scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Structure-Based Drug Design and Characterization of Sulfonyl-Piperazine Benzothiazinone Inhibitors of DprE1 from Mycobacterium tuberculosis

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Jérémie Piton ◽  
Anthony Vocat ◽  
Andréanne Lupien ◽  
Caroline S. Foo ◽  
Olga Riabova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Design ◽  
Antitubercular Activity ◽  
Cysteine Residue ◽  
Drug Candidate ◽  
Structure Based Drug Design ◽  
Content Type ◽  
Active Site Cysteine ◽  
Covalent Adducts

ABSTRACT Macozinone (MCZ) is a tuberculosis (TB) drug candidate that specifically targets the essential flavoenzyme DprE1, thereby blocking synthesis of the cell wall precursor decaprenyl phosphoarabinose (DPA) and provoking lysis of Mycobacterium tuberculosis. As part of the MCZ backup program, we exploited structure-guided drug design to produce a new series of sulfone-containing derivatives, 2-sulfonylpiperazin 8-nitro 6-trifluoromethyl 1,3-benzothiazin-4-one, or sPBTZ. These compounds are less active than MCZ but have a better solubility profile, and some derivatives display enhanced stability in microsomal assays. DprE1 was efficiently inhibited by sPBTZ, and covalent adducts with the active-site cysteine residue (C387) were formed. However, despite the H-bonding potential of the sulfone group, no additional bonds were seen in the crystal structure of the sPBTZ-DprE1 complex with compound 11326127 compared to MCZ. Compound 11626091, the most advanced sPBTZ, displayed good antitubercular activity in the murine model of chronic TB but was less effective than MCZ. Nonetheless, further testing of this MCZ backup compound is warranted as part of combination treatment with other TB drugs.

scholarly journals The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

2015 ◽  
Vol 59 (8) ◽  
pp. 4446-4452 ◽  
Author(s):  
Vadim Makarov ◽  
João Neres ◽  
Ruben C. Hartkoorn ◽  
Olga B. Ryabova ◽  
Elena Kazakova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitro Group ◽  
Covalent Bond ◽  
Antimycobacterial Activity ◽  
Content Type ◽  
Sar Studies ◽  
Covalent Bond Formation

ABSTRACT8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity againstMycobacterium tuberculosisin vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml againstM. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorablein vitroabsorption-distribution-metabolism-excretion/toxicity (ADME/T) andin vivopharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

scholarly journals Efflux Attenuates the Antibacterial Activity of Q203 in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Jichan Jang ◽  
Ryangyeo Kim ◽  
Minjeong Woo ◽  
Jinsun Jeong ◽  
Da Eun Park ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Ex Vivo ◽  
Drug Candidate ◽  
Content Type ◽  
Tb Treatment ◽  
Pump Activity ◽  
Efflux Pump Inhibitors ◽  
Combinatorial Strategy

ABSTRACT New and improved treatments for tuberculosis (TB) are urgently needed. Recently, it has been demonstrated that verapamil, an efflux inhibitor, can reduce bacterial drug tolerance caused by efflux pump activity when administered in combination with available antituberculosis agents. The aim of this study was to evaluate the effectiveness of verapamil in combination with the antituberculosis drug candidate Q203, which has recently been developed and is currently under clinical trials as a potential antituberculosis agent. We evaluated changes in Q203 activity in the presence and absence of verapamil in vitro using the resazurin microplate assay and ex vivo using a microscopy-based phenotypic assay for the quantification of intracellular replicating mycobacteria. Verapamil increased the potency of Q203 against Mycobacterium tuberculosis both in vitro and ex vivo, indicating that efflux pumps are associated with the activity of Q203. Other efflux pump inhibitors also displayed an increase in Q203 potency, strengthening this hypothesis. Therefore, the combination of verapamil and Q203 may be a promising combinatorial strategy for anti-TB treatment to accelerate the elimination of M. tuberculosis.

scholarly journals In Vitro Isolation and Characterization of Oxazolidinone-Resistant Mycobacterium tuberculosis

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Matthew B. McNeil ◽  
Devon D. Dennison ◽  
Catherine D. Shelton ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
23S Rrna ◽  
Clinical Settings ◽  
Content Type ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Ribosomal Protein L3 ◽  
Resistant Strains

ABSTRACT Oxazolidinones are promising candidates for the treatment of Mycobacterium tuberculosis infections. We isolated linezolid-resistant strains from H37Rv (Euro-American) and HN878 (East-Asian) strains; resistance frequencies were similar in the two strains. Mutations were identified in ribosomal protein L3 (RplC) and the 23S rRNA (rrl). All mutant strains were cross resistant to sutezolid; a subset was cross resistant to chloramphenicol. Mutations in rrl led to growth impairment and decreased fitness that may limit spread in clinical settings.

scholarly journals In VitroandIn VivoActivities of Three Oxazolidinones against Nonreplicating Mycobacterium tuberculosis

2014 ◽  
Vol 58 (6) ◽  
pp. 3217-3223 ◽  
Author(s):  
Ming Zhang ◽  
Claudia Sala ◽  
Neeraj Dhar ◽  
Anthony Vocat ◽  
Vasan K Sambandamurthy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Latent Tuberculosis ◽  
Drug Susceptibility ◽  
New Combination ◽  
Drug Candidate ◽  
Bacteriostatic Effect ◽  
Content Type ◽  
New Class ◽  
Modest Activity

ABSTRACTOxazolidinones represent a new class of antituberculosis drugs that exert their function by inhibiting protein synthesis. Here, we compared the activities of three oxazolidinones, linezolid, PNU-100480, and AZD5847, against latent tuberculosis using a simple model employing the streptomycin-starvedMycobacterium tuberculosisstrain 18b. Thein vitrodrug susceptibility results showed that the three oxazolidinones had a bacteriostatic effect against actively growing bacilli but potent bactericidal activity against nonreplicating cells. In the murine model of latent infection withM. tuberculosis18b, the efficacy of the three compounds varied greatly. Indeed, AZD5847 or its prodrug exhibited no activity or only modest activity, respectively, after 2 months of treatment, whereas both linezolid and PNU-100480 were effective against latent bacilli in mice and showed promising outcomes in combination therapy with rifampin. Moreover, the potency of PNU-100480 was significantly greater than that of linezolid, making it an attractive drug candidate in the development of new combination therapies for latent tuberculosis.

scholarly journals In VitroActivity of AZD5847 against Geographically Diverse Clinical Isolates of Mycobacterium tuberculosis

2014 ◽  
Vol 58 (7) ◽  
pp. 4222-4223 ◽  
Author(s):  
Jim Werngren ◽  
Maria Wijkander ◽  
Nasrin Perskvist ◽  
V. Balasubramanian ◽  
Vasan K. Sambandamurthy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lavage Fluid ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
Drug Candidate ◽  
Content Type ◽  
Geographical Regions ◽  
Lung Biopsies

ABSTRACTThe MIC of the novel antituberculosis (anti-TB) drug AZD5847 was determined against 146 clinical isolates from diverse geographical regions, including eastern Europe, North America, Africa, and Asia, using the automated Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system. These isolates originated from specimen sources such as sputum, bronchial alveolar lavage fluid, pleural fluid, abscess material, lung biopsies, and feces. The overall MIC90was 1.0 mg/liter (range, 0.125 to 4 mg/liter). The MICs of AZD5847 for isolates ofMycobacterium tuberculosiswere similar among drug-sensitive strains, multidrug-resistant (MDR) strains, and extensively drug resistant (XDR) strains. The goodin vitroactivity of AZD5847 againstM. tuberculosisand the lack of cross-resistance make this agent a promising anti-TB drug candidate.

scholarly journals Peptidoglycan Hydrolases RipA and Ami1 Are Critical for Replication and Persistence of Mycobacterium tuberculosis in the Host

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Claire Healy ◽  
Alexandre Gouzy ◽  
Sabine Ehrt
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Drug Target ◽  
New Drugs ◽  
Chemotherapeutic Regimen ◽  
Content Type ◽  
Cell Wall Polymer ◽  
Redundant Functions

ABSTRACT Synthesis and cleavage of the cell wall polymer peptidoglycan (PG) are carefully orchestrated processes and are essential for the growth and survival of bacteria. Yet, the function and importance of many enzymes that act on PG in Mycobacterium tuberculosis remain to be elucidated. We demonstrate that the activity of the N-acetylmuramyl-l-alanine amidase Ami1 is dispensable for cell division in M. tuberculosis in vitro yet contributes to the bacterium’s ability to persist during chronic infection in mice. Furthermore, the d,l-endopeptidase RipA, a predicted essential enzyme, is dispensable for the viability of M. tuberculosis but required for efficient cell division in vitro and in vivo. Depletion of RipA sensitizes M. tuberculosis to rifampin and to cell envelope-targeting antibiotics. Ami1 helps sustain residual cell division in cells lacking RipA, but the partial redundancy provided by Ami1 is not sufficient during infection, as depletion of RipA prevents M. tuberculosis from replicating in macrophages and leads to dramatic killing of the bacteria in mice. Notably, RipA is essential for persistence of M. tuberculosis in mice, suggesting that cell division is required during chronic mouse infection. Despite the multiplicity of enzymes acting on PG with redundant functions, we have identified two PG hydrolases that are important for M. tuberculosis to replicate and persist in the host. IMPORTANCE Tuberculosis (TB) is a major global heath burden, with 1.6 million people succumbing to the disease every year. The search for new drugs to improve the current chemotherapeutic regimen is crucial to reducing this global health burden. The cell wall polymer peptidoglycan (PG) has emerged as a very successful drug target in bacterial pathogens, as many currently used antibiotics target the synthesis of this macromolecule. However, the multitude of genes encoding PG-synthesizing and PG-modifying enzymes with apparent redundant functions has hindered the identification of novel drug targets in PG synthesis in Mycobacterium tuberculosis. Here, we demonstrate that two PG-cleaving enzymes are important for virulence of M. tuberculosis. In particular, the d,l-endopeptidase RipA represents a potentially attractive drug target, as its depletion results in the clearance of M. tuberculosis from the host and renders the bacteria hypersusceptible to rifampin, a frontline TB drug, and to several cell wall-targeting antibiotics.

scholarly journals Expression and Functional Characterization of the First Bacteriophage-Encoded Chaperonin

2012 ◽  
Vol 86 (18) ◽  
pp. 10103-10111 ◽  
Author(s):  
Lidia P. Kurochkina ◽  
Pavel I. Semenyuk ◽  
Victor N. Orlov ◽  
Johan Robben ◽  
Nina N. Sykilinda ◽  
...  
Keyword(s):  
Thermal Inactivation ◽  
Functional Characterization ◽  
Content Type ◽  
Chaperonin Groel ◽  
Phage Propagation ◽  
Physicochemical Methods ◽  
First Time

Chaperonins promote protein foldingin vivoand are ubiquitously found in bacteria, archaea, and eukaryotes. The first viral chaperonin GroEL ortholog, gene product 146 (gp146), whose gene was earlier identified in the genome of bacteriophage EL, has been shown to be synthesized during phage propagation inPseudomonas aeruginosacells. The recombinant gp146 has been expressed inEscherichia coliand characterized by different physicochemical methods for the first time. Using serum against the recombinant protein, gp146's native substrate, the phage endolysin gp188, has been immunoprecipitated from the lysate of EL-infected bacteria and identified by mass spectrometry.In vitroexperiments have shown that gp146 has a protective effect against endolysin thermal inactivation and aggregation, providing evidence of its chaperonin function. The phage chaperonin has been found to have the architecture and some properties similar to those of GroEL but not to require cochaperonin for its functional activity.

scholarly journals Characterization of Large Deletion Mutants of Mycobacterium tuberculosis Selected for Isoniazid Resistance

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Catherine Vilchèze ◽  
Rajagopalan Saranathan ◽  
Brian Weinrick ◽  
William R. Jacobs
Keyword(s):  
Oxidative Stress ◽  
Mycobacterium Tuberculosis ◽  
Large Deletion ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Isoniazid Resistance ◽  
Content Type ◽  
Genomic Deletions

ABSTRACT Large genomic deletions (LGDs) (6 to 63 kbp) were observed in isoniazid-resistant Mycobacterium tuberculosis mutants derived from four M. tuberculosis strains. These LGDs had no growth defect in vitro but could be defective in intracellular growth and showed various sensitivities toward oxidative stress despite lacking katG. The LGD regions comprise 74 genes, mostly of unknown function, that may be important for M. tuberculosis intracellular growth and protection against oxidative stress.

Sign in / Sign up

Export Citation Format

Share Document