scholarly journals Investigating β-lactam drug targets in Mycobacterium tuberculosis using chemical probes

2019 ◽  
Author(s):  
Samantha R. Levine ◽  
Kimberly E. Beatty
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Small Molecule ◽  
Drug Targets ◽  
Chemical Probes ◽  
Cell Wall Peptidoglycan ◽  
Small Molecule Probes ◽  
Protein Gel ◽  
Cross Links ◽  
Deadly Disease

AbstractTuberculosis is a deadly disease that requires a flexible arsenal of drugs to treat it. Although β-lactam antibiotics are rarely used to treat Mycobacterium tuberculosis (Mtb) infections today, the targets of these drugs are present in the bacterium. Moreover, the cell wall peptidoglycan of Mtb contains an abundance of unusual (3→3) cross-links. These cross-links are formed by enzymes called L,D-transpeptidases, which are susceptible to inhibition by the carbapenem class of antibiotics. We developed new small molecule probes to investigate the L,D-transpeptidases and other β-lactam drug targets in Mtb. We synthesized probes based on three classes of antibiotics, a monobactam, cephalosporin, and carbapenem. For the carbapenem, we synthesized a meropenem analogue conjugated to a far-red fluorophore. This probe was particularly useful in identifying active L,D-transpeptidases in protein gel-resolved lysates. Next we analyzed β-lactam targets in lysates from both hypoxic and actively-replicating cultures of Mtb. We identified numerous targets, including transpeptidases, carboxypeptidases, and the β-lactamase BlaC. Overall, we provide evidence that Mtb dynamically regulates the enzymes responsible for maintaining cell wall peptidoglycan and that meropenem is a good inhibitor of those enzymes.

scholarly journals Xanthine-Based Photoaffinity Probes Allow Assessment of Ligand Engagement by TRPC5 Channels

2020 ◽  
Author(s):  
Claudia Bauer ◽  
Aisling Minard ◽  
Isabelle Pickles ◽  
Matthew Burnham ◽  
Nikil Kapur ◽  
...  
Keyword(s):  
Small Molecule ◽  
Drug Targets ◽  
Direct Evidence ◽  
Binding Mode ◽  
Chemical Probes ◽  
Binding Interaction ◽  
Biological Studies ◽  
Photoaffinity Probes ◽  
Direct Binding ◽  
Trpc5 Channels

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

scholarly journals Molecular Investigation of Resistance to the Antituberculous Drug Ethionamide in Multidrug-Resistant Clinical Isolates ofMycobacterium tuberculosis

2010 ◽  
Vol 55 (1) ◽  
pp. 355-360 ◽  
Author(s):  
F. Brossier ◽  
N. Veziris ◽  
C. Truffot-Pernot ◽  
V. Jarlier ◽  
W. Sougakoff
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Nadh Dehydrogenase ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Cell Wall Biosynthesis ◽  
Molecular Investigation ◽  
Antituberculous Drug ◽  
Resistant Cells

ABSTRACTEthionamide (ETH) needs to be activated by the mono-oxygenase EthA, which is regulated by EthR, in order to be active againstMycobacterium tuberculosis. The activated drug targets the enzyme InhA, which is involved in cell wall biosynthesis. Resistance to ETH has been reported to result from various mechanisms, including mutations altering EthA/EthR, InhA and its promoter, the NADH dehydrogenase encoded byndh, and the MshA enzyme, involved in mycothiol biosynthesis. We searched for such mutations in 87 clinical isolates: 47 ETH-resistant (ETHr) isolates, 24 ETH-susceptible (ETHs) isolates, and 16 isolates susceptible to ETH but displaying an intermediate proportion of resistant cells (ETHSip; defined as ≥1% but <10% resistant cells). In 81% (38/47) of the ETHrisolates, we found mutations inethA,ethR, orinhAor its promoter, which mostly corresponded to new alterations inethAandethR. The 9 ETHrisolates without a mutation in these three genes (9/47, 19%) had no mutation inndh, and a single isolate had a mutation inmshA. Of the 16 ETHSipisolates, 7 had a mutation inethA, 8 had no detectable mutation, and 1 had a mutation inmshA. Finally, of the 24 ETHsisolates, 23 had no mutation in the studied genes and 1 displayed a yet unknown mutation in theinhApromoter. Globally, the mechanism of resistance to ETH remained unknown for 19% of the ETHrisolates, highlighting the complexity of the mechanisms of ETH resistance inM. tuberculosis.

scholarly journals Xanthine-Based Photoaffinity Probes Allow Assessment of Ligand Engagement by TRPC5 Channels

2020 ◽  
Author(s):  
Claudia Bauer ◽  
Aisling Minard ◽  
Isabelle Pickles ◽  
Matthew Burnham ◽  
Nikil Kapur ◽  
...  
Keyword(s):  
Small Molecule ◽  
Drug Targets ◽  
Direct Evidence ◽  
Binding Mode ◽  
Chemical Probes ◽  
Binding Interaction ◽  
Biological Studies ◽  
Photoaffinity Probes ◽  
Direct Binding ◽  
Trpc5 Channels

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

scholarly journals Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Matthew B. McNeil ◽  
Theresa O’Malley ◽  
Devon Dennison ◽  
Catherine D. Shelton ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
New Drugs ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Primary Mechanism ◽  
Resistant Mutants ◽  
Mycobacterial Cell Wall

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

scholarly journals A Microbiological, Toxicological, and Biochemical Study of the Effects of Fucoxanthin, a Marine Carotenoid, on Mycobacterium tuberculosis and the Enzymes Implicated in Its Cell Wall: A Link Between Mycobacterial Infection and Autoimmune Diseases

Marine Drugs ◽  
2019 ◽  
Vol 17 (11) ◽  
pp. 641 ◽  
Author(s):  
Miroslava Šudomová ◽  
Mohammad Shariati ◽  
Javier Echeverría ◽  
Ioana Berindan-Neagoe ◽  
Seyed Nabavi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Autoimmune Diseases ◽  
Drug Targets ◽  
Bacterial Infections ◽  
Animal Studies ◽  
Biochemical Study ◽  
Mycobacterial Infection ◽  
Susceptible Host ◽  
Standard Drug

This study explored the antitubercular properties of fucoxanthin, a marine carotenoid, against clinical isolates of Mycobacterium tuberculosis (Mtb). Two vital enzymes involved in Mtb cell wall biosynthesis, UDP-galactopyranose mutase (UGM) and arylamine-N-acetyltransferase (TBNAT), were selected as drug targets to reveal the mechanism underlying the antitubercular effect of fucoxanthin. The obtained results showed that fucoxanthin showed a clear bacteriostatic action against the all Mtb strains tested, with minimum inhibitory concentrations (MIC) ranging from 2.8 to 4.1 µM, along with a good degree of selectivity index (ranging from 6.1 to 8.9) based on cellular toxicity evaluation compared with standard drug isoniazid (INH). The potent inhibitory actions of fucoxanthin and standard uridine-5’-diphosphate against UGM were recorded to be 98.2% and 99.2%, respectively. TBNAT was potently inactivated by fucoxanthin (half maximal inhibitory concentration (IC50) = 4.8 µM; 99.1% inhibition) as compared to INH (IC50 = 5.9 µM; 97.4% inhibition). Further, molecular docking approaches were achieved to endorse and rationalize the biological findings along with envisaging structure-activity relationships. Since the clinical evidence of the last decade has confirmed the correlation between bacterial infections and autoimmune diseases, in this study we have discussed the linkage between infection with Mtb and autoimmune diseases based on previous clinical observations and animal studies. In conclusion, we propose that fucoxanthin could demonstrate great therapeutic value for the treatment of tuberculosis by acting on multiple targets through a bacteriostatic effect as well as by inhibiting UGM and TBNAT. Such outcomes may lead to avoiding or decreasing the susceptibility to autoimmune diseases associated with Mtb infection in a genetically susceptible host.

scholarly journals Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis

PLoS ONE ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e60143 ◽  
Author(s):  
Tulika Munshi ◽  
Antima Gupta ◽  
Dimitrios Evangelopoulos ◽  
Juan David Guzman ◽  
Simon Gibbons ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Wall Peptidoglycan

scholarly journals Novel drug targets for Mycobacterium tuberculosis: 2-heterostyrylbenzimidazoles as inhibitors of cell wall protein synthesis

2017 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohana Rao Anguru ◽  
Ashok Kumar Taduri ◽  
Rama Devi Bhoomireddy ◽  
Malathi Jojula ◽  
Shravan Kumar Gunda
Keyword(s):  
Cell Wall ◽  
Protein Synthesis ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Cell Wall Protein ◽  
Novel Drug ◽  
Novel Drug Targets
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