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2.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-327156

ABSTRACT

The main protease (M pro ) of SARS-CoV-2 is essential for viral replication and is considered to be one of the most promising SARS-CoV-2 drug targets. While M pro mutations have occurred in many variants, including Omicron (B.1.1.529), their structural and biochemical importance have, until this point, remained uncharacterized. Using X-ray crystallography, we show the Omicron M pro mutant (P132H) induces a small rearrangement of residues distal from the active site. While enzymatic activity and small-molecule inhibition appears unchanged, the melting temperature (T m ) of Omicron M pro is 2.6 ° C lower than Alpha and Delta M pro . Yet, when incubated with inhibitors, these enzymes have nearly identical T m values. The physiological importance of the P132H mutant is unclear;however, we show residue 132 is located at the interface of the dimerization and catalytic domains and is frequently mutated in other variants. Lower thermal stability may indicate increased flexibility that can potentially broaden substrate profile or alter inhibitor binding. Therefore, structural insights are key to anticipating future mutations that may promote drug-resistance and are especially important at present, given the recent approval of nirmatrelvir (PAXLOVID), an oral SARS-CoV-2 M pro inhibitor.

3.
Viruses ; 14(2)2022 01 25.
Article in English | MEDLINE | ID: covidwho-1667339

ABSTRACT

In 2014 and 2021, two nucleic-acid vaccine candidates named MAV E2 and VGX-3100 completed phase III clinical trials in Mexico and U.S., respectively, for patients with human papillomavirus (HPV)-related, high-grade squamous intraepithelial lesions (HSIL). These well-tolerated but still unlicensed vaccines encode distinct HPV antigens (E2 versus E6+E7) to elicit cell-mediated immune responses; their clinical efficacy, as measured by HSIL regression or cure, was modest when compared with placebo or surgery (conization), but both proved highly effective in clearing HPV infection, which should help further optimize strategies for enhancing vaccine immunogenicity, toward an ultimate goal of preventing malignancies in millions of patients who are living with persistent, oncogenic HPV infection but are not expected to benefit from current, prophylactic vaccines. The major roadblocks to a highly efficacious and practical product remain challenging and can be classified into five categories: (i) getting the vaccines into the right cells for efficient expression and presentation of HPV antigens (fusion proteins or epitopes); (ii) having adequate coverage of oncogenic HPV types, beyond the current focus on HPV-16 and -18; (iii) directing immune protection to various epithelial niches, especially anogenital mucosa and upper aerodigestive tract where HPV-transformed cells wreak havoc; (iv) establishing the time window and vaccination regimen, including dosage, interval and even combination therapy, for achieving maximum efficacy; and (v) validating therapeutic efficacy in patients with poor prognosis because of advanced, recurrent or non-resectable malignancies. Overall, the room for improvements is still large enough that continuing efforts for research and development will very likely extend into the next decade.


Subject(s)
Cancer Vaccines/therapeutic use , Cervical Intraepithelial Neoplasia/therapy , Neoplasms/therapy , Papillomavirus Infections/therapy , Papillomavirus Vaccines/therapeutic use , Uterine Cervical Neoplasms/therapy , Vaccines, DNA/therapeutic use , Animals , Cervical Intraepithelial Neoplasia/immunology , Clinical Trials as Topic , Female , Humans , Immunogenicity, Vaccine , Neoplasms/immunology , Neoplasms/virology , Papillomavirus Infections/immunology , Papillomavirus Vaccines/immunology , Squamous Intraepithelial Lesions of the Cervix/therapy , Uterine Cervical Neoplasms/virology , Vaccines, DNA/immunology , /therapeutic use
4.
J Am Chem Soc ; 143(49): 20697-20709, 2021 12 15.
Article in English | MEDLINE | ID: covidwho-1550253

ABSTRACT

The main protease (Mpro) is a validated antiviral drug target of SARS-CoV-2. A number of Mpro inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 Mpro inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates Jun9-62-2R (dichloroacetamide) and Jun9-88-6R (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to GC-376, these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent Mpro inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 Mpro with Jun9-62-2R and Jun9-57-3R reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.


Subject(s)
Acetamides/pharmacology , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemistry , Cathepsin L/antagonists & inhibitors , Drug Design , Drug Discovery , Enzyme Inhibitors/pharmacology , Humans , Models, Molecular , Molecular Dynamics Simulation , Structure-Activity Relationship , Substrate Specificity
5.
ACS Cent Sci ; 7(7): 1245-1260, 2021 Jul 28.
Article in English | MEDLINE | ID: covidwho-1387139

ABSTRACT

The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PLpro inhibitors including Jun9-72-2 and Jun9-75-4 with improved enzymatic inhibition and antiviral activity compared to GRL0617, which was reported as a SARS-CoV PLpro inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PLpro inhibitors in the BSL-2 setting. X-ray crystal structure of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation. Molecular dynamics simulations showed that Jun9-72-2 and Jun9-75-4 engaged in more extensive interactions than GRL0617. Overall, the PLpro inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PLpro assay is a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.

6.
Infect Drug Resist ; 14: 2823-2827, 2021.
Article in English | MEDLINE | ID: covidwho-1334840

ABSTRACT

OBJECTIVE: The present study aimed to investigate the correlation between obesity and virus persistence in patients with COVID-19. DESIGN AND METHODS: A total of 57 patients with laboratory-confirmed COVID-19 were admitted to two clinical centers, and data were analyzed retrospectively. Among them, 18 patients with body mass index (BMI) ≥ 25 kg/m2 were diagnosed with obesity, and dynamics of viral replication were compared. RESULTS: Eighteen patients were diagnosed with obesity. The correlations between BMI and white blood cell, C-reactive protein, and cycle threshold (Ct) values of ORF1ab were not significant (all P > 0.05). On day 7 after admission, virus clearance was achieved in 13 (33.3%) patients with BMI < 25 kg/m2 and 5 (27.8%) patients with BMI ≥ 25 kg/m2 (χ2 = 0.176, P =0.68). On day 14, the RNA tests were negative in 37 (94.9%) patients with BMI < 25 kg/m2 and 13 (72.2%) patients with BMI ≥ 25 kg/m2 (χ2 = 5.865, P = 0.03). Multivariable analysis showed that only BMI ≥ 25 kg/m2 (P = 0.02) was the independent risk factor for virus clearance on day 14. CONCLUSION: Obesity may affect the clearance of SARS-CoV-2, and BMI should be assessed in patients with COVID-19, although they are not seriously ill.

7.
J Med Chem ; 65(4): 2848-2865, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1199254

ABSTRACT

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most potent and selective noncovalent SARS-CoV-2 Mpro inhibitors reported to date, and a novel binding pocket in Mpro that can be explored for inhibitor design.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Drug Design , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/metabolism , Chlorocebus aethiops , Coronavirus 3C Proteases/isolation & purification , Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Proline/analogs & derivatives , Proline/chemical synthesis , Proline/chemistry , Proline/pharmacology , Pyrrolidines/chemical synthesis , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , SARS-CoV-2/enzymology , Sulfonic Acids/chemical synthesis , Sulfonic Acids/chemistry , Sulfonic Acids/pharmacology , Vero Cells
8.
Sci Adv ; 6(50)2020 12.
Article in English | MEDLINE | ID: covidwho-969082

ABSTRACT

The main protease (Mpro) of SARS-CoV-2 is a key antiviral drug target. While most Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently found that several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II and XII, which are also active against human cathepsin L, a host protease that is important for viral entry. In this study, we solved x-ray crystal structures of Mpro in complex with calpain inhibitors II and XII and three analogs of GC-376 The structure of Mpro with calpain inhibitor II confirmed that the S1 pocket can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. The structure of calpain inhibitor XII revealed an unexpected, inverted binding pose. Together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of dual inhibitors as SARS-CoV-2 antivirals.


Subject(s)
Cathepsin L/chemistry , Coronavirus 3C Proteases/chemistry , Drug Design , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Animals , Caco-2 Cells , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Dogs , Humans , Kinetics , Madin Darby Canine Kidney Cells , Models, Chemical , Molecular Structure , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Protein Domains , Vero Cells
9.
Cell Res ; 30(8): 678-692, 2020 08.
Article in English | MEDLINE | ID: covidwho-599672

ABSTRACT

A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/enzymology , Coronavirus Infections/metabolism , Glycoproteins/pharmacology , Pneumonia, Viral/metabolism , Proline/analogs & derivatives , Pyrrolidines/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Virus Replication/drug effects , A549 Cells , Animals , Antiviral Agents/chemistry , COVID-19 , Caco-2 Cells , Chlorocebus aethiops , Coronavirus 3C Proteases , Coronavirus Infections/virology , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Drug Discovery/methods , Humans , Inhibitory Concentration 50 , Kinetics , Pandemics , Pneumonia, Viral/virology , Proline/pharmacology , Protein Conformation , Pyrrolidines/chemistry , SARS-CoV-2 , Sulfonic Acids , Vero Cells , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
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