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1.
Microorganisms ; 10(7)2022 Jul 21.
Article in English | MEDLINE | ID: covidwho-1957393

ABSTRACT

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.

2.
Alergologia Polska - Polish Journal of Allergology ; 9(1):33-40, 2022.
Article in English | EMBASE | ID: covidwho-1818532

ABSTRACT

COVID-19 is an infectious disease that is caused by coronavirus SARS-CoV-2. As the time passed it became clear that, aside from the vaccines, other efficient methods of fighting the disease are urgently needed. Among the whole list of medicines used to treat COVID-19, those which are administered orally have a great advantage. The aim of this paper is to present the current knowledge about the recently investigated oral medications for COVID-19. The authors present molnupiravir (Lagevrio), baricitinib (Olumiant) and ritonavir+PF-07321332 (Paxlovid). All of these drugs have been registered recently or are waiting for authorization by EMA or FDA. During the study, the authors learned that huge progress was made, nevertheless, more studies are needed, especially in the field of side effects and drug-drug interactions of the considered substances.

3.
J Biol Chem ; 298(6): 101972, 2022 06.
Article in English | MEDLINE | ID: covidwho-1799877

ABSTRACT

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.


Subject(s)
COVID-19 , Lactams/chemistry , SARS-CoV-2 , Viral Protease Inhibitors/chemistry , COVID-19/drug therapy , COVID-19/virology , Coronavirus 3C Proteases , Cysteine Endopeptidases/metabolism , Humans , Leucine , Nitriles , Pandemics , Proline , SARS-CoV-2/drug effects , Viral Proteins/metabolism
4.
Embase; 2022.
Preprint in English | EMBASE | ID: ppcovidwho-333295

ABSTRACT

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). We evaluated the in vitro catalytic activity and in vitro potency of nirmatrelvir against the main protease (Mpro) of prevalent variants of concern (VOC) or variants of interest (VOI): Alpha (α, B.1.1.7), Beta (β, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (o, B.1.1.529) as well as the original Washington or wildtype strain. These VOC/VOI carry prevalent mutations at varying frequencies in the Mpro specifically for: α, β, γ (K90R), λ (G15S) and o (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrate that they are catalytically comparable to wildtype. 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 0.933nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the o, λ and β Mpro at 1.63 - 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 (1).

5.
J Virol ; 96(8): e0201321, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1779314

ABSTRACT

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.


Subject(s)
Lactams , Leucine , Nitriles , Peptide Hydrolases , Proline , Antiviral Agents/chemistry , Antiviral Agents/metabolism , COVID-19/drug therapy , Humans , Lactams/chemistry , Lactams/metabolism , Leucine/chemistry , Leucine/metabolism , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/enzymology , Nitriles/chemistry , Nitriles/metabolism , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Proline/chemistry , Proline/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , SARS Virus/chemistry , SARS Virus/enzymology , SARS-CoV-2/chemistry , SARS-CoV-2/enzymology
6.
Rev Esp Quimioter ; 35(3): 236-240, 2022 Jun.
Article in Spanish | MEDLINE | ID: covidwho-1698934

ABSTRACT

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.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Drug Combinations , Humans , Indoles , Lactams , Leucine , Nitriles , Peptide Hydrolases/metabolism , Proline , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , Pyrrolidinones , Ritonavir/therapeutic use
7.
Antibiotics (Basel) ; 11(2)2022 Feb 09.
Article in English | MEDLINE | ID: covidwho-1674435

ABSTRACT

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.

8.
ChemMedChem ; 17(9): e202200016, 2022 05 04.
Article in English | MEDLINE | ID: covidwho-1653198

ABSTRACT

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.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/chemistry , Coronavirus Papain-Like Proteases , Humans , Lactams , Leucine , Mass Spectrometry , Nitriles , Peptide Hydrolases , Proline , Protease Inhibitors/pharmacology
9.
Front Biosci (Landmark Ed) ; 26(12): 1723-1736, 2021 12 30.
Article in English | MEDLINE | ID: covidwho-1614664

ABSTRACT

Thousands of drugs, nutraceuticals and their combinations can be used to select candidate therapeutics for targeting SARS-CoV-2 and its symptoms in order to curb COVID-19. A comprehensive, multi-level strategy against COVID-19 should include drug targeting of biomolecules and biochemical pathways involved in the prevention and proliferation of the infection, and the fatal or serious symptoms following infection. Several drugs are routinely used in the treatment of different categories of seriously ill COVID-19 patients including tocilizumab, remdesivir and dexamethasone. The current risk/benefit assessment supports the emergency testing and approval of more drugs. The process for new drug selection could be based on the identification of one drug for one target, or of a multi-potent drug for many targets and drug combinations for one or more targets, that can cause a substantial reduction in the high mortality rate of COVID-19. Several drugs have been identified that can fit this potential role by targeting different stages of COVID-19 including baricitinib, molnupiravir and PF-07321332/ritonavir and also the combination of deferiprone with N-acetylcysteine for inhibiting the vicious circle of oxidative stress toxicity and endothelial cell damage. Most of these drugs are expected to be effective against all the SARS-CoV-2 variants including Omicron (B.1.1.529) and also the associated COVID-19 complications.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/therapeutic use , Drug Delivery Systems , Humans , Lactams , Leucine , Nitriles , Pandemics , Proline
10.
Rev Esp Quimioter ; 35(2): 115-130, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1567024

ABSTRACT

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.


Subject(s)
COVID-19 , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , Pandemics/prevention & control , SARS-CoV-2
11.
J Mol Graph Model ; 110: 108042, 2022 01.
Article in English | MEDLINE | ID: covidwho-1517349

ABSTRACT

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.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/therapeutic use , Coronavirus 3C Proteases , Humans , Lactams , Leucine , Molecular Docking Simulation , Molecular Dynamics Simulation , Nitriles , Proline , Protease Inhibitors
12.
Int J Mol Sci ; 22(17)2021 Aug 24.
Article in English | MEDLINE | ID: covidwho-1374423

ABSTRACT

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.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Protease Inhibitors/pharmacology , Lactams/pharmacology , Leucine/pharmacology , Nitriles/pharmacology , Proline/pharmacology , Antiviral Agents/therapeutic use , Catalytic Domain/drug effects , Coronavirus 3C Proteases/metabolism , Coronavirus Protease Inhibitors/therapeutic use , Humans , Lactams/therapeutic use , Leucine/therapeutic use , Lopinavir/pharmacology , Molecular Docking Simulation , Molecular Dynamics Simulation , Nitriles/therapeutic use , Proline/therapeutic use , Ritonavir/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology
13.
J Enzyme Inhib Med Chem ; 36(1): 1646-1650, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1320278

ABSTRACT

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.


Subject(s)
Antiviral Agents/chemistry , Lactams/chemistry , Leucine/chemistry , Molecular Dynamics Simulation , Nitriles/chemistry , Proline/chemistry , Protease Inhibitors/chemistry , Antiviral Agents/pharmacology , Humans , Lactams/pharmacology , Leucine/pharmacology , Ligands , Nitriles/pharmacology , Peptide Hydrolases/metabolism , Proline/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Software
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