title: 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: BACKGROUND
  In 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.
  
  
  METHODS
  A 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  In 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.
authors.forename: Antonia
authors.initials: A
authors.lastname: Efstathiou
authors.forename: Nicolas
authors.initials: N
authors.lastname: Gaboriaud-Kolar
authors.forename: Despina
authors.initials: D
authors.lastname: Smirlis
authors.forename: Vassilios
authors.initials: V
authors.lastname: Myrianthopoulos
authors.forename: Konstantina
authors.initials: K
authors.lastname: Vougogiannopoulou
authors.forename: Alexandros
authors.initials: A
authors.lastname: Alexandratos
authors.forename: Marina
authors.initials: M
authors.lastname: Kritsanida
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Mikros
authors.forename: Ketty
authors.initials: K
authors.lastname: Soteriadou
authors.affiliation: Laboratories of Pharmacognosy and Pharmaceutical Chemistry, Department of Pharmacy, University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece. skaltsounis@pharm.uoa.gr.
authors.forename: Alexios-Leandros
authors.initials: AL
authors.lastname: Skaltsounis
mesh_terms: D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology; D007891:Leishmania / Q000187:drug effects / Q000201:enzymology*; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries; D013045:Species Specificity
chemical_list: C438144:ASI1 protein, S cerevisiae; D007211:Indoles; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries; D042846:CDC2-CDC28 Kinases; D038362:Glycogen Synthase Kinase 3; C027185:indirubin