ESI-MS assay of M. tuberculosis cell wall antigen 85 enzymes permits substrate profiling and design of a mechanism-based inhibitor.

Mycobacterium tuberculosis Antigen 85 enzymes are vital to the integrity of the highly impermeable cell envelope and are potential therapeutic targets. Kinetic analysis using a label-free assay revealed both mechanistic details and a substrate profile that allowed the design and construction of a selective in vitromechanism-based inhibitor. M tuberculosis (Mtb), the causative agent of tuberculosis, is among the foremost causes of death and morbidity worldwide. It persists within the host with the aid of a complex and highly impermeable cell envelope containing a high content of long-chain fatty acids (mycolic acids) present as mycolate esters of arabino-galactan and of trehalose 1. 3 Trehalose diand monomycolates (TDM 2a and TMM 3a, respectively) are interconverted by the Antigen 85 a,b, and c (Ag85) mycolyltransferase isoforms (Figure 1). 6 The Ag85 enzymes play a vital role in the construction and maintenance of the cell envelope, and have recently been shown to display plasticity for substrates based upon the trehalose motif. Genetic ‘knockouts’ highlight Ag85c as the most active isoform; loss causes a 40% decrease inmycolate content of the cell wall. These enzymes are therefore suggested targets for novel antimycobacterial drugs, the development of which would benefit from a rapid and accurate assay of Ag85 activity and a thorough understanding of enzyme mechanism through the development of potential probes. However, despite the availability of several crystal structures and KM estimates measured under pseudosingle-substrate conditions or with heterogeneous substrates, no detailed kinetic analysis or structure activity relationship (SAR) studies have been reported. Current assays include nonspecific radiometric procedures for the detection of generic mycolyltransferase activity and coupled enzymatic assays for Ag85c. 6 Such methods preclude the possibility of readily automated screening or do not allow complete dissection of the steps of Ag85 acyl transfer, respectively. We report here the implementation of a label-free assay that allows full kinetic analysis as well as the potential for moderate-throughput screening; a panel of representative monosaccharides was used to map configurational structure activity relationships. Together these data allowed the design of a mechanism-based inhibitor probe of Ag85c. Mass spectrometry (MS) can be readily automated, and multiple natural substrates and products can be monitored simultaneously to give an unimpeded view of kinetic processes. 13 Kinetic analysis and substrate/inhibitor profiling were enabled by the development of a precise and quantitative MS assay of Ag85 activity based on total ion counts (TICs) of ions corresponding to trehalose, 6,60-dihexanoyltrehalose 2b (TDH), and 6-hexanoyltrehalose 3b (TMH) (Figure 1). This exploited our previously described calibration approach with a pseudointernal standard (hereN-acetyl-D-glucosamine) injected concurrently with the sample aliquot; calibration plots for each monitored mass allowed quantitative measurements of concentration. Full bi-substrate kinetic analysis was performed by determining initial rates of TMH 3b formation at varied TDH 2b and trehalose 1 concentrations. 13 These data were fitted by nonlinear regression methods to a range of plausible kinetic models, including modified rate equations that account for multiple acyl transfer pathways. Together these and double-reciprocal analysis (see Supporting Information [SI]) ruled out simple (e.g., bi-bi ping-pong) mechanisms that are typical of other Figure 1. Reaction timecourse catalyzed by Ag85. Conditions: 250 μM 1, 250 μM 2b, 2 μM Ag85c, 1 mM TEA buffer (pH 7.2), 37 C. Full kinetic analysis utilized the full range of conditions [Tre] = 5 125 μM, [2b] = 25 600 μM, [Ag85c] = 10 nM. Received: May 9, 2011