Document text
Principal Investigator: Matthew Hall
Organization: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
Fiscal Year: 2024
Award: $500,000
Funding agency: National Center for Advancing Translational Sciences
1.The Target-Based Assays 1 (Martinez) Group has identified and prioritized viral targets amenable for therapeutic intervention via small molecule modulation, including endonucleases, exonuclease, proteases, and methyltransferases. Candidate compounds resulting from each assay will be screened in live-virus assays by APP colleagues, and in silico approaches are used to support screening efforts. These viral targets include:
•Inhibitors of Bunyaviral endonucleases: High-throughput Foster resonance energy transfer (FRET)-based biochemical assays for RVFV, LACV, HANV, and PTV endonucleases have been developed and screened against small molecule libraries. Cell-based Split-luciferase cellular thermal shift assay (CETSA) have been developed to further characterize inhibitor-endonuclease engagement.
•Inhibitors of viral proteases: High-throughput FRET-based biochemical assays for Flavivirus NS2B-NS3 (WNV, ZIKV, DENV2, and YWF), Alphavirus NSP2 (CHKV), and Picornavirus 2A and 3C (EN68 and E71) proteases have been developed and screened.
•Inhibitors of viral Methyltransferases: A high-throughput screening platform utilizing recombinant MTases from families including Flaviviral and Coronaviral MTases has been developed. Flaviviral NS5 (DENV, WNV) has also been screened using a DNA-Encoded Library (DEL) platform and hits are being validated in MTase-Glo based and binding assays.
•Inhibitors of viral Exonuclease: a FRET-based assay was developed to identify inhibitors of Coronavirus NSP14/10 exonuclease activity and screened.
2.The Target-Based Assays 2 (Kales) group has developed assays to facilitate the study of mutations in key CoV-2 therapeutic targets and have leverage those technologies and platforms to target other viral families of pandemic potential, including Alphaviruses. The team has initiated the design and optimization of assays related to the following:
•Inhibitors of SARS-CoV-2 PLpro Protease: the team has completed screening over 100K compounds and has validated several novel chemotypes through a multi-assay format workflow, which includes several biochemical assays, a cell-based nanoluciferase reporter, and live virus testing with the Advanced Models and Cell-based Discovery Group.
•Inhibitors of SARS-CoV-2 3CL/Mpro Protease: to assess effects of mutations on current therapeutics, including Paxlovid, the team has developed a nanoluciferase cell-based reporter that can facilitate rapid testing of emerging 3CL mutations. To date, 13 mutant variant forms of SARS-CoV-2 3CL have been tested to assess activity and efficacy of existing therapeutics and to support the identification of novel therapeutics to emerging mutations.
•Non-Nucleoside Inhibitors (NNIs) of SARS-CoV-2 RNA-dependent RNA Polymerase: several HTS-amenable biochemical assays and multiple secondary assays have been developed to support screening for inhibitors against recombinant CoV-2 RdRp. This platform has since been pivoted to target other viral RdRps, including Enterovirus D68 (EVD68). To date, over 50K compounds have been screened against EVD68 RdRp and hit validation in a live virus assay is underway.
•Bunyavirales Minigenome: to support ongoing screening of Bunyavirales antivirals, our team has adopted two Minigenome Virus Reporters of Lassa virus (LASV) and has validated several active chemotypes from our in-house medicinal chemistry efforts. Building from this platform, our team is currently optimizing an Ebola Minigenome Reporter to expand our target portfolio.
•Inhibitors of Alphavirus nsp3/macrodomain: several activity and binding-based assays towards nsp3/macrodomain of Chikungunya (CHIKV) and Venezuelan Equine Encephalitis (VEEV) have been developed. To date, nearly 100K compounds have been screened and novel chemotypes have been identified that demonstrate no activity towards the human counter target, MacroD2. Off-DNA synthesis is underway and hit profiling will include other Alphavirus macrodomains currently in expression, including Eastern Equine Encephalitis Virus (EEEV) and Mayaro virus (MAYV).
3.The Advanced Models and Cell-Based Discovery (Lee) group works across a large panel of BSL-2 and BSL-3 viruses in both 2D and 3D assays for antiviral screening and development.
•Live viruses: More than 25 species of full-length or isolate human viruses have been onboarded across 10 viral families, including Bunyavirales, Arenaviridae JUNV, Peribunyaviridae CEV, LACV, Phenuiviridae RVFV-MP12 and PTV (strains -Balliet and -Adames), Nairoviridae HAZV; Coronaviridae SARS-CoV-2 (previous and current variants of concern), HCoV-OC43/GFP, HCoV-229E, HCoV-NL63;Flaviviridae DENV (serotypes 1-4), KUNV, YFV-17D, ZIKV (strains -MR766 and -PRVAB59); Paramyxoviridae PIV-3-GFP, PIV-1-GFP; Picornaviridae EV-D68; Pneumoviridae RSV; Togaviridae CHIKV 181/25/nluc, MAYV/nluc, ONNV-GFP, RRV-GFP, SINV-GFP (neuroinvasive and non-neuroinvasive strains), and VEEV-Tc83-GFP.
•HTS Assays: the team has 22 live virus 1536w or 384w HTS assays developed and 8 in development across 23 viral species, including Coronaviridae, Phenuiviridae (Bunyavirales), Peribunyaviridae (Bunyavirales), Arenaviridae (Bunyavirales), Flaviviridae, Paramyxoviridae, Picornaviridae and Togaviridae. In addition, the team continues to optimize and support the BSL-3 high-containment lab on NIH main campus containing liquid handlers, an automated confocal plate reader and an integrated screening system to support APP screening pipelines.
•Predictive complex in vitro models for antiviral evaluation: the team has been continuing work and establishment of models for antiviral testing in ALI lung models and small intestinal models for coronaviruses, neural spheroid infection and disease models for neurotropic viruses including viruses from Togaviridae and Bunyavirales (RVFV, CHIKV, CEV, JCV, LACV, MAYV, SINV, and VEEV) and hit compound validation, liver spheroid infection and disease models for a panel of viruses from Togaviridae, Bunyavirales, and Flaviviridae and hit compound validation, and brain endothelial vascular bed model that recapitulates damage induced by exposure to the viral protein NS1 from the flavivirus JEV
•Screening of NCATS libraries against a panel of viruses (CHIKV, MAYV, VEEV, ZIKV, KUNV, LACV, RVFV, EVD-68, FIPV) of outbreak or pandemic potential
•Critical support of multiple collaborations
4.The Medicinal Chemistry group is working simultaneously on multiple targets, to either validate hits that come out of the high throughput screening of NCATS or collaborator libraries, or to drive SAR on the validated targets. The aim of these efforts is to provide high quality leads as promising preclinical candidates.
•Papain like protease, SARS-CoV-2: SAR optimization of a literature lead has produced compounds with cell activity in a nano luciferase reporter virus assay.
•Endonuclease, BUNV: High throughput screening of a metal binding pharmacophore library via FRET-based biochemical endonuclease assays has been completed, and hits are being optimized through extensive SAR studies to drive down potency and understand in vivo exposure liabilities.
•Exonuclease, nsp14/10, SARS CoV2: A FRET-based exonuclease assay has been used for the high throughput screening of NCATS libraries to find potent inhibitors of the viral exonuclease, and several chemotypes have been identified for validation via synthesis and retesting. Single agent activity of these exonuclease inhibitors and the potential synergy with RdRp inhibitors is being evaluated in cell-based assays.
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