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. Because the tricarboxylic acid (TCA) cycle connects many pathways of cellular metabolism, preventing the function of this cycle through enzyme inhibition is an attractive strategy for targeting infectious agents (1). In Mycobacterium tuberculosis, experimental evidence (2) has suggested that fumarate hydratase, the essential enzyme responsible for the reversible conversion of fumarate to (L)-malate, is a vulnerable target. This vulnerability is in part due to the fact that, unlike other bacteria (such as Escherichia coli), M. tuberculosis expresses only one enzyme that performs this function (3). In addition to its role in metabolism under aerobic conditions, fumarate hydratase has also garnered interest because of the discovery of a flux toward the reverse TCA cycle under hypoxic conditions in nonreplicating M. tuberculosis (2, 4, 5). However, despite these discoveries, no small molecule inhibitor of the M. tuberculosis fumarate hydratase has been reported. The discovery of such an inhibitor would provide an important tool to begin probing the role of the TCA cycle in both actively replicating and nonreplicating bacteria.From the standpoint of drug development, however, targeting the M. tuberculosis fumarate hydratase poses a significant challenge, because the protein is highly evolutionarily conserved. In particular, the human and M. tuberculosis homologs share identical active site residues as well as 53% overall sequence identity (6, 7). Both homologs form a stable homotetramer containing four active sites, and every active site is composed of residues from three enzyme subunits. Each dumbbell-shaped subunit within the tetramer contains three domains: an N-terminal domain, a central domain, and a C-terminal domain (8-10). The N-and C-terminal domains are predominantly α-helical and linked by the central domain that consists of five tightly packed helices. The central domains of ...

Fumarase Deficiency Causes Protein and Metabolite Succination and Intoxicates Mycobacterium tuberculosis

“…Fumarase null mutants in other bacteria, and even eukaryotes, have been reported to be viable (Carls and Hanson, 1971; Mercado-Lubo et al, 2009; Song et al, 2013; Xu et al, 2013). Interestingly, a small molecule inhibitor of Fum that potently and selectively inhibits Mtb Fum, via binding to an allosteric regulatory site, was recently reported (Kasbekar et al, 2016). However, this compound only slowed growth and failed to kill Mtb , leaving the phenotypic consequences of Fum inactivation in Mtb unexplored.…”

Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase

“…The overall structure of Hs FH is very similar to all available models for class II fumarases: E. coli FumC (FumC FH, PDB code: , , , , , and ; ), Mycobacterium tuberculosis FH ( Mt FH, PDB code: , , , , , and ; ), Saccharomyces cerevisiae (strain ATCC 204508/S288c) FH ( Sc FH, PDB code: ; ), Thermus thermophilus FH ( Tt FH, PDB code: ; results not published), Rickettsia prowazekii FH ( Rp FH, PDB code: ; ), Burkholderia pseudomallei FH ( Bp FH, PDB code: ; results not published), Mycobacterium marinum FH ( Mm FH, PDB code: ; ), Rhizobium meliloti FH ( Rm FH, PDB code: ; results not published), Mycobacterium smegmatis FH ( Ms FH, PDB code: ; ). The superposition of Cα atoms between Hs FH and the structurally equivalent atoms shows in average (chain A) an RMSD of 0.5 Å for Hs FH, 1.09 Å for FumC FH, 2.01 Å for Mt FH, 1.22 Å for Sc FH, 1.56 Å for Tt FH, 2.06 Å for Rp FH, 0.96 Å for Bp FH, 1.55 Å for Mm FH, 0.76 Å for Rm FH and 1.7 Å for Ms FH.…”

“…The overall structure of HsFH is very similar to all available models for class II fumarases: E. coli FumC (FumC FH, PDB code: 1YFE, 1FUO, 1FUQ, 2FUS, 1KQ7, 1FUP and 1FUR; [5,28,32,33]), Mycobacterium tuberculosis FH (MtFH, PDB code: 4ADM, 4ADL, 4APA, 4APB, 3NO9, 5F91 and 5F92; [34,35]), Saccharomyces cerevisiae (strain ATCC 204508/S288c) FH (ScFH, PDB code: 1YFM; [36]), Thermus thermophilus FH (TtFH, PDB code: 1VDK; results not published), Rickettsia prowazekii FH (RpFH, PDB code: 3GTD; [37]), Burkholderia pseudomallei FH (BpFH, PDB code: 3TV2; results not published), Mycobacterium marinum FH (MmFH, PDB code: 3QBP; [38]), Rhizobium meliloti FH (RmFH, PDB code: 4HGV; results not published), Mycobacterium smegmatis FH (MsFH, PDB code: 3RD8; [38]). The superposition of 7).…”

“…), class II FHs share on average an RMSD of 1.9 Å with the main deviations occurring at both N‐ and C‐ extremities. In fact, the mobility of D3 (C‐terminal domain), in its open and closed conformations was described to be involved in the FH mechanism of catalysis and therefore, compounds that interfere with this hinge motion may function as allosteric inhibitors .…”

Identification of activators of human fumarate hydratase by quantitative high-throughput screening

“…Currently there are no biochemically characterized inhibitors of FH, and no known activator for this enzyme. Two series of inhibitors have been reported to inhibit the activity of human 11 or Mycobacterium tuberculosis FH 12 . In an attempt to identify nutrient-dependent cytotoxic compounds, Takeuchi et al 11 screened a collection of 6,000 small molecules and discovered a new class of inhibitors of human FH (compound 1-3 with pyrrolidinone structure).…”