Kazuki Inamori

Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging

We developed a peptide microarray based on surface plasmon resonance (SPR) imaging for monitoring protein kinase activities in cell lysates. The substrate peptides of kinases were tethered to the microarray surface modified with a self-assembled monolayer of an alkanethiol with triethylene glycol terminus to create a low nonspecific binding surface. The phosphorylation of the substrate peptides immobilized on the surface was detected with the following phosphate specific binders by amplifying SPR signals: anti-phosphotyrosine antibody for tyrosine kinases and Phos-tag biotin (a phosphate-specific ligand with biotin tag) for serine/threonine kinases. Using the microarray, 9 kinds of protein kinases were evaluated as a pattern of phosphorylation of 26 kinds of substrate peptides. The pattern was unique for each protein kinase. The microarray could be used to evaluate the inhibitory activities of kinase inhibitors. The microarray was applied successfully for kinase activity monitoring of cell lysates. The chemical stimuli responsive activity changes of protein kinases in cell lysates could also be monitored by the peptide microarray. Thus, the peptide microarray based on SPR imaging would be applicable to cell-based drug discovery, diagnosis using tissue lysates, and biochemical studies to reveal signal transduction pathways.

Optimal surface chemistry for peptide immobilization in on-chip phosphorylation analysis.

We investigated the optimal surface chemistry of peptide immobilization for on-chip phosphorylation analysis. In our previous study, we used a heterobifunctional cross-linker sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxalate (SSMCC) to immobilize cysteine-terminated peptides on an amine-modified gold surface. The study revealed that the phosphorylation efficiency and rate were low (only 20% at 2 h) comparing with the reaction in solution. In this study, to improve the phosphorylation efficiency, the kinase substrates were immobilized via poly(ethylene glycol) (PEG), a flexible, hydrophilic polymer. An improvement in cSrc phosphorylation was achieved (60% at 1 h) from using a PEG-inserted peptide and SSMCC. However, no phosphorylation could be detected when the peptide was immobilized with a PEG-containing cross-linker. Fluorescence-labeled peptide studies revealed that the use of longer cross-linkers resulted in lower immobilization density. We considered that the flexible PEG linker was preferable to secure high phosphorylation efficiency for the immobilized peptide, probably due to the improvement of cSrc accessibility and peptide mobility, but the immobilization protocol is critical for keeping high density of the peptide immobilization. In addition, such an accelerating effect of PEG linker against on-chip phosphorylation of an immobilized peptide may depend on kinase structures or the position of the active center, because no improvement of on-chip peptide phosphorylation was observed in protein kinase A. However, PEG linker also did not suppress the phosphorylation in protein kinase A. Thus, we concluded that SSMCC and PEGylated peptide will be a good combination for the surface chemistry of on-chip phosphorylation in peptide array.

Establishment of screening system toward discovery of kinase inhibitors using label-free on-chip phosphorylation assays

We describe a label-free method for the kinase inhibition assay toward discovery of kinase inhibitors. The surface plasmon resonance (SPR) imaging analysis using zinc(II) compound was adopted on the on-chip phosphorylation analysis. In this study, following three subjects were focused: (1) to monitor the inhibition of three inhibitors supporting by their specific inhibition mechanisms, (2) to quantify the inhibitory activities, and (3) to prove the reliability of the obtained 50% inhibition concentration (IC(50)) value. First, the inhibitory activities of Amide 5-24, H-89 and Gö6983 on PKA and PKCdelta were determined, and specific inhibitions for two kinases could be observed quantitatively. Second, the inhibition curves of Amide 5-24, Amide 14-22 and H-89 were obtained, and the results supported the two previous reports: (1) the inhibition efficiency of Amide 5-24 was much higher than that of Amide 14-22, and (2) the inhibitory activity of H-89 followed ATP-binding site blocking mechanism. Last, the obtained IC(50) values by the SPR imaging were almost corresponded to those by the solution assay, although on-chip phosphorylation efficiency was low (approximately 12%). In conclusion, validation of the on-chip phosphorylation analysis for kinase inhibitors was achieved. This label-free method might be applied for discovery of kinase inhibitors.

High-Throughput SPR Biosensor

Surface plasmon resonance (SPR) imaging technique is label free, real-time, and high-throughput analysis method for interaction studies with array format. The application of SPR imaging for the small molecule arrays, which were fabricated by photoaffinity crosslinking, can be the first screening step for reverse chemical genomics. The fabrication process of sugar array and sugar-lectin interaction study was demonstrated. The protocol of array fabrication did not require any chemical modifications of sugar chains for immobilizations. The biotinylated sugars were used to investigate signal ratios between lectin and antistreptavidin antibody binding. And it seemed that signal normalization could be achieved, even though the accurate densities of immobilized sugars were unclear.