Structural Analysis and Inhibitor Optimization of Cryptosporidium N-myristoyltransferase For Drug Discovery

Abstract Each year over 525,000 children under age five are killed by diarrhea caused by infectiousdisease. Cryptosporidiosis, the second most frequent cause of childhood diarrhea, is an infectioncaused by colonization of the intestines by the eukaryotic parasites, Cryptosporidium parvum orC. hominis, and particularly damages and kills malnourished children. Despite the high incidenceand significant impact on malnourished children, there are no effective preventive or therapeutictreatments for cryptosporidiosis in this population. N-myristoyltransferase (NMT) is an enzyme which transfers myristate, a 14-carbon fattyacid, to the N-terminus of glycyl-peptides and contributes to targeting the substrate protein tomembrane regions. NMT has been validated as a drug target in fungal and parasitic diseases,including malaria and leishmaniasis. Our collaborators previously screened the GSK Tres Cantosproprietary library of 3 million compounds against P. falciparum NMT and identified 8 uniqueclusters of compounds with anti NMT activity. We hypothesized that NMT inhibitors effectiveagainst Plasmodium would also be active against Cryptosporidium. To test this hypothesis, wescreened representative compounds from each cluster for their ability to inhibit CryptosporidiumNMT (CpNMT) activity (using an in vitro enzymatic assay) and parasite growth (using a cell cultureassay). We have identified three different scaffolds effective at inhibiting CpNMT, one of whichwas also effective at preventing replication in cell culture. Based on our preliminary results, we hypothesize that inhibitors targeting CpNMT willresult in parasite clearance in animal models of infection. We propose to test our hypothesisby examining our best hit compounds in a mouse model of infection. We will first expand ourlibrary of three hit scaffolds by synthesizing and acquiring structurally related compounds using astructure-guided approach. We will progress the compounds through a screening cascadecomposed of in vitro assays to characterize the compounds and determine structure activityrelationship (SAR) profiles of the hit scaffolds. Select compounds will then be tested in an animalmodel of infection to determine if the NMT inhibitors are active in vivo. In parallel, we will developstructural biology tools for determining the mode of binding and crystal structure of boundcompounds to the CpNMT enzyme.