Development of Selective Bisubstrate‐Based Inhibitors Against Protein Kinase C (PKC) Isozymes By Using Dynamic Peptide Microarrays

Abstract

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP-binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate-based inhibitors that target the more selective peptide-binding site in addition to the ATP-binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate-based leads. Dynamic peptide microarrays were used to find peptide-binding site inhibitors. These were linked with chemoselective click chemistry to an ATP-binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate-based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCtheta amongst three isozymes.

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

“…The lead compound had nanomolar K i (estimated from a sub-micromolar IC 50 value) and over 20-fold selectivity towards PKCq over PKCa and PKCz. [50] Another staurosporinepeptide conjugate with high potency (IC 50 = 2.6 nm) and selectivity for PKAc was prepared by applying the phage displayaided peptide library approach (compound 13). [51] The further optimization of this compound yielded the bivalent ligand with even higher (sub-nanomolar) affinity.…”

Cell-Penetrating Bisubstrate-Based Protein Kinase C Inhibitors

“…Interestingly, one of the differences observed is diminished phosphorylation of some PKC substrates found in our initial PKC profiling experiment, e.g., MARCKS. 21 In conclusion, CPPs were successfully applied for the intracellular delivery of selective bisubstrate-based PKC inhibitors. Confocal microscopy and FACS showed that CPPbisubstrates were taken up and tolerated by HeLa cells.…”

“…Figure 1, panel b shows a model of inhibitor 1 conjugated to the Tat-peptide bound to PKCθ based on several crystal structures of PKC−inhibitor complexes. The view is along the peptide binding groove containing the pseudosubstrate part 20,21 (white) with the ATP-competitive component (blue) in a deep binding pocket in the center. The CPP-part (red) is situated outside the kinase on the left.…”

“…21 This is furnished with an azido group for connecting an ATP-competitive part and an orthogonal attachment point for a CPP-component. In this example a disulfide linkage was used; however, other conjugation strategies may be employed, e.g., native chemical ligation.…”

Prediction of Response to Preoperative Chemoradiotherapy in Rectal Cancer by Multiplex Kinase Activity Profiling

“…Recognizing that tumor sensitivity to CRT in LARC likely implicates a multitude of signaling cascades, multiplex profiling of kinase activity in pretreatment tumor biopsy specimens was applied using peptide microarrays with tyrosine kinase substrates (Tyrosine Kinase PamChip96 Array; PamGene International B.V., 's-Hertogenbosch, The Netherlands) that allow measurement of phosphosubstrate signatures generated by biologic samples (14)(15)(16)(17)(18)(19)(20)(21)(22). The working hypothesis was that particular subsets of kinase activity profiles within pretreatment tumor biopsy specimens might correlate to tumor response to preoperative CRT in LARC.…”