Celia SchifferZvornicanin, Duggan, Sarah2025-07-282025-07-282025-07-3110.13028/238t-qj28https://hdl.handle.net/20.500.14038/54638SARS-CoV-2 main protease (Mpro) is essential for viral replication and a leading target for antiviral development against COVID-19. The direct-acting antiviral (DAA) in Paxlovid, nirmatrelvir, inhibits Mpro effectively, but design efforts thus far have not prioritized the avoidance of drug resistance. Emerging drug resistance to Paxlovid could result in massive public health implications, since no other DAAs against Mpro are approved for use at this time. This dissertation investigates how SARS-CoV-2 Mpro develops resistance, how to design inhibitors that prevent resistance, and what features are essential for broad-spectrum Mpro inhibition across other coronaviruses of pandemic potential. To identify inhibitor interactions most vulnerable to resistance, I calculated the substrate envelope of SARS-CoV-2 Mpro as the consensus shape of its substrates in the active site, and used the envelope to predict possible drug resistance mutations from current therapeutics. Using the substrate envelope, I predicted that E166 would be a hotspot of resistance arising from nirmatrelvir treatment. Thus, I characterized resistant variants E166A and E166V and discovered that L50F is not only hyperactive as a standalone variant but is also a compensatory mutation that restores some catalytic efficiency in combination with highly resistant E166 mutations. These findings offer actionable strategies for designing SARS-CoV-2 DAAs that mimic natural substrates, avoid resistance-prone interactions, and retain potency against resistant variants. To support the development of pan-coronaviral inhibitors, I investigated the evolutionary and structural diversity of coronavirus Mpros. This included solving crystal structures of Mpros from SW1 and HKU15, which revealed both conserved features that may be exploited for broad-spectrum design and divergent elements that may pose challenges for inhibitor generalizability. Finally, I investigated how Mpro coordinates cleavage between its two distal active sites, which exhibit positive cooperativity despite being ~40 Å apart. I designed novel heterodimeric cleavage assays and performed targeted mutagenesis to reveal a conserved network of residues that mediates communication between sites. This work yielded new insights and new questions about Mpro’s cleavage cycle—the sequence of events in dimerization, substrate binding, catalysis, and product release. Together, these findings improve our understanding of which features of Mpro are most vulnerable to resistance, and how to design inhibitors that avoid those liabilities while preserving potency. By calculating the substrate envelope, identifying compensatory and resistance-conferring mutations, solving divergent coronavirus Mpro crystal structures, and mapping the communication network between active sites, this work lays a foundation for designing resilient antivirals against both SARS-CoV-2 and future coronaviruses with pandemic potential. Plus, it highlights inhibitor design strategies which may be broadly applicable to many drug target enzymes.Copyright © 2025 Sarah Zvornicaninhttps://creativecommons.org/licenses/by/4.0/biochemistrySARS-CoV-2COVID-19cooperativitynirmatrelvirdrug resistanceprotease inhibitorstructure-based drug designstructural analysisDoctoral Dissertation0000-0003-0282-1809