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

Abstract

There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium 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 against C. parvum. To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential 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 DHFRdeficient 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 the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinant C. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. 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 the C. 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.

Efficacies of Lipophilic Inhibitors of Dihydrofolate Reductase against Parasitic Protozoa

“…The DHFR domains from two strains of C. parvum were used, one derived from an infected human (hCp-yeast) and the other from a bovine infection (bCp-yeast) (46). Although the two differ at nine positions, none of these differences occur in amino acids that would be expected to cause changes in drug sensitivity; we have detected no differences in this study or in a previous one (4,46). A set of yeast strains that expressed the DHFR domain from P. falciparum was also constructed (48).…”

“…The three compounds (compounds 36, 63, and 69) displayed in Fig. 5 4. Summary of patterns of drug activity observed on radial assay.…”

“…The radial assay used here has been described in detail by Sibley et al (44) and Brophy et al (4). Briefly, drugs were dissolved in dimethyl sulfoxide (DMSO) at 10 Ϫ2 M and stored at Ϫ70°C until use.…”

“…DHFR inhibitors function principally as competitive inhibitors of the enzyme. We have shown that the sensitivity of our engineered yeast strains to DHFR inhibitors depends on the interaction of the drug and the heterologous DHFR enzyme (4,44,48). For example, point mutations within the coding region of the P. falciparum DHFR gene can render the enzyme resistant to pyrimethamine.…”

“…Function of the endogenous dfr1 gene was eliminated from the yeast (15), and the defect was complemented by expression of a heterologous DHFR gene from P. falciparum, T. gondii, Pneumocystis carinii, Cryptosporidium parvum, or humans (4). DHFR inhibitors function principally as competitive inhibitors of the enzyme.…”