Exploration of P1 and P4 modifications of nirmatrelvir: Design, synthesis, biological evaluation, and X-ray structural studies of SARS-CoV-2 Mpro inhibitors.

We report the synthesis, biological evaluation, and X-ray structural studies of a series of SARS-CoV-2 Mpro inhibitors based upon the X-ray crystal structure of nirmatrelvir, an FDA approved drug that targets the main protease of SARS-CoV-2. The studies involved examination of various P4 moieties, P1 five- and six-membered lactam rings to improve potency. In particular, the six-membered P1 lactam ring analogs exhibited high SARS-CoV-2 Mpro inhibitory activity. Several compounds effectively blocked SARS-CoV-2 replication in VeroE6 cells. One of these compounds maintained good antiviral activity against variants of concern including Delta and Omicron variants. A high-resolution X-ray crystal structure of an inhibitor bound to SARS-CoV-2 Mpro was determined to gain insight into the ligand-binding properties in the Mpro active site.

Exploratory Studies of Effective Inhibitors against the SARS-CoV-2 Main Protease by Halogen Incorporation and Amide Bond Replacement.

In the development of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drugs, its main protease (Mpro), which is an essential enzyme for viral replication, is a promising target. To date, the Mpro inhibitors, nirmatrelvir and ensitrelvir, have been clinically developed by Pfizer Inc. and Shionogi & Co., Ltd., respectively, as orally administrable drugs to treat coronavirus disease of 2019 (COVID-19). We have also developed several potent inhibitors of SARS-CoV-2 Mpro that include compounds 4, 5, TKB245 (6), and TKB248 (7), which possesses a 4-fluorobenzothiazole ketone moiety as a reactive warhead. In compounds 5 and TKB248 (7) we have also found that replacement of the P1-P2 amide of compounds 4 and TKB245 (6) with the corresponding thioamide improved their pharmacokinetics (PK) profile in mice. Here, we report the design, synthesis and evaluation of SARS-CoV-2 Mpro inhibitors with replacement of a digestible amide bond by surrogates (9-11, 33, and 34) and introduction of fluorine atoms in a metabolically reactive methyl group on the indole moiety (8). As the results, these compounds showed comparable or less potency compared to the corresponding parent compounds, YH-53/5h (2) and 4. These results should provide useful information for further development of Mpro inhibitors.

Structure-Activity Relationship Studies of SARS-CoV-2 Main Protease Inhibitors Containing 4-Fluorobenzothiazole-2-carbonyl Moieties.

The main protease (Mpro) of SARS-CoV-2 is an attractive target for the development of drugs to treat COVID-19. Here, we report the design, synthesis, and structure-activity relationship (SAR) studies of highly potent SARS-CoV-2 Mpro inhibitors including TKB245 (5)/TKB248 (6). Since we have previously developed Mpro inhibitors (3) and (4), several hybrid molecules of these previous compounds combined with nirmatrelvir (1) were designed and synthesized. Compounds such as TKB245 (5) and TKB248 (6), containing a 4-fluorobenzothiazole moiety at the P1' site, are highly effective in the blockade of SARS-CoV-2 replication in VeroE6 cells. Replacement of the P1-P2 amide with the thioamide surrogate in TKB248 (6) improved its PK profile in mice compared to that of TKB245 (5). A new diversity-oriented synthetic route to TKB245 (5) derivatives was also developed. The results of the SAR studies suggest that TKB245 (5) and TKB248 (6) are useful lead compounds for the further development of Mpro inhibitors.

SARS-CoV-2 papain-like protease (PLpro) inhibitory and antiviral activity of small molecule derivatives for drug leads.

We report here the synthesis and biological evaluation of a series of small molecule SARS-CoV-2 PLpro inhibitors. We compared the activity of selected compounds in both SARS-CoV-1 and SARS-CoV-2 PLpro inhibitory and antiviral assays. We have synthesized and evaluated several new structural variants of previous leads against SARS-CoV-2 PLpro. The replacement of the carboxamide functionality with sulfonamide derivatives resulted in PLpro inhibitors with potent PLpro inhibitory and antiviral activity in VeroE6 cells similar to GRL0617. To obtain molecular insight, we created an optimized model of a potent sulfonamide derivative in the SARS-CoV-2 PLpro active site.

GRL-142 binds to and impairs HIV-1 integrase nuclear localization signal and potently suppresses highly INSTI-resistant HIV-1 variants.

Nuclear localization signal (NLS) of HIV-1 integrase (IN) is implicated in nuclear import of HIV-1 preintegration complex (PIC). Here, we established a multiclass drug-resistant HIV-1 variant (HIVKGD) by consecutively exposing an HIV-1 variant to various antiretroviral agents including IN strand transfer inhibitors (INSTIs). HIVKGD was extremely susceptible to a previously reported HIV-1 protease inhibitor, GRL-142, with IC50 of 130 femtomolar. When cells were exposed to HIVKGD IN-containing recombinant HIV in the presence of GRL-142, significant decrease of unintegrated 2-LTR circular cDNA was observed, suggesting that nuclear import of PIC was severely compromised by GRL-142. X-ray crystallographic analyses revealed that GRL-142 interacts with NLS's putative sequence (DQAEHLK) and sterically blocks the nuclear transport of GRL-142-bound HIVKGD's PIC. Highly INSTI-resistant HIV-1 variants isolated from heavily INSTI-experienced patients proved to be susceptible to GRL-142, suggesting that NLS-targeting agents would serve as salvage therapy agents for highly INSTI-resistant variant-harboring individuals. The data should offer a new modality to block HIV-1 infectivity and replication and shed light on developing NLS inhibitors for AIDS therapy.

Exploration of imatinib and nilotinib-derived templates as the P2-Ligand for HIV-1 protease inhibitors: Design, synthesis, protein X-ray structural studies, and biological evaluation.

Structure-based design, synthesis, X-ray structural studies, and biological evaluation of a new series of potent HIV-1 protease inhibitors are described. These inhibitors contain various pyridyl-pyrimidine, aryl thiazole or alkylthiazole derivatives as the P2 ligands in combination with darunavir-like hydroxyethylamine sulfonamide isosteres. These heterocyclic ligands are inherent to kinase inhibitor drugs, such as nilotinib and imatinib. These ligands are designed to make hydrogen bonding interactions with the backbone atoms in the S2 subsite of HIV-1 protease. Various benzoic acid derivatives have been synthesized and incorporation of these ligands provided potent inhibitors that exhibited subnanomolar level protease inhibitory activity and low nanomolar level antiviral activity. Two high resolution X-ray structures of inhibitor-bound HIV-1 protease were determined. These structures provided important ligand-binding site interactions for further optimization of this class of protease inhibitors.

Identification of SARS-CoV-2 Mpro inhibitors containing P1' 4-fluorobenzothiazole moiety highly active against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

Potent and biostable inhibitors of the main protease of SARS-CoV-2.

Potent and biostable inhibitors of the main protease (Mpro) of SARS-CoV-2 were designed and synthesized based on an active hit compound 5h (2). Our strategy was based not only on the introduction of fluorine atoms into the inhibitor molecule for an increase of binding affinity for the pocket of Mpro and cell membrane permeability but also on the replacement of the digestible amide bond by a surrogate structure to increase the biostability of the compounds. Compound 3 is highly potent and blocks SARS-CoV-2 infection in vitro without a viral breakthrough. The derivatives, which contain a thioamide surrogate in the P2-P1 amide bond of these compounds (2 and 3), showed remarkably preferable pharmacokinetics in mice compared with the corresponding parent compounds. These data show that compounds 3 and its biostable derivative 4 are potential drugs for treating COVID-19 and that replacement of the digestible amide bond by its thioamide surrogate structure is an effective method.

Generation of Angiotensin-Converting Enzyme 2/Transmembrane Protease Serine 2-Double-Positive Human Induced Pluripotent Stem Cell-Derived Spheroids for Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Drug Evaluation.

We newly generated two human induced pluripotent stem cell (hiPSC)-derived spheroid lines, termed Spheroids_4MACE2-TMPRSS2 and Spheroids_15M63ACE2-TMPRSS2, both of which express angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), which are critical for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Both spheroids were highly susceptible to SARS-CoV-2 infection, and two representative anti-SARS-CoV-2 agents, remdesivir and 5h (an inhibitor of SARS-CoV-2's main protease), inhibited the infectivity and replication of SARS-CoV-2 in a dose-dependent manner, suggesting that these human-derived induced spheroids should serve as valuable target cells for the evaluation of anti-SARS-CoV-2 activity. IMPORTANCE The hiPSC-derived spheroids we generated are more expensive to obtain than the human cell lines currently available for anti-SARS-CoV-2 drug evaluation, such as Calu-3 cells; however, the spheroids have better infection susceptibility than the existing human cell lines. Although we are cognizant that there are human lung (and colonic) organoid models for the study of SARS-CoV-2, the production of those organoids is greatly more costly and time consuming than the generation of human iPSC-derived spheroid cells. Thus, the addition of human iPSC-derived spheroids for anti-SARS-CoV-2 drug evaluation studies could provide the opportunity for more comprehensive interpretation of the antiviral activity of compounds against SARS-CoV-2.

Neutralising activity and antibody titre in 10 patients with breakthrough infections of the SARS-CoV-2 Omicron variant in Japan.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 has multiple amino acid mutations in its spike proteins, which may allow it to evade immunity elicited by vaccination. We examined the neutralising activity and S1-IgG titres in patients with breakthrough infections caused by the Omicron variant after two doses of vaccination. We found that neutralising activity was significantly lower for the Omicron variant than for the Wuhan strain. Two doses of vaccination might not induce sufficient neutralising activity for the Omicron variant.

Characterization and antiviral susceptibility of SARS-CoV-2 Omicron BA.2.

The recent emergence of SARS-CoV-2 Omicron (B.1.1.529 lineage) variants possessing numerous mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies and antiviral drugs for COVID-19 against these variants1,2. The original Omicron lineage, BA.1, prevailed in many countries, but more recently, BA.2 has become dominant in at least 68 countries3. Here we evaluated the replicative ability and pathogenicity of authentic infectious BA.2 isolates in immunocompetent and human ACE2-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone4, we observed similar infectivity and pathogenicity in mice and hamsters for BA.2 and BA.1, and less pathogenicity compared with early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from individuals who had recovered from COVID-19 and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987 plus REGN10933, COV2-2196 plus COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir and S-217622) can restrict viral infection in the respiratory organs of BA.2-infected hamsters. These findings suggest that the replication and pathogenicity of BA.2 is similar to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron BA.2 variants.

Characterization and antiviral susceptibility of SARS-CoV-2 Omicron/BA.2.

The recent emergence of SARS-CoV-2 Omicron variants possessing large numbers of mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies, and antiviral drugs for COVID-19 against these variants1,2. While the original Omicron lineage, BA.1, has become dominant in many countries, BA.2 has been detected in at least 67 countries and has become dominant in the Philippines, India, and Denmark. Here, we evaluated the replicative ability and pathogenicity of an authentic infectious BA.2 isolate in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone3, we observed similar infectivity and pathogenicity in mice and hamsters between BA.2 and BA.1, and less pathogenicity compared to early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from COVID-19 convalescent individuals and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987/REGN10933, COV2-2196/COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir, and S-217622) can restrict viral infection in the respiratory organs of hamsters infected with BA.2. These findings suggest that the replication and pathogenicity of BA.2 is comparable to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron/BA.2 variants.

Design, Synthesis and X-Ray Structural Studies of Potent HIV-1 Protease Inhibitors Containing C-4 Substituted Tricyclic Hexahydro-Furofuran Derivatives as P2 Ligands.

The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporate novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2' ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2' ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high-resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.

Chloropyridinyl Esters of Nonsteroidal Anti-Inflammatory Agents and Related Derivatives as Potent SARS-CoV-2 3CL Protease Inhibitors.

We report the design and synthesis of a series of new 5-chloropyridinyl esters of salicylic acid, ibuprofen, indomethacin, and related aromatic carboxylic acids for evaluation against SARS-CoV-2 3CL protease enzyme. These ester derivatives were synthesized using EDC in the presence of DMAP to provide various esters in good to excellent yields. Compounds are stable and purified by silica gel chromatography and characterized using 1H-NMR, 13C-NMR, and mass spectral analysis. These synthetic derivatives were evaluated in our in vitro SARS-CoV-2 3CLpro inhibition assay using authentic SARS-CoV-2 3CLpro enzyme. Compounds were also evaluated in our in vitro antiviral assay using quantitative VeroE6 cell-based assay with RNAqPCR. A number of compounds exhibited potent SARS-CoV-2 3CLpro inhibitory activity and antiviral activity. Compound 9a was the most potent inhibitor, with an enzyme IC50 value of 160 nM. Compound 13b exhibited an enzyme IC50 value of 4.9 µM. However, it exhibited a potent antiviral EC50 value of 24 µM in VeroE6 cells. Remdesivir, an RdRp inhibitor, exhibited an antiviral EC50 value of 2.4 µM in the same assay. We assessed the mode of inhibition using mass spectral analysis which suggested the formation of a covalent bond with the enzyme. To obtain molecular insight, we have created a model of compound 9a bound to SARS-CoV-2 3CLpro in the active site.

Indole Chloropyridinyl Ester-Derived SARS-CoV-2 3CLpro Inhibitors: Enzyme Inhibition, Antiviral Efficacy, Structure-Activity Relationship, and X-ray Structural Studies.

Here, we report the synthesis, structure-activity relationship studies, enzyme inhibition, antiviral activity, and X-ray crystallographic studies of 5-chloropyridinyl indole carboxylate derivatives as a potent class of SARS-CoV-2 chymotrypsin-like protease inhibitors. Compound 1 exhibited a SARS-CoV-2 3CLpro inhibitory IC50 value of 250 nM and an antiviral EC50 value of 2.8 µM in VeroE6 cells. Remdesivir, an RNA-dependent RNA polymerase inhibitor, showed an antiviral EC50 value of 1.2 µM in the same assay. Compound 1 showed comparable antiviral activity with remdesivir in immunocytochemistry assays. Compound 7d with an N-allyl derivative showed the most potent enzyme inhibitory IC50 value of 73 nM. To obtain molecular insight into the binding properties of these molecules, X-ray crystal structures of compounds 2, 7b, and 9d-bound to SARS-CoV 3CLpro were determined, and their binding properties were compared.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.

Characterization of a new SARS-CoV-2 variant that emerged in Brazil.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

Biochemical and Structural Properties of Entecavir-Resistant Hepatitis B Virus Polymerase with L180M/M204V Mutations.

Entecavir (ETV) is a widely used anti-hepatitis B virus (HBV) drug. However, the emergence of resistant mutations in HBV reverse transcriptase (RT) results in treatment failure. To understand the mechanism underlying the development of ETV resistance by HBV RT, we analyzed the L180M, M204V, and L180M/M204V mutants using a combination of biochemical and structural techniques. ETV-triphosphate (ETV-TP) exhibited competitive inhibition with dGTP in both wild-type (wt) RT and M204V RT, as observed using Lineweaver-Burk plots. In contrast, RT L180M or L180M/M204V did not fit either competitive, uncompetitive, noncompetitive, or typical mixed inhibition, although ETV-TP was a competitive inhibitor of dGTP. Crystallography of HIV RTY115F/F116Y/Q151M/F160M/M184V, mimicking HBV RT L180M/M204V, showed that the F115 bulge (F88 in HBV RT) caused by the F160M mutation induced deviated binding of dCTP from its normal tight binding position. Modeling of ETV-TP on the deviated dCTP indicated that a steric clash could occur between ETV-TP methylene and the 3'-end nucleoside ribose. ETV-TP is likely to interact primarily with HBV RT M171 prior to final accommodation at the deoxynucleoside triphosphate (dNTP) binding site (Y. Yasutake, S. Hattori, H. Hayashi, K. Matsuda, et al., Sci Rep 8:1624, 2018, https://doi.org/10.1038/s41598-018-19602-9). Therefore, in HBV RT L180M/M204V, ETV-TP may be stuck at M171, a residue that is conserved in almost all HBV isolates, leading to the strange inhibition pattern observed in the kinetic analysis. Collectively, our results provide novel insights into the mechanism of ETV resistance of HBV RT caused by L180M and M204V mutations. IMPORTANCE HBV infects 257 million people in the world, who suffer from elevated risks of liver cirrhosis and cancer. ETV is one of the most potent anti-HBV drugs, and ETV resistance mutations in HBV RT have been extensively studied. Nevertheless, the mechanisms underlying ETV resistance have remained elusive. We propose an attractive hypothesis to explain ETV resistance and effectiveness using a combination of kinetic and structural analyses. ETV is likely to have an additional interaction site, M171, beside the dNTP pocket of HBV RT; this finding indicates that nucleos(t)ide analogues (NAs) recognizing multiple interaction sites within RT may effectively inhibit the enzyme. Modification of ETV may render it more effective and enable the rational design of efficient NA inhibitors.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

SARS-CoV-2 encodes main protease (Mpro), an attractive target for therapeutic interventions. We show Mpro is susceptible to glutathionylation leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that a single modification with glutathione is sufficient to block dimerization and loss of activity. Proteolytic digestions of Mpro revealed Cys300 as a primary target of glutathionylation, and experiments using a C300S Mpro mutant revealed that Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of Cys300 and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to human disease and the pathophysiology of SARS-CoV-2 in bats, which develop oxidative stress during flight.