Globally, there are nearly 1.7 billion cases of diarrheal disease each year. Diarrheal diseases are a leading cause of malnutrition and death in children under five years old, causing 1 in 9 child deaths worldwide – more than malaria and AIDS combined. A 2013 study revealed cryptosporidiosis as a leading cause of non-viral diarrhea in young children, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates immune-dependent efficacy. Given the unmet medical need for a treatment with universal efficacy, we initiated a drug discovery effort to identify novel anti-cryptosporidial small molecule inhibitors. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized to 1536-well format and automated to enable high-throughput phenotypic screening against large, diverse libraries of small molecule compounds. A screen of more than 1.2M compounds identified nearly 400 anti-cryptosporidial hits with sub-micromolar activity. Formal hit assessment included counter-screens against Cryptosporidium hominis and mammalian cell lines to further establish compound specificity and selectivity. Subsequent hit-to-lead candidates were advanced for lead optimization following validation of efficacy in an INFγ-/- mouse model of acute cryptosporidiosis. From these efforts, two chemical series representing novel lead compounds were selected for medicinal chemistry optimization to improve efficacy and address liabilities to achieve the criteria set forth in the target product profile. Additional lead compounds have also been identified in the ReFRAME library, including clofazimine, a leprosy drug currently under investigation as a repurposed drug for cryptosporidiosis in a Phase 2A clinical trial in Malawi.

From phenotypic screening to lead optimization: Calibr cryptosporidiosis drug discovery

2019 Fall National Meeting

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