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

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.

FIG 1

Differential target-based screen of 4,030 compounds from various libraries. (A) Representation of the percentage of inhibition toward LmCK1.2 activity versus the percentage of inhibition toward SsCK1 activity. The compounds in sectors a and b are potent toward LmCK1.2 since they show more than 40% inhibition, whereas the compounds in sectors b and d are potent toward SsCK1. (B) A total of 336 hit compounds were identified in the screen, of which 245 inhibit SsCK1 (6.1% hit rate) and 128 inhibit LmCK1.2 (3.2% hit rate). Only 37 compounds showed equal potency against both CK1s. (C) Compounds were classified according to their specificity: compounds only potent against SsCK1 (only SsCK1), more potent against SsCK1 than LmCK1.2 (SsCK1 > LmaCK1.2), equally potent on both kinases (SsCK1 = LmCK1.2), more potent against LmCK1.2 than SsCK1 (SsCK1 < LmaCK1.2), and only potent on LmCK1.2 (only LmCK1.2). Compounds were also classified according to their percent inhibition: class 1 corresponds to compounds that inhibit the kinase activity between 80 and 100%, class 2 corresponds to compounds that inhibit the kinase activity between 60 and 80%, and class 3 corresponds to compounds that inhibit the kinase activity between 40 and 60%. A total of 23 compounds were more potent toward LmCK1.2 than SsCK1, and 68 compounds were specific to LmCK1.2 (the numbers in the histograms indicate the percentage of compounds in each category).

FIG 2

Determination of the IC₅₀ of the 45 compounds belonging to class 1 that have a percent inhibition above 90%. Each point represents the IC₅₀ of a particular compound toward LmCK1.2. Nonspecific compounds have a potency below 10 μM toward both kinases, whereas specific compounds have a potency below 10 μM only toward LmCK1.2.

FIG 3

Screening of the purine derivative library. (A) Structure of the purine backbone. R1, R2, and R3 represent different substitutions of the purines. (B) We performed a target-based screening of 588 derivatives. Each point represents the percent inhibition toward LmCK1.2 activity versus the percent inhibition toward SsCK1 activity of each compound. The compounds in the top left are more potent toward LmCK1.2, whereas the compounds in the bottom right are more potent toward SsCK1. (C) Compounds were classified according to their specificity: only potent on SsCK1, more potent on SsCK1 than LmCK1.2 (SsCK1 > LmCK1.2), equally on both kinases (SsCK1 = LmCK1.2), more potent on LmCK1.2 than SsCK1 (SsCK1 < LmCK1.2), and only potent on LmCK1.2. Compounds were also classified according to their % of inhibition: class 1 corresponds to compounds that inhibit kinases between 80 and 100%, class 2 corresponds to compounds that inhibit kinases between 60 and 80%, and class 3 corresponds to compounds that inhibit kinases between 40 and 60%. Only 4% of the compounds are more potent toward LmCK1.2 than SsCK1 or specific to LmCK1.2. (D) We determined the IC₅₀ of the 21 compounds belonging to class 1 that have a percent inhibition above 90%. Each point represents the IC₅₀ of a particular compound toward LmCK1.2 versus SsCK1. The IC₅₀ values are lower toward SsCK1 than LmCK1.2.

FIG 4

Screening of the indirubin derivative library. (A) Structure of the indirubin backbone. (B) Target-based screening of 400 derivatives. Each point represents the percent inhibition toward LmCK1.2 activity versus the percent inhibition toward SsCK1 activity for each compound. The compounds in the top left are more potent toward LmCK1.2, whereas the compounds in the bottom right are more potent toward SsCK1. (C) Compounds were classified according to their specificity: only potent on SsCK1, more potent on SsCK1 than LmCK1.2 (SsCK1 > LmaCK1.2), equally on both kinases (SsCK1 = LmCK1.2), more potent on LmCK1.2 than SsCK1 (SsCK1 < LmaCK1.2), and only potent on LmCK1.2. Compounds were also classified according to their percent inhibition: class 1 corresponds to compounds that inhibit the kinases between 80 and 100%, class 2 corresponds to compounds that inhibit the kinases between 60 and 80%, and class 3 corresponds to compounds that inhibit the kinases between 40 and 60%. Fifty-seven percent of the compounds are more potent toward LmCK1.2 than SsCK1, and 46% are specific to LmCK1.2. (D) IC₅₀s of the 55 compounds that are specific to LmCK1.2 or that belong to class 1 with a percent inhibition above 90%. Each point represents the IC₅₀ of a particular compound toward LmCK1.2 versus SsCK1. The IC₅₀ are lower against LmCK1.2 than SsCK1.

FIG 5

Comparison of the antileishmanial activity of compounds on cultured and intracellular parasites. We performed a screening of 88 compounds from the main, the purine and the indirubin libraries on cultures promastigotes, axenic amastigotes, and intracellular parasites. Each point represents the percentage of metabolically active promastigotes or amastigotes at 10 μM versus the percentage of parasite burden at 10 μM for each compound. Black squares correspond to the percentage of metabolically active promastigotes at 10 μM versus the percentage of parasite burden at 10 μM, and gray dots correspond to the percentage of metabolically active amastigotes at 10 μM versus the percentage of parasite burden at 10 μM. Sectors: a, compounds that are potent against intracellular parasites but not against cultured parasites; b, compounds that are not potent against intracellular and cultured parasites; c, compounds that are potent against intracellular and cultured parasites; and d, compounds that are not potent against intracellular parasites but potent against cultured parasites.

FIG 6

Parasite burden versus macrophage viability. Using a visual high-content phenotypic assay, we calculated the percentage of viable macrophages and the percentage of infected cells. We analyzed the antileishmanial effect of the selected compounds from the main library (A), the indirubin library (B), and the purine library (C) versus their toxicity against macrophages. Sectors: a, compounds that are not potent against intracellular parasites but cytotoxic; b, compounds that are not potent against intracellular parasites and not cytotoxic; c, compounds that are potent against intracellular parasites but cytotoxic; and d, compounds that are potent against intracellular parasites and not cytotoxic.

FIG 7

PP2 and compound 42 are the most specific compounds toward CK1.2. Competitive ATP affinity chromatography assays were performed on amastigote cell lysates in presence or not of D4476, PP2, 5′ITu (Iodo), NSC699479 (compound 73), and compound 42. (A) ATP-binding proteins (elution) were eluted with an excess of ATP, resolved by SDS-PAGE electrophoresis and stained by SYPRO Ruby. (B) CK1.2 was revealed by Western blotting with an anti-LmCK1.2 antibody (SY3535).