From Drug Screening to Target Deconvolution: a Target-Based Drug Discovery Pipeline Using Leishmania Casein Kinase 1 Isoform 2 To Identify Compounds with Antileishmanial Activity

Abstract

h Existing therapies for leishmaniases present significant limitations, such as toxic side effects, and are rendered inefficient by parasite resistance. It is of utmost importance to develop novel drugs targeting Leishmania that take these two limitations into consideration. We thus chose a target-based approach using an exoprotein kinase, Leishmania casein kinase 1.2 (LmCK1.2) that was recently shown to be essential for intracellular parasite survival and infectivity. We developed a four-step pipeline to identify novel selective antileishmanial compounds. In step 1, we screened 5,018 compounds from kinase-biased libraries with Leishmania and mammalian CK1 in order to identify hit compounds and assess their specificity. For step 2, we selected 88 compounds among those with the lowest 50% inhibitory concentration to test their biological activity on host-free parasites using a resazurin reduction assay and on intramacrophagic amastigotes using a high content phenotypic assay. Only 75 compounds showed antileishmanial activity and were retained for step 3 to evaluate their toxicity against mouse macrophages and human cell lines. The four compounds that displayed a selectivity index above 10 were then assessed for their affinity to LmCK1.2 using a target deconvolution strategy in step 4. Finally, we retained two compounds, PP2 and compound 42, for which LmCK1.2 seems to be the primary target. Using this four-step pipeline, we identify from several thousand molecules, two lead compounds with a selective antileishmanial activity.

The low complexity regions in the C-terminus are essential for the subcellular localisation of Leishmania casein kinase 1 but not for its activity

“…The identification of the interacting proteins that drive the localisation of CK1.2 to these different organelles will be instrumental for the precise characterisations of its functions in Leishmania as well as in other parasites. Moreover, our present and previous data demonstrate the similarity of localisation, structure, activity, regulation between Leishmania CK1.2 and human CK1s, highlighting that Leishmania CK1.2 is an excellent model to study mammalian CK1s (these data, [72] [13] [73]). Indeed, we uncovered novel localisations of CK1 family members including the nucleolus and our data provide the first analysis of CK1 regulatory domains in parasites and the first demonstration of the importance of LCRs for CK1 localisation in eukaryotes.…”

“…Escherichia coli Rosetta (DE3) pLysS Competent Cells (Merck Cat# 70956) containing pBAD-thio-topo-LmaCK1.2-V5-His 6 , pBAD-thio-topo-LmaCK1.2ΔC10-V5-His 6 , pBAD-thio-topo-LmaCK1.2ΔC43-V5-His 6 or pBAD-thio-topo-LmaCK1.2ΔN7-V5-His6 were grown at 37°C and induced with arabinose (0,02% final) for 4h at room temperature [13]. Cells were harvested by centrifugation at 10,000 g for 10 min at 4°C and the recombinant proteins were purified as described previously [13] [73]. The eluates were supplemented with 15% glycerol and stored at −80°C.…”

“…The eluates were supplemented with 15% glycerol and stored at −80°C. The kinase assays were performed as described previously [13] [73].…”

Repurposing of Human Kinase Inhibitors in Neglected Protozoan Diseases

“…When evaluated on cultured promastigotes and amastigotes, PP2 has shown low inhibitory activity with EC 50 values >10 and >50 μ m . Estimated EC 50 on intracellular parasites was ∼1 μ m . Similar results have also been published by Sanderson et al., where PP2 was inactive on both L. donovani species .…”

Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth

“…Cell-cycling promastigotes of both Leishmania species were taken from the logarithmic growth phase for viability assays. Anti-leishmanial activity of compounds was evaluated against host cell-free parasites using a resazurin reduction assay (adapted from (Durieu et al., 2016)) and on intramacrophagic L. amazonensis amastigotes using a High-Content phenotypic Assay (HCA) (Aulner et al., 2013). For the dye reduction assay, compounds were tested in quadruplicate at 20, 4 and 0.8 μM at 26 °C and 37 °C for promastigotes and amastigotes, respectively.…”