Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

Abstract

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-D-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å . Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.Tuberculosis remains a serious global health threat with over a million deaths per year. The recent emergence of multidrugresistant strains of Mycobacterium tuberculosis, the pathogen that causes the lung disease, highlights the need for rapid development of new antibacterial drugs to combat tuberculosis (1-4).3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) 4 is an important enzyme in M. tuberculosis and other pathogens. It catalyzes the first committed step in the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids and other essential aromatic metabolites in microorganisms, plants, and apicomplexan parasites (5-7). This pathway is absent in humans, and inhibitors of amino acid biosynthesis have been shown to be effective antimicrobial and herbicidal agents (8, 9). Gene disruption studies have demonstrated that M. tuberculosis is not viable if the shikimate pathway is not operational (10). These findings make DAH7PS an attractive target for drug development.DAH7PS catalyzes the aldol-like condensation of P-enolpyruvate and D-erythrose 4-phosphate (E4P) to yield 3-deoxy-Darabino-heptulusonate 7-phosphate (Fig. 1a). The reaction mechanism has been subject to extensive study, and many of the key details of the mechanism have been elucidated (11-16). The reactio...

Identification of Novel Polyphenolic Inhibitors of Shikimate Dehydrogenase (AroE)

“…1 The pathway has received considerable attention due in large part to the identification of one of its enzymes, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, as the cellular target of the herbicide, glyphosate (N-(phosphonomethyl) glycine). 2 While a number of studies have sought to identify inhibitors of other enzymes in the pathway, [3][4][5][6][7][8][9] a relatively small number of compounds have been identified that block the pathway's fourth reaction, the NADPH-dependent conversion of 3-dehydroshikimate to shikimate. [10][11][12] This reaction is catalyzed by shikimate dehydrogenase (AroE).…”

Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

“…These amino acids are essential nutrients in the animal diets, and their biosynthetic pathways are prime targets of antimicrobial drugs and plant herbicides (e.g., glyphosate) (Schönbrunn et al, 2001;Reichau et al, 2011). Phe and Tyr also serve as precursors of various aromatic natural products such as pigments, antibiotics, and alkaloids (Bentley, 1990;Maeda and Dudareva, 2012).…”

Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase – The importance of accommodating the active site water

“…The initial nucleophilic attack of PEP on E4P is predicted to be promoted by coordination of the E4P aldehydic oxygen to the essential divalent metal ion, giving an oxocarbenium ion intermediate 4 ( Fig. 1) [3,6]. The addition of an active site nucleophilic water results in a phosphohemiketal tetrahedral intermediate 5, which forms acyclic DAH7P on the elimination of phosphate.…”

“…In mammals these essential metabolites are obtained from dietary sources, and the enzymes of the shikimate pathway are absent. Therefore, the enzymes of this pathway have been identified as promising targets for the development of new antibacterial therapeutics [3][4][5].…”

“…1). Many of the key mechanistic details of the reaction have been elucidated through various labelling, structural and alternative-substrate studies [3,[6][7][8][9][10][11][12]. The ordered sequential reaction proceeds stereospecifically with respect to both substrates, with the si face of PEP attacking the re face of E4P.…”