Optimization of Lead BKIs for Cryptosporidiosis Therapy

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ABSTRACT: Optimization of Lead BKIs for Cryptosporidiosis Therapy The broad, long-term objective of this research is to develop a therapeutic for treatment of Cryptosporidium infection. Cryptosporidium infection causes persistent diarrhea (cryptosporidiosis) that is associated with increased morbidity and mortality in children and immunocompromised individuals. Cryptosporidium is one of the most important pathogens leading to poor outcomes in <2-yo malnourished children in resource poor countries, including >3-fold mortality and strong associations with stunting and impaired neurological development. The only available therapeutic, nitazoxanide, does not work in immunocompromised individuals and has <30% efficacy in malnourished children. New therapeutics for Cryptosporidium are badly needed. We have been developing bumped-kinase inhibitors (BKIs) that selectively target Cryptosporidium calcium- dependent protein kinase 1 (CDPK1) as therapeutics for cryptosporidiosis. In our program, our leads have performed very well, demonstrating efficacy at low oral dosages in mouse, calf, and piglet models of C. parvum and C. hominis, while retaining almost all the favorable safety aspects consistent with a late lead. However, safety issues with BKI leads have stopped us from developing current late leads. We now understand the safety issues associated with late BKI leads and have found these safety issues are not inextricably associated with the structure-activity relationship (SAR) of BKIs’ efficacy against cryptosporidiosis. In this proposal, we will use the efficacy and safety SAR to help drive medicinal chemistry towards a late pre-clinical lead with safety, pharmacokinetic, and efficacy properties that can be developed for treatment in the target population of <2-yo malnourished children and immunocompromised individuals. The Aims are: AIM 1) Establish late leads for cryptosporidiosis therapy, including Subaims 1A) In vitro efficacy and safety testing, 1B) IFN- γ -KO mouse nLuc-C. parvum efficacy testing, 1C) Mouse multidosing, 7d safety testing, 1D) Determine metabolites of BKIs, and, 1E) Design and synthesize new BKIs; AIM 2) Test novel late leads in calf clinical model for cryptosporidiosis; and, AIM 3) Assess advanced late leads for late safety testing, including subaims 3A) Bone safety testing, 3B) Pregnancy/developmental/fetal safety testing; 3C) Rat and dog cardiovascular (CV) safety testing, and, 3D) Pre-GLP safety and polymorph testing. At the end of the grant period, we expect to have a preclinical lead and at least one back up molecule that is ready to move into GLP safety testing, pre-GMP manufacturing scale up, and IND filing to move towards human trials for a BKI for therapy of cryptosporidiosis.