Duckworth Benjamin P, Wilson Daniel J, Nelson Kathryn M, Boshoff Helena I, Barry Clifton E, Aldrich Courtney C
Center for Drug Design and ‡Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.
ACS Chem Biol. 2012 Oct 19;7(10):1653-8. doi: 10.1021/cb300112x. Epub 2012 Jul 23.
MbtA is an adenylating enzyme from Mycobacterium tuberculosis that catalyzes the first step in the biosynthesis of the mycobactins. A bisubstrate inhibitor of MbtA (Sal-AMS) was previously described that displays potent antitubercular activity under iron-replete as well as iron-deficient growth conditions. This finding is surprising since mycobactin biosynthesis is not required under iron-replete conditions and suggests off-target inhibition of additional biochemical pathways. As a first step toward a complete understanding of the mechanism of action of Sal-AMS, we have designed and validated an activity-based probe (ABP) for studying Sal-AMS inhibition in M. tuberculosis. This probe labels pure MbtA as well as MbtA in mycobacterial lysate, and labeling can be completely inhibited by preincubation with Sal-AMS. Furthermore, this probe provides a prototypical core scaffold for the creation of ABPs to profile any of the other 66 adenylating enzymes in Mtb or the multitude of adenylating enzymes in other pathogenic bacteria.
MbtA是一种来自结核分枝杆菌的腺苷酸化酶，它催化分枝菌素生物合成的第一步。之前曾描述过一种MbtA的双底物抑制剂（Sal-AMS），该抑制剂在铁充足以及铁缺乏的生长条件下均表现出强大的抗结核活性。这一发现令人惊讶，因为在铁充足的条件下并不需要分枝菌素的生物合成，这表明它对其他生化途径存在脱靶抑制作用。作为全面了解Sal-AMS作用机制的第一步，我们设计并验证了一种基于活性的探针（ABP），用于研究结核分枝杆菌中Sal-AMS的抑制作用。该探针可标记纯MbtA以及分枝杆菌裂解物中的MbtA，并且通过与Sal-AMS预孵育可完全抑制标记。此外，该探针为创建ABP提供了一个典型的核心支架，用于分析结核分枝杆菌中其他66种腺苷酸化酶或其他致病细菌中的多种腺苷酸化酶。
