An inhibitor-driven study for enhancing the selectivity of indirubin derivatives towards leishmanial Glycogen Synthase Kinase-3 over leishmanial cdc2-related protein kinase 3

Abstract

BackgroundIn search of new antiparasitic agents for overcoming the limitations of current leishmaniasis chemotherapy, we have previously shown that 6-bromoindirubin-3'-oxime (6BIO) and several other 6-substituted analogues of indirubin, a naturally occurring bis-indole present in mollusks and plants, displayed reverse selectivity from the respective mammalian kinases, targeting more potently the leishmanial Cyclin-Dependent Kinase-1 (CDK1) homologue [cdc2-related protein kinase 3 (LCRK3)] over leishmanial Glycogen Synthase Kinase-3 (LGSK-3). This reversal of selectivity in Leishmania parasites compared to mammalian cells makes the design of specific indirubin-based LGSK-3 inhibitors difficult. In this context, the identification of compounds bearing specific substitutions that shift indirubin inhibition towards LGSK-3, previously found to be a potential drug target, over LCRK3 is imperative for antileishmanial targeted drug discovery.MethodsA new in-house indirubin library, composed of 35 compounds, initially designed to target mammalian kinases (CDKs, GSK-3), was tested against Leishmania donovani promastigotes and intracellular amastigotes using the Alamar blue assay. Indirubins with antileishmanial activity were tested against LGSK-3 and LCRK3 kinases, purified from homologous expression systems. Flow cytometry (FACS) was used to measure the DNA content for cell-cycle analysis and the mode of cell death. Comparative structural analysis of the involved kinases was then performed using the Szmap algorithm.ResultsWe have identified 7 new indirubin analogues that are selective inhibitors of LGSK-3 over LCRK3. These new inhibitors were also found to display potent antileishmanial activity with GI50 values of <1.5 μΜ. Surprisingly, all the compounds that displayed enhanced selectivity towards LGSK-3, were 6BIO analogues bearing an additional 3'-bulky amino substitution, namely a piperazine or pyrrolidine ring. A comparative structural analysis of the two aforementioned leishmanial kinases was subsequently undertaken to explain and rationalize the selectivity trend determined by the in vitro binding assays. Interestingly, the latter analysis showed that selectivity could be correlated with differences in kinase solvation thermo dynamics induced by minor sequence variations of the otherwise highly similar ATP binding pockets.ConclusionsIn conclusion, 3'-bulky amino substituted 6-BIO derivatives, which demonstrate enhanced specificity towards LGSK-3, represent a new scaffold for targeted drug development to treat leishmaniasis.

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

“…Compound 42 is our best lead compound, with an EC 50 on intracellular parasites of 60 Ϯ 5 nM and an SI of 50. Although several authors have already described the antileishmanial effect of indirubins (57)(58)(59), this particular derivative has not been previously tested on intracellular parasites. From previous published work, we already know some of the targets of the indirubins, such as Leishmania CRK3 or GSK3 (57,58).…”

Antiparasitic Efficacy of Artemisia ludoviciana Nutt. (Asteraceae) Essential Oil for Acanthamoeba castellanii, Leishmania infantum and Trichomonas vaginalis

“…34 The toxic side effects of anti-leishmanial medications used for treatment and the development of resistance of the parasite to these medications have led to studies related to definition and formulation of new molecules to date. 37 Different studies have been completed; [12][13][14][15][16][17][18] from a study showing the anti-leishmanial activity of ethanol extracts from Allium sativa (garlic) and Azadirachta indica (Neem), locally used for treatment of Cutaneous leishmaniasis in the Sudan, had no significant difference when compared to pentostam, 16 to studies emphasizing that the anti-leishmanial efficacy on promatigotes and amastigotes of Leishmania tropica was higher compared to two antibiotics in the macrolid group of azithromycin and clarithromycin. 38 The AL-EO presented in this study can be considered for use as an antileishmanial.…”

“…[12][13][14][15][16] Due to the lack of an effective prophylactic against the disease, the toxic effects of currently-used medications and the increasing resistance to these medications, the necessity for discovery and development of new therapeutic agents has been reported. 11,17,18 Trichomoniosis is a common infection everywhere in the world and the infection rates are reported to show great variations from country to country and society to society. Researchers have stated that the different results obtained by different people in different regions may be due to factors such as the use of different techniques for diagnosis, and deficient and mistaken assessments.…”

Lipophilic conformationally constrained spiro carbocyclic 2,6‐diketopiperazine‐1‐acetohydroxamic acid analogues as trypanocidal and leishmanicidal agents: An extended SAR study

“…The inhibitory activity and the cytotoxicity of the compounds were determined with the use of an MTT-based assay, the Alamar blue, as previously described. [14] Upon the incubation, the medium was removed, wells were thoroughly washed with PBS to remove free parasites, and 200 μl of fresh medium containing the different concentrations of the compounds was added. 20 μl of Alamar blue were added upon the 72-hr incubation and 24 hr later the colorimetric readings were performed (wavelength: 550 nm, reference wavelength: 620 nm).…”