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.
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.
Xingi
Evangelia
E
Laboratory of Molecular Parasitology, Department of Microbiology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521 Athens, Greece.
Smirlis
Despina
D
Myrianthopoulos
Vassilios
V
Magiatis
Prokopios
P
Grant
Karen M
KM
Meijer
Laurent
L
Mikros
Emmanuel
E
Skaltsounis
Alexios-Leandros
AL
Soteriadou
Ketty
K
D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology*; D007893:Leishmania donovani / Q000378:metabolism*; D007896:Leishmaniasis / Q000188:drug therapy* / Q000378:metabolism; D010091:Oximes / Q000494:pharmacology*
C483321:6-bromoindirubin-3'-oxime; D005456:Fluorescent Dyes; D007211:Indoles; D010091:Oximes; D018844:Cyclin-Dependent Kinases; D038362:Glycogen Synthase Kinase 3