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.

Surface plasmon resonance imaging measurements of caspase reactions on peptide microarrays.

Enzymatic activity monitoring of caspases, which are a class of cysteine protease, was performed by using peptide arrays based on surface plasmon resonance (SPR) imaging. The strategy of the detection is straightforward, using streptavidin to amplify the SPR signals of the surface-immobilized substrate peptides labeled with biotin at the C termini. Thus, the cleavage of the substrate peptides by caspases was detected as a signal decrease. Using this method, we succeeded in monitoring the activities of purified caspases and caspases in cell lysates. The SPR imaging-based peptide array would be applicable to cell-based drug screening and biochemical studies to reveal signal transduction processes.

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.

A peptide microarray for the detection of protein kinase activity in cell lysate.

DNA microarray enables the analysis of DNA or mRNA expression levels, but it has not been possible to completely understand life using obtained information. Consequently, protein or peptide arrays have attracted much interest. Since the development of a practical protein microarray is still far away in light of handling difficulties, the peptide microarray is a promising tool for analyzing protein functions. We have developed a peptide microarray to detect protein kinase activity in cell lysate. All substrate peptides for kinases were immobilized chemoselectively on amino-coated glass slides. After phosphorylation of the immobilized peptides, phosphorylation was detected by fluorescence imaging. We detected the protein kinase activities, including that in cell lysate, in response to drug stimulation. Therefore, this peptide microarray would be useful for a high-throughput kinase assay of intracellular signals and would be applicable to drug screening.

A protein kinase signal-responsive gene carrier modified RGD peptide.

We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using a cationic polymer. However, this polymer alone cannot deliver any gene into living cells. In the present work, we modified the signal-responsive polymer to the RGD peptide for the introduction of a polymer/DNA complex into living cells and succeeded in regulating the gene expression responding to intracellular PKA activation.

Development of a fluorescence peptide chip for the detection of caspase activity.

Proteases play a key role in cell functions, and it is very important to monitor their activities for drug screening and diagnosis of diseases. In the present study, a new class of fluorescence probe, into which a fluorophore and a quencher have been introduced, was developed and applied to the on-chip detection of caspase-3 activity. This probe is non fluorescent in the absence of caspase-3. However, when it is treated with active caspase-3, the fluorescence intensity increases dependent on the caspase-3 activity due to the cleavage of the quencher-containing moiety on a glass slide. This caspase-dependent increase in the fluorescence intensity was also detected when the glass slide immobilizing the probe peptide was treated with cell lysate stimulated by staurosporine (STP), which is an apoptosis-inducing agent. On the other hand, such an increase was not detected in the case of control cell lysate without STP-stimulation. The developed system is a rapid and sensitive method and is useful for the direct measurement of protease activity on a glass array.

An intracellular kinase signal-responsive gene carrier for disordered cell-specific gene therapy.

We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using cationic polymer. This cationic polymer (PAK) was a graft-type polymer with an oligopeptide that is a substrate for PKA and could regulate gene-expression in a cell-free system. In the present study, we carried out a detailed characterization of the PAK-DNA complex (AFM observation and DLS measurement) and tried to apply this polymer to living cells. In the unstimulated NIH 3T3 cells, transfection of the PAK-DNA complex showed no expression of the delivered gene. This means that PAK formed a stable complex with DNA in the normal cells to totally suppress gene expression. In contrast, significant expression was seen when the PAK-DNA complex was delivered to forskolin-treated cells. Thus, activated PKA disintegrates the complexes even in living cells, resulting in gene expression. Our results indicate that this type of intracellular signal-responsive polymer will be useful for the cell-specific release of genes.

A peptide sequence controls the physical properties of nanoparticles formed by peptide-polymer conjugates that respond to a protein kinase a signal.

We previously reported that poly(N-isopropylacrylamide) grafted with Peptide 1 (-GLRRASLG) and poly(ethylene glycol) changed its physical properties in response to an intracellular protein phosphorylation signal, protein kinase A (PKA) (Katayama, Y. et al. (2001) Macromolecules 34, 905). In this study, we investigated the effect of changing peptide structure on the lower critical solution temperature (LCST) of peptide-polymer conjugates, before and after phosphorylation with PKA. For Peptide 2 (Ac-LRRASL-), which has a formal net charge of +2 at physiological pH, the LCST of the conjugate decreased on phosphorylation. In contrast, the LCSTs of the conjugates with Peptide 3 (-ALRRASLE) and Peptide 4 (Ac-DWDALRRASL-), which have neutral net charges, were greatly increased. This suggests that the LCST of the polymer was mainly governed by two factors: the change in hydration around the polymer chain and the interpeptide electrostatic repulsion, resulting from phosphorylation. These polymers have potential for use as drug capsules that respond to cellular conditions.

Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule.

We describe herein a detection and quantification system for on-chip phosphorylation of peptides by surface plasmon resonance (SPR) imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(II) complex). The biotinylated compound is a dinuclear zinc(II) complex that is suitable for accessing phosphate anions as a bridging ligand on the two zinc(II) ions. The compound was exposed on the peptide array and detected with streptavidin (SA) via a biotin-SA interaction by SPR imaging. In the conventional method using antibody, both anti-phosphoserine and anti-phosphotyrosine antibodies were required for phosphoserine and phosphotyrosine detection, respectively. Detection of the phosphate group by the zinc(II) complex, however, was independent of the phosphorylated amino acid residues. The calibration curve for the phosphorylation ratios was established with a calibration chip, on which phosphoserine-containing peptide probes were immobilized. The peptide probes, which were phosphorylated on the surface by protein kinase A, were detected and quantified by SPR imaging using the zinc(II) complex, SA, and anti-SA antibody. The reaction rate and the kinetics of on-chip phosphorylation were also evaluated with the peptide array. The phosphorylation ratio was saturated at approximately 20% in 2 h in this study.

Mass-tag technology for monitoring of protein kinase activity using mass spectrometry.

Monitoring of intracellular protein kinase activity is very important for fields involving diagnosis and drug screening. However, current methods, such as radiometry using (32)P, or ELISA, are laborious and time-consuming. We have developed high-throughput assay system of protein kinase activity using mass-tagged substrate peptide probes and mass spectrometry. This assay system can easily evaluate target kinase activity and will potentially be able to simultaneously profile many protein kinase activities.

Intracellular signal-responsive artificial gene regulation for novel gene delivery.

We describe two types of artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) or caspase-3. These molecular systems use newly synthesized cationic polymers, PAK and PAC. The PAK polymer includes substrate oligopeptide for PKA, ARRASLG, as receptor of PKA signal, while the PAC polymer possesses oligopeptide that is comprised of a substrate sequence of caspase-3, DEVD, and a cationic oligolysine, KKKKKK. These polymers formed stable complexes with DNA to totally suppress the gene expression. However, PKA or caspase-3 signal disintegrates the PAK-DNA or the PAC-DNA complex, respectively. This liberates the DNA and activated the gene expression. These systems are the first concept of an intracellular signal-responsive gene-regulation system using artificial polymer. We expect that these systems can be applied to the novel highly cell specific gene delivery strategy that is involved in our previously proposed new drug delivery concept, the drug delivery system based on responses to cellular signals.