Novel Saccharomyces cerevisiae Screen Identifies WR99210 Analogues That Inhibit Mycobacterium tuberculosis Dihydrofolate Reductase

Abstract

The ongoing selection of multidrug-resistant strains of Mycobacterium tuberculosis has markedly reduced the effectiveness of the standard treatment regimens. Thus, there is an urgent need for new drugs that are potent inhibitors of M. tuberculosis, that exhibit favorable resistance profiles, and that are well tolerated by patients. One promising drug target for treatment of mycobacterial infections is dihydrofolate reductase (DHFR; EC 1.5.1.3), a key enzyme in folate utilization. DHFR is an important drug target in many pathogens, but it has not been exploited in the search for drugs effective against M. tuberculosis. The triazine DHFR inhibitor WR99210 has been shown to be effective against other mycobacteria. We show here that WR99210 is also a potent inhibitor of M. tuberculosis and Mycobacterium bovis BCG growth in vitro and that resistance to WR99210 occurred less frequently than resistance to either rifampin or isoniazid. Screening of drugs with M. tuberculosis cultures is slow and requires biosafety level 3 facilities and procedures. We have developed an alternative strategy: initial screening in an engineered strain of the budding yeast Saccharomyces cerevisiae that is dependent on the M. tuberculosis DHFR for its growth. Using this system, we have screened 19 compounds related to WR99210 and found that 7 of these related compounds are also potent inhibitors of the M. tuberculosis DHFR. These studies suggest that compounds of this class are excellent potential leads for further development of drugs effective against M. tuberculosis.The ongoing selection of multidrug-resistant strains of Mycobacterium tuberculosis has markedly reduced the effectiveness of the standard treatment regimens (27). Thus, there is an urgent need for new drugs that are potent inhibitors of M. tuberculosis, that exhibit favorable resistance profiles, and that are well tolerated by patients. One promising drug target for treatment of mycobacterial infections is dihydrofolate reductase (DHFR; EC 1.5.1.3), a key enzyme in folate utilization that catalyzes the reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate is necessary for the one-carbon transfer reactions used in the biosynthesis of nucleic and amino acids including thymidylate, adenine, methionine, glycine, and histidine. DHFR inhibitors act by halting the synthesis of these DNA, RNA, and protein subunits, thereby arresting cell growth. Specific competitive inhibitors of DHFR have been used for more than 50 years in combination with sulfa and sulfone compounds as inexpensive effective inhibitors of bacterial and parasitic infections (13, 32). DHFR has distinct advantages as a potential drug target. First, the biochemistry of the folate pathway and the DHFR enzyme are well characterized, and the crystal structures of 10 DHFR enzymes have been solved. Second, this long history means that the safety and selectivity of these inhibitors have been intensively studied (38) and compounds with a higher degree of selectivity for the microbial enzymes than for the human enzym...

Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

“…However, ThyA requires concomitant dihydrofolate reductase to regenerate tetrahydrofolate, so a combination of ThyX and DHFR inhibition might limit M. tuberculosis growth. Promising inhibitors of M. tuberculosis DHFR have been identified previously (7,8). Our detailed determination of the function of key residues in the ThyX enzyme should facilitate further study of this potential drug target and will help to better understand the complex reactions of this interesting enzyme.…”

Heterologous Expression of Mycobacterial Proteins in Saccharomyces cerevisiae Reveals Two Physiologically Functional 3-Hydroxyacyl-Thioester Dehydratases, HtdX and HtdY, in Addition to HadABC and HtdZ

“…The yeast Saccharomyces cerevisiae is a convenient model system for investigating the physiological function of mycobacterial proteins (15), since this organism undertakes mitochondrial fatty acid biosynthesis in a FASII-dependent manner (27). The equivalent HAD in yeast is represented by the aforementioned Htd2p (30).…”

Antitubercular Drugs Development: Recent Advances in Selected Therapeutic Targets and Rational Drug Design

“…While bacteria synthesize folate de novo, mammals must assimilate preformed folate derivatives through an active transport system (Mdluli & Spigelman, 2006). Dihydrofolate reductase, which catalyses the reduction of dihydrofolate to tetrahydrofolate, a key enzyme in folate utilization whose inhibition may affect the growth of M. tuberculosis (Gerum et al, 2002), and dehydropteroate synthase are validated targets of the widely used antibacterial sulfonamide, trimethoprim (Huovinen et al, 1995). Two enzymes involved in the de novo biosynthesis of NAD that affects the NADH/NAD + ratio upon which M. tuberculosis is dependent, have been studied as possible drug targets (Bellinzoni et al, 2002).…”