Binding of Protein Kinase Inhibitors to Synapsin I Inferred from Pair-Wise Binding Site Similarity Measurements

Enrico De Franchi,Fabio Benfenati,Claire Schalon,Mirko Messa,Franco Onofri,Didier Rognan,Floyd Romesberg

doi:10.1371/journal.pone.0012214

Enrico De Franchi, Fabio Benfenati + Show 5 more

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https://doi.org/10.1371/journal.pone.0012214

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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-γ35S 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 µM), 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-90α 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.

Highlights

Drug designers had been focusing on a single macromolecular target and a single or very few chemical series [1]
Outstanding efforts of structural genomic consortia to complete the structural proteome let us anticipate an acceptable coverage of the UniProt database [11] in only 15 years [7]
Among the list of possible hits, the sc-PDB entries were ranked by decreasing similarity to 1aux and corresponding proteins were ranked according to a Receiver Operating Characteristic (ROC) classifying scheme [40] from the statistically most similar (Pim-1 kinase) to the least similar

Summary

Introduction

Drug designers had been focusing on a single macromolecular target and a single or very few chemical series [1]. Global chemogenomic approaches [14] targeting arrays of ligands (rows) and proteins (columns) to generate huge two-dimensional binding matrices enlarge our vision of how chemical and biological spaces match [15]. Diverse descriptors and methods have already been validated on existing data [23,24,25] This approach led to the discovery of several off-targets for known drugs [20,21]. Pure ligand-based methods have two main drawbacks : (i) they are restricted by the incomplete coverage of target space by known ligands and cannot be applied to orphan proteins, (ii) the dogma stating that chemical similarity implies biological similarity is only true in 30% of test cases [26]

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