This report describes the discovery of a group of novel inhibitors of flavivirus replication that may provide pharmacological benefits for the medical treatment of flaviviruses. We have identified a series of compounds able to inhibit an enzyme essential for viral replication and that show cell culture activity similar to that of the known antiviral ribavirin.
Crystal structures of the capping enzyme with GTP bound have been previously solved (10
), and based on mutational studies, GTP association is shown to require π-π stacking interactions with Phe 24 and the formation of hydrogen bonds with Lys 13, Leu 16, Asn 17, Leu 19, Lys 28, Ser 150, Arg 213, and Ser 215. A water bridge also forms between the nitrogenous base and the backbone oxygen of Leu 19 that is suggested to play a role in affinity.
Based on the computational docking of BG-5 (), the compound in this series with the highest affinity for the enzyme, we suggest that the members of the thioxothiazolidin family of compounds interact with the enzyme in ways that mimic the association of GTP. Specifically, this compound appears to hydrogen bond with Lys 28 and Ser 150 and π-π stack with the aromatic side chain of Phe 24. In addition, BG-5, GTP, and other compounds with improved binding affinity appear to interact with two subpockets within the capping enzyme GTP binding site. Subpocket 1 allows for interactions between the small molecules and Phe 24 and Lys 21, while subpocket 2 allows for interactions between the small molecules and Asn 17, Leu 18, and Leu 19. Interaction with the two subpockets provides a rationale for the SAR shown in . First, compounds with increased bulk in the R2 to R6 positions are predicted to have increased interactions within one or both of the subpockets (for example, BG-322 and BG-323). Second, the addition of a carbon-carbon bond linking the acid moiety in the R1 position suggests that these compounds could protrude into these subpockets to a greater extent while still maintaining the ability of compounds to form hydrogen bonds with Ser 150 and Lys 28. Interestingly, the two subpockets also provide a structural explanation for the similar inhibitory effects of BG-318 and BG-328: based on docking studies, both compounds appear to interact with subpocket 2 to similar (nonoptimal) extents, and for this reason, affinity is not increased by altering the distance between the thiazolidin core and the acid moiety. Currently, BG-5 has the highest affinity for the capping enzyme, and its proposed orientation within the binding site () supports our hypothesis that compounds able to interact with either or both subpockets have an increased affinity for the binding site. Additional analogs need to be tested to further clarify how the thioxothiazolidins bind to the capping enzyme, although our preliminary pharmacophore presented in provides a sound basis for further SAR development.
Two compounds in this study, BG-5 and BG-323, displayed antiviral activity in the dengue virus replicon assay (). BG-323 displayed a TI of 6, which indicates that they are more efficacious than our ribavirin positive controls. Further testing of BG-323 showed that it was able to suppress the replication of West Nile (Kunjin) and yellow fever viruses by plaque assay, qRT-PCR analysis of medium samples, and Western blot analysis. These data suggest that BG-323 is able to reduce the amount of infectious virus that is released from infected cells and indicate that BG-323 has significant antiviral activity in culture. This finding is exciting, as BG-323 (shown docked to the yellow fever virus capping enzyme in ) has a structure similar to those of known FDA-approved drugs, such as the antidiabetes drug Epalrestat (22
), indicating that an optimized thioxothiazolidin may be developed into a drug clinically usable for the treatment of flavivirus infection. One concern is that thioxothiazolidins are considered to be somewhat promiscuous in HTS assays. However, the observation that BG-323 demonstrates antiviral activity against flaviviruses but not alphaviruses () suggests that BG-323 is not merely a “sticky” molecule but has some specificity against flavivirus replication, making it an interesting candidate for further development. Additionally, we have previously identified other core structures that may be substituted for the thioxothiazolidin core to mitigate any liabilities that the moiety may present (11
Fig 5 Molecular docking of BG-323 with the capping enzyme. On the left, a three-dimensional rendering of the predicted orientation of BG-323 within the yellow fever virus capping enzyme is presented. In the yellow fever virus protein, light blue represents (more ...)
The biochemical data we present demonstrate that BG-323 has activity against the capping enzyme guanylyltransferase in vitro, although it is possible that part of the antiviral effect observed in cells may be due to BG-323 interfering with the methyltransferase activity of the capping enzyme or interfering with a cellular protein such as aldose reductase (the target of Epalrestat). Further testing is needed to definitively show the mechanism of action of this series of compounds in cell culture. We have also tested if BG-323 interferes with other enzymatic assays, such as PCR, but have not observed any significant effect (data not shown), indicating that BG-323 is not broadly reactive toward enzymes.
While BG-323 does show promise as a novel anti-flaviviral therapeutic, there are a few issues that need to be addressed to increase its efficacy. The BG-323 TI of 6 is somewhat low, and increasing this value will be critical to further development. BG-323 binds to the capping enzyme with a Ki of ~10 μM and has an EC50 of ~30 μM, indicating that the molecule is able to pass cellular membranes relatively effectively and interfere with viral replication at a concentration not much higher than is inhibitory concentration in biochemical assays. Therefore, increasing binding affinity while maintaining cellular permeability may help lower the effective EC50 of the compound series to increase the TI of the molecule and bring its inhibitory activity into a more therapeutically useful range. In addition, BG-323 possesses a Michael acceptor group that may increase reactivity and lead to the weak toxicity we observed in cell culture. Several approaches to mitigate these issues and potentially increase the TI of BG-323 are currently being investigated, such as replacement of the propanoic acid group with the bioisostere tetrazol, conversion of the propanoic acid to an ethyl ester that may act as a prodrug, and/or reduction of the Michael acceptor to avoid undesired reactivity. Additionally, BG-323 does appear to lose activity during the course of longer experiments, suggesting that the compound may degrade over time or may bind albumin. Medicinal chemistry and pharmacokinetic studies are necessary to identify analogs or formulations that may increase the stability of the compound over time in vitro and in vivo. Further SAR development, including mutagenesis analysis of the capping enzyme and cocrystallization of BG-323 and the capping enzyme, will help clarify critical interactions and increase binding affinity.
BG-323 represents a valuable platform for the further development of antiviral inhibitors of flavivirus RNA replication. Future studies will focus on decreasing the EC50 of BG-323, improving bioavailability, and testing this family of molecules for in vitro and in vivo efficacy against a number of additional flaviviruses.