Amalgamating the tools of molecular biology, biochemistry, cell biology, genetics, structural biology, and pharmacology, this proposal offers an interdisciplinary dissection of the purine salvage pathway of Cryptosporidium parvum, a protozoan parasite that is an opportunistic pathogen of people with AIDS and an NIAID Biodefense Research Category B Priority Pathogen. C. parvum is the etiologic agent of cryptosporidiosis, a debilitating and potentially fatal disease for which no consistently effective chemotherapies exist, especially for immunocompromised individuals. Thus, more efficacious drugs, particularly those that target unique features in C. parvum, are urgently needed. Among the more striking metabolic discrepancies between C. parvum and its human host are the pathways for purine acquisition. C. parvum, unlike mammals, cannot synthesize purines de novo and is dependent upon a unique purine salvage mechanism to fulfill its nutritional needs. Inhibition of purine salvage, therefore, presents a selective therapeutic paradigm for treating C. parvum infections. In this proposal, we will investigate three imperative components of purine salvage;adenosine kinase (CpAK), adenylate deaminase (CpAMPDA), and guanylate synthetase (CpGMPS). There are two Specific Aims. Multi-component Specific Aim I delineates a detailed biochemical, biological, and functional characterization of all three enzymes and a structural analysis of CpAK. We will clone all three genes and functionally evaluate their gene products through biochemical studies of their encoded products and by rescue or complementation of genetic lesions in other organisms. The expression patterns of the CpAK, CpAMPDA, and CpGMPS genes and the levels and location of the three proteins in intact C. parvum parasites will be ascertained throughout biologically accessible life cycle stages. Finally, structure-function studies based upon a molecular model of CpAK will test the roles of key residues on CpAK that are projected to be involved in the catalytic mechanism, and crystallographic studies on CpAK will be initiated with the ultimate intention of determining its three-dimensional structure. The second Specific Aim has two parts. First, we will perform a structure-activity relationship analysis of CpAK against a battery of available nucleoside analogs. Second, we will optimize and implement a high throughput screen of a structurally diverse chemical repository using an E. coli-based assay with the purpose of discovering novel inhibitors of CpAK that could be potentially used for treating the parasitic disease. The "hits" from the screen will be further evaluated using target-based and cell-based assays and eventually in a mouse model that mimics the human disease. Overall, the proposed investigations constitute a logical step in the validation of these enzymes as potential therapeutic targets and in the implementation of a rational strategy of drug discovery for treatment of cryptosporidiosis.
The overall purpose of this proposal is to investigate components of the purine salvage pathway of Cryptosporidium parvum, a waterborne parasite that causes a devastating opportunistic infection in people with AIDS and is also a Category B priority pathogen of the National Institute of Allergy and Infectious Disease because the organism can be maliciously manipulated as a biological weapon. There is currently no vaccine and no consistently effective chemotherapy, so there is an urgent need for new drugs and new drug targets. Because purine salvage is essential for Cryptosporidium parvum and not for humans, our studies address the vital issue of validating new targets and discovering potentially novel drugs.