14 results on '"Michael Dominic Sacco"'
Search Results
2. Development of the Safe and Broad‐Spectrum Aldehyde and Ketoamide Mpro inhibitors Derived from the Constrained α, γ‐AA Peptide Scaffold
- Author
-
Lei Wang, Chunlong Ma, Michael Dominic Sacco, Songyi Xue, Mentalla Mahmoud, Laurent Calcul, Yu Chen, Jun Wang, and Jianfeng Cai
- Subjects
Organic Chemistry ,General Chemistry ,Catalysis - Published
- 2023
3. Discovery of Di- and Trihaloacetamides as Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity
- Author
-
Haozhou Tan, Xiangzhi Meng, Maura V Gongora, Michael Dominic Sacco, Fushun Zhang, Yanmei Hu, Yan Xiang, Zilei Xia, Yu Chen, Juliana Choza, Janice Jang, Michael T. Marty, Chunlong Ma, Jun Wang, Julia Alma Townsend, and Xiujun Zhang
- Subjects
Models, Molecular ,Proteases ,medicine.drug_class ,Cathepsin L ,medicine.medical_treatment ,Molecular Dynamics Simulation ,Antiviral Agents ,Biochemistry ,Article ,Catalysis ,Cathepsin B ,Substrate Specificity ,Structure-Activity Relationship ,Colloid and Surface Chemistry ,Acetamides ,Drug Discovery ,medicine ,Cathepsin K ,Animals ,Humans ,Protease Inhibitors ,Enzyme Inhibitors ,Coronavirus 3C Proteases ,Cathepsin ,Protease ,biology ,SARS-CoV-2 ,Chemistry ,Rational design ,Calpain ,General Chemistry ,Drug Design ,biology.protein ,Antiviral drug - 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.
- Published
- 2021
4. Discovery of SARS-CoV‑2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay
- Author
-
Julia Alma Townsend, George Lambrinidis, Michael T. Marty, Yu Chen, Chunlong Ma, Maura V Gongora, Antonios Kolocouris, Jun Wang, Fushun Zhang, Yan Xiang, Zilei Xia, Xiujun Zhang, Xiangzhi Meng, Mandy Ba, Tommy Szeto, Michael Dominic Sacco, and Yanmei Hu
- Subjects
Conformational change ,medicine.drug_class ,General Chemical Engineering ,High-throughput screening ,medicine.medical_treatment ,Cell ,papain-like protease ,Article ,chemistry.chemical_compound ,medicine ,QD1-999 ,chemistry.chemical_classification ,Reporter gene ,Protease ,SARS-CoV-2 ,Chemistry ,COVID-19 ,General Chemistry ,antiviral ,PLpro ,Papain ,medicine.anatomical_structure ,Förster resonance energy transfer ,Enzyme ,Biochemistry ,FlipGFP ,Antiviral drug ,Research Article - 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., A cell-based FlipGFP reporter assay was developed for the screening of SARS-CoV-2 papain-like protease inhibitors and was shown to have a positive correlation with antiviral activity.
- Published
- 2021
5. GpsB Coordinates Cell Division and Cell Surface Decoration by Wall Teichoic Acids in Staphylococcus aureus
- Author
-
Abigail Hough, Prahathees J. Eswara, Lauren R. Hammond, Michael Dominic Sacco, Yu Chen, Sebastian J. Khan, and Catherine Spanoudis
- Subjects
Microbiology (medical) ,Staphylococcus aureus ,Penicillin binding proteins ,Cell division ,Physiology ,Virulence Factors ,Bacillus subtilis ,Bacterial cell structure ,chemistry.chemical_compound ,Bacterial Proteins ,Cell Wall ,Genetics ,Humans ,FtsZ ,Teichoic acid ,biology ,General Immunology and Microbiology ,Ecology ,Divisome complex ,Cell Biology ,Cell cycle ,Staphylococcal Infections ,biology.organism_classification ,Cell biology ,Teichoic Acids ,Infectious Diseases ,chemistry ,biology.protein ,Cell Division - Abstract
Bacterial cell division is a complex and highly regulated process requiring the coordination of many different proteins. Despite substantial work in model organisms, our understanding of the systems regulating cell division in non-canonical organisms, including critical human pathogens, is far from complete. One such organism is Staphylococcus aureus, a spherical bacterium that lacks known cell division regulatory proteins. Recent studies on GpsB, a protein conserved within the Firmicutes phylum, have provided insight into cell division regulation in S. aureus and other related organisms. It has been revealed that GpsB coordinates cell division and cell wall synthesis in multiple species by interacting with Penicillin Binding Proteins (PBPs) and other partners. In S. aureus, we have previously shown that GpsB directly regulates FtsZ polymerization. In this study, using Bacillus subtilis as a tool, we isolated intragenic and extragenic spontaneous suppressor mutants that abrogate the lethality of S. aureus GpsB overproduction in B. subtilis. Through characterization of these mutants, we identified several key residues important for the function of GpsB. Furthermore, we discovered an additional role for GpsB in wall teichoic acid (WTA) biosynthesis in S. aureus. Specifically, we show that GpsB directly interacts with the wall teichoic acid export protein TarG using a bacterial two-hybrid analysis. We also identified a three-residue motif in GpsB that is crucial for this interaction. Based on the analysis of the localization of TagG in B. subtilis and its homolog TarG in S. aureus, it appears that WTA machinery is a part of the divisome complex. As such, we show additional evidence to the growing body of work that suggests that along with peptidoglycan synthesis, WTA biosynthesis and export may take place at the site of cell division. Taken together, this research illustrates how GpsB performs an essential function in S. aureus by directly linking the tightly regulated cell cycle processes of cell division and WTA-mediated cell surface decoration.IMPORTANCE/AUTHOR SUMMARYCytokinesis in bacteria involves an intricate orchestration of several key cell division proteins and other factors involved in building a robust cell envelope. One of the key factors that differentiates Gram-positive bacteria from Gram-negative bacteria is the presence of teichoic acids interlaced within the Gram-positive cell wall. By characterizing the role of Staphylococcus aureus GpsB, an essential cell division protein in this organism, we have uncovered an additional role for GpsB in wall teichoic acids (WTA) biosynthesis. We show that GpsB directly interacts with TarG of the WTA export complex. We also show this function of GpsB may be conserved in other GpsB homologs as GpsB and the WTA exporter complex follow similar localization patterns. It has been suggested that WTA acts as a molecular signal to control the activity of autolytic enzymes, especially during the separation of conjoined daughter cells. Thus, our results reveal that GpsB, in addition to playing a role in cell division, may also help coordinate WTA biogenesis.
- Published
- 2022
6. Expedited Approach toward the Rational Design of Noncovalent SARS-CoV-2 Main Protease Inhibitors
- Author
-
Jun Wang, Chunlong Ma, Xiujun Zhang, Yanmei Hu, David C. Schultz, Michael Dominic Sacco, Naoya Kitamura, Xiangzhi Meng, Adis Kukuljac, Mandy Ba, Tommy Szeto, Julia Alma Townsend, Michael T. Marty, Fushun Zhang, Yu Chen, Yan Xiang, and Sara Cherry
- Subjects
Models, Molecular ,Proteases ,Pyrrolidines ,Proline ,Stereochemistry ,medicine.drug_class ,medicine.medical_treatment ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Microbial Sensitivity Tests ,Cysteine Proteinase Inhibitors ,Antiviral Agents ,01 natural sciences ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Boceprevir ,Chlorocebus aethiops ,Drug Discovery ,medicine ,Animals ,Humans ,Binding site ,Vero Cells ,Coronavirus 3C Proteases ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Protease ,Dose-Response Relationship, Drug ,Molecular Structure ,SARS-CoV-2 ,Rational design ,COVID-19 ,COVID-19 Drug Treatment ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Enzyme ,chemistry ,Drug Design ,Molecular Medicine ,Sulfonic Acids ,Antiviral drug - Abstract
The main protease (M(pro)) of SARS-CoV-2 is a validated antiviral drug target. Several M(pro) inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, 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 non-covalent inhibitors 23R (Jun8-76-3A) that is structurally distinct from the canonical M(pro) inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially towards host proteases. The co-crystal structure of SARS-CoV-2 M(pro) 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 non-covalent SARS-CoV-2 M(pro) inhibitors reported to date, and a novel binding pocket in M(pro) that can be explored for inhibitor design.
- Published
- 2021
7. The P132H mutation in the main protease of Omicron SARS-CoV-2 decreases thermal stability without compromising catalysis or small-molecule drug inhibition
- Author
-
Michael Dominic Sacco, Yanmei Hu, Maura Verenice Gongora, Flora Meilleur, Michael Trent Kemp, Xiujun Zhang, Jun Wang, and Yu Chen
- Subjects
SARS-CoV-2 ,Mutation ,COVID-19 ,Humans ,Cell Biology ,Molecular Biology ,Catalysis ,Peptide Hydrolases - Abstract
The ongoing SARS-CoV-2 pandemic continues to be a significant threat to global health. First reported in November 2021, the Omicron variant (B.1.1.529) is more transmissible and can evade immunity better than previous SARS-CoV-2 variants, fueling an unprecedented surge in cases. To produce functional proteins from this polyprotein, SARS-CoV-2 relies on the cysteine proteases Nsp3/papain-like protease (PLpro) and Nsp5/Main Protease (Mpro)/3C-like protease to cleave at three and more than 11 sites, respectively.1 Therefore, Mpro and PLpro inhibitors are considered to be some of the most promising SARS-CoV-2 antivirals. On December 22, 2021, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for PAXLOVID, a ritonavir-boosted formulation of nirmatrelvir. Nirmatrelvir is a first-in-class orally bioavailable SARS-CoV-2 Mpro inhibitor.2 Thus, the scientific community must vigilantly monitor potential mechanisms of drug resistance, especially because SARS-CoV-2 is naïve to Mpro inhibitors. Mutations have been well identified in variants to this point.3 Notably, Omicron Mpro (OMpro) harbors a single mutation– P132H. In this study we characterize the enzymatic activity, drug inhibition, and structure of OMpro while evaluating the past and future implications of Mpro mutations.
- Published
- 2022
8. Rational Design of Hybrid SARS-CoV-2 Main Protease Inhibitors Guided by the Superimposed Cocrystal Structures with the Peptidomimetic Inhibitors GC-376, Telaprevir, and Boceprevir
- Author
-
Tommy Szeto, Michael Dominic Sacco, Xiangzhi Meng, Yu Chen, Yanmei Hu, Jun Wang, Fushun Zhang, Yan Xiang, Chunlong Ma, and Zilei Xia
- Subjects
Proteases ,Peptidomimetic ,medicine.drug_class ,medicine.medical_treatment ,viruses ,Article ,Telaprevir ,Cathepsin L ,3CL protease ,medicine ,Pharmacology (medical) ,Pharmacology ,Protease ,biology ,Chemistry ,SARS-CoV-2 ,Rational design ,virus diseases ,COVID-19 ,Cysteine protease ,antiviral ,Biochemistry ,main protease ,biology.protein ,Antiviral drug ,medicine.drug - Abstract
SARS-CoV-2 main protease (Mpro) is a cysteine protease that mediates the cleavage of viral polyproteins and is a validated antiviral drug target. Mpro is highly conserved among all seven human coronaviruses, with certain Mpro inhibitors having broad-spectrum antiviral activity. In this study, we designed two hybrid inhibitors UAWJ9-36-1 and UAWJ9-36-3 based on the superimposed X-ray crystal structures of SARS-CoV-2 Mpro with GC-376, telaprevir, and boceprevir. Both UAWJ9-36-1 and UAWJ9-36-3 showed potent binding and enzymatic inhibition against the Mpro's from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-229E, and HCoV-HKU1. Cell-based Flip-GFP Mpro assay results show that UAWJ9-36-1 and UAWJ9-36-3 inhibited the intracellular protease activity of SARS-CoV-2 Mpro. In addition, UAWJ9-36-1 and UAWJ9-36-3 had potent antiviral activity against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E, with UAWJ9-36-3 being more potent than GC-376 in inhibiting SARS-CoV-2. Selectivity profiling revealed that UAWJ9-36-1 and UAWJ9-36-3 had an improved selectivity index over that of GC-376 against host cysteine proteases calpain I and cathepsin L, but not cathepsin K. The X-ray crystal structures of SARS-CoV-2 Mpro with UAWJ9-36-1 and UAWJ9-36-3 were both solved at 1.9 A, which validated our design hypothesis. Overall, hybrid inhibitors UAWJ9-36-1 and UAWJ9-36-3 are promising candidates to be further developed as broad-spectrum coronavirus antivirals.
- Published
- 2021
9. An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity
- Author
-
Naoya Kitamura, Chunlong Ma, Xiangzhi Meng, Michael Dominic Sacco, Jun Wang, Sara Cherry, Julia Alma Townsend, Yu Chen, Fushun Zhang, Yan Xiang, Xiujun Zhang, David C. Schultz, Michael T. Marty, Yanmei Hu, and Adis Kukuljac
- Subjects
chemistry.chemical_classification ,Thermal shift assay ,Protease ,Chemistry ,medicine.drug_class ,Drug discovery ,Stereochemistry ,medicine.medical_treatment ,In vitro ,Enzyme ,medicine ,Binding site ,Antiviral drug ,IC50 - 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, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent Mpro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 μM and EC50 of 1.27 μM, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the α-methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.
- Published
- 2020
10. N-Hydroxyformamide LpxC inhibitors, their in vivo efficacy in a mouse Escherichia coli infection model, and their safety in a rat hemodynamic assay
- Author
-
John P. O'Donnell, Janelle Comita-Prevoir, Mark Sylvester, Samir H. Moussa, Camilo V. Vega, Alita A. Miller, Daniel Hines, Jan Antoinette C. Romero, Adam B. Shapiro, Nicole M. Carter, Yu Chen, Takeru Furuya, Jing Zhang, April Chen, Thomas F. Durand-Reville, Seth D. Ribe, Eric J. Kuenstner, Michael Dominic Sacco, and Tommasi Ruben A
- Subjects
Male ,Clinical Biochemistry ,Pharmaceutical Science ,Pharmacology ,Molecular Dynamics Simulation ,medicine.disease_cause ,Crystallography, X-Ray ,Biochemistry ,Amidohydrolases ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Mice ,Structure-Activity Relationship ,In vivo ,Drug Discovery ,medicine ,Escherichia coli ,Potency ,Animals ,Enzyme Inhibitors ,Molecular Biology ,Escherichia coli infection ,Escherichia coli Infections ,Hydroxamic acid ,Binding Sites ,biology ,Formamides ,Pseudomonas aeruginosa ,Organic Chemistry ,Hemodynamics ,biology.organism_classification ,In vitro ,Anti-Bacterial Agents ,Rats ,Disease Models, Animal ,chemistry ,Molecular Medicine ,Female ,Bacteria ,Half-Life - Abstract
UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase (LpxC), the zinc metalloenzyme catalyzing the first committed step of lipid A biosynthesis in Gram-negative bacteria, has been a target for antibacterial drug discovery for many years. All inhibitor chemotypes reaching an advanced preclinical stage and clinical phase 1 have contained terminal hydroxamic acid, and none have been successfully advanced due, in part, to safety concerns, including hemodynamic effects. We hypothesized that the safety of LpxC inhibitors could be improved by replacing the terminal hydroxamic acid with a different zinc-binding group. After choosing an N-hydroxyformamide zinc-binding group, we investigated the structure-activity relationship of each part of the inhibitor scaffold with respect to Pseudomonas aeruginosa and Escherichia coli LpxC binding affinity, in vitro antibacterial potency and pharmacological properties. We identified a novel, potency-enhancing hydrophobic binding interaction for an LpxC inhibitor. We demonstrated in vivo efficacy of one compound in a neutropenic mouse E. coli infection model. Another compound was tested in a rat hemodynamic assay and was found to have a hypotensive effect. This result demonstrated that replacing the terminal hydroxamic acid with a different zinc-binding group was insufficient to avoid this previously recognized safety issue with LpxC inhibitors.
- Published
- 2020
11. Discovery of an Orally Available Diazabicyclooctane Inhibitor (ETX0282) of Class A, C, and D Serine β-Lactamases
- Author
-
Alita A. Miller, Sushmita D. Lahiri, Tommasi Ruben A, Janelle Comita-Prevoir, Sarah M. McLeod, Michael Dominic Sacco, Frank Wu, Jeroen C. Verheijen, Adam B. Shapiro, April Chen, Thomas F. Durand-Reville, Jan Antoinette C. Romero, Robert A. Giacobbe, Tricia L. May-Dracka, Nicole M. Carter, John P. O'Donnell, Yu Chen, Wu Xiaoyun, Jing Zhang, and John P. Mueller
- Subjects
medicine.drug_class ,Antibiotics ,Drug Evaluation, Preclinical ,Administration, Oral ,Drug Annotation ,Microbial Sensitivity Tests ,Pharmacology ,Gram-Positive Bacteria ,01 natural sciences ,Skin Diseases ,beta-Lactamases ,Serine ,03 medical and health sciences ,Mice ,Structure-Activity Relationship ,Drug Discovery ,Gram-Negative Bacteria ,medicine ,Structure–activity relationship ,Animals ,Humans ,Penicillin-Binding Proteins ,Prodrugs ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Cefpodoxime Proxetil ,Chemistry ,3. Good health ,0104 chemical sciences ,Bioavailability ,Anti-Bacterial Agents ,Rats ,Multiple drug resistance ,010404 medicinal & biomolecular chemistry ,Enzyme ,Drug Design ,Molecular Medicine ,Fluoroacetate ,beta-Lactamase Inhibitors ,Azabicyclo Compounds ,Half-Life ,Protein Binding - Abstract
Multidrug resistant Gram-negative bacterial infections are an increasing public health threat due to rapidly rising resistance toward β-lactam antibiotics. The hydrolytic enzymes called β-lactamases are responsible for a large proportion of the resistance phenotype. β-Lactamase inhibitors (BLIs) can be administered in combination with β-lactam antibiotics to negate the action of the β-lactamases, thereby restoring activity of the β-lactam. Newly developed BLIs offer some advantage over older BLIs in terms of enzymatic spectrum but are limited to the intravenous route of administration. Reported here is a novel, orally bioavailable diazabicyclooctane (DBO) β-lactamase inhibitor. This new DBO, ETX1317, contains an endocyclic carbon-carbon double bond and a fluoroacetate activating group and exhibits broad spectrum activity against class A, C, and D serine β-lactamases. The ester prodrug of ETX1317, ETX0282, is orally bioavailable and, in combination with cefpodoxime proxetil, is currently in development as an oral therapy for multidrug resistant and carbapenem-resistant Enterobacterales infections.
- Published
- 2020
12. Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis
- Author
-
Michael Dominic Sacco, Brooke R Tomlinson, Prahathees J. Eswara, Robert S Brzozowski, Judy J. Chen, Anika N. Ali, Lindsey N. Shaw, and Yu Chen
- Subjects
Molecular Biology and Physiology ,Cell division ,gpsb ,Mutant ,ved/biology.organism_classification_rank.species ,lcsh:QR1-502 ,peptidoglycan ,Cell morphology ,Microbiology ,cell shape ,mreb ,lcsh:Microbiology ,03 medical and health sciences ,Bacterial Proteins ,FtsZ ,Model organism ,Molecular Biology ,Gene ,030304 developmental biology ,Suppressor mutation ,cell morphology ,0303 health sciences ,biology ,030306 microbiology ,ved/biology ,Staphylococcal Infections ,Phenotype ,QR1-502 ,Cell biology ,filamentation ,pbp ,Mutation ,biology.protein ,ftsz ,slog ,Cell Division ,Research Article ,Bacillus subtilis - Abstract
Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated., Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.
- Published
- 2020
13. Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L
- Author
-
Ang Gao, Julia Alma Townsend, Jun Wang, Brett L. Hurst, Yan Xiang, Xiujun Zhang, Antonios Kolocouris, Bart Tarbet, Yanmei Hu, Chunlong Ma, Naoya Kitamura, Panagiotis Lagarias, Xiangzhi Meng, Michael Dominic Sacco, Peter H. Dube, Michael T. Marty, and Yu Chen
- Subjects
Peptidomimetic ,Cathepsin L ,medicine.medical_treatment ,Crystallography, X-Ray ,01 natural sciences ,Madin Darby Canine Kidney Cells ,3CL protease ,chemistry.chemical_compound ,Structural Biology ,Chlorocebus aethiops ,boceprevir ,skin and connective tissue diseases ,Research Articles ,Coronavirus 3C Proteases ,0303 health sciences ,Multidisciplinary ,Molecular Structure ,biology ,Chemistry ,SciAdv r-articles ,Calpain ,Biochemistry ,Research Article ,medicine.drug_class ,Stereochemistry ,Protein domain ,Molecular Dynamics Simulation ,calpain inhibitors ,Article ,Cell Line ,03 medical and health sciences ,Dogs ,Protein Domains ,Viral entry ,Virology ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Protease Inhibitors ,Vero Cells ,030304 developmental biology ,Cathepsin ,Protease ,010405 organic chemistry ,SARS-CoV-2 ,fungi ,COVID-19 ,GC-376 ,0104 chemical sciences ,body regions ,Coronavirus ,Kinetics ,Models, Chemical ,Cell culture ,main protease ,Drug Design ,biology.protein ,Caco-2 Cells ,Norvaline ,Antiviral drug - Abstract
X-ray crystal structures of SARS-CoV-2 Mpro with dual inhibitors provide a new direction for the designing of COVID-19 antivirals., 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.
- Published
- 2020
14. Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease
- Author
-
Tommy Szeto, Michael Dominic Sacco, Michael T. Marty, Jun Wang, Xiujun Zhang, Yanmei Hu, Julia Alma Townsend, Brett L. Hurst, Bart Tarbet, Chunlong Ma, and Yu Chen
- Subjects
Peptidomimetic ,Viral protein ,medicine.medical_treatment ,Biology ,calpain inhibitors ,medicine.disease_cause ,Virus ,Article ,3CL protease ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Boceprevir ,medicine ,boceprevir ,Humans ,Molecular Biology ,030304 developmental biology ,Coronavirus ,chemistry.chemical_classification ,0303 health sciences ,Protease ,SARS-CoV-2 ,COVID-19 ,Cell Biology ,Virology ,GC-376 ,High-Throughput Screening Assays ,Förster resonance energy transfer ,Enzyme ,Pharmaceutical Preparations ,Mechanism of action ,chemistry ,Viral replication ,main protease ,medicine.symptom ,Structural biology ,030217 neurology & neurosurgery ,Peptide Hydrolases - Abstract
A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), 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 6.96% as of May 4, 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 Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three monomers 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.
- Published
- 2020
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.