A slow, tight-binding inhibitor of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis

InhA, the enoyl-ACP reductase in Mycobacterium tuberculosis is an attractive target for the development of novel drugs against tuberculosis, a disease that kills more than two million people each year. InhA is the target of the current first line drug isoniazid (INH) for the treatment of tuberculosis infections. Compounds that directly target InhA and do not require activation by the mycobacterial catalase-peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. Previously we reported the synthesis of several diphenyl ethers with nM affinity for InhA. However, these compounds are rapid reversible inhibitors of the enzyme, and based on the knowledge that long drug-target residence times are an important factor for in vivo drug activity, we set out to generate a slow onset inhibitor of InhA using structure based drug design. 2-(oTolyloxy)-5-hexylphenol (PT70) is a slow, tight binding inhibitor of InhA with a K1 value of 22 pM. PT70 binds preferentially to the InhA-NAD + complex and has a residence time of 24 minutes on the target, which is 14,000 times longer than that of the rapid-reversible inhibitor from which it is derived. The 1.8 Å crystal structure of the ternary complex between InhA, NAD + and PT70 reveals the molecular details of enzyme-inhibitor recognition and supports the hypothesis that slow onset inhibition is coupled to ordering of an active site loop, which leads to the closure of the substrate binding pocket. Mycobacterium tuberculosis is the causative agent of tuberculosis, an infectious disease that is spread all over the world. Two billion people, one third of the world’s population, are infected with tuberculosis and M. tuberculosis is responsible for 8.8 million new infections and 1.6 million deaths each year (1). The emergence of multi-drug resistant M. tuberculosis strains, that are resistant against the current frontline drugs isoniazid and rifampicin, contribute to the spread and worsen the situation by lengthening the treatment considerably from 6 months to nearly two years and thereby increasing the cost for therapy 20 fold. Extensively drug resistant strains, that are almost untreatable with current chemotherapeutics, threaten both developing and industrialized countries (2). Novel drugs with activity against drug resistant strains are therefore urgently needed to restrain the disease that was once thought to be under control. One of the most effective and widely used drugs for the treatment of tuberculosis is isoniazid (INH). INH is a prodrug that has to be activated by KatG, the mycobacterial catalase-peroxidase, to form together with NAD(H) the INH-NAD adduct (3). This adduct is a slow onset inhibitor of InhA, the enoyl-ACP reductase of the mycobacterial type II fatty acid biosynthesis pathway (4,5). In this pathway, very long chain fatty acids are generated that act as precursors for mycolic acids, which in turn are essential building blocks of the waxy cell http://www.jbc.org/cgi/doi/10.1074/jbc.M109.090373 The latest version is at JBC Papers in Press. Published on March 3, 2010 as Manuscript M109.090373