Crystal Structure of SARS-CoV-2 Main Protease in Complex with the Non-Covalent Inhibitor ML188.
ABSTRACT: Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (Mpro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 Mpro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 Mpro at 2.5 µM, which is more potent than against SAR-CoV-1 Mpro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 Mpro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19.
Project description:Severe diseases such as the ongoing COVID-19 pandemic, as well as the previous SARS and MERS outbreaks, are the result of coronavirus infections and have demonstrated the urgent need for antiviral drugs to combat these deadly viruses. Due to its essential role in viral replication and function, 3CL<sup>pro</sup> (main coronaviruses cysteine-protease) has been identified as a promising target for the development of antiviral drugs. Previously reported SARS-CoV 3CL<sup>pro</sup> non-covalent inhibitors were used as a starting point for the development of covalent inhibitors of SARS-CoV-2 3CL<sup>pro</sup>. We report herein our efforts in the design and synthesis of submicromolar covalent inhibitors when the enzymatic activity of the viral protease was used as a screening platform.
Project description:The main protease (M<sup>pro</sup>) is a validated antiviral drug target of SARS-CoV-2. A number of M<sup>pro</sup> inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 M<sup>pro</sup> inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates <b>Jun9-62-2R</b> (dichloroacetamide) and <b>Jun9-88-6R</b> (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to <b>GC-376</b>, these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent M<sup>pro</sup> inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 M<sup>pro</sup> with <b>Jun9-62-2R</b> and <b>Jun9-57-3R</b> reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.
Project description:The spread of SARS-CoV-2 has affected human health globally. Hence, it is necessary to rapidly find the drug-candidates that can be used to treat the infection. Since the main protease (M<sup>pro</sup>) is the key protein in the virus's life cycle, M<sup>pro</sup> is served as one of the critical targets of antiviral treatment. We employed virtual screening tools to search for new inhibitors to accelerate the drug discovery process. The hit compounds were subsequently docked into the active site of SARS-CoV-2 main protease and ranked by their binding energy. Furthermore, in-silico ADME studies were performed to probe for adoption with the standard ranges. Finally, molecular dynamics simulations were applied to study the protein-drug complex's fluctuation over time in an aqueous medium. This study indicates that the interaction energy of the top ten retrieved compounds with COVID-19 main protease is much higher than the interaction energy of some currently in use protease drugs such as ML188, nelfinavir, lopinavir, ritonavir, and ?-ketoamide. Among the discovered compounds, Pubchem44326934 showed druglike properties and was further analyzed by MD and MM/PBSA approaches. Besides, the constant binding free energy over MD trajectories suggests a probable drug possessing antiviral properties. MD simulations demonstrate that GLU166 and GLN189 are the most important residues of M<sup>pro,</sup> which interact with inhibitors.
Project description:The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently needs an effective cure. 3CL protease (3CL<sup>pro</sup>) is a highly conserved cysteine proteinase that is indispensable for coronavirus replication, providing an attractive target for developing broad-spectrum antiviral drugs. Here we describe the discovery of myricetin, a flavonoid found in many food sources, as a non-peptidomimetic and covalent inhibitor of the SARS-CoV-2 3CL<sup>pro</sup>. Crystal structures of the protease bound with myricetin and its derivatives unexpectedly revealed that the pyrogallol group worked as an electrophile to covalently modify the catalytic cysteine. Kinetic and selectivity characterization together with theoretical calculations comprehensively illustrated the covalent binding mechanism of myricetin with the protease and demonstrated that the pyrogallol can serve as an electrophile warhead. Structure-based optimization of myricetin led to the discovery of derivatives with good antiviral activity and the potential of oral administration. These results provide detailed mechanistic insights into the covalent mode of action by pyrogallol-containing natural products and a template for design of non-peptidomimetic covalent inhibitors against 3CL<sup>pro</sup>s, highlighting the potential of pyrogallol as an alternative warhead in design of targeted covalent ligands.
Project description:Covalent inhibitors targeting the main protease (M<sup>pro</sup>, or 3CLpro) of SARS-CoV-2 have shown promise in preclinical investigations. Herein, we report the discovery of two new series of molecules that irreversibly bind to SARS-CoV-2 M<sup>pro</sup>. These acrylamide containing molecules were discovered using our covalent DNA-encoded library (DEL) screening platform. Following selection against SARS-CoV-2 M<sup>pro</sup>, off-DNA compounds were synthesized and investigated to determine their inhibitory effects, the nature of their binding, and to generate preliminary structure-activity relationships. LC-MS analysis indicates a 1:1 (covalent) binding stoichiometry between our hit molecules and SARS-CoV-2 M<sup>pro</sup>. Fluorescent staining assay for covalent binding in the presence of cell lysate suggests reasonable selectivity for SARS-CoV-2 M<sup>pro</sup>. And lastly, inhibition of enzymatic activity was also observed against a panel of 3CLpro enzymes from different coronavirus strains, with IC<sub>50</sub> values ranging from inactive to single digit micromolar. Our results indicate that DEL selection is a useful approach for identifying covalent inhibitors of cysteine proteases.
Project description:A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure-activity relationships within S(1'), S(1), and S(2) enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action.
Project description:The main protease (3CL M<sup>pro</sup>) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. To date, no small-molecule clinical drugs are available that specifically inhibit SARS-CoV-2 M<sup>pro</sup>. To aid rational drug design, we determined a neutron structure of M<sup>pro</sup> in complex with the α-ketoamide inhibitor telaprevir at near-physiological (22 °C) temperature. We directly observed protonation states in the inhibitor complex and compared them with those in the ligand-free M<sup>pro</sup>, revealing modulation of the active-site protonation states upon telaprevir binding. We suggest that binding of other α-ketoamide covalent inhibitors can lead to the same protonation state changes in the M<sup>pro</sup> active site. Thus, by studying the protonation state changes induced by inhibitors, we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 M<sup>pro</sup>.
Project description:The main protease, M<sup>pro</sup> (or 3CL<sup>pro</sup>) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the M<sup>pro</sup> from both SARS-CoV and SARS-CoV-2 with IC<sub>50</sub> values in the nanomolar range. Crystal structures of SARS-CoV-2 M<sup>pro</sup> with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.
Project description:The outbreak of coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has turned into a pandemic. The enzyme 3C-like protease (3CL<sup>pro</sup>) is essential for the maturation of viral polyproteins in SARS-CoV-2 and is therefore regarded as a key drug target for treating the disease. To identify 3CL<sup>pro</sup> inhibitors that can suppress SARS-CoV-2 replication, we performed a virtual screening of 500,282 compounds in a Korean compound bank. We then subjected the top computational hits to inhibitory assays against 3CL<sup>pro</sup> in vitro, leading to the identification of a class of non-covalent inhibitors. Among these inhibitors, compound 7 showed an EC<sub>50</sub> of 39.89 μM against SARS-CoV-2 and CC<sub>50</sub> of 453.5 μM. This study provides candidates for the optimization of potent 3CL<sup>pro</sup> inhibitors showing antiviral effects against SARS-CoV-2.
Project description:The SARS-CoV-2 main protease (M<sup>pro</sup>) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 M<sup>pro</sup> with a known Michael acceptor (peptidyl) inhibitor, <b>N3</b>. The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods. The simulations show a two-step mechanism, and give structures and calculated barriers in good agreement with experiment. Using these results and information from our previous investigation on the proteolysis reaction of SARS-CoV-2 M<sup>pro</sup>, we design two new, synthetically accessible <b>N3</b>-analogues as potential inhibitors, in which the recognition and warhead motifs are modified. QM/MM modelling of the mechanism of inhibition of M<sup>pro</sup> by these novel compounds indicates that both may be promising candidates as drug leads against COVID-19, one as an irreversible inhibitor and one as a potential reversible inhibitor.