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

Int J Parasitol. 2009 Oct;39(12):1289-303. doi: 10.1016/j.ijpara.2009.04.005. Epub 2009 May 13.


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 (IC(50)) values < or =1.2 microM). 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-3beta, 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-3beta 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis*
  • Cell Cycle / drug effects
  • Cells, Cultured
  • Cyclin-Dependent Kinases / antagonists & inhibitors*
  • Cyclin-Dependent Kinases / chemistry
  • Cyclin-Dependent Kinases / metabolism
  • Drug Evaluation, Preclinical
  • Flow Cytometry
  • Fluorescent Dyes
  • Glycogen Synthase Kinase 3 / antagonists & inhibitors*
  • Glycogen Synthase Kinase 3 / chemistry
  • Glycogen Synthase Kinase 3 / metabolism
  • Humans
  • Immunoblotting
  • Indoles / pharmacology*
  • Leishmania donovani / metabolism*
  • Leishmaniasis / drug therapy*
  • Leishmaniasis / metabolism
  • Oximes / pharmacology*


  • 6-bromoindirubin-3'-oxime
  • Fluorescent Dyes
  • Indoles
  • Oximes
  • Cyclin-Dependent Kinases
  • Glycogen Synthase Kinase 3