| One third of the world's population is infected with M. tuberculosis, the causative agent of tuberculosis (TB) that claims the life of approximately 2 million people every year. The rapid spread of multidrug-resistance tuberculosis as well the emergence of extensively drug resistance strains that are resistant to virtually any known antibiotic, has transformed this once curable disease into a major public health challenge. To regain control of this disease and the associated mortality and morbidity new antibiotics are urgently needed.;Pyrazinamide is a frontline drug in the treatment of tuberculosis with unique sterilizing properties. Even though, pyrazinamide monotherapy leads to rapid resistance, the combination of pyrazinamide and isoniazid has reduced the period of chemotherapy necessary for a durable cure from 9 to 6 months. Although pyrazinamide has been used clinically for over 50 years the mechanism of action of this drug is poorly understood.;From work in our laboratory, it has been shown that pyrazinamide, the pharmacologically active metabolite pyrazinoic acid as well as other pyrazinamide derivatives inhibit M. tuberculosis fatty acid synthase I (FAS I). We propose that like pyrazinamide, pyrazinamide analogs that target specific enzymatic processes of FAS I are potential anti-mycobacterial agents. Herein, the characterization of the binding and inhibition mechanisms of pyrazinamide and analogs of this compound was investigated.;FAS I was isolated and purified from a recombinant M. smegmatis construct where the native fas1 gene was replaced by the M. tuberculosis fas1 gene. Saturation Transfer Difference NMR spectroscopy (STD-NMR) was used to definitively establish the interaction of pyrazinamide and related compounds with FAS I as well as to study the structural requirements of effective FAS I inhibition.;The present study unequivocally establishes that PZA as well as PZA analogs target Mtb FAS I. Furthermore, it was found that PZA and 5-Cl-PZA bind at the protein NADPH binding site in a reversible manner. Since Mtb FAS I contains two distinct catalytic sites that utilize NADPH as a reductant namely, the beta-ketoacyl reductase and the beta-enoyl reductase, it is proposed that PZA and PZA analogs target either one of these two catalytic domains. |
