Murine Efficacy and Pharmacokinetic Evaluation of the Flaviviral NS5 Capping Enzyme 2-Thioxothiazolidin-4-One Inhibitor BG-323

Abstract

Arthropod-borne flavivirus infection continues to cause significant morbidity and mortality worldwide. Identification of drug targets and novel antiflaviviral compounds to treat these diseases has become a global health imperative. A previous screen of 235,456 commercially available small molecules identified the 2-thioxothiazolidin-4-one family of compounds as inhibitors of the flaviviral NS5 capping enzyme, a promising target for antiviral drug development. Rational drug design methodologies enabled identification of lead compound BG-323 from this series. We have shown previously that BG-323 potently inhibits NS5 capping enzyme activity, displays antiviral effects in dengue virus replicon assays and inhibits growth of West Nile and yellow fever viruses with low cytotoxicity in vitro. In this study we further characterized BG-323’s antiviral activity in vitro and in vivo. We found that BG-323 was able to reduce replication of WNV (NY99) and Powassan viruses in culture, and we were unable to force resistance into WNV (Kunjin) in long-term culture experiments. We then evaluated the antiviral activity of BG-323 in a murine model. Mice were challenged with WNV NY99 and administered BG-323 or mock by IP inoculation immediately post challenge and twice daily thereafter. Mice were bled and viremia was quantified on day three. No significant differences in viremia were observed between BG-323-treated and control groups and clinical scores indicated both BG-323-treated and control mice developed signs of illness on approximately the same day post challenge. To determine whether differences in in vitro and in vivo efficacy were due to unfavorable pharmacokinetic properties of BG-323, we conducted a pharmacokinetic evaluation of this small molecule. Insights from pharmacokinetic studies indicate that BG-323 is cell permeable, has a low efflux ratio and does not significantly inhibit two common cytochrome P450 (CYP P450) isoforms thus suggesting this molecule may be less likely to cause adverse drug interactions. However, the T1/2 of BG-323 was suboptimal and the percent of drug bound to plasma binding proteins was high. Future studies with BG-323 will be aimed at increasing the T1/2 and determining strategies for mitigating the effects of high plasma protein binding, which likely contribute to low in vivo efficacy.

Powassan virus, a scoping review of the global evidence

“…(), Bullard et al. (), Chernesky (,b), Chernesky & Whittaker‐Haines (), Ebel et al. (), Flint et al.…”

“…Two studies using small molecule compounds; 6‐azauridine, 2‐C‐methylcytidine and interferon alpha 2a did not report the magnitude of POWV inhibition (Flint et al., ; Osolodkin et al., ). However, two other potential antiflaviviral agents, the NS5 capping enzyme inhibitor BG‐323 (Bullard et al., ) and adenosine analog NITD008 (Yin et al., ), significantly reduced POWV titres in vitro in their respective studies. An I. scapularis organ culture model was developed to study the effects of POWV localization and replication to ultimately enhance screening for potential vaccine candidates and proteins for therapeutic use (Grabowski et al., ).…”

Structural & functional characterization of the dengue virus non-structural protein 5 (NS5)

“…This was supported by an earlier SAXS analysis which showed that the full length NS5 from DENV-3 could assume multiple conformations in solution, from compact to more extended forms(254). In the crystal structure of DENV-3 NS5, a well-ordered linker region (residues[263][264][265][266][267][268][269][270][271][272] between the MTase and RdRp domains was fully resolved for the first time.The linker comprises a short 310-helix (residues 263-266) which probably acts as a swivel, making structural transition to confer conformational flexibility of NS5 to perform its enzymatic functions and for interaction with viral and host proteins. Given the sequence heterogenicity of the linker residues across flaviviruses (Figure3.6) despite a conservation in the length of the linker region, we probed the relevance of their unique amino acid composition in our linker swapping experiment using DENV-2 subgenomic replicon system.…”

“…BG-323 was shown to decrease guanylation activity in vitro, present antiviral efficacy in DENV replicon assay and inhibit replication of WNV and YFV. Moreover, resistance of WNV to the compound was not detected after long-term culture experiment (267). However, it is probable that the antiviral effect detected may be attributed to the compound interfering with the MTase activity of the capping enzyme or intervening with aldose reductase since BG-323 has a structure that resembles the known FDA-approved drug, Epalrestat, that targets this cellular protein for improving the conditions of patients with diabetic neuropathy (268).…”

“…Additional experiments need to be carried out to confirm the mechanism of action for this series of compounds. The same group tested the antiviral effects of BG-323 in murine model and observed a lack of in vivo efficacy which may be due to its short half-life (T1/2) in mice and human plasma protein binding (267). Future studies would be necessary to ameliorate the effects of plasma protein binding and improve compound stability and half-life in vivo.…”