Cyclipostins and cyclophostin analogs inhibit the antigen 85C from Mycobacterium tuberculosis both in vitro and in vivo

Abstract

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 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 CyC_7β, CyC_8β, 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 CyC_8β disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both trehalose dimycolate 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.

Keywords: Ag85 complex; Mycobacterium tuberculosis; cell wall; crystal structure; cyclipostins; cyclophostin; inhibition mechanism; trehalose dimycolate; trehalose monomycolate; triacylglycerol.

Figure 1.

Ag85 complex mycolyltransferase activity is inhibited by CyC analogs in vivo. Exponentially growing M. tuberculosis mc²6230 was incubated with increasing concentrations of CyC₁₇ or CyC_7β in 7H9^{OADC/Tween 80} at 37 °C with agitation for 1 h. Subsequently, bacteria were labeled with sodium [2-¹⁴C]acetate for 6 h at 37 °C with agitation. The cultures were split, and from the first volume were extracted the total methyl esters of mycolates (MAME) and fatty acids (FAME). From the second volume, apolar and polar lipid fractions were obtained before derivatization of arabinogalactan MAME. A, chemical structures of the CyC analogs used in this study. B and C, effect of CyC₁₇ (B) or CyC_7β (C) treatment on the mycolic acid profiles of M. tuberculosis mc²6230. Equal counts (50,000 cpm) of MAME + FAME fraction were loaded on a TLC plate and resolved once using the solvent system hexane/ethyl acetate (95:5, v/v) run twice (far left). The apolar fraction was loaded (50,000 cpm), and TMM and TDM were visualized on a 1D TLC plate using the solvent system chloroform/methanol/water (40:8:1, v/v/v) (middle left). Equal volumes of arabinogalactan MAME fraction were loaded, and α, methoxy, and keto mycolic acids were visualized on a 1D TLC plate using the solvent system hexane/ethyl acetate (95:5, v/v) run twice (middle right). Densitometric analysis (far right) was performed on the TLCs shown in the left panels. Histograms and error bars, means and S.D. values calculated from at least two independent experiments.

Figure 2.

Inhibition of the Ag85C mycolyltransferase activity is mediated by the covalent binding of CyC analogs. A, the enzymatic activity of Ag85C was tested using a fluorescence-based assay in the presence of different concentrations of CyC_7β, CyC_8β, and CyC₁₇. The inhibitory effect was determined at the maximum rate of the reaction. Error bars, S.D. calculated from three independent experiments. Curves for CyC_7β, CyC_8β, and CyC₁₇ were fitted using the EC₅₀ shift non-linear regression model in GraphPad Prism with R² values of 0.9675, 0.9508, and 0.9415, respectively. B, equal amounts of either Ag85C or Ag85C^S124A were pretreated with CyC_7β, CyC_8β, and CyC₁₇; incubated with TAMRA-FP, separated by SDS-PAGE; and visualized by Coomassie Blue staining (top) or in-gel fluorescence visualization (middle). The merged image is shown at the bottom. TAMRA labeling of Ag85C is prevented by the covalent binding of the CyC analogs to the catalytic Ser¹²⁴. No TAMRA-FP labeling is seen for the Ag85C^S124A variant, confirming Ser¹²⁴ as the TAMRA-binding site. C and D, global mass modification of Ag85C (C) and Ag85C^S124A (D) preincubated with CyC_7β, CyC_8β, and CyC₁₇ as determined using an Ultraflex III mass spectrometer (Bruker Daltonics) in linear mode with the LP_66 kDa method. The mechanism of action of the phosphonates CyC_7β and CyC_8β and of the phosphate analog CyC₁₇ based on mass spectrometry analyses is illustrated in C. a.u., arbitrary units.

Figure 3.

DGAT activity of the antigen 85 complex and inhibition by CyC analogs. A, chemical reaction occurring while determining the DGAT activity. DTNB reacts with the free-thiol group coming from the release of SH-CoA during the formation of TAG from 1,2-dipalmitoylglycerol (DAG) and a molecule of acyl-CoA. B, comparison of the DGAT activity of Ag85A, Ag85B, Ag85C, and MPT51. Enzymatic activity was determined by the colorimetry-based assay illustrated in A. Inset, activity of the wildtype and S124A Ag85C proteins using palmitoyl-CoA (C16) as acyl donor molecule. Error bars, S.D. calculated from three independent experiments. C, inhibitory effect of CyC analogs on Ag85C DGAT activity. Inhibition was performed with increasing concentrations of CyC_7β, CyC_8β, and CyC₁₇ using the colorimetry-based assay illustrated in A. The inhibitory effect was determined after 1 h of reaction. Error bars, S.D. calculated from three independent experiments. Curves for CyC_7β, CyC_8β, and CyC₁₇ were fitted using the EC₅₀ shift non-linear regression model on GraphPad with R² values of 0.9755, 0.9641, and 0.9422, respectively. D, comparison of the DGAT activity of Ag85C and Tgs1 in the absence or presence of CyC_7β, CyC_8β, and CyC₁₇.

Figure 4.

Biosynthesis of TAG in M. tuberculosis is inhibited by CyC₁₇ and is dependent upon Ag85C expression. A, quantitative real-time PCR analysis showing the -fold increase in the Ag85C transcripts in M. tuberculosis mc²6230 containing either pMV261 (ctrl), pMV261-Ag85C, or pMV261-Ag85C^S124A (left). Western blotting using the 32/15 and 17/4 monoclonal antibodies probed against purified Ag85A/B/C and crude lysates of M. tuberculosis mc²6230 containing either pMV261, pMV261-Ag85C, or pMV261-Ag85C^S124A (right). B, Nile Red staining of M. tuberculosis strains growing exponentially (left) with the corresponding fluorescence quantification (right). Fluorescence quantification was performed on 30 bacilli of each group. Shown are the mean fluorescence and S.D. values. Means were compared by the two-tailed Mann–Whitney test. ns, non-significant; **, p < 0.01. Results shown are representative of two independent experiments. C, cultures were exposed to increasing concentrations of CyC₁₇ in 7H9^{OADC/Tween 80} and labeled with sodium [2-¹⁴C]acetate for 4 h at 37 °C with agitation. The apolar fraction was extracted to analyze de novo synthesis of TAG. Equal counts (50,000 cpm) of apolar fraction were loaded, and TAG was visualized on a 1D TLC plate using the solvent system petroleum ether/diethyl ether (90:10, v/v) (left). Right, densitometric analysis of TLCs. Histograms and error bars, means and S.D. values calculated from four independent experiments.

Figure 5.

Structural basis for Ag85C inhibition by CyC_8β. A, crystal structure of Ag85C in complex with CyC_8β. The figure displays the overall asymmetric unit with the two monomers represented as blue and magenta schematics. CyC_8β is shown as sticks and colored in yellow. B, simulated annealing F_o − F_c OMIT map contoured at 3σ attesting to the presence of two CyC_8β that could be entirely modeled for one molecule and partially for the second one. The map also reveals the presence of an extra, but non-interpretable, electron density in the vicinity of the CyC_8β molecule. C, surface representation of the Ag85C structure bound to CyC_8β. The hydrophobic residues are colored in blue, and the catalytic Ser¹²⁴ is shown in green. D, CyC_8β binding site. Ag85C residues involved in CyC_8β recognition are displayed as blue sticks for those involved in hydrogen bond (black dashes) formation. Residues in orange are involved in hydrophobic interactions with the acyl chain of CyC_8β, and Arg⁴¹ in gray contributes to the recognition of the CyC_8β headgroup by van der Waals interaction. Ser¹²⁴, Glu²²⁸, and His²⁶⁰ form the catalytic triad. Red spheres, water molecules.

Figure 6.

Mode of inhibition of the Ag85 complex by CyC_8β. A, superposition of the Ag85B-trehalose (PDB code 1F0P, blue) and Ag85C-CyC_8β (gray) crystal structures. The headgroup of CyC_8β (yellow) occupies the same site as trehalose (green). B, Ag85C residues (gray) involved in the recognition of CyC_8β are all strictly conserved in Ag85B (cyan) and Ag85A (magenta). C, superposition of the Ag85C-ebselen (PDB code 4QDU; blue) and Ag85C-CyC_8β (gray) crystal structures. CyC_8β binds far away from the ebselen-binding site and does not trigger structural rearrangement of helix α9. D, superposition of the Ag85C-DEP (PDB code 1DQY; cyan) and Ag85C-CyC_8β (gray) crystal structures. CyC_8β presents a similar mode of inhibition as DEP (cyan stick), a nonspecific σ/β hydrolase inhibitor.