title: An evaluation of indirubin analogues as phosphorylase kinase inhibitors.
abstract: Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360μM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8μM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work.
authors.affiliation: School of Physical Sciences & Computing, Division of Chemistry, University of Central Lancashire, Preston PR1 2HE, United Kingdom; School of Forensic & Investigative Sciences, University of Central Lancashire, Preston PR1 2HE, United Kingdom.
authors.forename: Jaida
authors.initials: J
authors.lastname: Begum
authors.affiliation: Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Str., 41221 Larissa, Greece.
authors.forename: Vassiliki T
authors.initials: VT
authors.lastname: Skamnaki
authors.affiliation: School of Forensic & Investigative Sciences, University of Central Lancashire, Preston PR1 2HE, United Kingdom.
authors.forename: Colin
authors.initials: C
authors.lastname: Moffatt
authors.affiliation: Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 116 35 Athens, Greece.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bischler
authors.affiliation: Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 116 35 Athens, Greece.
authors.forename: Josephine
authors.initials: J
authors.lastname: Sarrou
authors.affiliation: Division of Pharmacognosy, Department of Pharmacy, University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece.
authors.forename: Alexios-Leandros
authors.initials: AL
authors.lastname: Skaltsounis
authors.affiliation: Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Str., 41221 Larissa, Greece.
authors.forename: Demetres D
authors.initials: DD
authors.lastname: Leonidas
authors.affiliation: Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 116 35 Athens, Greece.
authors.forename: Nikos G
authors.initials: NG
authors.lastname: Oikonomakos
authors.affiliation: School of Physical Sciences & Computing, Division of Chemistry, University of Central Lancashire, Preston PR1 2HE, United Kingdom; School of Forensic & Investigative Sciences, University of Central Lancashire, Preston PR1 2HE, United Kingdom. Electronic address: jhayes@uclan.ac.uk.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Hayes
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D051357:Cyclin-Dependent Kinase 2 / Q000737:chemistry; D051360:Cyclin-Dependent Kinase 5 / Q000737:chemistry; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry; D000071679:Glycogen Synthase Kinase 3 beta; D057166:High-Throughput Screening Assays; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry*; D008024:Ligands; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010091:Oximes / Q000737:chemistry*; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014584:User-Computer Interface
chemical_list: C514974:7-bromoindirubin-3'-oxime; D007004:Hypoglycemic Agents; D007211:Indoles; D008024:Ligands; D010091:Oximes; D047428:Protein Kinase Inhibitors; D010764:Phosphorylase Kinase; D051360:Cyclin-Dependent Kinase 5; D000071679:Glycogen Synthase Kinase 3 beta; C495894:CDK2 protein, human; C495905:CDK5 protein, human; D051357:Cyclin-Dependent Kinase 2; D038362:Glycogen Synthase Kinase 3; C448021:glycogen synthase kinase 3 alpha; C027185:indirubin
keywords: Glycogen phosphorylase; Indirubins; Kinase inhibitors; QM/MM-PBSA; Sigma-hole; Type 2 diabetes