ABSTRACT
Analogs of pyrazinamide (=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.
Subject(s)
Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Bacterial Proteins/metabolism , Fatty Acid Synthases/metabolism , Mycobacterium tuberculosis/enzymology , NADP/metabolism , Pyrazinamide/analogs & derivatives , Pyrazinamide/pharmacology , Bacterial Proteins/antagonists & inhibitors , Fatty Acid Synthases/antagonists & inhibitors , Humans , Mycobacterium tuberculosis/drug effects , Protein Binding/drug effects , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Fatty Acid Synthases/antagonists & inhibitors , Mycobacterium tuberculosis/enzymology , Pyrazinamide/pharmacology , Binding Sites/drug effects , Binding, Competitive/drug effects , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , NADP/chemistry , NADP/pharmacology , Pyrazinamide/analogs & derivatives , Pyrazinamide/chemistry , Stereoisomerism , Structure-Activity RelationshipABSTRACT
An analog of pyrazinamide (PZA), 5-chloropyrazinamide (5-Cl-PZA), has previously been shown to inhibit mycobacterial fatty acid synthase I (FASI). FASI has been purified from a recombinant strain of M. smegmatis (M. smegmatis Deltafas1 attB::M. tuberculosis fas1). Following purification, FASI activity and inhibition were assessed spectrophotometrically by monitoring NADPH oxidation. The observed inhibition was both concentration and structure dependent, being affected by both substitution at the 5 position of the pyrazine nucleus and the nature of the ester or N-alkyl group. Under the conditions studied, both 5-Cl-PZA and PZA exhibited concentration and substrate dependence consistent with competitive inhibition of FASI with K(i)s of 55 to 59 microM and 2,567 to 2,627 microM, respectively. The results were validated utilizing a radiolabeled fatty acid synthesis assay. This assay showed that FASI was inhibited by PZA and pyrazinoic acid as well as by a series of PZA analogs.