Antivirals for Rare, Emerging and Endemic Viruses of the Genus Alphavirus

National Center For Advancing Translational Sciences

Investigators

Abstract

Several binding assays towards nsp3/macrodomain of Chikungunya (CHIKV) and Venezuelan Equine Encephalitis (VEEV) have been developed and optimized. These include HTRF and AlphaLISA assays using a novel peptide substrate as well as NanoDSF and SPR. Activity-based assays are in development as well as a cell-based assay to bridge the gap between binding and activity with live virus. Four screening strategies are being pursued: 1) HTS of NCATS compound libraries (nearly 100K compounds) with novel chemotypes identified that demonstrate no activity towards the human counter target, MacroD2; 2) DNA encoded library (DEL) screening at a CRO, with off-DNA synthesis and hit confirmation completed; 3) High throughput structure-based fragment screening at the Diamond Light Source (UK) with hits informing the outputs from the other assays; 4) NMR-based fragment screen using in-house core facilities, with confirmed hits feeding into ongoing med chem/fragment exploration. This assay can also further validate hits from the other approaches. To complement hit triage, NanoDSF and SPR-based assays have been developed to confirm compound binding, and activity-based assays are currently being optimized. Hit profiling will include other Alphavirus macrodomains, which have been expressed and purified by the Protein Expression Lab (Frederick) for testing, including Eastern Equine Encephalitis Virus (EEEV) and Mayaro virus (MAYV). This combined platform includes representative species of Old and New World Alphavirus families. Other biophysical methods including CETSA and X-ray crystallography are being explored to complement this workflow. Full-length or isolate human viruses have been onboarded across the genus, including: CHIKV 181/25/nluc, MAYV/nluc, ONNV-GFP, RRV-GFP, SINV-GFP (neuroinvasive and non-neuroinvasive strains), and VEEV-Tc83-GFP. Live virus 1536 well or 384 well HTS assays have been developed and NCATS libraries have been screened against a panel of viruses (CHIKV, MAYV, VEEV). Finally, the team has been continuing work on predictive complex in vitro neural and liver spheroid infection and disease models for the neurotropic Alphaviruses (CHIKV, MAYV, SINV, and VEEV). The team is optimizing leads against members of the genus derived from the HTS and DEL confirmed hits, informed by the structures from the fragment screen, NMR solution structural studies, and molecular modeling, and with valuation of drug metabolism and pharmacokinetic (DMPK) properties. The team has identified an attractive low molecular weight lead class with balanced, sub-micromolar activity against CHIKV and VEEV.