with it, there is no approved vaccine or antiviral treatment available at the moment. Therefore, the development of potent antiviral drugs against CHIKV is urgently needed.

We showed that prostratin, a potent activator of protein kinases C (PKC), reduced CHIKV RNA and infectious particles in a dose-dependent manner at a post-entry step during virus replication. The antiviral effect of the compound was cell-dependent, with potent antiviral activity observed in human skin fibroblasts cells, the primary target cells of CHIKV infection. The antiviral activity of prostratin was markedly reduced in the presence of PKC inhibitors, therefore confirming that the antiviral effect results from an activation of PKCs and that PKCs are potential targets for inhibition of CHIKV replication.

In the same context, we identified that salicylate-based analogs of bryostatin, another PKC activator, protected cells from CHIKV-induced cell death. Interestingly, ‘capping’ a hydroxyl group of a lead bryostatin analog, which is crucial for binding to PKCs, did not abrogate the anti-CHIKV activity of the scaffold, putatively indicating the involvement of a PKC-independent pathway. A detailed comparative analysis of the effect of the non-capped versus the two capped bryostatin analogs revealed that the non-capped analog acts both at early and late stages in CHIKV replication cycle, while the capped analogs only interfere with a later stage process. Co-dosing with PKC inhibitors counteracted the antiviral activity of non-capped analog without affecting that of capped analogs. Thus, the capped bryostatin analogs can inhibit CHIKV replication through a novel, yet still elusive, non-PKC dependent pathway. Interestingly, treatment of CHIKV-infected cells with a combination of a non-capped and a capped analog resulted in a pronounced synergistic antiviral effect.

Favipiravir (T-705) is a broad-spectrum antiviral agent with a potent inhibitory effect on CHIKV replication in cell culture. We evaluated the potential protective effect of favipiravir , during both the acute and chronic phase of the infection in a CHIKV arthritis mouse model. Interestingly, favipiravir treatment during the acute phase of CHIKV infection completely inhibited systemic viral spread and reduced the viral RNA levels in the ankles of inoculated feet. On the other hand, favipiravir treatment during the late stage of infection did not result in significant differences in viral RNA levels among the treated and untreated mice in the ankles. cDNA amplification of the major parts of the CHIKV coding region of the viral RNA extracted from both the acute and chronic phase ankle samples revealed that the band for the viral RNA-dependent RNA polymerase (RdRp) amplicon is missing and the bands representing some viral proteins have a smaller size than expected. These results gave an initial indication that the viral RNA detected during the chronic phase may be a defective viral genome or degraded RNA and not the genome of actively replicating virus.

A previous study in our lab showed that the K291R substitution in the CHIKV RdRp is implicated in low-level resistance against favipiravir in vitro. Interestingly, this lysine is a highly conserved amino acid in the RdRp of positive-sense, single-stranded RNA [+ssRNA] viruses. To obtain insights into the unique broad-spectrum antiviral activity of favipiravir, we explored the role of this lysine using the coxsackievirus B3 (CVB3). Introduction of the corresponding K-to-R substitution in the CVB3 RdRp (K159R) resulted in a non-viable virus. Replication competence was restored after spontaneous acquisition of an A239G substitution in the RdRp. A mutagenesis analysis at position K159 resulted in only one additional viable variant (K159M) that also acquired the A239G mutation. The K159 mutations markedly decreased the RdRp efficiency in polymerase assays, which was restored by the introduction of the A239G mutation. The K159R-A239G and K159M-A239G viruses were more susceptible to favipiravir than wild-type virus (WT) and exhibited lower fidelity in polymerase assays. Furthermore, the K159R-A239G virus showed a highly attenuated phenotype in mice. These results thus corroborated that this conserved lysine of the viral RdRp is a key residue for both the functionality of this enzyme as well as for the broad-spectrum mechanism of action of favipiravir.

Besides CHIKV, many other viruses lack an available antiviral treatment, including enteroviruses (Picornaviridae family). Although infections with enteroviruses are usually mild, they can occasionally result in severe diseases such as paralytic poliomyelitis by polioviruses (PVs) and myocarditis by CVBs.

We identified a novel and selective benzene sulfonamide derivative inhibitor (i.e. compound 17) of CVB3 replication which was in silico designed to inhibit the viral 3C protease. We showed that compound 17 inhibited the replication of a panel of group B enteroviruses, but had no antiviral activity against representative viruses from other enterovirus groups. In contrast to what was expected for a protease inhibitor, a time-of-drug-addition study revealed that compound 17 interfered with an early step in the virus replication cycle. This observation was corroborated by the results of a thermostability study, which provided additional evidence for an interaction between the compound and the viral capsid. Subsequently, this latter target was confirmed by the genotyping of independently-selected compound 17-resistant CVB3 variants, which were all found to carry a mutation in the VP1 gene. Interestingly, the mutated VP1 residues are not located inside the classical drug-binding pocket for capsid binders such as pleconaril. On the other hand, both the VP1 glutathione binding pocket as well as the ion channel at the 5-fold axis of the VP1 pentamer appear to be implicated in the mechanism of action of compound 17.

Finally, we showed that glutathione (GSH), an essential host factor for stabilization of the enterovirus capsid during morphogenesis, has a pronounced heat-stabilizing effect for the three types of PV Sabin vaccine strains. CVB3 was also used as a surrogate for PV to assess whether the GSH-stabilized virus would still be infectious in mice (a requirement for the live-attenuated vaccine). Indeed, whereas heat-inactivated CVB3 was no longer infectious in highly susceptible SCID mice, GSH-stabilized virus remained as infectious as non-heated virus. Several studies are ongoing to generate genetically stable Sabin polioviruses to be used as a safe, oral live attenuated vaccine during the post-polio eradication era. The fact that GSH is naturally present at high concentration in the human body makes it a safe and efficient candidate stabilizer for these new oral poliomyelitis vaccine (OPV) formulations.