Pharmacological Assessment Defines Leishmania donovani Casein Kinase 1 as a Drug Target and Reveals Important Functions in Parasite Viability and Intracellular Infection

Abstract

ABSTRACTProtein kinase inhibitors have emerged as new drugs in various therapeutic areas, including leishmaniasis, an important parasitic disease. Members of theLeishmaniacasein kinase 1 (CK1) family represent promising therapeutic targets.Leishmaniacasein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins, thus exerting a potential immune-suppressive action on infected host cells. Moreover, its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as a therapeutic target in the disease-relevant amastigote stage remain to be established. In this study, we used a multidisciplinary approach combining bioinformatics, biochemical, and pharmacological analyses with a macrophage infection assay to characterize and defineLeishmaniaCK1.2 as a valid drug target. We show that recombinant and transgenicLeishmaniaCK1.2 (i) can phosphorylate CK1-specific substrates, (ii) is sensitive to temperature, and (iii) is susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect stage and the vertebrate amastigote stage. We further demonstrated that reduction of CK1 activity by specific inhibitors, such as D4476, blocks promastigote growth, strongly compromises axenic amastigote viability, and decreases the number of intracellularLeishmania donovaniandL. amazonensisamastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles compared to those of mammalian CK1 kinases opens new opportunities forLeishmaniaCK1.2 antileishmanial drug development. Our report provides the first chemical validation ofLeishmaniaCK1 protein kinases, required for amastigote intracellular survival, as therapeutic targets.