Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, ON, K7L 3N6, Canada.
Chembiochem. 2020 Feb 3;21(3):368-372. doi: 10.1002/cbic.201900379. Epub 2019 Nov 8.
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 Ldt , which is suitable for high-throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with Ldt , 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 β-lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of β-lactams are more potent Ldt inhibitors than other β-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.
结核分枝杆菌 L、d-转肽酶（Ldts）参与细胞壁生物合成，已成为治疗结核病的有希望的靶点。然而，目前还没有一种有效的方法来测试这些酶的抑制作用。我们提出了一种基于荧光的 Ldt 检测方法，适用于高通量筛选。我们确定了两种荧光探针，它们与 Ldt 反应后释放荧光团，因此可以在抑制剂存在的情况下评估催化位点的可用性。该测定方法应用于一组β-内酰胺抗生素和相关抑制剂；结果验证了这样的观察结果，即（碳青霉烯）类β-内酰胺比其他β-内酰胺类具有更强的 Ldt 抑制作用，尽管观察到了意想不到的效力变化。该方法将能够对 Ldts 进行系统的构效关系研究，从而有助于鉴定对结核分枝杆菌有活性的新型抗生素。
