Theoretical insights into the binding interaction of Nirmatrelvir with SARS-CoV-2 Mpro mutants (C145A and C145S): MD simulations and binding free-energy calculation to understand drug resistance.

Mpro, the main protease and a crucial enzyme in SARS-CoV-2 is the most fascinating molecular target for pharmacological treatment and is also liable for viral protein maturation. For antiviral therapy, no drugs have been approved clinically to date. Targeting the Mpro with a compound having inhibitory properties against it can hinder viral replication. The therapeutic potential of the antiviral compound Nirmatrelvir (NMV) against SARS-CoV-2 Mpro was investigated using a systematic approach of molecular docking, MD simulations, and binding free energy calculation based on the MM-GBSA method. NMV, a covalent inhibitor with a recently revealed chemical structure, is a promising oral antiviral clinical candidate with significant in vitro anti-SARS-CoV-2 action in third-phase clinical trials. To explore the therapeutic ability and possible drug resistance, the Mpro system was studied for WT and two of its primary mutants (C145A & C145S). The protein-ligand (Mpro/NMV) complexes were further examined through long MD simulations to check the possible drug resistance in the mutants. To understand the binding affinity, the MM-GBSA method was applied to the Mpro/NMV complexes. Moreover, PCA analysis confirms the detachment of the linker region from the major domains in C145S and C145A mutants allowing for conformational alterations in the active-site region. Based on the predicted biological activities and binding affinities of NMV to WT and mutant (C145A & C145S) Mpro, it can be stipulated that NMV may have conventional potency to act as an anti-viral agent against WT Mpro, while the catalytic-dyad mutations may show substantial mutation-induced drug resistance.Communicated by Ramaswamy H. Sarma.

Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation.

ABSTRACTThe main protease (3-chymotrypsin-like protease, 3CLpro) of SARS-CoV-2 has become a focus of anti-coronavirus research. Despite efforts, drug development targeting 3CLpro has been hampered by limitations in the currently available activity assays. Additionally, the emergence of 3CLpro mutations in circulating SARS-CoV-2 variants has raised concerns about potential resistance. Both emphasize the need for a more reliable, sensitive, and facile 3CLpro assay. Here, we report an orthogonal dual reporter-based gain-of-signal assay for measuring 3CLpro activity in living cells. It builds on the finding that 3CLpro induces cytotoxicity and reporter expression suppression, which can be rescued by its inhibitor or mutation. This assay circumvents most limitations in previously reported assays, especially false positives caused by nonspecific compounds and signal interference from test compounds. It is also convenient and robust for high throughput screening of compounds and comparing the drug susceptibilities of mutants. Using this assay, we screened 1789 compounds, including natural products and protease inhibitors, with 45 compounds that have been reported to inhibit SARS-CoV-2 3CLpro among them. Except for the approved drug PF-07321332, only five of these inhibit 3CLpro in our assays: GC376; PF-00835231; S-217622; Boceprevir; and Z-FA-FMK. The susceptibilities of seven 3CLpro mutants prevalent in circulating variants to PF-07321332, S-217622, and GC376 were also assessed. Three mutants were identified as being less susceptible to PF-07321322 (P132H) and S-217622 (G15S, T21I). This assay should greatly facilitate the development of novel 3CLpro-targeted drugs and the monitoring of the susceptibility of emerging SARS-CoV-2 variants to 3CLpro inhibitors.

Stabilization of the Dimeric State of SARS-CoV-2 Main Protease by GC376 and Nirmatrelvir.

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1' and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.

Structural basis of main proteases of HCoV-229E bound to inhibitor PF-07304814 and PF-07321332.

PF-07321332 and PF-07304814, inhibitors against SARS-CoV-2 developed by Pfizer, exhibit broad-spectrum inhibitory activity against the main protease (Mpro) from various coronaviruses. Structures of PF-07321332 or PF-07304814 in complex with Mpros of various coronaviruses reveal their inhibitory mechanisms against different Mpros. However, the structural information on the lower pathogenic coronavirus Mpro with PF-07321332 or PF-07304814 is currently scarce, which hinders our comprehensive understanding of the inhibitory mechanisms of these two inhibitors. Meanwhile, given that some immunocompromised individuals are still affected by low pathogenic coronaviruses, we determined the structures of lower pathogenic coronavirus HCoV-229E Mpro with PF-07321332 and PF-07304814, respectively, and analyzed and defined in detail the structural basis for the inhibition of HCoV-229E Mpro by both inhibitors. Further, we compared the crystal structures of multiple coronavirus Mpro complexes with PF-07321332 or PF-07304814 to illustrate the differences in the interaction of Mpros, and found that the inhibition mechanism of lower pathogenic coronavirus Mpro was more similar to that of moderately pathogenic coronaviruses. Our structural studies provide new insights into drug development for low pathogenic coronavirus Mpro, and provide theoretical basis for further optimization of both inhibitors to contain potential future coronaviruses.

Nirmatrelvir and COVID-19: development, pharmacokinetics, clinical efficacy, resistance, relapse, and pharmacoeconomics.

Nirmatrelvir/ritonavir (N/R) is one of the most effective antiviral drugs against SARS-CoV-2. The preclinical development, pharmacodynamics and pharmacokinetics of N/R are reviewed herein. Randomized clinical trials have been conducted exclusively with pre-Omicron variants of concern, but in vitro studies show that efficacy against all Omicron sublineages is preserved, as confirmed by post-marketing observational studies. Nevertheless, investigations of large viral genome repositories have shown that mutation in the main protease causing resistance to N/R are increasingly frequent. In addition, virological and clinical rebounds after N/R discontinuation have been reported in immunocompetent patients. This finding is of concern when translated to immunocompromised patients, in whom N/R efficacy has not been formally investigated in clinical trials. Economical sustainability and perspectives for this therapeutic arena are discussed.

Developmental safety of nirmatrelvir in zebrafish (Danio rerio) embryos.

BACKGROUND: Nirmatrelvir, in combination with ritonavir, is one of the first orally available antiviral treatment for coronavirus disease 2019 (COVID-19). Symptomatic pregnant women are at increased risk for severe illness and complications that can affect the developing baby. No malformations or lower embryo-fetal survival have been observed when nirmatrelvir were administered to pregnant rats and rabbits. Safety evaluation of drugs used for treating COVID-19 also in pregnancy is urgent for public health, then in this study we further investigated nirmatrelvir developmental toxicity using zebrafish as in vivo model. MATERIAL AND METHODS: Using the standardized Fish Embryo Toxicity (FET) test, we first determined the lethal concentration 50 (LC50), exposing embryos from gastrula stage up to 120 hr post fertilization (hpf) and daily recording lethality. Then, we exposed embryos to five doses comprising the human therapeutic one and up to the LC50 (25 µM). Morphology was evaluated at 72 and 120 hpf. RESULTS: Nirmatrelvir did not affect survival rate and did not induce morphological defects up to the human therapeutic dose. Exposure at higher doses (2.4× and 3× the human Cmax ) however resulted in decreased hatching rate, reduced growth, slower heartbeat with pericardial edema, reduction of eye dimension, absence of the swim bladder and disruption of the anterior-posterior axis, with lack of tail detachment, spinal curvature and straight and smaller head. CONCLUSIONS: Our findings in zebrafish embryos add further information about developmental nirmatrelvir safety. Further studies are needed for pharmacological safety assessment of nirmatrelvir exposure during pregnancy.

Structural Basis for the Inhibition of SARS-CoV-2 Mpro D48N Mutant by Shikonin and PF-07321332.

Preventing the spread of SARS-CoV-2 and its variants is crucial in the fight against COVID-19. Inhibition of the main protease (Mpro) of SARS-CoV-2 is the key to disrupting viral replication, making Mpro a promising target for therapy. PF-07321332 and shikonin have been identified as effective broad-spectrum inhibitors of SARS-CoV-2 Mpro. The crystal structures of SARS-CoV-2 Mpro bound to PF-07321332 and shikonin have been resolved in previous studies. However, the exact mechanism regarding how SARS-CoV-2 Mpro mutants impact their binding modes largely remains to be investigated. In this study, we expressed a SARS-CoV-2 Mpro mutant, carrying the D48N substitution, representing a class of mutations located near the active sites of Mpro. The crystal structures of Mpro D48N in complex with PF-07321332 and shikonin were solved. A detailed analysis of the interactions between Mpro D48N and two inhibitors provides key insights into the binding pattern and its structural determinants. Further, the binding patterns of the two inhibitors to Mpro D48N mutant and wild-type Mpro were compared in detail. This study illustrates the possible conformational changes when the Mpro D48N mutant is bound to inhibitors. Structural insights derived from this study will inform the development of new drugs against novel coronaviruses.

The Combination of Molnupiravir with Nirmatrelvir or GC376 Has a Synergic Role in the Inhibition of SARS-CoV-2 Replication In Vitro.

Introduction: The development of effective vaccines has partially mitigated the trend of the SARS-CoV-2 pandemic; however, the need for orally administered antiviral drugs persists. This study aims to investigate the activity of molnupiravir in combination with nirmatrelvir or GC376 on SARS-CoV-2 to verify the synergistic effect. Methods: The SARS-CoV-2 strains 20A.EU, BA.1 and BA.2 were used to infect Vero E6 in presence of antiviral compounds alone or in combinations using five two-fold serial dilution of compound concentrations ≤EC90. After 48 and 72 h post-infection, viability was performed using MTT reduction assay. Supernatants were collected for plaque-assay titration. All experiments were performed in triplicate, each being repeated at least three times. The synergistic score was calculated using Synergy Finder version 2. Results: All compounds reached micromolar EC90. Molnupiravir and GC376 showed a synergistic activity at 48 h with an HSA score of 19.33 (p < 0.0001) and an additive activity at 72 h with an HSA score of 8.61 (p < 0.0001). Molnupiravir and nirmatrelvir showed a synergistic activity both at 48 h and 72 h with an HSA score of 14.2 (p = 0.01) and 13.08 (p < 0.0001), respectively. Conclusion: Molnupiravir associated with one of the two protease-inhibitors nirmatrelvir and GC376 showed good additive-synergic activity in vitro.

Nirmatrelvir más ritonavir (Paxlovid) una potente combinación inhibidora de la proteasa 3CLpro del SARS-CoV-2 / Nirmatrelvir plus ritonavir (Paxlovid) a potent SARS-CoV-2 3CLpro protease inhibitor combination

Todos los coronavirus, incluido el SARS-CoV-2, codificandos proteasas necesarias para el procesado de las poliproteínaspp1a y pp1ab. La proteasa principal 3CL (quimiotripsina-like) dalugar a la formación de las proteínas nsp11/16. La proteasa 3CLse ha constituido como una de las posibles dianas terapéuticaspara el desarrollo de fármacos antivirales frente al SARS-CoV-2debido a su secuencia y estructura altamente conservada entretodos los coronavirus. Durante la pandemia del SARS-CoV-1 seidentificó un derivado hidroximetilcetona (PF-00835231) conuna intensa actividad inhibidora frente a la proteasa 3CL. Lasmodificaciones químicas posteriores dieron lugar al derivadoPF-07321332 (nirmatrelvir) que ha mostrado una elevada eficacia antiviral frente al SARS-CoV-2. Los datos de la compañíaindican que es capaz de reducir un 89% el riesgo de hospitalización y fallecimiento de los pacientes infectados con apenasefectos adversos. Su eficacia mejora si se administra por vía oralen las primeras 24-48 horas y la duración del tratamiento se haestablecido entre 3-5 días. La forma comercial lleva asociadael antiviral ritonavir que ha mostrado enlentecer el metabolismo de nirmatrelvir, alargando su vida media. Este antiviral seríaeficaz frente a las actuales y futuras variantes virales, ya quela 3CL no se modifica en ellas. La FDA aprobó este antiviral ennoviembre de 2021 y la EMA está en fase de evaluación final. (AU)

All coronavirus, including SARS-CoV-2, encode two proteases needed for the processing of PP1A and PP1AB polyproteins. The main protease 3CL (chemotripsine-like) gives rise tothe formation of NSP11/16 proteins. The 3CL protease has beenconstituted as one of the possible therapeutic targets for thedevelopment of antiviral drugs against SARS-COV-2 due to itshighly conserved sequence and structure among all coronaviruses. During the SARS-COV-1 pandemic, a hydroxymethyl ketone derivative (PF-00835231) was identified with an intenseinhibitory activity against the 3CL protease. Subsequent chemical modifications gave rise to derivative PF-07321332 (nirmatrelvir) which has shown a high antiviral efficacy againstSARS-COV-2. The company’s data indicate that it is capable ofreducing 89% the risk of hospitalization and death of patientsinfected with hardly adverse effects. Its effectiveness improvesif it is administered orally in the first 24-48 hours and the duration of treatment has been established between 3-5 days. Thecommercial form has been associated with the antiviral ritonavir that has shown the metabolism of nirmatrelvir, lengtheningits average life. This antiviral would be effective against currentand future viral variants, since 3CL is not modified in them. TheFDA approved this antiviral in November 2021 and EMA is inthe final evaluation phase. (AU)

Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants.

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (ß, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, ß, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and ß Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.

Potential Resistance of SARS-CoV-2 Main Protease (Mpro) against Protease Inhibitors: Lessons Learned from HIV-1 Protease.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome 2 (SARS-CoV-2), has been one of the most devastating pandemics of recent times. The lack of potent novel antivirals had led to global health crises; however, emergence and approval of potent inhibitors of the viral main protease (Mpro), such as Pfizer's newly approved nirmatrelvir, offers hope not only in the therapeutic front but also in the context of prophylaxis against the infection. By their nature, RNA viruses including human immunodeficiency virus (HIV) have inherently high mutation rates, and lessons learnt from previous and currently ongoing pandemics have taught us that these viruses can easily escape selection pressure through mutation of vital target amino acid residues in monotherapeutic settings. In this paper, we review nirmatrelvir and its binding to SARS-CoV-2 Mpro and draw a comparison to inhibitors of HIV protease that were rendered obsolete by emergence of resistance mutations, emphasizing potential pitfalls in the design of inhibitors that may be of important relevance to the long-term use of novel inhibitors against SARS-CoV-2.

Structural Basis of the Main Proteases of Coronavirus Bound to Drug Candidate PF-07321332.

The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genera of coronaviruses is the substrate-binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332, developed by Pfizer, is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here, we report three crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of the main protease harbors multiple inhibitor-binding sites, where PF-07321332 occupies subsites S1, S2, and S4 and appears more restricted than other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals. IMPORTANCE The current pandemic of multiple variants has created an urgent need for effective inhibitors of SARS-CoV-2 to complement vaccine strategies. PF-07321332, developed by Pfizer, is the first orally administered coronavirus-specific main protease inhibitor approved by the FDA. We solved the crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and MERS-CoV that bound to the PF-07321332, suggesting PF-07321332 is a broad-spectrum inhibitor for coronaviruses. Structures of the main protease inhibitor complexes present an opportunity to discover safer and more effective inhibitors for COVID-19.

Pharmacological treatment of COVID-19: an opinion paper / Tratamiento farmacológico del COVID-19: un documento de opinión

The precocity and efficacy of the vaccines developed so far against COVID-19 has been the most significant and saving advance against the pandemic. The development of vaccines has not prevented, during the whole period of the pandemic, the constant search for therapeutic medicines, both among existing drugs with different indications and in the development of new drugs. The Scientific Committee of the COVID-19 of the Illustrious College of Physicians of Madrid wanted to offer an early, simplified and critical approach to these new drugs, to new developments in immunotherapy and to what has been learned from the immune response modulators already known and which have proven effective against the virus, in order to help understand the current situation. (AU)

La precocidad y la eficacia de las vacunas desarrolladas hasta ahora frente al COVID-19, ha sido el avance más significativo y salvador frente a la pandemia. El desarrollo vacunal no ha impedido, durante todo el periodo de la pandemia, la búsqueda constante de remedios terapéuticos, tanto entre los medicamentos ya existentes y con indicaciones diversas, como en el desarrollo de nuevos fármacos. Sobre estos nuevos fármacos, sobre las novedades en la inmunoterapia y sobre lo aprendido de los moduladores de la respuesta inmune ya conocidos y que se han mostrado eficaces frente al virus, el Comité Científico del COVID-19 del Ilustre Colegio de Médicos de Madrid ha querido ofrecer una aproximación precoz, simplificada y critica que pueda ayudar a comprender la situación actual. (AU)

Oral Nirmatrelvir/Ritonavir Therapy for COVID-19: The Dawn in the Dark?

Nirmatrelvir/ritonavir (Paxlovid™) is an effective and safe antiviral drug that inhibits the main protease (Mpro), 3CL protease, of SARS-CoV-2. A reduction in COVID-19-related hospitalization or death was observed in patients treated with nirmatrelvir/ritonavir within five days of symptom onset. Moreover, good oral availability enables the usage of nirmatrelvir/ritonavir, not only in hospitalized patients, but also among outpatients. Nirmatrelvir (PF-07321332) has been demonstrated to stop the spread of COVID-19 in animal models. Despite frequent mutations in the viral genomes of SARS-CoV-2, nirmatrelvir shows an effective antiviral effect against recent coronavirus mutants. Despite the promising antiviral effect of nirmatrelvir, there are several unresolved concerns. First, the final results of large-scale clinical trials for early therapy of mild cases of COVID-19 are not yet published. Second, the effectiveness of nirmatrelvir against upcoming variants in the coming years requires close monitoring. Considering the promising preliminary results of the EPIC-HR trial, nirmatrelvir/ritonavir in conjunction with vaccines and non-pharmacological interventions, may represent the dawn in the dark of the COVID-19 pandemic.

[Nirmatrelvir plus ritonavir (Paxlovid) a potent SARS-CoV-2 3CLpro protease inhibitor combination]. / Nirmatrelvir más ritonavir (Paxlovid) una potente combinación inhibidora de la proteasa 3CLpro del SARS-CoV-2.

All coronavirus, including SARS-CoV-2, encode two proteases needed for the processing of PP1A and PP1AB polyproteins. The main protease 3CL (chemotripsine-like) gives rise to the formation of NSP11/16 proteins. The 3CL protease has been constituted as one of the possible therapeutic targets for the development of antiviral drugs against SARS-COV-2 due to its highly conserved sequence and structure among all coronaviruses. During the SARS-COV-1 pandemic, a hydroxymethyl ketone derivative (PF-00835231) was identified with an intense inhibitory activity against the 3CL protease. Subsequent chemical modifications gave rise to derivative PF-07321332 (nirmatrelvir) which has shown a high antiviral efficacy against SARS-COV-2. The company's data indicate that it is capable of reducing 89% the risk of hospitalization and death of patients infected with hardly adverse effects. Its effectiveness improves if it is administered orally in the first 24-48 hours and the duration of treatment has been established between 3-5 days. The commercial form has been associated with the antiviral ritonavir that has shown the metabolism of nirmatrelvir, lengthening its average life. This antiviral would be effective against current and future viral variants, since 3CL is not modified in them. The FDA approved this antiviral in November 2021 and EMA is in the final evaluation phase.

Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS-CoV-2 Papain-Like Protease.

The two SARS-CoV-2 proteases, i. e. the main protease (Mpro ) and the papain-like protease (PLpro ), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PLpro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PLpro inhibitors and selected Mpro inhibitors on PLpro catalysis. The results reveal that some, but not all, PLpro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing Mpro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PLpro . Less selective Mpro inhibitors, e. g. auranofin, inhibit PLpro , highlighting the potential for dual PLpro /Mpro inhibition. MS-based PLpro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.

Pharmacological treatment of COVID-19: an opinion paper.

The precocity and efficacy of the vaccines developed so far against COVID-19 has been the most significant and saving advance against the pandemic. The development of vaccines has not prevented, during the whole period of the pandemic, the constant search for therapeutic medicines, both among existing drugs with different indications and in the development of new drugs. The Scientific Committee of the COVID-19 of the Illustrious College of Physicians of Madrid wanted to offer an early, simplified and critical approach to these new drugs, to new developments in immunotherapy and to what has been learned from the immune response modulators already known and which have proven effective against the virus, in order to help understand the current situation.

Characterization of the non-covalent interaction between the PF-07321332 inhibitor and the SARS-CoV-2 main protease.

We have studied the non-covalent interaction between PF-07321332 and SARS-CoV-2 main protease at the atomic level using a computational approach based on extensive molecular dynamics simulations with explicit solvent. PF-07321332, whose chemical structure has been recently disclosed, is a promising oral antiviral clinical candidate with well-established anti-SARS-CoV-2 activity in vitro. The drug, currently in phase III clinical trials in combination with ritonavir, relies on the electrophilic attack of a nitrile warhead to the catalytic cysteine of the protease. Nonbonded interaction between the inhibitor and the residues of the binding pocket, as well as with water molecules on the protein surface, have been characterized using two different force fields and the two possible protonation states of the main protease catalytic dyad HIS41-CYS145. When the catalytic dyad is in the neutral state, the non-covalent binding is likely to be stronger. Molecular dynamics simulations seems to lend support for an inhibitory mechanism in two steps: a first non-covalent addition with the dyad in neutral form and then the formation of the thiolate-imidazolium ion pair and the ligand relocation for finalising the electrophilic attack.

Exploring the Binding Mechanism of PF-07321332 SARS-CoV-2 Protease Inhibitor through Molecular Dynamics and Binding Free Energy Simulations.

The novel coronavirus disease, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), rapidly spreading around the world, poses a major threat to the global public health. Herein, we demonstrated the binding mechanism of PF-07321332, α-ketoamide, lopinavir, and ritonavir to the coronavirus 3-chymotrypsin-like-protease (3CLpro) by means of docking and molecular dynamic (MD) simulations. The analysis of MD trajectories of 3CLpro with PF-07321332, α-ketoamide, lopinavir, and ritonavir revealed that 3CLpro-PF-07321332 and 3CLpro-α-ketoamide complexes remained stable compared with 3CLpro-ritonavir and 3CLpro-lopinavir. Investigating the dynamic behavior of ligand-protein interaction, ligands PF-07321332 and α-ketoamide showed stronger bonding via making interactions with catalytic dyad residues His41-Cys145 of 3CLpro. Lopinavir and ritonavir were unable to disrupt the catalytic dyad, as illustrated by increased bond length during the MD simulation. To decipher the ligand binding mode and affinity, ligand interactions with SARS-CoV-2 proteases and binding energy were calculated. The binding energy of the bespoke antiviral PF-07321332 clinical candidate was two times higher than that of α-ketoamide and three times than that of lopinavir and ritonavir. Our study elucidated in detail the binding mechanism of the potent PF-07321332 to 3CLpro along with the low potency of lopinavir and ritonavir due to weak binding affinity demonstrated by the binding energy data. This study will be helpful for the development and optimization of more specific compounds to combat coronavirus disease.

Supervised Molecular Dynamics (SuMD) Insights into the mechanism of action of SARS-CoV-2 main protease inhibitor PF-07321332.

The chemical structure of PF-07321332, the first orally available Covid-19 clinical candidate, has recently been revealed by Pfizer. No information has been provided about the interaction pattern between PF-07321332 and its biomolecular counterpart, the SARS-CoV-2 main protease (Mpro). In the present work, we exploited Supervised Molecular Dynamics (SuMD) simulations to elucidate the key features that characterise the interaction between this drug candidate and the protease, emphasising similarities and differences with other structurally related inhibitors such as Boceprevir and PF-07304814. The structural insights provided by SuMD will hopefully be able to inspire the rational discovery of other potent and selective protease inhibitors.