Cryptosporidium Thymidylate Synthase-Dihydrofolate Reductase as Drug Target

Nelson, Richard

Abstract

Cryptosporidium parvum is a coccidian parasite that infects intestinal epithelial cells and caused a serve, life-threatening diarrheal disease in AIDS and other immunocompromised patients. Currently, there is no specific chemotherapy for cryptosporidiosis and effective therapeutic an prophylactic drugs are urgently needed. Although antifolate chemotherapy is an effective treatment for infections caused by related parasites (P. falciparum, T. gondii, I. belii, and Eimeria spp. it is neither curative nor suppressive for cryptosporidiosis. The reason for this is unknown; it may be that the clinically-available antifolates are simply poor inhibitors of the target Cryptosporidium enzymes, that these specific antifolates do not reach the unusual intracellular-extracytoplasmic niche occupied by the parasite or that C. parvum possesses unique salvage pathways that by pass the metabolic blocks imposed by the drugs. The targets of antifolate drugs are dihydropteroate synthase (DHPS) and, protozoa, the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR); their activities are essential for folate biosynthesis and homeostasis and thymidylate biosynthesis. Inhibition of any of these enzymes results in the cessation of thymidylate and DNA synthesis and, therefore, thymineless death. A large number of substrate analogs have been synthesized and tested for inhibition of DHPS, TS and DHFR; many of them are clinically efficacious drugs used in the treatment of infectious diseases (sulfonamides, diaminopyrimidines) and in cancer chemotherapy (nucleoside and folate analogs). The inhibitory potency of these drugs varies dramatically for the DHFRs of different microbial genera and species yet there has been to attempt to access their efficacy in inhibiting the target Cryptosporidium enzyme. This application proposes to biochemically and structurally characterize C. parvum Ts-DHFR as a guide to drug discovery. We have cloned and completely sequenced the C. parvum TS-DHFR gene and have engineered a parent expression constructed for facile transfer of the complete TS-DHFR coding cassette into essentially and expression vector. This investigation proposes to: (1) Express enzymatically active, recombinant TS-DHFR in bacterial, yeast and/or insect cell host-vector systems and biochemically purify the recombinant enzyme. (2) Determine the enzymes' kinetic constants with the natural substrates and quantitate its inhibition by DHFR inhibitors that have failed in empirical clinical attempt to treat cryptosporidiosis. (3) Model the three dimensional structure of the TS-DHFR domains by fitting the predicted amino acid sequences to known crystal structures using computer graphic approaches and use the model structures and computer-assisted drug design technology to suggest lead compounds to assay with the recombinant enzyme and refine through reiteration. (4) Purify sufficient amounts of the recombinant, bifunctional enzyme for crystallization and determination of the atomic structure by X-ray crystallography; employ a model based on the atomic coordinates to design specific inhibitors using computer graphic and computation chemistry approaches.