Progress in Developing Inhibitors of SARS-CoV-2 3C-Like Protease.
ABSTRACT: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral outbreak started in late 2019 and rapidly became a serious health threat to the global population. COVID-19 was declared a pandemic by the World Health Organization in March 2020. Several therapeutic options have been adopted to prevent the spread of the virus. Although vaccines have been developed, antivirals are still needed to combat the infection of this virus. SARS-CoV-2 is an enveloped virus, and its genome encodes polyproteins that can be processed into structural and nonstructural proteins. Maturation of viral proteins requires cleavages by proteases. Therefore, the main protease (3 chymotrypsin-like protease (3CLpro) or Mpro) encoded by the viral genome is an attractive drug target because it plays an important role in cleaving viral polyproteins into functional proteins. Inhibiting this enzyme is an efficient strategy to block viral replication. Structural studies provide valuable insight into the function of this protease and structural basis for rational inhibitor design. In this review, we describe structural studies on the main protease of SARS-CoV-2. The strategies applied in developing inhibitors of the main protease of SARS-CoV-2 and currently available protein inhibitors are summarized. Due to the availability of high-resolution structures, structure-guided drug design will play an important role in developing antivirals. The availability of high-resolution structures, potent peptidic inhibitors, and diverse compound scaffolds indicate the feasibility of developing potent protease inhibitors as antivirals for COVID-19.
Project description: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 (M<sup>pro</sup>) 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 M<sup>pro</sup>, 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 M<sup>pro</sup> at 2.5 µM, which is more potent than against SAR-CoV-1 M<sup>pro</sup>. We determined the crystal structure of ML188 in complex with SARS-CoV-2 M<sup>pro</sup> 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:Coronaviruses are responsible for multiple pandemics and millions of deaths globally, including the current pandemic of coronavirus disease 2019 (COVID-19). Development of antivirals against coronaviruses, including the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) responsible for COVID-19, is essential for containing the current and future coronavirus outbreaks. SARS-CoV-2 proteases represent important targets for the development of antivirals because of their role in the processing of viral polyproteins. 3-Chymotrypsin-like protease (3CLpro) is one such protease. The cleavage of SARS-CoV-2 polyproteins by 3CLpro is facilitated by a Cys145-His41 catalytic dyad. We here characterized the catalytic roles of the cysteine-histidine pair for improved understanding of the 3CLpro reaction mechanism, to inform the development of more effective antivirals against Sars-CoV-2. The catalytic dyad residues were substituted by site-directed mutagenesis. All substitutions tested (H41A, H41D, H41E, C145A, and C145S) resulted in a complete inactivation of 3CLpro, even when amino acids with a similar catalytic function to that of the original residues were used. The integrity of the structural fold of enzyme variants was investigated by circular dichroism spectroscopy to test if the catalytic inactivation of 3CLpro was caused by gross changes in the enzyme secondary structure. C145A, but not the other substitutions, shifted the oligomeric state of the enzyme from dimeric to a higher oligomeric state. Finally, the thermodynamic stability of 3CLpro H41A, H41D, and C145S variants was reduced relative the wild-type enzyme, with a similar stability of the H41E and C145A variants. Collectively, the above observations confirm the roles of His41 and Cys145 in the catalytic activity and the overall conformational fold of 3CLpro SARS-CoV-2. We conclude that the cysteine-histidine pair should be targeted for inhibition of 3CLpro and development of antiviral against COVID-19 and coronaviruses.
Project description:Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (M<sup>pro</sup>) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the M<sup>pro</sup> substrate-binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 M<sup>pro</sup> protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their M<sup>pro</sup> inhibiting activities did not correlate with their antiviral activities. This conundrum is resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PL<sup>pro</sup>). HCV drugs that inhibit PL<sup>pro</sup> synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir's antiviral activity as much as 10-fold, while those that only inhibit M<sup>pro</sup> do not synergize with remdesivir.
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:Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CL<sup>pro</sup>) and papain-like protease (PL<sup>pro</sup>) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease. In this study, 3CL<sup>pro</sup> and PL<sup>pro</sup> assay platforms were established, and their substrate specificities were characterized. The assays were used to screen collections of 1,068 and 2,701 FDA-approved drugs. After excluding the externally used drugs which are too toxic, we totally identified 12 drugs as 3CL<sup>pro</sup> inhibitors and 36 drugs as PL<sup>pro</sup> inhibitors active at 10 μM. Among these inhibitors, six drugs were found to suppress SARS-CoV-2 with the half-maximal effective concentration (EC<sub>50</sub>) below or close to 10 μM. This study enhances our understanding on the proteases and provides FDA-approved drugs for prevention and/or treatment of COVID-19.
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), similar to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which belong to the same Betacoronavirus genus, induces severe acute respiratory disease that is a threat to human health. Since the outbreak of infection by SARS-CoV-2 began, which causes coronavirus disease 2019 (COVID-19), the disease has rapidly spread worldwide. Thus, a search for effective drugs able to inhibit SARS-CoV-2 has become a global pursuit. The 3C-like protease (3CL<sup>pro</sup>), which hydrolyses viral polyproteins to produce functional proteins, is essential for coronavirus replication and is considered an important therapeutic target for diseases caused by coronaviruses, including COVID-19. Many 3CL<sup>pro</sup> inhibitors have been proposed and some new drug candidates have achieved success in preclinical studies. In this review, we briefly describe recent developments in determining the structure of 3CL<sup>pro</sup> and its function in coronavirus replication and summarise new insights into 3CL<sup>pro</sup> inhibitors and their mechanisms of action. The clinical application prospects and limitations of 3CL<sup>pro</sup> inhibitors for COVID-19 treatment are also discussed.
Project description:The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.
Project description:We prepared the three-dimensional model of the SARS-CoV-2 (aka 2019-nCoV) 3C-like protease (3CL <sup>pro</sup>) using the crystal structure of the highly similar (96% identity) ortholog from the SARS-CoV. All residues involved in the catalysis, substrate binding and dimerisation are 100% conserved. Comparison of the polyprotein PP1AB sequences showed 86% identity. The 3C-like cleavage sites on the coronaviral polyproteins are highly conserved. Based on the near-identical substrate specificities and high sequence identities, we are of the opinion that some of the previous progress of specific inhibitors development for the SARS-CoV enzyme can be conferred on its SARS-CoV-2 counterpart. With the 3CL <sup>pro</sup> molecular model, we performed virtual screening for purchasable drugs and proposed 16 candidates for consideration. Among these, the antivirals ledipasvir or velpatasvir are particularly attractive as therapeutics to combat the new coronavirus with minimal side effects, commonly fatigue and headache. The drugs Epclusa (velpatasvir/sofosbuvir) and Harvoni (ledipasvir/sofosbuvir) could be very effective owing to their dual inhibitory actions on two viral enzymes.
Project description:SARS-CoV-2 main protease (M<sup>pro</sup>) is a cysteine protease that mediates the cleavage of viral polyproteins and is a validated antiviral drug target. M<sup>pro</sup> is highly conserved among all seven human coronaviruses, with certain M<sup>pro</sup> inhibitors having broad-spectrum antiviral activity. In this study, we designed two hybrid inhibitors <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> based on the superimposed X-ray crystal structures of SARS-CoV-2 M<sup>pro</sup> with GC-376, telaprevir, and boceprevir. Both <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> showed potent binding and enzymatic inhibition against the M<sup>pro</sup>'s from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-229E, and HCoV-HKU1. Cell-based Flip-GFP M<sup>pro</sup> assay results show that <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> inhibited the intracellular protease activity of SARS-CoV-2 M<sup>pro</sup>. In addition, <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> had potent antiviral activity against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E, with <b>UAWJ9-36-3</b> being more potent than GC-376 in inhibiting SARS-CoV-2. Selectivity profiling revealed that <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> had an improved selectivity index over that of GC-376 against host cysteine proteases calpain I and cathepsin L, but not cathepsin K. The X-ray crystal structures of SARS-CoV-2 M<sup>pro</sup> with <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> were both solved at 1.9 Å, which validated our design hypothesis. Overall, hybrid inhibitors <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> are promising candidates to be further developed as broad-spectrum coronavirus antivirals.
Project description:The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL M<sup>pro</sup> due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL M<sup>pro</sup>, revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.