Analogs of the Antituberculous Agent Pyrazinamide Are Competitive Inhibitors of NADPH Binding to M. tuberculosis Fatty Acid Synthase I by Halimah Sayahi a ), Kaitlin M. Pugliese a ), Oren Zimhony b ), William R. Jacobs Jr. c ), Alexander Shekhtman a ), and John T. Welch* a ) a )Departmentof Chemistry, University at Albany, SUNY, 1400 Washington Ave, Albany, NY 12222, USA (phone: þ 1-518-442-4455; fax: þ 1-518-442-3462; e-mail: jwelch@albany.edu) b ) Infectious Diseases Division, Kaplan Medical Center, affiliated to the School of Medicine, Hebrew University and Hadassah Jerusalem, P.O. Box 1, Rehovot 76100, Israel c ) Department of Microbiology and Immunology, Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA Dedicated to Professor Dieter Seebach on the occasion of his 75th birthday Analogs of pyrazinamide ( ¼ pyrazine-2-carboxamide; PZA), an essential component of short- course antituberculous chemotherapy, such as 5-chloropyrazinamide (5-Cl-PZA) act as competitive inhibitors of NADPH binding to purified mycobacterial fatty acid synthase I (FAS I) as shown by Saturation Transfer Difference (STD) NMR studies. In addition, pyrazinoic acid esters (POE) and 5-Cl- POE reversibly bind to FAS I with the relatively greater affinity of longer-chain esters for FAS I, clear from the STD amplification factors. The competitive binding of PZA and 5-Cl-PZA clearly illustrates that both agents bind FAS. In contrast to PZA, at low NADPH concentrations 5-Cl-PZA is a cooperative inhibitor of NADPH binding. Introduction. – Pyrazinamide ( ¼ pyrazine-2-carboxamide; PZA), a nicotinamide analog, is an essential component of short-course antituberculous chemotherapy [1]. First synthesized in 1936, the anti-tuberculosis properties of PZA were recognized only in 1952 [2] . Even though PZA is central to short-course tuberculosis chemotherapy [ 3] , the mechanism of action of the drug remains poorly understood. Recently, we reported that PZA is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis Fatty Acid Synthase I ( Mtb FAS I) [4]. At the same time, we have also found that pyrazinoic acid ( ¼ pyrazine-2-carboxylic acid; POA), the metabolite of PZA that accumulates intracellularly, binds to FAS I albeit at a different site from that of PZA and NADPH. These findings prompted an investigation of the FAS I binding of the significantly more potent antimycobacterial agents derived from 5-halo-substituted pyrazinoic acid or from PZA derivatives where the amide moiety was replaced with an ester. In culture, PZA is active against Mycobacterium tuberculosis ( Mtb) only at pH values < 6 [5]. Mtb takes up PZA by passive diffusion, whereupon a pyrazinamidase ( pncA) can convert PZA to POA [6] . The fate of POA, as well as the exact biochemical function inhibited by POA, is controversial. Intracellular accumulation of POA under acidic pH has been suggested to disrupt cellular membrane energetics [3] [7]. Other mechanisms have been proposed [7b] this includes the influence of PZA on the action CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 2582  2012 Verlag Helvetica Chimica Acta AG, Zürich