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Antimicrobial Agents and Chemotherapy, April 2000, p. 1019-1028, Vol. 44, No. 4
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Victoria Hertle Brophy,¹ John Vasquez,² Richard G. Nelson,² John R. Forney,³ Andre Rosowsky,⁴ and Carol Hopkins Sibley^1,*

Department of Genetics, University of Washington, Seattle, Washington 98195-7360¹; Division of Infectious Diseases, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, San Francisco, California 94143-0811²; Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100³; and Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115⁴

Received 10 June 1999/Returned for modification 20 November 1999/Accepted 27 December 1999

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 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 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.

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^* Corresponding author. Mailing address: Department of Genetics, Box 357360, University of Washington, Seattle, WA 98195-7360. Phone: (206) 685-9378. Fax: (206) 543-0754. E-mail: sibley{at}genetics.washington.edu.

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Antimicrobial Agents and Chemotherapy, April 2000, p. 1019-1028, Vol. 44, No. 4
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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