Structural Biology and Computational Modeling Core

The Structural Biology (SB) Core headed by Larry Shapiro and Dinshaw Patel will provide Research Project teams with biochemical, biophysical, structural and computational expertise and support towards identification and optimization of small-molecule inhibitors that individually and combinatorially target single- and multisubunit nonstructural proteins (Nsps) encoded by the SARS-CoV-2 viral genome. These targets include cysteine proteases 3CLpro (Nsp3) and PLpro (Nsp5) and RNA-dependent RNA polymerase (RdRp, Nsp12-Nsp7-Nsp82) critical for viral RNA transcription and replication, as well as the proofreading exonuclease (ExoN, Nsp14), the capping methylases N7-G-MTase (Nsp14-Nsp10) and 2’-O-MTase (Nsp16-Nsp10), and the multisubunit replication-transcription (RTC) complex critical for RNA capping machinery and synthesis fidelity. Based on ongoing and anticipated collaborations, the Shapiro lab will support research on projects 1, 2 and 5 focused primarily on SARS-CoV-2 inhibitors targeting 3CLpro protease and project 6 focused on inhibitors identified from metagenome screens, while the Patel lab will support research on project 3 focused on SARS- CoV-2 protease and methylase inhibitors and project 4 on inhibitors of viral RNA synthesis, including the RTC complex. The SB Core anticipates solving 6 to 9 X-ray based structures and 3 to 5 cryo-EM-based structures per year as part of this effort, while also providing computational expertise guiding iterative cycles of structure determination and chemical modification leading to optimization of the most promising candidates. Aim 1: The SB Core will assist Project research efforts in protein expression and purification, as well as RNA transcription and purification, followed by a detailed thermodynamic (ITC and SPR) and structural (X-ray and cryo-EM) characterization of small molecule leads identified from experimental and in silico screens targeted to Nsps of SARS-CoV-2 and metagenomic screens targeted to the intact virus. Aim 2: The SB Core will analyze inhibitor-target Nsp interactions and structure-activity (SAR) relationships to facilitate computation-based structure-guided lead compound optimization involving iterative cycles of structure determination and chemical derivatization. This effort, involving close collaboration between SB, Medicinal and Pharmacology Cores, will be targeted to identification and characterization of optimized leads that exhibit improved in vitro ADMET and in vivo pharmacokinetic properties against a range of SARS-CoV-2 Nsp targets, including the RTC complex. Aim 3: The SB Core will assist Project research efforts to extend ongoing studies on inhibitors targeting SARS- CoV-2 Nsps to analogous members of the flavivirus and alphavirus families. A comparison of structure-derived pocket architectures and intermolecular contacts for inhibitor-protein complexes amongst virus families should determine to what extent inhibitors targeted to Nsps of SARS-CoV-2 need additional modifications for targeting their analogous Nsps in flavivirus and alphavirus family members.