scholarly journals A competitive activity-based protein profiling platform yields cell wall synthesis inhibitors active against replicating and non-replicating Mycobacterium tuberculosis

2021 ◽  
Author(s):  
Michael Li ◽  
Hiren V. Patel ◽  
Armand B. Cognetta ◽  
Trever C. Smith ◽  
Ivy Mallick ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Biological Activity ◽  
Protein Profiling ◽  
Computational Docking ◽  
Competitive Activity ◽  
Form Part ◽  
Major Bottleneck ◽  
Potential False Positive ◽  
Activity Series

ABSTRACTThe identification and validation of a small molecule’s targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule’s targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here we studied the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening ~200 compounds, we focused on two inhibitors active against both exponentially replicating and hypoxia-induced drug-tolerant Mtb that form part of a four-compound structure-activity series. The compound with negligible activity revealed potential false positive targets not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirmed that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings showed that ABPP identifies the targets most likely relevant to a compound’s antibacterial activity.

scholarly journals Molecular Docking, Dynamics Simulation, and Scanning Electron Microscopy (SEM) Examination of Clinically Isolated Mycobacterium tuberculosis by Ursolic Acid: A Pentacyclic Triterpenes

2019 ◽  
Vol 19 (2) ◽  
pp. 328
Author(s):  
Dian Ayu Eka Pitaloka ◽  
Sophi Damayanti ◽  
Aluicia Anita Artarini ◽  
Elin Yulinah Sukandar
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Scanning Electron Microscopy ◽  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Ursolic Acid ◽  
Resistant Strain ◽  
Dynamics Simulation ◽  
Computational Docking ◽  
Scanning Electron

The purpose of this study was to analyze the inhibitory action of ursolic acid (UA) as an antitubercular agent by computational docking studies and molecular dynamics simulations. The effect of UA on the cell wall of Mycobacterium tuberculosis (MTB) was evaluated by using Scanning Electron Microscopy (SEM). UA was used as a ligand for molecular interaction and investigate its binding activities to a group of proteins involved in the growth of MTB and the biosynthesis of the cell wall. Computational docking analysis was performed by using autodock 4.2.6 based on scoring functions. UA binding was confirmed by 30 ns molecular dynamics simulation using gromacs 5.1.1. H37Rv sensitive strain and isoniazid-resistant strain were used in the SEM study. UA showed to have the optimum binding affinity to inhA (Two-trans-enoyl-ACP reductase enzyme involved in elongation of fatty acid) with the binding energy of -9.2 kcal/mol. The dynamic simulation showed that the UA-inhA complex relatively stable and found to establish hydrogen bond with Thr196 and Ile194. SEM analysis confirms that UA treatment in both sensitive strain and resistant strain affected the morphology cell wall of MTB. This result indicated that UA could be one of the potential ligands for the development of new antituberculosis drugs.

scholarly journals A screen of covalent inhibitors in Mycobacterium tuberculosis identifies serine hydrolases involved in lipid metabolism as potential therapeutic targets

2021 ◽  
Author(s):  
Brett M. Babin ◽  
Laura J. Keller ◽  
Yishay Pinto ◽  
Veronica L Li ◽  
Andrew Eneim ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Protein Profiling ◽  
Serine Hydrolases ◽  
Antibiotic Resistant ◽  
Competitive Activity ◽  
Covalent Inhibitors ◽  
New Antibiotics ◽  
Effective Therapeutic Strategy ◽  
Simultaneous Inhibition

The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screened a library of small molecules containing serine-reactive electrophiles and identified narrow spectrum inhibitors of M. tuberculous growth. Using these lead molecules, we performed competitive activity-based protein profiling and identified multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures revealed an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis revealed a path to resistance via the synthesis of mycocerates, but not through mutations to target enzymes. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M. tuberculosis infections.

scholarly journals Differential Role of Threonine and Tyrosine Phosphorylation in the Activation and Activity of the Yeast MAPK Slt2

2021 ◽  
Vol 22 (3) ◽  
pp. 1110
Author(s):  
Gema González-Rubio ◽  
Ángela Sellers-Moya ◽  
Humberto Martín ◽  
María Molina
Keyword(s):  
Cell Wall ◽  
Biological Activity ◽  
Biological Significance ◽  
Mitogen Activated Protein Kinase ◽  
Activation Loop ◽  
High Increase ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Full Activation

The Mitogen-Activated Protein Kinase (MAPK) Slt2 is central to signaling through the yeast Cell Wall Integrity (CWI) pathway. MAPKs are regulated by phosphorylation at both the threonine and tyrosine of the conserved TXY motif within the activation loop (T190/Y192 in Slt2). Since phosphorylation at both sites results in the full activation of MAPKs, signaling through MAPK pathways is monitored with antibodies that detect dually phosphorylated forms. However, most of these antibodies also recognize monophosphorylated species, whose relative abundance and functionality are diverse. By using different phosphospecific antibodies and phosphate-affinity (Phos-tag) analysis on distinct Slt2 mutants, we determined that Y192- and T190-monophosphorylated species coexist with biphosphorylated Slt2, although most of the Slt2 pool remains unphosphorylated following stress. Among the monophosphorylated forms, only T190 exhibited biological activity. Upon stimulation, Slt2 is first phosphorylated at Y192, mainly by the MAPKK Mkk1, and this phosphorylation is important for the subsequent T190 phosphorylation. Similarly, dephosphorylation of Slt2 by the Dual Specificity Phosphatase (DSP) Msg5 is ordered, with dephosphorylation of T190 depending on previous Y192 dephosphorylation. Whereas Y192 phosphorylation enhances the Slt2 catalytic activity, T190 is essential for this activity. The conserved T195 residue is also critical for Slt2 functionality. Mutations that abolish the activity of Slt2 result in a high increase in inactive Y192-monophosphorylated Slt2. The coexistence of different Slt2 phosphoforms with diverse biological significance highlights the importance of the precise detection of the Slt2 phosphorylation status.

1,3-Diarylpyrazolyl-acylsulfonamides as Potent Anti-tuberculosis Agents Targeting Cell Wall Biosynthesis in Mycobacterium tuberculosis

2021 ◽  
Vol 64 (17) ◽  
pp. 12790-12807
Author(s):  
Lutete Peguy Khonde ◽  
Rudolf Müller ◽  
Grant A. Boyle ◽  
Virsinha Reddy ◽  
Aloysius T. Nchinda ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Wall Biosynthesis

scholarly journals Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Development of a Microtiter Plate-Based Screen for UDP-Galactopyranose Mutase and Identification of an Inhibitor from a Uridine-Based Library

2003 ◽  
Vol 47 (1) ◽  
pp. 378-382 ◽  
Author(s):  
Michael S. Scherman ◽  
Katharine A. Winans ◽  
Richard J. Stern ◽  
Victoria Jones ◽  
Carolyn R. Bertozzi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targeting ◽  
Enzyme Inhibitor ◽  
Microtiter Plate ◽  
Biosynthetic Enzyme ◽  
Cell Wall Synthesis ◽  
Chemical Library ◽  
Microtiter Plate Assay ◽  
Plate Assay

ABSTRACT A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.

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