Identification of Cryptosporidium parvum Dihydrofolate Reductase Inhibitors by Complementation in Saccharomyces cerevisiae

There is a pressing need for drugs effective against the opportunistic protozoan pathogenCryptosporidium parvum . Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested againstC. parvum . To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors ofC. parvum DHFR using the model eukaryote,Saccharomyces cerevisiae . We expressed the DHFR genes ofC. parvum ,Plasmodium falciparum ,Toxoplasma gondii ,Pneumocystis carinii , and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on theC. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinantC. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressingC. parvum DHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by theC. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis.