Dataset Information

A Fluorescence-Based Assay for Screening ?-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase LdtMt2.

ABSTRACT: Mycobacterium tuberculosis l,d-transpeptidases (Ldts), which are involved in cell-wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence-based assay for LdtMt2 , which is suitable for high-throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with LdtMt2 , thus making it possible to assess the availability of the catalytic site in the presence of inhibitors. The assay was applied to a panel of ?-lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of ?-lactams are more potent Ldt inhibitors than other ?-lactam classes, though unexpected variations in potency were observed. The method will enable systematic structure-activity relationship studies on Ldts, thereby facilitating the identification of new antibiotics active against M.?tuberculosis.

SUBMITTER: de Munnik M

PROVIDER: S-EPMC7028133 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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A Fluorescence-Based Assay for Screening β-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase LdtMt2.

de Munnik Mariska M Lohans Christopher T CT Langley Gareth W GW Bon Corentin C Brem Jürgen J Schofield Christopher J CJ

Chembiochem : a European journal of chemical biology 20191108 3

Mycobacterium tuberculosis l,d-transpeptidases (Ldts), which are involved in cell-wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence-based assay for LdtMt2 , which is suitable for high-throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with LdtMt2 , thus making it po ...[more]

PMID: 31322798

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Project description:BackgroundThe emergence and spread of drug resistance in Trichomonas vaginalis parasites has become an important concern in trichomoniasis treatment. Fast and reliable growth assessment is critical for validating in vitro drug susceptibility and high-throughput screening of newly developed drugs.MethodsModified media without yeast extract were evaluated for their ability to support the growth of T. vaginalis parasites. The potential of the nucleic acid-binding dye SYBR Green I for detecting T. vaginalis drug resistance was characterized, and seeding parasite concentration and incubation time were optimized. The fluorescence assay based on SYBR Green I was further validated in four T. vaginalis isolates with different susceptibilities to the antibiotics metronidazole, tinidazole, ornidazole and secnidazole, and compared with the traditional method that detects minimum lethal concentrations (MLCs).ResultsA modified medium consisting of RPMI 1640 and Tryptone Plus as replacements for yeast extract and tryptone, respectively, in traditional trypticase-yeast extract-maltose (TYM) medium exhibited similar performance as TYM medium in maintaining T. vaginalis growth, while it showed much lower background fluorescent signals. The T. vaginalis SYBR Green I-based fluorescence (TSF) drug assay was found to have to satisfy one of two conditions to demonstrate the 50% inhibitory concentration of metronidazole for the sensitive isolate TV-334: (i) a seeding density of 3 × 104 parasites/ml and an incubation time of 48 h; or (ii) a seeding density of 1 × 104 parasites/ml and an incubation time of 72 h. Subsequent validation experiments revealed that the 48-h incubation/3 × 104 parasites/ml seeding density condition had a greater sensitivity to detect drug resistance than the 72-h condition. The TSF assay also exhibited high efficiency in identifying parasite drug resistance, as evidenced by its strong correlation with the standard MLC assay results (P = 0.003).ConclusionsThis study presents a robust TSF assay that has the potential to facilitate high-throughput, automated in vitro anti-trichomoniasis susceptibility testing for drug resistance monitoring and drug development. In comparison to the standard MLC method, this assay offers the advantages of reduced labor and elimination of subjective examination.

Dataset Information

A Fluorescence-Based Assay for Screening ?-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase LdtMt2.

Publications

A Fluorescence-Based Assay for Screening β-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase Ldt<sub>Mt2</sub>.

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