Introduction. We have developed the comprehensive analysis technology for the phosphorylation using the peptide array, especially in

Transcription

1 Introduction We have developed the comprehensive analysis technology for the on-chip phosphorylation using the peptide array, especially in surface plasmon resonance (SR) imaging or autoradiography. In this study, we made a detection and quantification system for on-chip phosphorylation of peptides by SR imaging techniques. The selection of the phosphate capture molecule was important. The conventional method for phosphorylation detection, (e.g., anti- phospho-ser/thr antibody) was not sufficient for both the specificity and sensitivity. Furthermore, we supposed to apply this technology to drug discovery, especially to the screening of protein kinase inhibitors. We examined the inhibition assay system for the on-chip phosphorylation using protein kinase inhibitors. MultiSRinter Surface lasmon Resonance Imaging System R SR Imaging Instrument Automated Spotter Multiple spots Surface lasmon Resonance Interaction

2 Aim of ur Study Detection of n-chip hosphorylated eptides for Comprehensive Analysis of rotein Kinase Activity Using SR Imaging or Autoradiography hosphorylation Immobilization of eptide robes as a rotein Kinase Substrate on the Chip Application for Drug Discovery

3 MultiSRinter White Light Source rinciple of SR Imaging Technique rism R (TYB) CCD Camera SR Image Au-Chip Analyte Ligand Continuous flow 6 6 Incidence Angle () Label-Free, Real-Time and Multiple Interaction Analysis Reflected Light Intensity %R elson, B.. et al., Anal. Chem. 999, 7,, Brockman, J. M. et al., Annu.. Rev. hys. Chem.,,,

4 Cys Strategy of n-chip hosphorylation Detection of Immobilized Substrate eptide Scheme A Biotinylated Zn L 3+ Streptavidin (SA) KA or csrc AT Cys Cys Cys Au Scheme B [- 3 ]AT for RI assay Autoradiography (RI Assay) H H Zn + hosphate Binding Site S H H H H Zn + - SR Imaging (on-labeled Assay) hos-tag BTL-4 We would like to thank rof. Koike and rof. Kinoshita in Hiroshima University who developed the biotinylated zinc(ii) complex. E. Kinoshita et al., Dalton Trans. 4, 8,,

5 Surface Chemistry of eptide Array EG-SH hotopattern 8-AT Au EG UV Irradiation HS (CH CH ) 3 H 8-amino--octanethiol, octanethiol, EG 3 -H alkanethiol hydrochloride Au Au EG Au Au H SH SSMCC Cysteine Terminated eptide H H EG H H Mal Mal EG Mal Mal EG - 3 S C CH Sulfosuccinimidyl-4-(-maleimidomethyl) maleimidomethyl) cyclohexane--carboxalate carboxalate Automated Spotter M. Kyo et al., Genes Cells. 4,9,, Au EG H Mal eptide solution was spotted on the gold chip. Bare Gold EG Surface (hydrophilic) Amino Group Introduction Maleimido Group Introduction eptide Immobilization

6 Detection of n-chip hosphorylation of Immobilized eptide robes by KA KA hosphorylation for 4 hours at 3 robe robe robe 3 robe 4 robe Kinase KA KA KA csrc csrc eptide sequence CGGLRRA LRRASLG-H CGGLRRA LRRAALG-H CGGLRRA LRRAS( 3 ) LG-H CGIYG G EFKKK-H CGIY( 3 ) G EFKKK-H Detection of on-chip phosphorylation of immobilized peptide by KA. SR difference image (A) and signal change (B) by Zn L 3+ pretreatment and streptavidin exposure on the on- chip phosphorylated peptide array. (C) Autoradiography image of the peptide array, which was on-chip phosphorylated in the presence of [ - 3 ]-AT. (D) The array pattern of the immobilized peptide probes. K. Inamori et al., Anal. Chem., 77,,

7 Quantification of n-chip hosphorylation Efficiency A B A B C D E F G H A B - C D E F G H C Ratio of SR Signal [%] Mixture Ratio [%] We fabricated a peptide array on which a mixture of %-phosphorylated and %-phosphorylated peptides was immobilized in various mixing ratios.. We hypothesized d that the SR signal ratio corresponds to the phosphorylation ratio on the chip. the intensity of the SR signal was saturated at approximately 4% phosphorylation. K. Inamori et al., Anal. Chem., 77,,

8 hosphorylation Kinetic Study of Immobilized eptide robe hosphorylation Ratio [%] KA Reaction Time [min] The on-chip phosphorylation ratio was saturated at around % in hour. The mass spectra revealed that phosphorylation ratio became 8 % at 3 min. This means that the phosphorylation rate on chip was much lower than that in solution. K. Inamori et al., Anal. Chem., 77,,

9 Examples of rotein Kinase Inhibitors rotein Kinase A Inhibitor Amide -4 Inhibition by Blocking the Substrate Binding Site rotein Kinase A Inhibitor: H-89H -[-(p-bromocinnamylamino)ethyl] Bromocinnamylamino)ethyl]--isoquinolinesulfonamideisoquinolinesulfonamide S H(CH ) HCH CC Br Inhibition by Blocking the AT Binding Site Thr-Thr Thr-Tyr-Ala-Asp-he-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg- Arg-Asn Asn-Ala-Ile-His-Asp rotein Kinase C Inhibitor: Gö6983 G -[-(3-dimethylaminopropyl) dimethylaminopropyl)--methoxyindol-3-yl] yl]-3- (H-indol indol-3-yl) maleimide 9

10 Inhibition Assay for n-chip KA hosphorylation Using eptide Inhibitor Amide -4 hosphorylation by KA for 4 hours at 3 Amide -4 SR Imaging M A B C D E F G H Autoradiography A B C D E F G H Array attern A B C D E F G H M 6 M 4 M robe robe robe 3 robe 4 robe Kinase KA KA KA csrc csrc eptide sequence CGGLRRASLG-H CGGLRRAALG-H CGGLRRAS( 3 ) LG-H CGIYG EFKKK-H CGIY( 3 ) G EFKKK-H

11 Effect of Length of eptide Inhibitor on Inhibition Efficiency of n-chip KA hosphorylation rotein Kinase A Inhibitor Amide -4 Thr-Thr Thr-Tyr-Ala-Asp-he-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp 6- Thr-Tyr Tyr-Ala-Asp-he-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile 4- Gly-Arg Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile Autoradiography min at 3 SR 6min at 3 Ratio of 33 Uptake % Ratio of SR Signal % E-.E-.E-8.E-6.E-4 KA Inhibitor Amide concentration (M) Amide-4 Amide6- Amide4-.E-.E-.E-8.E-6.E-4 KA Inhibitor Amide concentration (M) Amide-4 Amide6- Amide4- The inhibition effect was changed with the amino acid sequence. The amide -4 showed the strongest inhibition efficiency.

12 Effect of AT Concentration on Inhibition of n-chip KA hosphorylation by H-89H Autoradiography min at 3 SR 6min at 3 Ratio of 33 Uptake % Ratio of SR Signal %.E-.E-.E-8.E-6.E-4 H-89 concentration (M).E-.E-.E-8.E-6.E-4.E- H-89 concentration (M) µm AT µm AT µm AT Compared with peptide inhibitor, amide -4 in the same molar, the inhibition effect by H-89 was stronger. In SR analysis, the inhibition effect by H-89 H was influenced by the AT concentration in the reaction.

13 Relative Fluorecence Units Inhibition Analysis for KA hosphorylation in Solution by Superquenching-based Universal Assay Substate eptide Quencher QTL Sensor M III QTL Sensor: fluorescent.e-.e-.e-.e-9.e-8.e-7.e-6.e-.e-4 KA Inhibitor Amide conc. (M) Amide -4 Amide 6- Amide 4- Kinase hosphatase Relative fluorecence Units hosphate Group M III hosphate Group Binding Carrier Fluorescence quenched.e-.e-.e-.e-9.e-8.e-7.e-6.e-.e-4 KA Inhibitor H-89 conc. (M) AT µm AT µm AT µm As the results of superquenching-based universal assay in solution condition, the similar tendency with immobilized peptide for the inhibition effect was shown. 3

14 n-chip Inhibition Assay for KA/KC KC Reaction () Autoradiography KA KC SR Imaging KA KC Control Control Amide-4 H-89 Gö6983 Amide-4 H-89 Gö6983 : CGGLSRTLSV : CGGLARALAV 3: CGGLRRTLSVA 4: CGGLRRALAVA : CGGLRRASLG LG 6: CGGLRRAALG LG : CGGLRRASLG LG : CGGLRRAALG LG 3: CGGLRRApS pslg 4: CGGLRRTLSVA :CGGLRRALAVA 6: blank Inhibitor Concentration: - 6 M hosphorylation: : 6min at 3 4

15 n-chip Inhibition Assay for KA/KC KC Reaction () Autoradiography SR Imaging KA Inhibition Efficiency (%) CGGLRRTLSVA CGGLRRASLG Inhibition Efficiency (%) CGGLRRTLSVA CGGLRRASLG Amide-4 H-89 Gö6983 Amide-4 H-89 Gö6983 KC Inhibition Efficiency (%) CGGLRRTLSVA CGGLRRASLG Inhibition Efficiency (%) CGGLRRTLSVA CGGLRRASLG Amide-4 H-89 Gö6983 Amide-4 H-89 Gö6983

16 Conclusions We have developed the comprehensive analysis technology for the on-chip phosphorylation using the peptide array, especially in surface plasmon resonance (SR) imaging or autoradiography. A detection and quantification system for on-chip phosphorylation of peptides by SR imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(ii) ) complex) was established. KA phosphorylation ratio on the chipwas saturated at around % in hour. Using commercially available protein kinase inhibitors, the inhibition effect of the on-chip phosphorylation by KA or KCd in the immobilized peptides was detected by SR imaging and autoradiography analysis. Three inhibitors, -4 amide. H-89, H and Gö6983 G were compared in the inhibition of KA and KCd reaction. The selectivity of inhibition effect by each inhibitor r was shown in SR imaging and autoradiography. The inhibition effect by peptide inhibitor, -4 amide was changed with the amino acid sequence. In the small-molecule molecule inhibitor, H-89, H the inhibition effect was influenced by the AT concentration in the phosphorylation reaction. As the results of the assay in the solution condition, the same tendency y was verified. We propose that our detection system for the on-chip phosphorylation is very valuable for the screening of a new drug, such as a protein kinase inhibitor. Corresponding to: Kazuki Inamori Kazuki_inamori@bio.toyobo.co.jp This work was supported by project committed from ew Energy and Industrial Technology Development rganization (ED). 6