Identification of Cryptosporidium parvum dihydrofolate reductase inhibitors by complementation in Saccharomyces cerevisiae

There is a pressing need for drugs effective against the opportunistic prot ozoan pathogen Cryptosporidium parvum. Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), hav e been widely exploited as chemotherapeutic targets. Although many DHFR inh ibitors have been synthesized, only a few have been tested against C. parvu m. To expedite and facilitate the discovery of effective anti-Cryptosporidi um antifolates, we have developed a rapid and facile method to screen poten tial inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae. We expressed the DHFR genes of C. parvum, Plasmodium falciparum , Toxoplasma gondii, Pneumocystis carinii, and humans in the same DHFR-defi cient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the compleme ntation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzy me. These same compounds were also potent or selective inhibitors of the pu rified recombinant C. parvum DHFR enzyme. Six novel lipophilic DHFR inhibit ors potently inhibited the growth of yeast expressing C. parvum DHFR Howeve r, the inhibition was nonselective, as these compounds also strongly inhibi ted the growth of yeast dependent on the human enzyme. Conversely, the anti bacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. par vum enzyme. Future chemical refinement of the potent and selective lead com pounds identified in this study may allow the design of an efficacious anti folate drug for the treatment of cryptosporidiosis.