Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

Abstract

Enzymes in essential metabolic pathways are attractive targets for the treatment of bacterial diseases, but in many cases, the presence of homologous human enzymes makes them impractical candidates for drug development. Fumarate hydratase, an essential enzyme in the tricarboxylic acid (TCA) cycle, has been identified as one such potential therapeutic target in tuberculosis. We report the discovery of the first small molecule inhibitor, to our knowledge, of the Mycobacterium tuberculosis fumarate hydratase. A crystal structure at 2.0-Å resolution of the compound in complex with the protein establishes the existence of a previously unidentified allosteric regulatory site. This allosteric site allows for selective inhibition with respect to the homologous human enzyme. We observe a unique binding mode in which two inhibitor molecules interact within the allosteric site, driving significant conformational changes that preclude simultaneous substrate and inhibitor binding. Our results demonstrate the selective inhibition of a highly conserved metabolic enzyme that contains identical active site residues in both the host and the pathogen.

Keywords: Mycobacterium tuberculosis; TCA cycle; allosteric regulation; fumarate hydratase; selective inhibition.

Fig. 1.

A high-throughput screen identifies inhibitors of the M. tuberculosis fumarate hydratase. (A) Schematic of the primary fluorescence-based high-throughput screening assay used to monitor fumarate hydratase activity. Fumarate (1) is used to initiate the reaction, and (l)-malate (2), oxaloacetate (3), and citrate (4) are produced. The assay is monitored by measuring the conversion of resazurin (5) to resorufin (6). (B) The saturation curve, fit using the Michaelis–Menten model, for the fumarate hydratase enzyme as measured by the assay shown in A. Data are reported as an average of replicates (n = 3), and error bars indicate SEMs. (C) The structure and dose–response profiles of the most potent compound (7) from the screen. Inhibition is observed in both the primary assay (black) and a variation of the assay in which citrate synthase is removed (red). (D) The dose–response curve obtained for 7 (black; n = 3). The compound shows no inhibitory effect on the human fumarate hydratase (red; n = 2). Data are reported as an average of replicates, and error bars indicate SEMs.

Fig. 2.

A crystal structure of the M. tuberculosis fumarate hydratase at 2.0-Å resolution reveals the existence of an allosteric site. (A) A ribbon/surface representation of the overall structure of the homotetrameric M. tuberculosis fumarate hydratase with 7 bound to an allosteric site. The dashed circles indicate the location of the four active sites, and the solid circles indicate the location of the two allosteric sites. Colors represent the subunits of the tetramer: green, subunit A; blue, subunit B; purple, subunit C; red, subunit D. (B) Fourier difference density (green mesh; contoured at 3σ) calculated before 7 was added to the model. (C) Superimposition of the fumarate hydratase enzyme bound with formate (gray) or 7 (colors). An arrow indicates the conformational change in the C-terminal domain of subunit C on binding of 7. The solid circle indicates the location of the bound inhibitor at the allosteric site. (D) The stabilizing stacking interaction (indicated by a dashed line) of the central phenyl rings of 7 within the allosteric site. (E) Snapshot of several key interactions between 7 and surrounding amino acid residues. A hydrogen bond between the backbone oxygen atom of Leu429 and the 2-position of the quinazolinone ring of 7 is shown with a dashed line. Arg432 hydrogen bonds to the amide oxygen atom of 7 and also forms a stacking interaction with the quinazolinone ring.

Fig. 3.

Structural data and enzyme kinetics support a competitive mode of inhibition. (A) Cartoon representation of fumarate hydratase in the presence of substrate and the allosteric inhibitor, 7. (Left) In the absence of substrate or substrate mimic, the C-terminal domain of subunit C adopts the open conformation. (Center) When (l)-malate or formate is bound to the active site, the C-terminal domain of subunit C adopts the closed conformation. (Right) In the presence of the allosteric inhibitor, subunit C shifts to the open conformation. The electron density for the formate ions is lost, suggesting that substrate and inhibitor cannot be simultaneously bound. (B) A depiction of the fumarate hydratase active site and allosteric site when formate (gray structure; formate is in yellow) or 7 is bound (colored structure). On binding of 7, the C-terminal domain of subunit C (purple) rotates into the open conformation. (Upper Inset) At the allosteric site, Leu429C and Arg432C shift toward the inhibitor. (Lower Inset) At the active site, the binding of 7 results in the loss of electron density of the loop residues 316A–323A as well as the movement of active site residues Lys324A and Ser139D. These active site residues form critical hydrogen bonds (dashed lines) to the substrate-mimicking formate ions in the formate-bound structure. (C) The equilibrium binding model that accounts for two molecules of inhibitor (I) binding to the enzyme (E) or enzyme–substrate complex (ES) in the presence of substrate (S) and product (P). (D) The saturation curves of the fumarate hydratase enzyme in the presence of different concentrations of 7, fit using the model shown in C. Data are reported as an average of replicates (n = 2), and error bars indicate SEMs.

Fig. 4.

Compound 7 shows inhibition of bacterial growth rate under aerobic conditions. Treatment of M. tuberculosis H37Rv with the fumarate hydratase inhibitor 7 under aerobic conditions results in a decrease in bacterial growth. The fluorescence of cells constitutively expressing GFP was measured 12 d after dosing. Cultures were grown in 7H9 medium supplemented with glucose and casitone. The control cultures were treated with DMSO (black column), and 7 was dosed at concentrations up to 250 μM (blue columns). The relative fluorescence units (RFU) are represented as an average of replicates (n = 4), and error bars indicate SEMs.