Apoptotic Marker Expression in the Absence of Cell Death in Staurosporine-Treated Leishmania donovani

Abstract

The protozoan parasite Leishmania donovani undergoes several developmental transitions in its insect and vertebrate hosts that are induced by environmental changes. The roles of protein kinases in these adaptive differentiation steps and their potential as targets for antiparasitic intervention are only poorly characterized. Here, we used the generic protein kinase inhibitor staurosporine to gain insight into how interference with phosphotransferase activities affects the viability, growth, and motility of L. donovani promastigotes in vitro. Unlike the nonkinase drugs miltefosine and amphotericin B, staurosporine strongly reduced parasite biosynthetic activity and had a cytostatic rather than a cytotoxic effect. Despite the induction of a number of classical apoptotic markers, including caspase-like activity and surface binding of annexin V, we determined that, on the basis of cellular integrity, staurosporine did not cause cell death but caused cell cycle arrest and abrogated parasite motility. In contrast, targeted inhibition of the parasite casein kinase 1 (CK1) protein family by use of the CK1-specific inhibitor D4476 resulted in cell death. Thus, pleiotropic inhibition of L. donovani protein kinases and possibly other ATP-binding proteins by staurosporine dissociates apoptotic marker expression from cell death, which underscores the relevance of specific rather than broad kinase inhibitors for antiparasitic drug development. Leishmaniasis is an important parasitic disease affecting over 12 million people worldwide and causing a variety of pathologies, ranging from self-healing cutaneous lesions to fatal visceral infection causing hepatosplenomegaly (http://apps.who.int/tdr/svc /diseases/leishmaniasis). Treatments available for visceral leishmaniasis include pentavalent antimony (Sb V ) compounds as firstline drugs and pentamidine and amphotericin B (AmpB) as second-line drugs, the uses of which are limited by toxicity and availability. In addition, the clinical value of antimony therapy is threatened by the emergence of drug resistance. Recently, miltefosine (hexadecylphosphocholine [HePC]), an alkylphosphocholine originally developed as an anticancer drug, was proven to be effective and safe for use against visceral leishmaniasis in India (1) and was successfully applied to treat patients infected with antimony-resistant parasites. However, the therapeutic window of this drug might be very short, given the appearance of drug resistance in vitro (2). Thus, in the absence of vaccination and given the limitations of current therapies in cost, efficacy, and safety, there is an urgent need for the identification of novel targets and new chemical entities with antileishmanial activity.Parasite-specific signaling pathways have recently attracted increasing attention as potential drug targets (3). Biochemical and genetic studies revealed important roles for trypanosomatid protein kinases in parasite growth and infectivity (4, 5), and as a result this class of proteins is the subject of several ongoing drug develop...

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“…Viability of L. infantum was susceptible to EO of A. ha with an IC 50 of 0.0725  μ L/mL, which corresponds to 68  μ g/mL and to EO of A. c with an IC 50 of 0.05  μ L/mL (=44  μ g/mL), at 24 h exposure. Positive control was assessed by amphotericin B under the same conditions and IC 50 value against LV49 strain was 0.03  μ g/mL, which is similar to the ones reported in the literature [40]. …”

Alkyl Galactofuranosides Strongly Interact with Leishmania donovani Membrane and Provide Antileishmanial Activity

“…In our hands, the CC 50 values obtained for THP-1 and HMØ were 6-to 12-fold lower than those with HePC, which showed cytotoxic levels similar to those described earlier by others (26,43). Compound 1 had the most interesting SI, which was 4-fold higher than that of HePC.…”

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

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