Inhibition of Ag 85 C by Cyclipostins and Cyclophostin 1 Cyclipostins and Cyclophostin analogs inhibit the antigen 85 C from Mycobacterium tuberculosis both in vitro and in vivo

An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of Cyclipostins and Cyclophostin (CyC) analogs exhibiting potent activity against Mycobacterium tuberculosis both in vitro and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex (i.e. Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate (TDM) and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC7β, CyC8β, and CyC17 bind covalently to the catalytic Ser124 residue in Ag85C, inhibit mycolyltransferase activity, i.e. the transfer of a fatty acid molecule onto trehalose, and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC8β disclosed that this inhibitor occupies Ag85C’s substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both TDM synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex in vitro and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against M. tuberculosis. http://www.jbc.org/cgi/doi/10.1074/jbc.RA117.000760 The latest version is at JBC Papers in Press. Published on January 4, 2018 as Manuscript RA117.000760 Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jauary 6, 2018 hp://w w w .jb.org/ D ow nladed from Inhibition of Ag85C by Cyclipostins and Cyclophostin 2 INTRODUCTION With 10.4 million new cases and 1.8 million deaths in 2016, tuberculosis (TB) continues to be a major global health problem. TB is caused by Mycobacterium tuberculosis, a resilient microorganism that persists through long courses of antibiotics and years of dormancy within the host. The emergence of multi drug-resistant (MDR) and extensively drug-resistant (XDR) TB contribute to the difficulties in treating this bacterial infection (1). Chemotherapeutic treatments against TB remain very challenging and complicated, essentially because of the slow rate of growth of the bacilli and the presence of a thick, greasy and relatively drug-impermeable cell wall (2). This mycobacterial cell wall consists of a complex skeleton comprising covalently linked macromolecules such as peptidoglycan, arabinogalactan and mycolic acids, in which noncovalently associated glycolipids are interspersed (3). The mycolic acid portion of the envelope is composed of very long fatty acids (C70–90) that are either covalently attached to the arabinan moiety of the arabinogalactan (AG) polymer or found esterified to trehalose as trehalose monomycolate (TMM) or trehalose dimycolate (TDM). Because several key antitubercular drugs such as isoniazid, SQ109, delamanid, or ethambutol target different aspects of the biosynthetic steps responsible for the cell wall attachment of mycolic acids (4–7), this pathway is of particular interest from a drug discovery perspective. The three functionally and structurally related members of the antigen 85 complex, designated Ag85A, –B and –C, are among the most abundantly secreted proteins in M. tuberculosis (8). These enzymes are responsible for the biosynthesis of TMM and TDM as well as the covalent attachment of mycolic acids to AG (9– 11). Deletion of fbpC2, encoding Ag85C, resulted in a 40% decrease in the AG-bound mycolic acids but failed to affect the production of noncovalently linked mycolates (10), while deletion of fbpA or fbpB, encoding Ag85A and Ag85B, respectively, lead to reduced TDM levels (12–14), implying that although a level of functional redundancy exist in vivo between the three members, the contribution of each member is significant. The lack of double and triple knockout mutants might indicate that the loss of two or more Ag85 enzymes is detrimental to M. tuberculosis viability. An additional isoform, designated Ag85D or MPT51, has been characterized but found to be inactive due to the lack of catalytic elements required for mycolyltransferase activity (11, 15, 16). Ag85A/B/C share the same mycolic acid donor TMM and their crystal structures present a highly conserved catalytic site which further support their similar enzymatic role (17–19). Due to their importance in mycolic acid metabolism, the Ag85 enzymes have often been proposed as attractive targets for future chemotherapeutic developments against TB (9, 20–22). Because of their high structural conservation, it can be inferred that a single compound may inhibit all three enzymes of the complex at the same time and would make improbable the development of resistance to inhibitors, since resistant mutants would require the simultaneous acquisition of mutations in at least two fbp genes. In addition, because these proteins are secreted, targeting the Ag85 complex will minimize the effect of efflux mechanisms that may result in resistance phenotypes. Early inhibitors such as trehalose analogs were first designed as Ag85 inhibitors but were found to exhibit relatively poor activity on whole mycobacterial cells (9, 23). Another potentially selective fluorophosphonate ,-D-trehalose inhibitor of the three antigen 85 enzymes has been reported to form a stable, covalent complex with the Ag85 enzyme following nucleophilic attack on the phosphorous atom of the catalytic Ser124 (24). In the same manner, the 2-amino-6propyl-4,5,6,7-tetrahydro-1-benzothiophene-3carbonitrile, designated I3-AG85, inhibits Ag85C and exposure of M. tuberculosis to this compound was associated with reduced survival rates in broth medium and in infected primary macrophages. Moreover, I3-AG85 was active against a panel of MDR/XDR strains although it exhibited an MIC of 100 μM (25). By combining fragment-based drug discovery with early whole cell antibacterial screening, tetrahydro-1by gest on Jauary 6, 2018 hp://w w w .jb.org/ D ow nladed from Inhibition of Ag85C by Cyclipostins and Cyclophostin 3 benzothiophene analogs were discovered as potent Ag85C inhibitory molecules against drugsusceptible and drug-resistant M. tuberculosis strains (26). The selenazole compound ebselen (2phenyl-1,2-benzisoselenazol-3(2H)-one) was found to inhibit the activity of Ag85C through an original mechanism by reacting with the conserved Cys209 residue located near the active site of the enzyme but not involved in the catalytic activity (27, 28). Ebselen was shown to directly impede the production of TDM and mycolylation of AG (27). Recently, Cyclipostins and Cyclophostin (CyC), representing a new class of monocyclic enolphosph(on)ate compounds, have been discovered to act as powerful antitubercular agents affecting growth of M. tuberculosis both in vitro and in infected macrophages (29). Among the set of 27 CyC analogs previously evaluated against M. tuberculosis H37Rv, 8 compounds exhibited potent anti-tubercular activities, particularly the Cyclophostin analogs CyC7 and CyC8 as well as the Cyclipostins-related molecule CyC17. While CyC7 exhibited a strong activity against extracellular and intracellular mycobacteria (MIC50 of 16.6 and 3.1 μM, respectively), CyC8was mostly found to be active against intracellular bacteria (MIC50 ≈11.7 μM). In contrast, CyC17 was a potent inhibitor of in vitro growth (MIC50 ≈ 0.5 μM) but failed to show activity against intracellular bacilli (29). To identify the putative target(s) of the CyC inhibitors, an activity-based protein profiling approach was used based on TAMRA-FP and Desthiobiotin-FP probes and mass spectrometry analyses. This led to the capture of several active serine/cysteine enzymes in a complex proteome prior to mass spectrometry identification, among which Ag85A (Rv3804c) and Ag85C (Rv0129c) were identified. The present study was undertaken to further explore and validate, through a combination of biochemical and structural approaches, the specificity of inhibition of the Ag85 activity by the CyC analogs, to determine their mode of action and to describe how they affect the mycolic acid profile in M. tuberculosis. RESULTS CyC analogs inhibit TDM biosynthesis and transfer of mycolic acids to arabinogalactan in M. tuberculosis. CyCs are a new class of compounds demonstrating potent antitubercular activity, presumably involving inhibition of the Ag85 activity (29). The chemical structures of the Cyclophostin analogs CyC7 and CyC8 and the Cyclipostins CyC17 used in this study are provided in Fig. 1A. To test whether treatment with these CyCs alters the mycolic acid composition of M. tuberculosis mc6230, cultures were exposed to increasing concentrations of CyC17 or CyC7-the two inhibitors most active against extracellularreplicating M. tuberculosis (29) followed by metabolic labeling with sodium [2-C]-acetate and lipid analysis. Extraction and separation of the total mycolic acid methyl esters (MAME) by thin layer chromatography (TLC) revealed that neither CyC17 nor CyC7 altered the de novo biosynthesis of mycolic acid (Fig. 1B and 1C, left panel). In contrast, separation of the apolar lipid fraction by TLC showed a dose-dependent decrease in TDM levels associated with a concomitant increase in the production of TMM, which is the natural substrate of the Ag85 proteins (Fig. 1B and 1C, middle panels). To address whether CyC treatment impacts also the cell wall-bound mycolic acids, radiolabeled mycolic acids we