Versatile Imidazole Scaffold with Potent Activity against Multiple Apicomplexan Parasites

Malaria, toxoplasmosis, and cryptosporidiosis are caused by apicomplexan parasites <i>Plasmodium</i> spp., <i>Toxoplasma gondii</i>, and <i>Cryptosporidium parvum</i>, respectively, and pose major health challenges. Their therapies are inadequate, ineffective or threatened by drug resistance. The development of novel drugs against them requires innovative and resource-efficient strategies. We exploited the kinome conservation of these parasites to determine the cellular targets and effects of two <i>Plasmodium falciparum</i> inhibitors in <i>T. gondii</i> and <i>C. parvum</i>. The imidazoles, (<i>R</i>)-RY-1-165 and (<i>R</i>)-RY-1-185, were developed to target the cGMP dependent protein kinase of <i>P. falciparum</i> (PfPKG), orthologs of which are present in <i>T. gondii</i> and <i>C. parvum</i>. Using structural and modeling approaches we determined that the molecules bind stereospecifically and interact with PfPKG in a manner unique among described inhibitors. We used enzymatic assays and mutant <i>P. falciparum</i> expressing PfPKG with a substituted "gatekeeper" residue to determine that cellular activity of the molecules is mediated through targets additional to PfPKG. These likely include <i>P. falciparum</i> calcium dependent protein kinase 1 and 4 (PfCDPK-1, -4), kinases that, like PfPKG, have small amino acids at the "gatekeeper" position. The molecules are active against <i>T. gondii</i> and <i>C. parvum</i>, with <i>T. gondii</i> tachyzoites being particularly sensitive. Using mutant parasites, enzyme assays and modeling studies we demonstrate that targets in <i>T. gondii</i> include TgPKG, TgCDPK1, TgCDPK4 and the mitogen activated kinase-like 1 (MAPKL-1). Our results suggest that this scaffold holds promise for the development of new toxoplasmosis drugs.