Structure-Based Optimization of ML300-Derived, Noncovalent Inhibitors Targeting the Severe Acute Respiratory Syndrome Coronavirus 3CL Protease (SARS-CoV-2 3CLpro).

Starting from the MLPCN probe compound ML300, a structure-based optimization campaign was initiated against the recent severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (3CLpro). X-ray structures of SARS-CoV-1 and SARS-CoV-2 3CLpro enzymes in complex with multiple ML300-based inhibitors, including the original probe ML300, were obtained and proved instrumental in guiding chemistry toward probe compound 41 (CCF0058981). The disclosed inhibitors utilize a noncovalent mode of action and complex in a noncanonical binding mode not observed by peptidic 3CLpro inhibitors. In vitro DMPK profiling highlights key areas where further optimization in the series is required to obtain useful in vivo probes. Antiviral activity was established using a SARS-CoV-2-infected Vero E6 cell viability assay and a plaque formation assay. Compound 41 demonstrates nanomolar activity in these respective assays, comparable in potency to remdesivir. These findings have implications for antiviral development to combat current and future SARS-like zoonotic coronavirus outbreaks.

Inhibition Mechanism of SARS-CoV-2 Main Protease with Ketone-Based Inhibitors Unveiled by Multiscale Simulations: Insights for Improved Designs*.

We present the results of classical and QM/MM simulations for the inhibition of SARS-CoV-2 3CL protease by a hydroxymethylketone inhibitor, PF-00835231. In the noncovalent complex the carbonyl oxygen atom of the warhead is placed in the oxyanion hole formed by residues 143 to 145, while P1-P3 groups are accommodated in the active site with interactions similar to those observed for the peptide substrate. According to alchemical free energy calculations, the P1' hydroxymethyl group also contributes to the binding free energy. Covalent inhibition of the enzyme is triggered by the proton transfer from Cys145 to His41. This step is followed by the nucleophilic attack of the Sγ atom on the carbonyl carbon atom of the inhibitor and a proton transfer from His41 to the carbonyl oxygen atom mediated by the P1' hydroxyl group. Computational simulations show that the addition of a chloromethyl substituent to the P1' group may lower the activation free energy for covalent inhibition.

Pseudo-Dipeptide Bearing α,α-Difluoromethyl Ketone Moiety as Electrophilic Warhead with Activity against Coronaviruses.

The synthesis of α-fluorinated methyl ketones has always been challenging. New methods based on the homologation chemistry via nucleophilic halocarbenoid transfer, carried out recently in our labs, allowed us to design and synthesize a target-directed dipeptidyl α,α-difluoromethyl ketone (DFMK) 8 as a potential antiviral agent with activity against human coronaviruses. The ability of the newly synthesized compound to inhibit viral replication was evaluated by a viral cytopathic effect (CPE)-based assay performed on MCR5 cells infected with one of the four human coronaviruses associated with respiratory distress, i.e., hCoV-229E, showing antiproliferative activity in the micromolar range (EC50 = 12.9 ± 1.22 µM), with a very low cytotoxicity profile (CC50 = 170 ± 3.79 µM, 307 ± 11.63 µM, and 174 ± 7.6 µM for A549, human embryonic lung fibroblasts (HELFs), and MRC5 cells, respectively). Docking and molecular dynamics simulations studies indicated that 8 efficaciously binds to the intended target hCoV-229E main protease (Mpro). Moreover, due to the high similarity between hCoV-229E Mpro and SARS-CoV-2 Mpro, we also performed the in silico analysis towards the second target, which showed results comparable to those obtained for hCoV-229E Mpro and promising in terms of energy of binding and docking pose.

Polyketone metabolites isolated from Rhodiola tibetica endohytic fungus Alternaria sp. HJT-Y7 and their SARS-CoV-2 virus inhibitory activitives.

Six new polyketone metabolites, compounds (1-6) and seven known polyketone compounds (7-13) were isolated from Rhodiola tibetica endophytic fungus Alternaria sp. The structural elucidation of five new polyketone metabolites were elucidated on the basis of spectroscopic including 2D NMR and HRMS and spectrometric analysis. Inhibition rate evaluation revealed that compounds 1(EC50 = 0.02 mM), 3(EC50 = 0.3 mM), 6(EC50 = 0.07 mM), 8(EC50 = 0.1 mM) and 9(EC50 = 0.04 mM) had inhibitory effect on the SARS-CoV-2 virus.

3CL Protease Inhibitors with an Electrophilic Arylketone Moiety as Anti-SARS-CoV-2 Agents.

The novel coronavirus, SARS-CoV-2, has been identified as the causative agent for the current coronavirus disease (COVID-19) pandemic. 3CL protease (3CLpro) plays a pivotal role in the processing of viral polyproteins. We report peptidomimetic compounds with a unique benzothiazolyl ketone as a warhead group, which display potent activity against SARS-CoV-2 3CLpro. The most potent inhibitor YH-53 can strongly block the SARS-CoV-2 replication. X-ray structural analysis revealed that YH-53 establishes multiple hydrogen bond interactions with backbone amino acids and a covalent bond with the active site of 3CLpro. Further results from computational and experimental studies, including an in vitro absorption, distribution, metabolism, and excretion profile, in vivo pharmacokinetics, and metabolic analysis of YH-53 suggest that it has a high potential as a lead candidate to compete with COVID-19.

Peptidomimetic α-Acyloxymethylketone Warheads with Six-Membered Lactam P1 Glutamine Mimic: SARS-CoV-2 3CL Protease Inhibition, Coronavirus Antiviral Activity, and in Vitro Biological Stability.

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e, 15h, and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.

Field-Template, QSAR, Ensemble Molecular Docking, and 3D-RISM Solvation Studies Expose Potential of FDA-Approved Marine Drugs as SARS-CoVID-2 Main Protease Inhibitors.

Currently, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has infected people among all countries and is a pandemic as declared by the World Health Organization (WHO). SARS-CoVID-2 main protease is one of the therapeutic drug targets that has been shown to reduce virus replication, and its high-resolution 3D structures in complex with inhibitors have been solved. Previously, we had demonstrated the potential of natural compounds such as serine protease inhibitors eventually leading us to hypothesize that FDA-approved marine drugs have the potential to inhibit the biological activity of SARS-CoV-2 main protease. Initially, field-template and structure-activity atlas models were constructed to understand and explain the molecular features responsible for SARS-CoVID-2 main protease inhibitors, which revealed that Eribulin Mesylate, Plitidepsin, and Trabectedin possess similar characteristics related to SARS-CoVID-2 main protease inhibitors. Later, protein-ligand interactions are studied using ensemble molecular-docking simulations that revealed that marine drugs bind at the active site of the main protease. The three-dimensional reference interaction site model (3D-RISM) studies show that marine drugs displace water molecules at the active site, and interactions observed are favorable. These computational studies eventually paved an interest in further in vitro studies. Finally, these findings are new and indeed provide insights into the role of FDA-approved marine drugs, which are already in clinical use for cancer treatment as a potential alternative to prevent and treat infected people with SARS-CoV-2.

Michael Acceptors Tuned by the Pivotal Aromaticity of Histidine to Block COVID-19 Activity.

The question of whether COVID protease (SARS-CoV-2 Mpro) can be blocked by inhibitors has been examined, with a particularly successful performance exhibited by α-ketoamide derivative substrates like 13b of Hilgenfeld and co-workers (Zhang, L., et al. Science 2020, 368, 409-412). After the biological characterization, here density functional theory calculations explain not only how inhibitor 13b produces a thermodynamically favorable interaction but also how to reach it kinetically. The controversial and unprovable concept of aromaticity here enjoys being the agent that rationalizes the seemingly innocent role of histidine (His41 of Mpro). It has a hydrogen bond with the hydroxyl group and is the proton carrier of the thiol of Cys145 at almost zero energy cost that favors the interaction with the inhibitor that acts as a Michael acceptor.

Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2*.

The global pandemic caused by SARS-CoV-2 calls for the fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high-throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including unnatural amino acids that targets the nonprimed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within a human proteome without background. We expect that these reagents will be useful in the drug-development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses.