Methyl-Thiazoles: A Novel Mode of Inhibition with the Potential to Develop Novel Inhibitors Targeting InhA in Mycobacterium tuberculosis Pravin S. Shirude,* ,† Prashanti Madhavapeddi, ‡ Maruti Naik, † Kannan Murugan, † Vikas Shinde, † Radha Nandishaiah, ‡ Jyothi Bhat, ‡ Anupriya Kumar, † Shahul Hameed, † Geoffrey Holdgate, # Gareth Davies, # Helen McMiken, # Naina Hegde, ‡ Anisha Ambady, ‡ Janani Venkatraman, ‡ Manoranjan Panda, † Balachandra Bandodkar, † Vasan K. Sambandamurthy, ‡ and Jon A. Read* ,# † Department of Medicinal Chemistry, ‡ Department of Biosciences, AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore-560024, India # Discovery Sciences, AstraZeneca, Alderley Park, Macclesfield SK10 4TG, U.K. *S Supporting Information ABSTRACT: InhA is a well validated Mycobacterium tuberculosis (Mtb) target as evidenced by the clinical success of isoniazid. Translating enzyme inhibition to bacterial cidality by targeting the fatty acid substrate site of InhA remains a daunting challenge. The recent disclosure of a methyl-thiazole series demonstrates that bacterial cidality can be achieved with potent enzyme inhibition and appropriate physicochemical properties. In this study, we report the molecular mode of action of a lead methyl-thiazole, along with analogues with improved CYP inhibition profile. We have identified a novel mechanism of InhA inhibition characterized by a hitherto unreported “Y158-out” inhibitor-bound conformation of the protein that accommodates a neutrally charged “warhead”. An additional novel hydrophilic interaction with protein residue M98 allows the incorporation of favorable physicochemical properties for cellular activity. Notably, the methyl-thiazole prefers the NADH-bound form of the enzyme with a K d of ∼13.7 nM, as against the NAD + -bound form of the enzyme. ■ INTRODUCTION Tuberculosis (TB) continues to be a major global cause of morbidity and mortality due to the infectious pathogen Mycobacterium tuberculosis (Mtb). The emergence of Mtb strains resistant to first line and second line TB drugs adds to the challenge in global efforts to control this infection. 1 The bacterial fatty acid biosynthesis pathway represents a validated and yet relatively unexploited target for drug discovery. 2 Fatty acids are essential for bacterial growth, however, they cannot be scavenged from the host and must be synthesized de novo. 3,4 In Mtb, Enoyl-acyl carrier protein reductase (ACPER), known as InhA, is encoded by the inhA gene as an essential NADH dependent enzyme in the mycolic acid biosynthetic pathway. 4,5 Mycolic acids are linked to the cell wall and form a waxy protective coating around the bacterial cell, which serves as a permeability barrier. The bacterial fatty acid biosynthetic pathway (FAS-II) is fundamentally distinct from the multi- enzyme FAS-I complex found in mammals. This combination results in a molecular target which is both essential in Mtb as well as sufficiently different from human enzymes to be an attractive target for small molecule drug discovery. InhA is a clinically validated target based on the success of isoniazid (INH) in treating TB patients. 5−7 INH is a pro-drug and is activated by KatG, a catalase−peroxidase enzyme. This enzyme oxidizes INH to an acyl radical which then forms a covalent adduct (INH-NAD) with nicotinamide adenine dinucleotide (NAD). 6 The active drug is the INH-NAD covalent adduct (1) and one of the resistance mechanisms to INH is via a specific mutation in the KatG gene. 6 A number of additional drugs such as ethionamide and propionamide also target InhA via an adduct with cofactors. 8 Additionally, the existence of Mtb clinical isolates that harbor mutations in the inhA structural gene or inhA promoter region that confer resistance to INH have been reported. These observations imply that identifying direct inhibitors of InhA would have tremendous clinical value in combating TB, not least due to the likelihood of being devoid of cross-resistance with current therapies that target InhA via a pro-drug mechanism. 7 Multiple molecular modes of action have been attempted to target InhA. The early success of INH in inhibiting InhA relied on targeting the NAD adduct formation which competes kinetically with the cofactor NADH. A similar mode of action has been attempted with boronate NAD adducts with limited success. 9 Mimicking the molecular mode of action of INH could be achieved either using a new pro-drug or by discovering Received: June 26, 2013 Article pubs.acs.org/jmc © XXXX American Chemical Society A dx.doi.org/10.1021/jm4012033 | J. Med. Chem. XXXX, XXX, XXX−XXX