Each year over 525,000 children under age five are killed by diarrhea caused by infectious disease. Cryptosporidiosis, the second most frequent cause of childhood diarrhea, is an infection caused by colonization of the intestines by the eukaryotic parasites, Cryptosporidium parvum or C. hominis, and particularly damages and kills malnourished children. Despite the high incidence and significant impact on malnourished children, there are no effective preventive or therapeutic treatments for cryptosporidiosis in this population. N-myristoyltransferase (NMT) is an enzyme which transfers myristate, a 14-carbon fatty acid, to the N-terminus of glycyl-peptides and contributes to targeting the substrate protein to membrane 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 Cantos proprietary library of 3 million compounds against P. falciparum NMT and identified 8 unique clusters of compounds with anti NMT activity. We hypothesized that NMT inhibitors effective against Plasmodium would also be active against Cryptosporidium. To test this hypothesis, we screened representative compounds from each cluster for their ability to inhibit Cryptosporidium NMT (CpNMT) activity (using an in vitro enzymatic assay) and parasite growth (using a cell culture assay). We have identified three different scaffolds effective at inhibiting CpNMT, one of which was also effective at preventing replication in cell culture. Based on our preliminary results, we hypothesize that inhibitors targeting CpNMT will result in parasite clearance in animal models of infection. We propose to test our hypothesis by examining our best hit compounds in a mouse model of infection. We will first expand our library of three hit scaffolds by synthesizing and acquiring structurally related compounds using a structure-guided approach. We will progress the compounds through a screening cascade composed of in vitro assays to characterize the compounds and determine structure activity relationship (SAR) profiles of the hit scaffolds. Select compounds will then be tested in an animal model of infection to determine if the NMT inhibitors are active in vivo. In parallel, we will develop structural biology tools for determining the mode of binding and crystal structure of bound compounds to the CpNMT enzyme.
The investigators have identified preliminary evidence that a new series of compounds may be effective for treatment of cryptosporidiosis, a neglected diarrheal disease. The project will conduct hit validation and characterization studies using in vitro and in vivo assays. Synthetic chemistry will be guided by structure-based rational design and final compounds will be tested for proof-of-concept in an animal model.
