The emergence of SARS-CoV-2 variants and drug-resistant mutants calls for additional oral antivirals. The SARS-CoV-2 papain-like protease (PL ^pro ) is a promising but challenging drug target. We designed and synthesized 85 noncovalent PL ^pro inhibitors that bind to a recently discovered ubiquitin binding site and the known BL2 groove pocket near the S4 subsite. Leads inhibited PL ^pro with the inhibitory constant K _i values from 13.2 to 88.2 nanomolar. The co-crystal structures of PL ^pro with eight leads revealed their interaction modes. The in vivo lead Jun12682 inhibited SARS-CoV-2 and its variants, including nirmatrelvir-resistant strains with EC ₅₀ from 0.44 to 2.02 micromolar. Oral treatment with Jun12682 improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting that PL ^pro inhibitors are promising oral SARS-CoV-2 antiviral candidates.

The ever-evolving SARS-CoV-2 variants necessitate the development of additional oral antivirals. This study presents the systematic design of quinoline-containing SARS-CoV-2 papain-like protease (PL^pro) inhibitors as potential oral antiviral drug candidates. By leveraging the recently discovered Val70^Ub binding site in PL^pro, we designed a series of quinoline analogs demonstrating potent PL^pro inhibition and antiviral activity. Notably, the X-ray crystal structures of 6 lead compounds reveal that the 2-aryl substitution can occupy either the Val70^Ub site as expected or the BL2 groove in a flipped orientation. The in vivo lead Jun13296 exhibits favorable pharmacokinetic properties and potent inhibition against SARS-CoV-2 variants and nirmatrelvir-resistant mutants. In a mouse model of SARS-CoV-2 infection, oral treatment with Jun13296 significantly improves survival, reduces body weight loss and lung viral titers, and prevents lung tissue damage. These results underscore the potential of quinoline PL^pro inhibitors as promising oral SARS-CoV-2 antiviral candidates, instilling hope for the future of SARS-CoV-2 treatment.

The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial enzymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein, Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld's ability to work with diverse partners, we solved its crystal structure. We observed the canonical flavodoxin (Fld) fold and features common to other long-chain Flds but not all the surface loops thought to recognize partner proteins. Moreover, some of the loops display alternative structures and dynamics. To advance studies of protein-protein associations and conformational consequences, we assigned the ¹⁹F NMR signals of all five tyrosines (Tyrs). Our electrochemical measurements show that incorporation of 3-¹⁹F-Tyr in place of Tyr has only a modest effect on Rp9Fld's redox properties even though Tyrs flank the flavin on both sides. Meanwhile, the ¹⁹F probes demonstrate the expected paramagnetic effect, with signals from nearby Tyrs becoming broadened beyond detection when the flavin semiquinone is formed. However, the temperature dependencies of chemical shifts and linewidths reveal dynamics affecting loops close to the flavin and regions that bind to partners in a variety of systems. These coincide with patterns of amino acid type conservation but not retention of specific residues, arguing against detailed specificity with respect to partners. We propose that the loops surrounding the flavin adopt altered conformations upon binding to partners and may even participate actively in electron transfer.