title: Binding of protein kinase inhibitors to synapsin I inferred from pair-wise binding site similarity measurements.
abstract: Predicting off-targets by computational methods is getting increasing importance in early drug discovery stages. We herewith present a computational method based on binding site three-dimensional comparisons, which prompted us to investigate the cross-reaction of protein kinase inhibitors with synapsin I, an ATP-binding protein regulating neurotransmitter release in the synapse. Systematic pair-wise comparison of the staurosporine-binding site of the proto-oncogene Pim-1 kinase with 6,412 druggable protein-ligand binding sites suggested that the ATP-binding site of synapsin I may recognize the pan-kinase inhibitor staurosporine. Biochemical validation of this hypothesis was realized by competition experiments of staurosporine with ATP-gamma(35)S for binding to synapsin I. Staurosporine, as well as three other inhibitors of protein kinases (cdk2, Pim-1 and casein kinase type 2), effectively bound to synapsin I with nanomolar affinities and promoted synapsin-induced F-actin bundling. The selective Pim-1 kinase inhibitor quercetagetin was shown to be the most potent synapsin I binder (IC50 = 0.15 microM), in agreement with the predicted binding site similarities between synapsin I and various protein kinases. Other protein kinase inhibitors (protein kinase A and chk1 inhibitor), kinase inhibitors (diacylglycerolkinase inhibitor) and various other ATP-competitors (DNA topoisomerase II and HSP-90alpha inhibitors) did not bind to synapsin I, as predicted from a lower similarity of their respective ATP-binding sites to that of synapsin I. The present data suggest that the observed downregulation of neurotransmitter release by some but not all protein kinase inhibitors may also be contributed by a direct binding to synapsin I and phosphorylation-independent perturbation of synapsin I function. More generally, the data also demonstrate that cross-reactivity with various targets may be detected by systematic pair-wise similarity measurement of ligand-annotated binding sites.
authors.affiliation: Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, Genova, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Defranchi
authors.forename: Enrico
authors.initials: E
authors.lastname: De Franchi
authors.forename: Claire
authors.initials: C
authors.lastname: Schalon
authors.forename: Mirko
authors.initials: M
authors.lastname: Messa
authors.forename: Franco
authors.initials: F
authors.lastname: Onofri
authors.forename: Fabio
authors.initials: F
authors.lastname: Benfenati
authors.forename: Didier
authors.initials: D
authors.lastname: Rognan
mesh_terms: D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D019295:Computational Biology / Q000379:methods*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D000090063:Proto-Oncogene Mas; D051573:Proto-Oncogene Proteins c-pim-1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D019311:Staurosporine / Q000378:metabolism / Q000494:pharmacology; D016704:Synapsins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000199:Actins; D008024:Ligands; C000715470:MAS1 protein, human; D047428:Protein Kinase Inhibitors; D000090063:Proto-Oncogene Mas; D016704:Synapsins; D000255:Adenosine Triphosphate; D051573:Proto-Oncogene Proteins c-pim-1; D019311:Staurosporine
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