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

Abstract

Keywords drug design; enzyme inhibition; isothermal titration calorimetry; protein structures; tuberculosis 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 B 5 ) 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- Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Accession codes:The atomic coordinates and structure factors for the apoenzyme and enzyme-ligand complexes have been deposited in the Protein Data Bank with the following ID codes: 3cov (apo-PS), 3cow (PS-2), 3coy (PS-3) and 3coz (PS-4).

Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery

“…The goal was to form in situ pantoyladenylate, the intermediate in the reaction catalyzed by Pts (27). There is no report on the affinity of pantoyladenylate to Pts, however an isosteric sulfonamide analog has a reported K d determined by ITC of 125 nM (32). Once the pantoyladenylate intermediate is formed, it would be tightly bound to Pts, occupying both the adenine and pantoate pocket and effectively competing with the fragment for the pantoate pocket.…”

“…The 17 fragment hits were then moved forward to protein X-ray crystallography. Pts can be readily crystallized as a dimeric apoenzyme, yielding high-resolution diffraction data (27,28,32). To locate the fragment binding sites and gain insight into the nature of protein-ligand interactions, fragments were soaked in Pts crystals overnight, at concentrations ranging from 50 to 200 mM, depending on the solubility of the fragments.…”

“…Expression, purification, tag removal, and crystallization of Pts were as described (27,28,32). Final Pts buffer was 50 mM Hepes pH 7.6, 50 mM NaCl, and 5 mM MgCl 2 .…”

“…The thermal shift assay was performed as described previously (32). Briefly, a stock solution containing 4 μM Pts, 10× Sypro Orange (Invitrogen), and 1× Pts buffer was prepared, from which a volume of 90 μL was dispensed into each of a 96-well plate.…”

“…The enzyme performs the condensation of pantoate and β-alanine, by using ATP as cofactor, to form the vitamin pantothenate (25)(26)(27)(28). Several approaches have been used to develop inhibitors, including high-throughput screening (29,30), mimicking the reaction intermediate (31,32), and more recently, we have demonstrated the application of dynamic combinatorial chemistry (33) as well as fragment growing and fragment linking (34,35). These further justify the choice of Pts as a model system to benchmark our biophysical fragment screening strategy.…”