Identification of KasA as the cellular target of an anti-tubercular scaffold

Abstract

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.

Figure 1. The anti-tubercular activity of GSK3011724A.

(a) Chemical structure of GSK3011724A. (b,c) GSK3011724A was evaluated in an acute (b) and chronic (c) model of murine TB infection. Each symbol represents data from a single mouse. A two-fold log c.f.u. reduction equates to a 2 log c.f.u. difference compared with untreated mice at the end of the treatment period. Untreated mice gave a log c.f.u. count of 7.1±0.1 (mean±s.d., n=5 mice) in the acute assay and 5.8±0.26 (mean±s.d., n=7 mice for the INH experiment) or 6.0±0.5 (mean±s.d., n=5 mice for GSK3011724A experiment) in the chronic assay. (d) Comparison of the in vivo efficacy of GSK3011724A and INH against M. tuberculosis H37Rv wild-type (H37Rv WT) and M. tuberculosis KasA mutant strains. Mice were infected with each strain and administered with INH or GSK3011724A at different doses during the acute phase (days 1 to 8 after infection). Each dot represents data from a single mouse. Log c.f.u. counts are shown as the difference with respect to the untreated group infected with each strain (Δlog c.f.u. for each mouse).

Figure 2. Inhibition of mycolic acid biosynthesis by GSK3011724A.

(a) M. bovis BCG cultures, labelled with [¹⁴C]-acetate, were treated with GSK3011724A, TLM and INH. The total FAMEs and MAMEs were extracted and analysed by autoradiography-TLC using equal counts (25,000 c.p.m.) for each lane, respectively (left and middle panels). In addition, cell wall-bound MAMEs were isolated and an equal aliquot (5%) was analysed by autoradiography-TLC for each lane, respectively (right panel). (b) M. bovis BCG total cellular lipids containing TDM/TMM were extracted following labelling and drug treatment (GSK3011724A, TLM and INH) and analysed by autoradiography-TLC using equal counts (25,000 c.p.m.) for each lane, respectively. (c) Impact on the MIC of GSK3011724A upon the overexpression of members of FAS-II in M. bovis BCG. The overexpression constructs of M. tuberculosis enzymes (Mt) pMV261-Mt-kasA, pMV261-Mt-kasB, pMV261-Mt-fabH, pMV261-Mt-inhA, pMV261-Mt-hadABC and pMV261-Mt-mabA were electroporated into M. bovis BCG and the MIC of GSK3011724A was evaluated with reference to M. bovis BCG pMV261. The outer and inner spots represent 10³ and 10² cells plated, respectively.

Figure 3. Chemoproteomics profiling of GSK3011724A.

(a) A propylamine-tagged derivative of GSK3011724A (1, inset) was synthesized and covalently immobilized to NHS-activated sepharose. Beads were incubated with M. bovis BCG extract either in the presence of vehicle (DMSO) or GSK3011724A (10 μM, 40 μM). Proteins captured by the beads in both conditions were quantified by LC–MS/MS analysis. KasA, Pks10 and Pks11 were identified as potential targets of GSK3011724A by virtue of their reduced capturing in the presence of excess GSK3011724A. (b) Generation of IC₅₀ values for KasA, Pks10 and Pks11. The chemoproteomic experiment was performed as in a but over a range of concentrations of the competing ‘free' inhibitor GSK3011724A (2–0.003 μM for KasA, 40–0.16 μM for Pks10 and Pks11) and a structurally related inactive analogue, 2 (40–0.16 μM). Apparent dissociation constants for GSK3011724A were determined from two independent experiments.

Figure 4. X-ray crystallographic analysis of KasA complexed with GSK3011724A.

(a) Co-crystal structure of KasA dimer (yellow, blue) with GSK3011724A (green space filled) and PEG (green sticks) bound with the open conformation of the acyl channel. The active site cysteine C171 is shown in line format, as are an overlay of the TLM and a phospholipid ligand taken from PDB entry 4C72 (ref. 25). (b) GSK3011724A and PEG both shown in stick format filling the acyl channel. (c) Hydrogen bonds between GSK3011724A and E199 within the binding site.

Figure 5. Impact of resistance-conferring mutations on the KasA-GSK3011724A crystal structure and structural comparison of KasA with KasB and FabH.

(a) Mapping of residues where resistance mutations occur onto the dimeric KasA-GSK3011724A X-ray crystallographic complex. GSK3011724A (green stick) marks the acyl channel, whereas TLM (magenta) from PDB entry 4C72 (ref. 25) marks the malonyl pocket. (b) Residues P201 and P206 that confer resistance by direct interactions with GSK3011724A are shown, as are the proximity of other resistance positions visible. (c) A ribbon representation of the open and closed forms of KasA with resistance mutations highlighted. The open KasA-GSK3011724A dimer is shown in yellow and blue (chain A, B respectively). The apo, closed conformation (PDB entry 2WGD (ref. 26)) is shown in orange and purple. The HTH arm formed by α5–α6 and the α'2 from its dimer partner move substantially on transition between the open and closed states. Residues within these two regions of movement where resistance mutations are found are shown in stick format. GSK3011724A, PEG and other residues sensitive to resistance mutations are shown in line format. (d) Surface around Chain A within KasA-GSK3011724A complex shown to illustrate the role of M277 in the open and closed states. Colouring and representations identical to c. (e) Hydrogen bonding interactions around T114 within the open KasA-GSK3011724A complex. (f) Structural alignment of KasA-GSK3011724A (yellow and blue) with KasB (PDB entry 2GP6 (ref. 54)) (magenta). The indazole ring of GSK3011724A sits favourably over G200 and P201 in KasA. In KasB, the respective residues of T199 and R200 would not be able to accommodate this favourable binding mode. (g) Structural overlay of KasA-GSK3011724A (yellow and blue) with FabH (PDB entry 2QNZ (ref. 55)) (orange). In FabH, T145 would directly clash with the indazole ring of GSK3011724A, accounting for the compound selectivity for KasA.