Inhibition of Isolated Mycobacterium tuberculosis Fatty Acid Synthase I by Pyrazinamide Analogs

Abstract

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 ⌬fas1 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 M and 2,567 to 2,627 M, 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.

Mutually Exclusive Genotypes for Pyrazinamide and 5-Chloropyrazinamide Resistance Reveal a Potential Resistance-Proofing Strategy

“…Strains were grown to mid-log phase (OD 600 , 0.5), cells were harvested by centrifugation (3,450 ϫ g; 10 min; 4°C), resuspended to an OD 600 of 0.2 in 10 ml 7H9 medium (pH 5.1) containing 2.5 mg ml Ϫ1 PZA, and incubated for 2 h at 37°C. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetate (1 Ci ml Ϫ1 ) was added, and the cultures were incubated for an additional 2 h. Cells were harvested by centrifugation (3,450 ϫ g; 10 min; 4°C). Fatty acid preparation and analysis were performed as previously described (28).…”

“…Based on the pK a and acidic-pH dependence of PA, it has been suggested that this weak acid works via a proton ionophore-type mechanism that directly causes uncoupling of the cellular membrane potential (22,25). However, it is important to note that PZA and many of its derivatives, such as n-propyl pyrazinoate, PA, and 5-Cl PZA, inhibit mycobacterial type 1 fatty acid synthase (FAS I) in whole-cell and cell-free assays (14,27). Moreover, synthetic overexpression of mycobacterial FAS I in M. smegmatis confers resistance to 5-Cl PZA (26).…”

The Antituberculosis Drug Pyrazinamide Affects the Course of Cutaneous Leishmaniasis In Vivo and Increases Activation of Macrophages and Dendritic Cells

“…PZA and related analogs have also demonstrated activity against other Mycobacterium spp. (9,21,27). In myco-bacteria, we found that PZA inhibits the enzyme fatty acid synthase I (FASI) (33) by competitive inhibition of a NADPH binding site (25).…”

Pyrazinoic Acid Decreases the Proton Motive Force, Respiratory ATP Synthesis Activity, and Cellular ATP Levels

“…PZA is a sterilizing drug that efficiently kills populations of Mycobacterium tuberculosis residing in acidic environments, as found during active inflammation (1,7,10,11,20,21,28,32). Despite the importance of PZA, no cellular target proteins have been clearly identified (4,23,30,41), and its mechanism of action is probably the least understood among all first-and second-line antituberculosis drugs. A better understanding of PZA action may help in development of new drugs to further shorten tuberculosis treatment.…”