LAUSR

Significance Malaria and cryptosporidiosis are major burdens to both global health and economic development in many countries. Malaria caused >400,000 deaths in 2017, and cryptosporidiosis is estimated to cause >200,000 deaths a year. The spread of drug resistance is a growing concern for malaria treatment, and there is no effective treatment for malnourished or immunocompromised children infected with Cryptosporidium. New treatments with novel mechanisms of action are needed for both diseases. We present a selective inhibitor of both Plasmodium and Cryptosporidium lysyl-tRNA synthetase capable of clearing parasites from mouse models of malaria and cryptosporidiosis infection. This provides very strong validation of lysyl-tRNA synthetase as a drug target in these organisms and a lead for further drug discovery. Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.