No abstract

Competitive inhibitors of dihydrofolate reductase (DHFR) are used in chemotherapy or prophylaxis of many microbial pathogens, including the eukaryotic parasites Plasmodium falciparum and Toxoplasma gondii. Unfortunately, point mutations in the DHFR gene can confer resistance to inhibitors specific to these pathogens. We have developed a rapid system for testing inhibitors of DHFRs from a variety of parasites. We replaced the DHFR gene from the budding yeast Saccharomyces cerevisiae with the DHFR-coding region from humans, P. falciparum, T. gondii, Pneumocystis carinii, and bovine or human-derived Cryptosporidium parvum. We studied 84 dicyclic and tricyclic 2,4-diaminopyrimidine derivatives in this heterologous system and identified those most effective against the DHFR enzymes from each of the pathogens. Among these compounds, six tetrahydroquinazolines were effective inhibitors of every strain tested, but they also inhibited the human DHFR and were not selective for the parasites. However, two quinazolines and four tetrahydroquinazolines were both potent and selective inhibitors of the P. falciparum DHFR. These compounds show promise for development as antimalarial drugs.The treatment of diseases caused by eukaryotic pathogens is particularly difficult because of the similarity between their cell biology and that of their human host. The selection for pathogens resistant to currently effective drugs and the increase in immunocompromised individuals have added urgency to the search for new therapies directed specifically against these pathogens. One fruitful avenue for identification of chemotherapeutic drugs is to screen compounds that have already been synthesized in order to identify those that might be active against these increasingly important pathogens. We have adopted this strategy and screened a large library of compounds that are directed against the enzyme dihydrofolate reductase (DHFR) (EC 1.5.1.3). DHFR is a central enzyme in nucleic acid and amino acid synthesis in all cells, but the active sites of enzymes from different organisms show subtle differences that allow the identification of inhibitors specific for a particular species (3,(16)(17)(18)24). For example, pyrimethamine is a selective inhibitor that is effective in the nanomolar range against the DHFRs from Plasmodium falciparum and Toxoplasma gondii, but the human enzyme is relatively insensitive to the drug (8,14,24). Thus, pyrimethamine has been used in malaria and toxoplasmosis therapy for many years (9, 49).We have designed an easy and inexpensive system to test in budding yeast (Saccharomyces cerevisiae) potential DHFR inhibitors against the enzymes from a variety of parasites. 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. We have shown that the sensitivity of our engi...

No abstract