@article{3002066,
    title = "6-Br-5methylindirubin-3′oxime (5-Me-6-BIO) targeting the leishmanial glycogen synthase kinase-3 (GSK-3) short form affects cell-cycle progression and induces apoptosis-like death: Exploitation of GSK-3 for treating leishmaniasis",
    author = "Xingi, E. and Smirlis, D. and Myrianthopoulos, V. and Magiatis, P. and Grant, K.M. and Meijer, L. and Mikros, E. and Skaltsounis, A.-L. and Soteriadou, K.",
    journal = "International Journal for Parasitology",
    year = "2009",
    volume = "39",
    number = "12",
    pages = "1289-1303",
    issn = "0020-7519",
    doi = "10.1016/j.ijpara.2009.04.005",
    keywords = "6 bromo 5 methylindirubin 3' oxime;  6 bromo indirubin 3' acetoxime;  6 bromo indirubin 3' oxime;  cyclin dependent kinase;  glycogen synthase kinase 3;  indirubin;  unclassified drug, cell organelle;  drug;  inhibition;  inhibitor;  parasite;  phenotype;  substitution, amastigote;  animal cell;  antiprotozoal activity;  apoptosis;  article;  cell count;  cell cycle G2 phase;  cell cycle M phase;  cell cycle progression;  cell cycle S phase;  cell viability;  controlled study;  drug potency;  drug selectivity;  drug structure;  drug targeting;  enzyme active site;  enzyme assay;  enzyme inhibition;  flow cytometry;  fluorescence activated cell sorting;  gene overexpression;  IC 50;  Leishmania donovani;  Leishmania infantum;  leishmaniasis;  mammal;  molecular docking;  mouse;  nick end labeling;  nonhuman;  nucleotide sequence;  phenotype;  prediction;  promastigote;  Trypanosoma brucei, Animals;  Apoptosis;  Cell Cycle;  Cells, Cultured;  Cyclin-Dependent Kinases;  Drug Evaluation, Preclinical;  Flow Cytometry;  Fluorescent Dyes;  Glycogen Synthase Kinase 3;  Humans;  Immunoblotting;  Indoles;  Leishmania donovani;  Leishmaniasis;  Oximes, Leishmania donovani;  Mammalia",
    abstract = "Indirubins known to target mammalian cyclin-dependent kinases (CDKs) and glycogen synthase kinase (GSK-3) were tested for their antileishmanial activity. 6-Br-indirubin-3′-oxime (6-BIO), 6-Br-indirubin-3′acetoxime and 6-Br-5methylindirubin-3′oxime (5-Me-6-BIO) were the most potent inhibitors of Leishmania donovani promastigote and amastigote growth (half maximal inhibitory concentration (IC50) values ≤1.2 μM). Since the 6-Br substitution on the indirubin backbone greatly enhances the selectivity for mammalian GSK-3 over CDKs, we identified the leishmanial GSK-3 homologues, a short (LdGSK-3s) and a long one, focusing on LdGSK-3s which is closer to human GSK-3β, for further studies. Kinase assays showed that 5-Me-6-BIO inhibited LdGSK-3s more potently than CRK3 (the CDK1 homologue in Leishmania), whilst 6-BIO was more selective for CRK3. Promastigotes treated with 5-Me-6-BIO accumulated in the S and G2/M cell-cycle phases and underwent apoptosis-like death. Interestingly, these phenotypes were completely reversed in parasites over-expressing LdGSK-3s. This finding strongly supports that LdGSK-3s is: (i) the intracellular target of 5-Me-6-BIO, and (ii) involved in cell-cycle control and in pathways leading to apoptosis-like death. 6-BIO treatment induced a G2/M arrest, consistent with inhibition of CRK3 and apoptosis-like death. These effects were partially reversed in parasites over-expressing LdGSK-3s suggesting that in vivo 6-BIO may also target LdGSK-3s. Molecular docking of 5-Me-6-BIO in CRK3 and 6-BIO in human GSK-3β and LdGSK-3s active sites predict the existence of functional/structural differences that are sufficient to explain the observed difference in their affinity. In conclusion, LdGSK-3s is validated as a potential drug target in Leishmania and could be exploited for the development of selective indirubin-based leishmanicidals. © 2009 Australian Society for Parasitology Inc."
}