GTP and RNA cap analog competition analysis with CHIKV nsP1.

<p>GTP and RNA cap analog competition analysis with CHIKV nsP1.</p

A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

<div><p>Chikungunya virus (CHIKV) is a mosquito-borne <i>Alphavirus</i> that causes severe and debilitating disease symptoms. Alarmingly, transmission rates of CHIKV have increased dramatically over the last decade resulting in 1.7 million suspected cases in the Western hemisphere alone. There are currently no antivirals for treatment of CHIKV infection and novel anti-alphaviral compounds are badly needed. nsP1 is the alphavirus protein responsible for the methyltransferase and guanylyltransferase activities necessary for formation of the 5’ type 0 cap structure added to newly formed viral RNA. Formation of this cap depends on nsP1 binding GTP and transferring a methylated GMP to nascent viral RNA. We have developed a fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time. Determining the relative affinities of 15 GTP analogs for nsP1 GTP revealed important structural aspects of GTP that will inform identification of inhibitors able to outcompete GTP for the nsP1 binding site. Validation of the assay for HTS was completed and a secondary orthogonal assay that measures guanylation activity was developed in order to evaluate hits from future drug screens. This platform provides an avenue for identification of potent nsP1 inhibitors, which would potentially provide compounds capable of treating disease caused by CHIKV infection.</p></div

Validation of CHIKV nsP1 purity and identity.

<p>Recombinant CHIKV nsP1 was purified with 6X his-mediated affinity chromatography followed by size exclusion chromatography. (A) Early attempts to purify CHIKV nsP1 resulted in highly aggregated protein that eluted exculsively in the void volume (eluted around 55 mL) during gel filtration, while protein purified after optimization of expression and purification conditions eluted around 72 mL. (B) Eluates resulting from size exclusion chromatography were concentrated and analyzed with SDS-PAGE gel electrophoresis and Coomassie staining. (C) This band was cut out, digested with trypsin and analysed further with orbitrap mass spectrometry analysis.</p

GTP and RNA cap analog competition analysis with CHIKV nsP1.

<p>GTP and RNA cap analog competition analysis with CHIKV nsP1.</p

Optimization of FP assay for CHIKV nsP1.

<p>In order to determine the optimal pH for the FP assay, purified CHIKV nsP1 was incubated in the indicated buffer types with 10 nM GTP-Bodipy in black plates for 1 hour at 25°C in the dark. (A) Plates were then scanned for FP and signal windows were calculated for each buffer condition. (B) To determine the effect of DMSO concentration on FP assay signal window, purified CHIKV nsp1 was incubated with 10 nM GTP-Bodipy and various concentrations of DMSO ranging from 0.01%-10%. Plates were incubated for 1 hour at 25°C in the dark and then read for FP. (C) In order to determine the optimal assay volume, purified nsP1 was incubated with 10 nM GTP-Bodipy in FP binding buffer with 0.2% DMSO and plated in black plates in decreasing volumes from 65 μL to 10 μL. Plates were incubated for 1 hour at 25°C in the dark, scanned for FP and signal windows were calculated for each volume. (D) In order to determine the stability of the signal window over time, CHIKV nsP1 was incubated with 10 nM GTP-Bodipy at 25°C in the dark. Plates were scanned every 15 minutes for 120 minutes and signal windows were calculated at each time point. n = 3.</p

Inhibition of guanylation by GTP analogs.

<p>nsP1 was incubated with GTP-680 and 100 μM of each GTP analog for 1 hour. Reactions were resolved on SDS-PAGE gels, scanned for fluorescence on an Odyssey Clx infared imager, and then stained with Coomassie. Guanylation was quantified and normalized to protein amount using ImageJ and Image Studio software. Percent of control (containing no GTP analog) was calculated for each inhibitor. n = 3.</p

Purified CHIKV nsP1 is enzymatically active.

<p>Purified CHIKV nsP1 was incubated with GTP-680 and either 100 μM GTP, 100 μM SAM or 100 μM SAM and GTP for 1 hour. Reactions were resolved on SDS page gels and gels were scanned for fluorescence on an Odyssey Clx Infrared Imaging System before being stained with Commassie. (A) Guanylation was quantified and normalized to protein quantity using ImageJ and Image Studio software. Analysis of guanylation signal indicated a robust signal in Control and SAM only wells and depressed guanylation signals in GTP and GTP + SAM wells (gel shown is a representative gel). (B) Percent of control was calculated for Control, SAM, GTP and GTP + SAM wells. n = 3.</p

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

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

Metabolic stability in human liver microsomes.

<p>BG-323 was incubated with pooled human liver microsomes. After reaction initiation with NADPH, reactions were sampled at times 0, 10, 30 and 60 minutes. BG-323 concentrations were determined by LC-MS/MS at each time point (n = 1).</p