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.

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.

Molecular evidence of the involvement of the nucleotide excision repair (NER) system in the repair of the mono(ADP-ribosyl)ated DNA adduct produced by pierisin-1, an apoptosis-inducing protein from the cabbage butterfly.

Pierisin-1 is a potent apoptosis-inducing protein found in the pupal extract of the cabbage white butterfly. Pierisin-1 catalyzes the mono(ADP-ribosyl)ation of the 2'-deoxyguanosine residue and produces a bulky adduct, N2-(ADP-ribos-1-yl)-2'-deoxyguanosine (N2-ADPR-dG) in DNA. Here, we examined the involvement of the nucleotide excision repair (NER) system in the removal of N2-ADPR-dG in Escherichia coli (E. coli) and human cells. The results of mobility shift gel electrophoresis assays using a 50-mer oligodeoxynucleotide containing a single N2-ADPR-dG showed that E. coli UvrAB proteins bound to the N2-ADPR-dG in vitro. Incubation of the adducted oligodeoxynucleotides with UvrABC resulted in the incision of the oligonucleotides in vitro. The results of filter binding and gel mobility shift assays using human XPA protein showed that XPA bound to DNA containing N2-ADPR-dGs in vitro. Finally, we introduced plasmids containing N2-ADPR-dGs into E. coli and human cells. N2-ADPR-adducted plasmids replicated l0 times and 20 times less efficiently in NER-deficient E. coli and human cells than in their wild-type counterparts, respectively. More mutations were induced in the plasmid propagated in NER-deficient cells than that in wild-type human cells. These results indicate the involvement of the NER system in the repair of N2-ADPR-dG in both E. coli and human cells.

Detection of genistein as an estrogenic contaminant of river water in Osaka.

The estrogenic activity in water at various localities on Lake Biwa-Yodo River, a representative watershed in Japan, was measured using a recombinant yeast that expresses the human estrogen receptor. The yeast bioassay revealed that the activities of 13 water samples had an average value of 14 pmol/L (3.8 ng/L) (17beta-estradiol equivalent) with a very wide range from 0 to 72 pmol/L (0-19.6 ng/ L), and two of the samples had prominent levels of activity (72 pmol/L (19.6 ng/L) and 56 pmol/L (15.2 ng/L)). We analyzed these two samples with instrumental approaches. A high-performance liquid chromatogram profile showed that the strong activity in one sample, which was collected just downstream of a sewage-treatment plant, would be due to 17beta-estradiol and estrone, whose source is considered to be human urine contained in the effluent of the plant. The activity in the other sample, which was obtained from a tributary river in a primarily residential area with some industrial development (i.e., Osaka City), however, did not correspond to 17beta-estradiol, estrone, or synthetic chemicals known as estrogenic. Analysis of a fraction with estrogenic activity by liquid chromatography-mass spectrometry (LC-MS) provided evidence that the activity in the water sample resulted from the presence of genistein, an isoflavone compound of plant origin.

Analysis of HPRT and supF mutations caused by pierisin-1, a guanine specific ADP-ribosylating toxin derived from the cabbage butterfly.

Pierisin-1, an ADP-ribosylating toxin derived from the cabbage butterfly, Pieris rapae, induces apoptosis in various mammalian cell lines. We recently reported that the target for ADP ribosylation by pierisin-1 is the 2'-deoxyguanosine residue in DNA. To examine whether pierisin-1 would induce mutations in mammalian cell genes, we conducted a mutational analysis for the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in pierisin-1-treated Chinese hamster lung (CHL) cells. N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine was detected by the (32)P-postlabeling method in CHL cells after treatment with pierisin-1 at doses of 2-32 ng/mL; adduct levels were 1.1-12.0 per 10(6) nucleotides. Pierisin-1 induced mutations in the HPRT gene dose-dependently, and the frequency was 38 times higher than the control, at a dose of 32 ng/mL. To confirm that mono(ADP-ribosyl)ated dG itself leads to mutations, the pierisin-1-treated DNA of plasmid pMY189 bearing the supF gene was used for mutational analysis. The mutation frequency of the supF gene treated with 2-8 micro g/mL of pierisin-1 was 17-40-fold the control value. Mutation spectrum analysis showed that single base substitutions dominated in both HPRT and supF genes. Among these, transversions were predominant, and more than 70% of the base substitutions occurred at G:C base pairs in both genes. The most frequent mutations were G:C to C:G, followed by G:C to T:A in HPRT gene, whereas G:C to T:A transversions dominated in the supF gene. Our results indicate that pierisin-1 produced N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine and this guanine-adduct could lead to mutations in the HPRT and supF genes. These findings could provide very useful information for understanding the biological significance of pierisin-1.