Inhibition of Mycobacterium tuberculosis Pantothenate Synthetase by Analogues of the Reaction Intermediate

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects approximately two billion people worldwide, and an estimated nine million of these develop TB each year.[1,2] TB is currently the leading cause of infectious disease mortality in the world by a bacterial pathogen, and claimed an estimated 1.7 million deaths in 2006.[3] As a result of the increasing manifestation of multiple-drug-resistant strains of M. tuberculosis and of the limitations of the current anti-TB therapies, the development of safe and effective new drugs with novel modes of action is urgently needed.[4] Pantothenate (vitamin B5) is the essential precursor to coenzyme A and acyl carrier proteins. The de novo biosynthetic pathway to pantothenate is present in many bacteria, fungi and plants and comprises four enzymes, encoded by panB, panE, panD and panC.[5] Bioinformatics analyses have identified this pathway as a potential target for antimicrobial agents.[6] The absence of each enzyme in mammals further suggests that inhibitors could be selective with a reduced risk of side effects. Crucially, genetic studies have shown that a pantothenate auxotroph of M. tuberculosis defective in the panC and panD genes fails to establish virulence in a mouse model of infection.[7] An attenuated strain of M. tuberculosis that deletes both panCD and the primary attenuating mutations of the bacille Calmette–Guerin (BCG) strain is now being considered as a human vaccine candidate for protection against TB.[8] A potential pitfall of inhibiting pantothenate biosynthesis as a general antimicrobial strategy is the ability of several bacteria, including Escherichia coli, to acquire pantothenate from the environment through pantothenate permase (panF).[9] However, to date, no panF homologues have been identified in the M. tuberculosis genome. Furthermore, it has been suggested that rescue of pantothenate through a putative salvage pathway might only be sufficient for M. tuberculosis to survive but not to cause disease.[7,10] The pantothenate pathway is therefore an attractive target for inhibitors that could provide lead compounds for novel antitubercular drugs. We have chosen to target M. tuberculosis pantothenate synthetase (PS, E.C. 6.3.2.1), the product of the panC gene. Pantothenate synthetase catalyzes the final step in the biosynthesis of pantothenate through a Bi Uni Uni Bi Ping Pong kinetic mechanism that consists of two consecutive steps.[11,12] The first reaction, which occurs upon sequential binding of ATP and pantoate, is the Mg2+-dependent formation of a tightly-bound pantoyl adenylate intermediate (1) followed by the release of pyrophosphate. In the second reaction, nucleophilic attack of β-alanine on the activated carbonyl group of 1 leads to formation of AMP and pantothenate (Scheme 1A). Several crystal structures of M. tuberculosis pantothenate synthetase in complex with substrates and products bound have been solved.[13,14] These structures provide informative snapshots of the enzyme in action during catalysis.[14] Despite the extensive structural and mechanistic information available, no inhibitors of M. tuberculosis pantothenate synthetase have been developed to date by using rational drug design. Nevertheless, increasing interest in pantothenate synthetase as an antitubercular target has led to the recent identification of potential inhibitors from high-throughput screens.[15,16] Scheme 1 A) Reaction catalyzed by pantothenate synthetase; the scheme shows the structure of pantoyl adenylate intermediate 1. B) Structures of sulfamoyl analogues 2–4 used in this study. Reaction intermediate 1 is assumed to bind tightly in the active site. Consequently, it was expected that nonreactive analogues of 1 would be potent inhibitors of the enzyme. This approach has precedence in the inhibition of aminoacyl-tRNA synthetases, which are structurally related to pantothenate synthetase, by sulfamoyl adenylate analogues that mimic the aminoacyl adenylate intermediate.[17,18] A similar strategy has been employed to develop potent inhibitors of the adenylation enzyme MbtA, which catalyses the first step in the biosynthetic pathway to the siderophore mycobactin in M. tuberculosis. A sulfamoyl adenylate mimic of the salicyl adenylate intermediate in the MbtA reaction exhibited nanomolar potency in vitro and showed activity against M. tuberculosis in cell-based assays at low micromolar concentrations.[19–21]