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.

Indirubins are bisindole alkaloids naturally occurring in indigo-bearing plants or in mollusks from the Muricidae family. They belong to the rather small family of indigoids, which has nevertheless found an extreme importance in the fields of dyes and medicinal chemistry. Indirubin has been found to be the active ingredient of a traditional Chinese Medicine used to treat the symptoms of leukemia. Further biological explorations revealed the ability of indirubin to bind cyclin-dependent kinases and 6-bromoindirubin, extracted from mollusks, to bind glycogen synthase kinase-3. The high affinity displayed by the two natural products has opened a vast field of research and triggered the development of hundred of derivatives with biological activities. The traditional use of indirubin for the treatment of leukemia has prompted different research groups to study the cytotoxic effect of indirubin derivatives on both solid tumors and leukemia. Moreover, the affinity of indirubins for kinases also allowed the exploration of their activity towards stem cells. The derivatives presented are in accordance with first discoveries and establish the close relation between activity and kinase inhibition. New derivatives have been patented and new interferences in signaling pathways are described. However, few in vivo studies have been performed and more efficient solutions are needed to unravel the major issue of solubility.

A high performance liquid chromatography (HPLC) method combined with spectrophotometric UV‐Vis detection is developed for the separation and identification of seven indigoid coloring compounds: indigotin, which is commercially available, and indirubin, 6‐bromoindigotin, 6′‐bromoindirubin, 6‐bromoindirubin, 6,6′‐dibromoindigotin, and 6,6′‐dibromoindirubin, which are synthesized to be used as reference compounds in the HPLC analysis. The chromatographic method is employed for the identification of the blue/purple coloring compounds in samples extracted from four mollusks, which have been used for the production of Tyrian Purple since antiquity: Hexaplex trunculus, Murex brandaris, Nucella lapillus, and Thais haemastoma. The composition of the analyzed samples, with respect to the reference materials, is compared and discussed. All seven indigoids are identified in Hexaplex trunculus and some of them are identified in the other three purple extracts.