The resurgence of tuberculosis (TB) coupled with the emergence of multi-drug resistant strains (MDR-TB) necessitates that novel therapies be developed. One strategy for the discovery of novel drugs is to identify the targets of effective agents. Pyrazinamide (PZA), one such agent, has a unique sterilizing activity. It is the inclusion of PZA with isoniazid and rifampin in current treatment regimens that constitutes the basis for 6-month short course therapy for M. tuberculosis (Mtb). PZA efficacy depends upon conversion of PZA to pyrazinoic acid, (POA) the active agent. The lack of pyrazinamidase, an enzyme that converts PZA to POA, confers resistance to PZA. PZA is effective only against Mtb among the mycobacterial species, and then is effective only at an acidic pH. Although the mechanism of PZA activation and resistance is known the precise mycobacterial function that is inhibited remains unknown. Using 5-Cl-PZA as a selective agent for mycobacteria, we have found that 5-Cl-PZA and PZA inhibit fatty acid synthetase I FASI in Mtb. FASI has been purified from M. smegmatis 2700 mc2, a recombinant strain where the native fas 1 gene has been deleted and replaced with Mtb fas 1 gene. Following purification, FASI enzymatic activity was measured using a spectrometric assay which monitors NADPH oxidation. Both 5-Cl-PZA and PZA showed concentration and substrate dependence consistent with competitive inhibition of FASI. These results were validated by a radiolabeled fatty acid assay. Preliminary studies have shown improved activity of PZA analogs against Mtb in infected human monocyte-derived macrophages and in murine models of infection.
A number of substituted pyrazinoic acid esters (POE) have been prepared providing considerable insight into the effect of structure on activity. These modifications have been very successful in expanding the activity of pyrazinamide to include M. avium and M. kansasii, organisms normally not susceptible to PZA. Several of these compounds (2'-octyl 5-chloropyrazinoate, n-octyl 5-chloropyrazinoate and n-propyl 5-chloropyrazinoate) have activities 100 to 1000 fold greater than that of pyrazinamide against M. tuberculosis. To afford a better comparison between the substituent effects of the POE and PZA, a number of substituted PZA analogs have been prepared (Table 1). Interestingly, some of these compounds not only show an expanded spectrum of antimycobacterial activity, e.g., they are active against M. avium, but they are also active against the PZA-resistant strain of M. tuberculosis, ATCC 35828. Particularly, remarkable was the activity of 5-chloropyrazinamide against the PZA-resistant organisms. Comparison of the activity of 5-chloropyrazinamide, PZA, POA and 5-chloropyrazinoic acid, revealed that 5-chloropyrazinamide is considerably more active than PZA against a number of different isolates. Most significantly the lower activity of 5-chloropyrazinoic acid relative to POA suggests that the activity of the 5-chloropyrazinamide against PZA resistant organisms may not be related to an increase in the potency of the acid formed on amidase action.
This work is collaborative with M. H. Cynamon, VA Syracuse, NY, O. Zimhony Kaplan Medical Center, The Hebrew University , Rehovot Israel, W. R. Jacobs, Albert Einstein College of Medicine, Bronx, NY and A. Shekhtman of the Chemistry Department, University at Albany.