3CLpro Research Articles

In silicoDrug Repurposing for the Identification of Antimalarial Drugs as Candidate Inhibitors of SARS-CoV-2

Background:Coronavirus disease (COVID-19) is a severe acute respiratory condition that has affected millions of people worldwide, indicating a global health emergency. Despite the deteriorating trends of COVID-19, no drugs are validated to have substantial efficacy in the potential treatment of COVID-19 patients in large-scale trials.Methods:This study aimed at identifying potential antimalarial candidate molecules for the treatment of COVID and evaluating the possible mechanism of action by in silico screening method. In silico screening studies on various antimalarial compounds, like amodiaquine, chloroquine, hydroxychloroquine, mefloquine, primaquine, and atovaquone, were conducted using PyRx and AutoDoc 1.5.6 tools against ACE 2 receptor, 3CL protease, hemagglutinin esterase, spike protein of SARS HR1 motif, and papain-like protease virus proteins.Results:Based on PyRx results, mefloquine and atovaquone were found to have higher docking affinity scores against virus proteins compared to other antimalarial compounds. Screening report of atovaquone exhibited affirmative inhibition constant for spike protein of SARS HR1 motif, 3CL protease, and papain-like protease.Conclusion:In silico analysis reported atovaquone as a promising candidate for COVID 19 therapy.

Progress on SARS-CoV-2 3CLpro Inhibitors: Inspiration from SARS-CoV 3CLpro Peptidomimetics and Small-Molecule Anti-Inflammatory Compounds.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) currently poses a threat to human health. 3C-like proteinase (3CLpro) plays an important role in the viral life cycle. Hence, it is considered an attractive antiviral target protein. Whole-genome sequencing showed that the sequence homology between SARS-CoV-2 3CLpro and SARS-CoV 3CLpro is 96.08%, with high similarity in the substrate-binding region. Thus, assessing peptidomimetic inhibitors of SARS-CoV 3CLpro could accelerate the development of peptidomimetic inhibitors for SARS-CoV-2 3CLpro. Accordingly, we herein discuss progress on SARS-CoV-2 3CLpro peptidomimetic inhibitors. Inflammation plays a major role in the pathophysiological process of COVID-19. Small-molecule compounds targeting 3CLpro with both antiviral and anti-inflammatory effects are also briefly discussed in this paper.

Bioactive Components of Houttuynia cordata Thunb and Their Potential Mechanisms Against COVID-19 Using Network Pharmacology and Molecular Docking Approaches.

We investigated the molecular mechanism by which Houttuynia cordata Thunb (HCT) may intervene in coronavirus disease 2019 (COVID-19) and COVID-19-induced cytokine storms using network pharmacology and molecular docking approaches. Using the Traditional Chinese medicine Systems Pharmacology Database and Analysis Platform (TCMSP), a "component-target-pathway" topology map of HCT for COVID-19 treatment was constructed using Cytoscape. Core target genes were analyzed using the STRING database, and the signal pathway map and biological mechanism of COVID-19 therapy were obtained using cluster profilers. Active components of HCT were docked with severe respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease (3CLpro) and RNA-dependent RNA polymerase (RdRp) using AutoDockTools. Data visualization and statistical analysis were conducted using the R program. A molecular dynamic simulation was carried out with the Groningen Machine for Chemical Simulation program. HCT had six active anti-COVID-19 ingredients and 45 molecular targets. Their crucial target proteins for COVID-19 treatment were the RELA (nuclear factor kappa B [NF-κB] p65 subunit), interleukin 6, and mitogen-activated protein kinase 1. In functional enrichment analysis, the potential molecular targets of active components of HCT for COVID-19 treatment belonged to 18 signaling pathways (adjusted P = 2.12E-11). Gene ontology obtained by Kyoto Encyclopedia of Genes and Genome enrichment screening showed that the primary mechanism of COVID-19 treatment was upregulation of protein kinase C followed by downregulations of T cell differentiation and proliferation and NF-κB signaling. Molecular docking showed that the active components of HCT (quercetin and kaempferol) had similar binding affinities for SARS-CoV-2 3CLpro and SARS-CoV-2 RdRp, primary COVID-19 target proteins as did clinically used drugs. These results were confirmed with molecular dynamics simulation. In conclusion, multiple components of HCT, especially quercetin and kaempferol, have the potential to treat COVID-19 infection and COVID-19-induced cytokine storm by targeting multiple proteins.

Targeting natural products against SARS-CoV-2.

The human coronavirus disease (COVID-19) pandemic is caused by a novel coronavirus; the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2). Natural products, secondary metabolites show positive leads with antiviral and immunotherapy treatments using genomic studies in silico docking. In addition, it includes the action of a mechanism targeting the SARS-CoV-2. In this literature, we aimed to evaluate the antiviral movement of the NT-VRL-1 unique terpene definition to Human coronavirus (HCoV-229E). The effects of 19 hydrolysable tannins on the SARS-CoV-2 were therefore theoretically reviewed and analyzed utilising the molecular operating surroundings for their C-Like protease 3CLpro catalytic dyad residues Angiotensin converting enzyme-2 (MOE 09). Pedunculagin, tercatan, and castalin were detected as interacting strongly with SARS-receptor Cov-2’s binding site and catalytic dyad (Cys145 and His41). SARS-CoV-2 methods of subunit S1 (ACE2) inhibit the interaction of the receiver with the s-protein once a drug molecule is coupled to the s-protein and prevent it from infecting the target cells in alkaloids. Our review strongly demonstrates the evidence that natural compounds and their derivatives can be used against the human coronavirus and serves as an area of research for future perspective.

Genomic evolution of the Coronaviridae family

The current outbreak of coronavirus disease-2019 (COVID-19) caused by SARS-CoV-2 poses unparalleled challenges to global public health. SARS-CoV-2 is a Betacoronavirus, one of four genera belonging to the Coronaviridae subfamily Orthocoronavirinae. Coronaviridae, in turn, are members of the order Nidovirales, a group of enveloped, positive-stranded RNA viruses. Here we present a systematic phylogenetic and evolutionary study based on protein domain architecture, encompassing the entire proteomes of all Orthocoronavirinae, as well as other Nidovirales. This analysis has revealed that the genomic evolution of Nidovirales is associated with extensive gains and losses of protein domains. In Orthocoronavirinae, the sections of the genomes that show the largest divergence in protein domains are found in the proteins encoded in the amino-terminal end of the polyprotein (PP1ab), the spike protein (S), and many of the accessory proteins. The diversity among the accessory proteins is particularly striking, as each subgenus possesses a set of accessory proteins that is almost entirely specific to that subgenus. The only notable exception to this is ORF3b, which is present and orthologous over all Alphacoronaviruses. In contrast, the membrane protein (M), envelope small membrane protein (E), nucleoprotein (N), as well as proteins encoded in the central and carboxy-terminal end of PP1ab (such as the 3C-like protease, RNA-dependent RNA polymerase, and Helicase) show stable domain architectures across all Orthocoronavirinae. This comprehensive analysis of the Coronaviridae domain architecture has important implication for efforts to develop broadly cross-protective coronavirus vaccines.

Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.

SARS-CoV-2 3CLpro displays faster self-maturation in vitro than SARS-CoV 3CLpro due to faster C-terminal cleavage.

The coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has become a worldwide pandemic. The 3C-like protease (3CLpro ), which cleaves 11 sites including its own N- and C-termini on the viral polyproteins, is essential for SARS-CoV-2 replication. In this study, we constructed the full-length inactive 3CLpro with N- and C-terminal extensions as substrates for monitoring self-cleavage by wild-type 3CLpro . We found that the rate-limiting C-terminal self-cleavage rate of SARS-CoV-2 3CLpro was 35-fold faster than that of SARS-CoV 3CLpro using the Trx/GST-tagged C145A 3CLpro substrates. Since self-cleavage of 3CLpro is the initial step for maturation of other viral proteins, our study suggests more facile SARS-CoV-2 replication than that of SARS-CoV.

Atorvastatin Effectively Inhibits Ancestral and Two Emerging Variants of SARS-CoV-2 in vitro.

This article evaluated the in vitro antiviral effect of atorvastatin (ATV) against SARS-CoV-2 and identified the interaction affinity between this compound and two SARS-CoV-2 proteins. The antiviral activity of atorvastatin against this virus was evaluated by three different treatment strategies [(i) pre-post treatment, (ii) pre-infection treatment, and (iii) post-infection treatment] using Vero E6 and Caco-2 cells. The interaction of atorvastatin with RdRp (RNA-dependent RNA polymerase) and 3CL protease (3-chymotrypsin-like protease) was evaluated by molecular docking. The CC50s (half-maximal cytotoxic concentrations) obtained for ATV were 50.3 and 64.5 μM in Vero E6 and Caco-2, respectively. This compound showed antiviral activity against SARS-CoV-2 D614G strain in Vero E6 with median effective concentrations (EC50s) of 15.4, 12.1, and 11.1 μM by pre-post, pre-infection, and post-infection treatments, respectively. ATV also inhibited Delta and Mu variants by pre-post treatment (EC50s of 16.8 and 21.1 μM, respectively). In addition, ATV showed an antiviral effect against the D614G strain independent of the cell line (EC50 of 7.4 μM in Caco-2). The interaction of atorvastatin with SARS-CoV-2 RdRp and 3CL protease yielded a binding affinity of −6.7 kcal/mol and −7.5 kcal/mol, respectively. Our study demonstrated the in vitro antiviral activity of atorvastatin against the ancestral SARS-CoV-2 D614G strain and two emerging variants (Delta and Mu), with an independent effect of the cell line. A favorable binding affinity between ATV and viral proteins by bioinformatics methods was found. Due to the extensive clinical experience of atorvastatin use, it could prove valuable in the treatment of COVID-19.

In Silico Multi-Target Approach Revealed Potential Lead Compounds as Scaffold for the Synthesis of Chemical Analogues Targeting SARS-CoV-2.

Simple SummaryCOVID-19 is an infectious disease caused by SARS-CoV-2. The virus has rapidly spread to humans, causing the ongoing coronavirus pandemic. Enormous progress in finding therapies has been made, but an effective therapy is still absent. In this study, we propose a computational strategy aimed at identifying novel multi-target scaffolds against the virus. The proposed study was performed using bioinformatics methods, such as homology modeling, virtual screening and classical molecular dynamics, where 3D structures of targets were reconstructed and compounds with potential inhibitory activity against different virus targets were identified. Furthermore, a potential mechanism of action was proposed. Our study could provide new insights and approaches for the rapid identification of novel multi-target inhibitors of SARS-CoV-2.Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infectious disease that spreads rapidly in humans. In March 2020, the World Health Organization (WHO) declared a COVID-19 pandemic. Identifying a multi-target-directed ligand approach would open up new opportunities for drug discovery to combat COVID-19. The aim of this work was to perform a virtual screening of an exclusive chemical library of about 1700 molecules containing both pharmacologically active compounds and synthetic intermediates to propose potential protein inhibitors for use against SARS-CoV-2. In silico analysis showed that our compounds triggered an interaction network with key residues of the SARS-CoV-2 spike protein (S-protein), blocking trimer formation and interaction with the human receptor hACE2, as well as with the main 3C-like protease (3CLpro), inhibiting their biological function. Our data may represent a step forward in the search for potential new chemotherapeutic agents for the treatment of COVID-19.

Oridonin Inhibits SARS-CoV-2 by Targeting Its 3C-Like Protease.

The current COVID‐19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), is an enormous threat to public health. The SARS‐CoV‐2 3C‐like protease (3CLpro), which is critical for viral replication and transcription, has been recognized as an ideal drug target. Herein, it is identified that three herbal compounds, Salvianolic acid A (SAA), (–)‐Epigallocatechin gallate (EGCG), and Oridonin, directly inhibit the activity of SARS‐CoV‐2 3CLpro. Further, blocking SARS‐CoV‐2 infectivity by Oridonin is confirmed in cell‐based experiments. By solving the crystal structure of 3CLpro in complex with Oridonin and comparing it to that of other ligands with 3CLpro, it is identified that Oridonin binds at the 3CLpro catalytic site by forming a C—S covalent bond, which is confirmed by mass spectrometry and kinetic study, blocking substrate binding through a nonpeptidomimetic covalent binding mode. Thus, Oridonin is a novel candidate to develop a new antiviral treatment for COVID‐19.

Efficient Identification of Anti-SARS-CoV-2 Compounds Using Chemical Structure- and Biological Activity-Based Modeling.

Identification of anti-SARS-CoV-2 compounds through traditional high-throughput screening (HTS) assays is limited by high costs and low hit rates. To address these challenges, we developed machine learning models to identify compounds acting via inhibition of the entry of SARS-CoV-2 into human host cells or the SARS-CoV-2 3-chymotrypsin-like (3CL) protease. The optimal classification models achieved good performance with area under the receiver operating characteristic curve (AUC-ROC) values of >0.78. Experimental validation showed that the best performing models increased the assay hit rate by 2.1-fold for viral entry inhibitors and 10.4-fold for 3CL protease inhibitors compared to those of the original drug repurposing screens. Twenty-two compounds showed potent (<5 μM) antiviral activities in a SARS-CoV-2 live virus assay. In conclusion, machine learning models can be developed and used as a complementary approach to HTS to expand compound screening capacities and improve the speed and efficiency of anti-SARS-CoV-2 drug discovery.

In silico discovery of 3 novel quercetin derivatives against papain-like protease, spike protein, and 3C-like protease of SARS-CoV-2

BackgroundThe derivatives of quercetin is known for their immune-modulating antiviral, anti-blood clotting, antioxidant, and also for its anti-inflammatory efficacy. The current study was therefore conducted to examine the noted novel derivatives of quercetin present in plant sources as an immune modulator and as an antiviral molecule in the COVID-19 disease and also to study their affinity of binding with potential three targets reported for coronavirus, i.e., papain-like protease, spike protein receptor-binding domain, and 3C-like protease.Based on the high-positive drug-likeness score, the reported derivatives of quercetin obtained from an open-source database were further filtered. Compounds with positive and high drug-likeness scores were further predicted for their potential targets using DIGEP-Pred software, and STRING was used to evaluate the interaction between modulated proteins. The associated pathways were recorded based on the Kyoto Encyclopedia of Genes and Genomes pathway database. Docking was performed finally using PyRx having AutoDock Vina to identify the efficacy of binding between quercetin derivatives with papain-like protease, spike protein receptor-binding domain, and 3C-like protease. The ligand that scored minimum binding energy was chosen to visualize the interaction between protein and ligand. Normal mode analysis in internal coordinates was done with normal mode analysis to evaluate the physical movement and stability of the best protein-ligand complexes using the iMODS server. ResultsForty bioactive compounds with the highest positive drug-likeness scores were identified. These 40 bioactives were responsible for regulating different pathways associated with antiviral activity and modulation of immunity. Finally, three lead molecules were identified based on the molecular docking and dynamics simulation studies with the highest anti-COVID-19 and immunomodulatory potentials. Standard antiviral drug remdesivir on docking showed a binding affinity of − 5.8 kcal/mol with PLpro, − 6.4 kcal/mol with 3CLpro, and − 8.6 kcal/mol with spike protein receptor-binding domain of SARS-CoV-2, the discovered hit molecules quercetin 3-O-arabinoside 7-O-rhamnoside showed binding affinity of − 8.2 kcal/mol with PLpro, whereas quercetin 3-[rhamnosyl-(1- > 2)-alpha-L-arabinopyranoside] and quercetin-3-neohesperidoside-7-rhamnoside was predicted to have a binding affinity of − 8.5 kcal/mol and − 8.8 kcal/mol with spike protein receptor-binding domain and 3CLpro respectively ConclusionDocking study revealed quercetin 3-O-arabinoside 7-O-rhamnoside to possess the highest binding affinity with papain-like protease, quercetin 3-[rhamnosyl-(1- > 2)-alpha-L-arabinopyranoside] with spike protein receptor-binding domain, and quercetin-3-neohesperidoside-7-rhamnoside with 3C-like protease and all the protein-ligand complexes were found to be stable after performing the normal mode analysis of the complexes in internal coordinates. Graphical Abstract▪

An in silico discovery of potential 3CL protease inhibitors of SARS-CoV-2 based upon inactivation of the cysteine 145-Histidine 41 catalytic dyad

Coronavirus disease 19 (COVID19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, several countries are at risk of the pandemic caused by this virus. In the absence of any vaccine or virus-specific antiviral treatments, the need is to fast track search for potential drug candidates to combat the virus. Though there are known drugs that are being repurposed to fight against the SARS-CoV-2, there is a requirement for the virus-specific drugs at the earliest. One of the main drug targets of SARS-CoV-2 is an essential non-structural protein, 3CL protease, critical for the life cycle of the virus. We have used molecular docking studies to screen a chemically diverse set of small molecules to identify potential drug candidates to target this protein. Of the 22,630 molecules from varied small molecule libraries, based on the binding affinities and physicochemical properties, we finalized 30 molecules to be potential drug candidates. Eight of these molecules bind in a manner allowing for the scope of a nearly orthogonal backside nucleophilic attack on their suitably placed electrophilic carbonyl groups by the thiol group of cysteine residue 145, while remaining inside a 4 Ǻ distance range. It is interesting since carbonyl groups are known to be attacked in a similar fashion by external nucleophiles and can be relevant when considering these molecules as potential mechanism-based irreversible inhibitors of the 3CLPro. Further, ADMET analysis and Molecular dynamics simulations and available bioactive assays led to the identification of three molecules with high potential to be explored as drug candidates/lead molecules to target 3CLPro of SARS-CoV-2. Communicated by Ramaswamy H. Sarma

&lt;i&gt;In Vitro&lt;/i&gt; and &lt;i&gt;in Silico&lt;/i&gt; Studies of Potential Coronavirus-Specific 3C-Like Protease Inhibitors

We investigated similar compounds to ebselen and tideglusib, which exhibit strong activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using Molecular ACCess System (MACCS) keys. Four candidate compounds were identified. One of them, phenyl-benzothiazol-3-one, showed coronavirus-specific 3C-like (3CL) protease inhibitory activity. The results indicated that a similarity score above 0.81 is a good indicator of activity for ebselen-and-tideglusib-like compounds. Subsequently, we simulated the ring-cleavage Michael reaction of ebselen at the Se center, which is responsible for its 3CL protease inhibitory activity, and determined the activation free energy of the reaction. The results showed that reaction simulation is a useful tool for estimating the activity of inhibitory compounds that undergo Michael addition reactions with the relevant cysteine S atom of 3CL proteases.

Replication-transcription complex of coronaviruses: functions of individual viral non-structural subunits, properties and architecture of their complexes.

Coronaviruses (CoVs) belong to the subfamily Orthocoronavirinae of the family Coronaviridae. CoVs are enveloped (+) RNA viruses with unusually long genomes. Severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and the novel coronavirus (2019-nCoV, SARS-CoV-2) have been identif ied as causing global pandemics. Clinically tested vaccines are widely used to control rapidly spreading, acute, and often severe infections; however, effective drugs are still not available. The genomes of SARS-CoV-2 and SARS-CoV are approximately 80 % identical, while the genomes of SARS-CoV-2 and MERS-CoV are approximately 50 % identical. This indicates that there may be common mechanisms of coronavirus pathogenesis and, therefore, potential therapeutic targets for each virus may be the same. The enzymes and effector proteins that make up the replication-transcription complex (RTC) of coronaviruses are encoded by a large replicase gene. These enzymes and effector proteins represent promising targets for potential therapeutic drugs. The enzyme targets include papain- and 3C-like cysteine proteinases that process two large viral polyproteins, RNA-dependent RNA polymerase, RNA helicase, viral genome-modifying enzymes, and enzymes with 3’–5’ exoribonuclease or uridylate-specif ic endonuclease activity. Currently, there are many studies investigating the complex molecular mechanisms involved in the assembly and function of the RTC. This review will encompass current, modern studies on the properties and complexes of individual non-structural subunits of the RTC, the structures of individual coronavirus RTC subunits, domain organization and functions of subunits, protein-protein interactions, properties and architectures of subunit complexes, the effect of mutations, and the identif ication of mutations affecting the viability of the virus in cell culture. Key words: non-structural proteins CoVs; subunits of replicase CoVs; replication-transcription complex of CoVs; architecture of non-structural protein complexes CoVs.

Antiviral Potential of Indian Medicinal Plants Against Influenza and SARS-CoV: A Systematic Review

The COVID-19 pandemic has posed a significant threat to human health due to the lack of drugs that can potentially act against SARS-CoV -2. Also, even after the emergency approval of WHO, the vaccines’ efficacy is still a question, and people are getting reinfections. Previous studies have demonstrated the efficacy of traditional medicinal plants against influenza and SARS coronavirus. The present article aims to review potential phytochemicals from Indian medicinal plants that may be used against SARS-CoV-2. Articles published in the English language between 1992 and 2021 were retrieved from Embase, PubMed, and Google scholar using relevant keywords, and the scientific literature on efficacies of Indian medicinal plants against SARS-CoV and influenza virus were analyzed. The initial search revealed 1304 studies, but, on subsequent screening, 115 eligible studies were reported. Twenty research articles investigating traditional medicinal plant extracts and metabolites against SARS-CoV and influenza A virus in in vitro and in vivo systems satisfied the search criteria. The studies reported that plant extracts and active compounds such as glycyrrhizin, 14-α-lipoyl andrographolide, and curcumin from medicinal plants such as Yashtimadhu ( Glycyrrhiza glabra), Bhunimba ( Andrographis paniculata), and Haridra ( Curcuma longa) are effective against the various phases of the virus life cycle, viz., virus-host cell attachment, viral replication, 3CL protease activity, neuraminidase activity, adsorption and penetration of the virus. As per ancient Indian literature, plants in Ayurveda possess Rasayana (revitalizing) and Jwara hara (antipyretic, anti-inflammatory) properties. This evidence may be used to conduct experimental and clinical trials to study the underlying mechanisms and efficacy of antiviral properties of Indian medicinal plants against SARS-CoV-2.

Application of Acoustic Ejection MS System to High-Throughput Screening for SARS-CoV-2 3CL Protease Inhibitors.

MS is a powerful methodology for chemical screening to directly quantify substrates and products of enzymes, but its low throughput has been an issue. Recently, an acoustic liquid-handling apparatus (Echo®) used for rapid nano-dispensing has been coupled to a high-sensitivity mass spectrometer to create the Echo® MS system, and we applied this system to screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease inhibitors. Primary screening of 32033 chemical samples was completed in 12 h. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.

Nirmatrelvir plus ritonavir (Paxlovid) a potent SARS-CoV-2 3CLpro protease inhibitor combination

Todos los coronavirus, incluido el SARS-CoV-2, codifican dos proteasas necesarias para el procesado de las poliproteínas pp1a y pp1ab. La proteasa principal 3CL (quimiotripsina-like) da lugar a la formación de las proteínas nsp11/16. La proteasa 3CL se ha constituido como una de las posibles dianas terapéuticas para el desarrollo de fármacos antivirales frente al SARS-CoV-2 debido a su secuencia y estructura altamente conservada entre todos los coronavirus. Durante la pandemia del SARS-CoV-1 se identificó un derivado hidroximetilcetona (PF-00835231) con una intensa actividad inhibidora frente a la proteasa 3CL. Las modificaciones químicas posteriores dieron lugar al derivado PF-07321332 (nirmatrelvir) que ha mostrado una elevada eficacia antiviral frente al SARS-CoV-2. Los datos de la compañía indican que es capaz de reducir un 89% el riesgo de hospitalización y fallecimiento de los pacientes infectados con apenas efectos adversos. Su eficacia mejora si se administra por vía oral en las primeras 24-48 horas y la duración del tratamiento se ha establecido entre 3-5 días. La forma comercial lleva asociada el antiviral ritonavir que ha mostrado enlentecer el metabolismo de nirmatrelvir, alargando su vida media. Este antiviral sería eficaz frente a las actuales y futuras variantes virales, ya que la 3CL no se modifica en ellas. La FDA aprobó este antiviral en noviembre de 2021 y la EMA está en fase de evaluación final.

Discovery of SARS-CoV-2 3CLPro Peptidomimetic Inhibitors through the Catalytic Dyad Histidine-Specific Protein-Ligand Interactions.

As the etiological agent for the coronavirus disease 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenges the ongoing efforts of vaccine development and drug design. Due to the accumulating cases of breakthrough infections, there are urgent needs for broad-spectrum antiviral medicines. Here, we designed and examined five new tetrapeptidomimetic anti-SARS-CoV-2 inhibitors targeting the 3C-Like protease (3CLPro), which is highly conserved among coronaviruses and essential for viral replications. We significantly improved the efficacy of a ketoamide lead compound based on high-resolution co-crystal structures, all-atom simulations, and binding energy calculations. The inhibitors successfully engaged the catalytic dyad histidine residue (H41) of 3CLPro as designed, and they exhibited nanomolar inhibitory capacity as well as mitigated the viral loads of SARS-CoV-2 in cellular assays. As a widely applicable design principle, our results revealed that the potencies of 3CLPro-specific drug candidates were determined by the interplay between 3CLPro H41 residue and the peptidomimetic inhibitors.

Disposition of Nirmatrelvir, an Orally Bioavailable Inhibitor of SARS-CoV-2 3C-Like Protease, across Animals and Humans.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor PF-07321332 (nirmatrelvir), in combination with ritonavir (Paxlovid), was recently granted emergency use authorization by multiple regulatory agencies for the treatment of coronavirus disease 2019 (COVID-19) in adults and pediatric patients. Disposition studies on nirmatrelvir in animals and in human reagents, which were used to support clinical studies, are described herein. Plasma clearance was moderate in rats (27.2 ml/min per kg) and monkeys (17.1 ml/min per kg), resulting in half-lives of 5.1 and 0.8 hours, respectively. The corresponding oral bioavailability was moderate in rats (34%-50%) and low in monkeys (8.5%), primarily due to oxidative metabolism along the gastrointestinal tract in this species. Nirmatrelvir demonstrated moderate plasma protein binding in rats, monkeys, and humans with mean unbound fractions ranging from 0.310 to 0.478. The metabolism of nirmatrelvir was qualitatively similar in liver microsomes and hepatocytes from rats, monkeys, and humans; prominent metabolites arose via cytochrome P450 (CYP450)-mediated oxidations on the P1 pyrrolidinone ring, P2 6,6-dimethyl-3-azabicyclo[3.1.0]hexane, and the tertiary-butyl group at the P3 position. Reaction phenotyping studies in human liver microsomes revealed that CYP3A4 was primarily responsible (fraction metabolized = 0.99) for the oxidative metabolism of nirmatrelvir. Minor clearance mechanisms involving renal and biliary excretion of unchanged nirmatrelvir were also noted in animals and in sandwich-cultured human hepatocytes. Nirmatrelvir was a reversible and time-dependent inhibitor as well as inducer of CYP3A activity in vitro. First-in-human pharmacokinetic studies have demonstrated a considerable boost in the oral systemic exposure of nirmatrelvir upon coadministration with the CYP3A4 inhibitor ritonavir, consistent with the predominant role of CYP3A4 in nirmatrelvir metabolism. SIGNIFICANCE STATEMENT: The manuscript describes the preclinical disposition, metabolism, and drug-drug interaction potential of PF-07321332 (nirmatrelvir), an orally active peptidomimetic-based inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease, which has been granted emergency use authorization by multiple regulatory agencies around the globe for the treatment of coronavirus disease 2019 (COVID-19) in COVID-19-positive adults and pediatric patients who are at high risk for progression to severe COVID-19, including hospitalization or death.