Development of dual effective kinase inhibitors as syndromic treatment of Giardiasis and Cryptosporidiosis - Project Summary/Abstract The potential for devastating consequences of cryptosporidiosis or giardiasis diarrhea in immunocompromised patients and malnourished children emphasizes the need for a single effective therapy that could be used syndromically where diagnosis may be delayed or uncertain. This will also be applicable for asymptomatic presentations of both diseases. The etiologic parasites colonize and reproduce in the small intestines of mammalian hosts where they are associated with epithelial cells microvilli. Hence, potential for finding dual therapeutic is high based on common anatomical site of infection. Protein kinase inhibitors have attracted considerable attention as potential therapeutics since a number of them have been released as drugs in recent years and many are in various phases of clinical trials. In a semi-High throughput screening of MMV Pathogen box and Celgene Global Health programs library, we identified dual hitting kinase inhibitors from 2 chemical scaffolds. The scaffolds offer new medicinal chemistry opportunities for development of effective multi-parasite therapeutics against cryptosporidiosis and giardiasis. This research proposal capitalizes on these preliminary findings to hypothesize that effective single agent therapy that could be used syndromically for treatment of either cryptosporidiosis or giardiasis is possible. Analogues of the 2 scaffolds already in hand and newly synthesized in Aims 1a will be screened in Aims 2a to determine in vitro efficacy in blocking parasites growth using direct phenotypic screening to develop structural activity relationship. We will also be collecting other standard in vitro ADMET and physicochemical parameters such as solubility, stability in simulated gastric/intestinal fluids, and mouse/human microsome stabilities, in-house hERG assay etc as Go/NoGo filters. Criteria for moving forward to in vivo studies will include dual parasites killing efficiency, selectivity, static vs. cidal, stability in both gastric fluids and gut Phase 1 metabolism, and ease of medicinal chemistry development. Since necessary factors for clinical relevance have not been established in these scaffolds, we will use early leads from Aims 2b to define the PK/PD properties necessary for optimum animal in vivo efficacy in Aim 2b. We will perform further in vitro safety profiling for early leads focused on further improving selectivity including quantitative proteomic analysis of offtarget safety kinases, cytochrome P450 isoenzyme activities and metabolite identification in Aims 1c. Using medicinal chemistry based iterative reasoning to synthesize analogs, we will do lead optimization for efficacy, selectivity, PK/ADMET properties, in vivo efficacy and resistance under Aim 1b. In Aim 3; optimized leads will be evaluated for clinical usefulness with experiments to define any liabilities related to CYP activities, mammalian kinome profile, AMES test, micronucleus assay and a safety panel of human receptors and ion channels. Other experiments will include final toxicology studies, additional resistance studies and metronidazole combination studies. The product of this research study would be useful as parasitic diarrheal therapeutic with the potential for broad spectrum activity. It will have significant public health benefits in many developing regions of the world.
