Investigating β-lactam drug targets in<i>Mycobacterium tuberculosis</i>using chemical probes

Beatty, Kimberly E.; Levine, Samantha

doi:10.1101/2019.12.19.881631

Cited by 3 publications

(2 citation statements)

References 62 publications

(91 reference statements)

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“…Before publication we learned that Prof. Erin Carlson’s group (UMinn) had independently synthesized the Meropenem-alkyne compound. Therefore, we coordinated with her to deposit both papers simultaneously online (BioRxiv, Dec. 2019 , ).…”

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Investigating β-Lactam Drug Targets in Mycobacterium tuberculosis Using Chemical Probes

Levine

Beatty

2021

ACS Infect. Dis.

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Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), infects 10 million people a year. An estimated 25% of humans harbor latent TB infections, an asymptomatic form of the disease. In both active and latent infections, Mtb relies on cell wall peptidoglycan for viability. In the current work, we synthesized fluorescent analogues of β-lactam antibiotics to study two classes of enzymes that maintain Mtb’s peptidoglycan: penicillin-binding proteins (PBPs) and l,d-transpeptidases (LDTs). This set of activity-based probes included analogues of three classes of β-lactams: a monobactam (aztreonam-Cy5), a cephalosporin (cephalexin-Cy5), and a carbapenem (meropenem-Cy5). We used these probes to profile enzyme activity in protein gel-resolved lysates of Mtb. All three out-performed the commercial reagent Bocillin-FL, a penam. Meropenem-Cy5 was used to identify β-lactam targets by mass spectrometry, including PBPs, LDTs, and the β-lactamase BlaC. New probes were also used to compare PBP and LDT activity in two metabolic states: dormancy and active replication. We provide the first direct evidence that Mtb dynamically regulates the enzymes responsible for maintaining peptidoglycan in dormancy. Lastly, we profiled drug susceptibility in lysates and found that meropenem inhibits PBPs, LDTs, and BlaC.

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Investigating β-Lactam Drug Targets in Mycobacterium tuberculosis Using Chemical Probes

Levine

Beatty

2021

ACS Infect. Dis.

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“…Indeed, previous work by us and others to identify PBP-selective inhibitors has yielded β-lactam-based ABPs. 21,22,65,66 There is currently only one report in the literature describing the use of a similar assay in a non-hypersusceptible organism, the PA01 strain of Pseudomonas aeruginosa, that investigated the Bocillin-FL labeling profiles with dual-combination treatments. 67 We have also observed Bocillin-FL labeling of the entire PBP complement in P. aeruginosa (Figure S4), and it is unclear why these results were obtained without the need of cell permeabilization.…”

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Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655

2022

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Penicillin-binding proteins (PBPs) make up an essential class of bacterial enzymes that carry out the final steps of peptidoglycan synthesis and regulate the recycling of this polymeric structure. PBPs are an excellent drug target and have been the most clinically relevant antibacterial target since the 1940s with the introduction of β-lactams. Despite this, a large gap in knowledge remains regarding the individual function and regulation of each PBP homologue in most bacteria. This can be attributed to a lack of chemical tools and methods that enable the study of individual PBPs in an activity-dependent manner and in their native environment. The development of such methods in Gram-negative bacteria has been particularly challenging due to the presence of an outer membrane and numerous resistance mechanisms. To address this, we have developed an optimized live-cell assay for screening inhibitors of the PBPs in Escherichia coli MG1655. We utilized EDTA to permeabilize Gram-negative cells, enabling increased penetration of our readout probe, Bocillin-FL, and subsequent analysis of PBP-inhibition profiles. To identify scaffolds for future development of PBP-selective activity-based probes, we screened ten β-lactams, one diazabicyclooctane, and one monobactam for their PBP-selectivity profiles in E. coli MG1655. These results demonstrate the utility of our assay for the screening of inhibitors in live, non-hypersusceptible Gram-negative organisms.

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The Prospect of Repurposing Immunomodulatory Drugs for Adjunctive Chemotherapy against Tuberculosis: A Critical Review

Lee

Bhakta

2021

Antibiotics

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Tuberculosis (TB) remains a global health emergency, with an estimated 2 billion people infected across the world, and 1.4 million people dying to this disease every year. Many aspects of the causative agent, Mycobacterium tuberculosis, make this disease difficult for healthcare and laboratory researchers to fight against, such as unique pathophysiology, latent infection and long and complex treatment regimens, thus causing patient non-compliance with the treatment. Development of new drugs is critical for tackling these problems. Repurposing drugs is a promising strategy for generating an effective drug treatment whilst circumventing many of the challenges of conventional drug development. In this regard, the incorporation of immunomodulatory drugs into the standard regimen to potentiate frontline drugs is found to be highly appealing. Drugs of diverse chemical classes and drug categories are increasingly being evidenced to possess antitubercular activity, both in vitro and in vivo. This article explores and discusses the molecular entities that have shown promise in being repurposed for use in anti-TB adjunctive therapy and aims to provide the most up-to-date picture of their progress.

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Investigating β-lactam drug targets inMycobacterium tuberculosisusing chemical probes

Cited by 3 publications

References 62 publications

Investigating β-Lactam Drug Targets in Mycobacterium tuberculosis Using Chemical Probes

Investigating β-Lactam Drug Targets in Mycobacterium tuberculosis Using Chemical Probes

Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655

The Prospect of Repurposing Immunomodulatory Drugs for Adjunctive Chemotherapy against Tuberculosis: A Critical Review

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