Ubiquitination assay

Abstract

The present application relates to a method of assaying ubiquitination in a sample by combining ubiquitin together with a substrate in a sample containing UBE1, UbcH3, Skp2-isoform 1, Skp1, Cull, Rbx1, Cks1, CDK2 and Cyclin E1 under conditions suitable for ubiquitination to take place, exposing the sample to a labelled binding partner which is specific for the ubiquitin, and measuring the amount of ubiquitin bound to the substrate.

Claims

1. A method of assaying ubiquitination in a sample comprising: (a) combining ubiquitin together with a substrate in a sample under conditions suitable for ubiquitination to take place and in the presence of a solid surface, wherein the sample includes the following components: UBE1; UbcH3; Skp2-isoform 1; Skp1; Cul1; Rbx1; Cks1; CDK2; and Cyclin E1, wherein each of the components and the substrate comprise an immobilisation tag which facilitates immobilisation of the substrate or component onto the solid surface, wherein each immobilisation tag is different, and wherein the Skp2-isoform 1 has the sequence comprising SEQ ID NO:3; (b) exposing the sample to a labeled binding partner which is specific for the ubiquitin; and (c) measuring the amount of labeled ubiquitin bound to any one of the substrate and components in the sample; wherein step (c) is performed two or more times in a single assay in order to measure the amount of labeled ubiquitin bound to two or more of the substrate and components in the sample, wherein each of the measured substrate and measured components are separated by being immobilized on the solid surface and the level of ubiquitination of two or more of the substrate and components can be measured simultaneously and/or sequentially to determine the amount of labeled ubiquitin bound to two or more of the measured substrate and measured components in the sample without changing the assay composition.

2. The method according to claim 1, wherein step (a) further comprises combining a potential modulator of ubiquitination.

3. The method according to claim 1, wherein the substrate, CDK2 and Cyclin E1 are combined prior to combination with the other components of (a).

4. The method of claim 1, wherein the substrate is P27.

5. The method of claim 1, wherein the UbcH3 is present at a final concentration between 750-1250 nM.

6. The method of claim 1, wherein measuring the amount of labeled ubiquitin bound to the substrate comprises Western blot analysis, Homogenous Time Resolved Fluorescence (HTRF), or electrochemiluminescence (ECL).

7. The method according to claim 1, wherein the UBE1 is added to a final concentration of 3-7 nM.

8. The method according to claim 1, wherein the Cks1 is added to a final concentration of 20-30 nM.

9. The method according to claim 1, wherein the CDK2 is added to a final concentration of 20-30 nM.

10. The method according to claim 1, wherein the Cyclin E1 is added to a final concentration of 20-30 nM.

11. The method according to claim 1, wherein the ubiquitin comprises a tag.

12. The method according to claim 11, wherein the ubiquitin tag is capable of interacting with the labeled binding partner to produce a detectable signal.

13. The method according to claim 12, wherein the ubiquitin tag and labeled binding partner form a FRET pair.

14. The method according to claim 1, wherein any one of the components and/or the substrate of (a) are immobilised on the solid surface.

15. The method according to claim 14, wherein the solid surface is an electrode.

16. The method according to claim 14 which comprises an additional wash step, (b), which occurs between steps (b) and (c) and removes unbound components or substrate from the solid surface.

17. The method according to claim 1, wherein the Skp2 isoform 1, Skp1, Cul1 and Rbx1 are combined prior to combination with the other components of (a).

18. The method according to claim 17, wherein the Skp2-isoform 1, Skp1, Cul1 and Rbx1 are added as a tetramer to a final concentration of 20-30 nM.

19. The method according to claim 1, wherein step (a) further comprises combining ATP.

20. The method according to claim 19, wherein the ATP is added to a final concentration of 75-125 M.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: List of complexes, proteins, manufacturing names, expression systems and accession numbers used throughout this study.

(2) FIG. 2: Ubiquitin biotinylation. Success of biotinylation and optimal conditions determined by ELISA (Streptavidin plate, anti-Ub primary antibody (R+D Systems, MAB701, 0.1-2 g/ml), anti species-HRP secondary (Sigma, A5278, 1 g/ml)

(3) FIG. 3: Ube1 and Ube1/UbcH3-Gel based. Incubated E1/Ub/ATP and E1/E2/Ub/ATP (100 hr incubation) tested using SDS PAGE gels. A band shift indicative of the presence of Ubiquitinated Ube1 has been observed in both E1 and E1/E2 assays. No band associated with Ubiquitinated UbcH3 is visible. This band shift is ATP dependent. Antibody used: Mono- and poly-Ubiquitinated conjugates, mouse mAb horseradish peroxidise conjugate (FK2H) Enzo Life Sciences, Cat no. PW0150, Batch no. X08036, supplied at 1 mg/ml, used at 1/1000 dilution.

(4) FIG. 4: Pre-incubated Ube1/UbcH3 HTRF. Pre-incubated E1/Ub/ATP and E1/E2/Ub/ATP (20, 40 and 100 hour incubation) tested using HTRF platform. Anti-6His-K was employed for E1 and E2 detection; Anti-FLAG-K for E2; Biotinylated Ub; and Streptavidin XLent! for biotin. FRET signal indicative of the presence of Ubiquitinated Ube1 and UbcH3 has been observed. This signal is enzyme and ATP dependent for the E1/E2 assays, as well as time dependent for the E1 assay.

(5) FIG. 5: Live Ube1 assay (60 minutes incubation)HTRF. Live assays (60 minute incubation) for Ube1 at higher concentrations (>250 nM) show a good signal which is dependent on the Ub-biotin concentration. Anti-6His-K was employed for E1 detection; biotinylated Ub; and Streptavidin XLent! for Biotin. At lower E1 concentrations, the excess of Ub-biotin inhibits the detection of a signal.

(6) FIG. 6: Pre-incubated Ube1/UbcH3-ECL. Pre-incubated E1/Ub/ATP and E1/E2/Ub/ATP (100 hour incubation) tested using ECL platform. Used Anti-His capture for E1 (Millipore, 16-255, 1/500-1/4000 dil); anti-FLAG capture for E2 (Millipore, MAB3118, 1/500-1/4000); biotinylated Ub; and streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Signal of the presence of ubiquitinated Ube1 and HbcH3 has been observed.

(7) FIG. 7: Live Ube1 assay (60 minutes inc.)ECL. Initial live assays (60 minute incubation) for Ube1 show good signal (>1000000) but very high background level (>250000) (Max signal/background 4-5 fold). Subsequent Ubiquitin only controls indicates that at elevated concentrations of ubiquitin, there is non-specific ubiquitin binding and consequently high background.

(8) FIG. 8: Live Ube1 assay (60 minutes inc.)ECL. Live assays (60 minute incubation) for Ube1 at lower concentrations of Ub (2 M) still show good signal (>1000000) and lower background (<10000) (Max signal/background 100 fold). Observed lag in signal increase with increasing Ube1 concentration. Signal is low at low concentrations of Ube1 (<25 mM). Approximately linear increase in signal after 25 nM.

(9) FIG. 9: ATP and Ubiquitin titration (Ube1 Assay)ECL. Live Ube1 assay E1/Ub/ATP, varying ATP and Ubiquitin concentrations. Uses Anti-His capture for E1 (Millipore, 16-255, 1/1000 dil); 100 nM Ube1; biotinylated Ub; streptavidin Sulfo tag (MSD, R32AD-1, 1 g/ml). Saturating ubiquitin and ATP conditions2 M Ub/500 M ATP.

(10) FIG. 10: Ube1 timecourseECL. E1 assay appears to reach completion after a very short time period. Impossible to monitor in current format. Could reduce the amounts of ATP/Ub or decrease reaction temperature to see reaction progress.

(11) FIG. 11: Ube1 inhibitor study PYR41ECL. Inhibitor study. Titration of PYR41 (Calbiochem 662105) from 30004 to 10 nM. Used anti-His capture for E1 (Millipore, 16-255, 1/1000 dil); 50 nM E1; 2 M Ub; 500 M ATP; 30-minute pre-incubation with cpd.; 30 minute reaction time. IC.sub.50 of 1.3 MSimilar to literature IC.sub.50 value: BiogenNova quote IC.sub.50 value as 5 M.

(12) FIG. 12 (a): Pre-incubated UbH3-ECL. Used pre-incubated His-E1/FLAG, c-Myc, HA-E2/Ub/ATP (24 hour incubation) and was tested using ECL platform. Used Anti-FLAG, c-Myc and HA capture for E2 (Millipore, MAB3118 (FLAG), 05274 (c-Myc), 05-904 (HA), all 1/500-1/4000); biotinylated Ub (1 M); ATP 500 M; E1:E2 pre inc. 1 M:1 M; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Signal indicative of the presence of Ubiquitinated UbcH3 has been observed.

(13) FIG. 12 (b): Live UbcH3ECL. Used Anti-HA capture for E2 (Millipore, 05-904(HA), 1/500); 12.5 to 200 nM E1; 6.25 to 200 mM E2; 2 M Ub; 500 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Initial live assays (60 minute incubation) for UbcH3 show good signal (>1500000) at high E1 concentrations. Max signal appears to be dependent on the amount of E1 in the assay. HA and c-Myc UbcH3's perform better than the FLAG version.

(14) FIG. 13: Live UbcH3 AssayECL. Used Anti-HA or Anti-c-Myc capture for E2 (Millipore, 05-904(HA), 1/500) (Millipore, 05-274 (c-Myc), 1/500); 3.125 to 25 nM E1; 6.25 to 200 mM E2; 2 M Ub; 500 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Some indication that non-specific binding of Ube1 leads to high signal. Live UbcH3 assay repeated using low concentrations of Ube1.

(15) FIG. 14 (a) Pre-incubated SCF.sup.skp2ECL. Used pre-incubated E1/E2/E3/p27/Ub/ATP (24 hour incubation) tested using ECL platform. Used anti-FLAG capture for p27 (Millipore, MAB3118 (FLAG), 1/500 to 1/4000); E1:E2:E3 based on Xu publication (40 nM E1:5 M E2:40 nM E3; biotinylated Ub (10 m), 500 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Signal indicative of the presence of Ubiquitinated p27 has been observed.

(16) FIG. 14 (b) Live SCF.sup.skp2ECL (b). Used live E1/E21E3/p27/Ub/ATP (60 minute incubation) tested using ECL platform. Used c-Myc and HA capture for p27 (05274 (c-Myc), 05-904 (HA), 1/500); 5 nM E1; 4 to 1000 nM E2 (HA or c-Myc); 50 nM E3; 3.125 to 100 nM p27; 2 M Ub; 500 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Signal indicative of the presence of ubiquitinated p27 has been observed.

(17) FIG. 15: Ube1 and Ube1+UbcH3 assay timecourseHTRF. E1 assay: 1 M NiNTA Ube1+1 M Ub+2 mM ATP. E1+E2 assay: 1 M NiNTA Ube1+1 M Flag UbcH3+1 M Ub+2 mM ATP. Both the E1 and E1+E2 assays appear to reach completion after a very short time period, which is in line with the ECL data. Used Anti-6HisK for E1+E2 detection; Anti-FLAG-K for E2; biotinylated Ub; streptavidin XLent! for biotin. Impossible to monitor in current format. Samples were diluted to 10 nM E1 in 1 reaction buffer (RB).

(18) FIG. 16: E2 and ubiquitin titration (Ube1+UbcH3 assay)HTRF. Live Ube1+UbcH3 assay100 nM E1/varying [E2]/200 nM Ub/2 mM ATP. 60 minute room temperature incubation. Live Ube1+UbcH3 assay 100 nM E1/1 M E2/varying [Ub]/2 mM ATP, 60 min room temperature incubation. Samples diluted to 10 nM E1 in 1 reaction buffer. Used Anti-6His-K for E1+E2 detection; anti FLAG K for E2 detection; biotinylated Ub; streptavidin XLent! for biotin. Optimal E1, E2 and Ubiquitin conditions are 100 nM Ube1/1 M UbcH3/400 nM Ub.

(19) FIG. 17: Ube1 and UbcH3 construct comparison (E1+E2 assay)HTRF. Live Ube1 and UbcH3 assay: E1/E2/Ub/2 mM ATP, varying E1 and E2 constructs at: 100 nM E1+1 M E2+400 nM Ub. Used anti-6His-K for E1+E2 detection; anti FLAG-K, anti HA-K or anti cMyc-K for E2 detection; biotinylated Ub; streptavidin XLent! for biotin. NiNTA-Ube1 and HA-UbcH3 in a ratio of 100 nM E1+1 M E2+400 nM Ub assayed at 2 mM ATP; and diluted 10-fold gives the best FRET-signal.

(20) FIG. 18: Ube1 Inhibitor study PYR41 in E1 and E1+E2 assay HTRF. Inhibitor study: 30 minutes pre-incubation with PYR-41. Assays were immediately diluted followed by addition of 2 mM ATP. Titration of PYR-41 (Calbiochem 662105) from 300 M to 0.01 M, IC.sub.50=43 ME1 (1 M E1, 2 M Ub (Anti-6His-K). IC.sub.50=16 M E1/E2 (100 nM E1, 1 M E2, 400 nM Ub (Anti-6-HIS-K)) IC.sub.50=20 M E1/E2 (as above Anti-FLAG-K). BiogenNova quote IC.sub.50 value as 5 M.

(21) FIG. 19: Western blotAnti phospho p27E3 assay. Incubated E1/E2/E3/p27Ub/ATP, Anti-phospho-p27 antibody (Millipore 06-996 1/1000). Anti species HRP (Cell Signalling #7074 1/1000). Definite shift in the position of the phospho-p27 band consistent with the poly-ubiquitination of p27.

(22) FIG. 20: Western blotAnti Phospho p27. Incubated FLAG-p27/CDK2/cyclinE with CDK2/cyclinE (0.1:0.1 mg/ml) and Mg-ATP (2 M), anti phospho-p27 antibody (Millipore 06-996 1/1000). Anti species HRP (Cell signalling #7074 1/1000). Incubation of p27/CDK2/CyclinE with CDK2/cyclinE and ATP leads to a significant increase in the amount of phospho p27 as detected by Western blot. Max. signal is seen even after 20 minutes.

(23) FIG. 21: Live SCF.sup.Skp2/Cks1 AssayECL. Live E1/E2/E3/p27/Ub/ATP (60 minute incubation) tested using ECL platform. No FLAG tagged E2/p27 due to problems encountered in E2 assay. Used c-Myc and HA capture for p27 (Millipore 05-274 (c-Myc), 05-904(HA), 1.500); 5 nM E1; 4 to 1000 nM E2 (HA or c-Myc); 50 nM E3/Cks1; 3.125 to 100 nM p27 (1-IA of c-Myc); 2 M Ub; 500 M ATP; streptavidin sulfo tag (MSD, R32AD-1 1 g/ml). Signal indicative of the presence of Ubiquitinated p27 has been observed. c-Myc tagged p27 gives significantly higher signal. Optimum signal at 50 nM p27, 1 M E2.

(24) FIG. 22: Live SCF.sup.Skp2/Cks1 AssayECL E1 vs. E2 titration. Used 60 minute incubation; 25 nM c-Myc p27; anti c-Myc capture for p27 (Millipore 05-274, 1/500); 0.78 to 50 nM E1; 15 to 1000 nM E2 (HA); 25 nM E3/Cks1; 2 M Ub; 500 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). E1hyperbolic increase in signal with [E1]. Optimum signal after 25 nM. However, optimum signal/background at 6.25 nM E1. E2linear increase in signal with [E2]. Final assay concentration set at 1 M.

(25) FIG. 23 (a): Live SCF.sup.Skp2/Cks1 AssayECL ubiquitin titration. Used 60 minute incubation; 25 nM c-Myc p27; anti c-Myc capture for p27 (Millipore 05-274, 1/500); 5 nM E1; 1 M E2 (HA); 25 nM E3/Cks1; 5 to 0.08 M Ub; 1000 to 15 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Hyperbolic increase in signal with [Ub]. Optimum signal after 2 M ubiquitin. Largely dependent of ATP concentration. K.sub.m values between 0.46 and 0.58 M. Final assay concentration is set at 2 M.

(26) FIG. 23 (b): Live SCF.sup.Skp2/Cks1 AssayECL ATP titration. Used 60 minute incubation; 25 nM c-Myc p27; anti c-Myc capture for p27 (Millipore 05-274, 1/500); 5 nM E1; 1 M E2 (HA); 25 nM E3/Cks1; 2 M Ub; 1000 to 7 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Initial experiments indicated a high signal which was independent of ATP concentration (lowest [ATP] of 21 M) indicative of tight binding. Repeat experiment showed similar profile with only a moderate decrease at 7 M ATP. K.sub.m estimated at 4 M. Final assay concentration set at 100 M.

(27) FIG. 24: Live SCF.sup.Skp2/Cks1 AssayECL E3 tetramer/Cks1 titration. Used 60 minute incubation; 25 nM c-Myc p27; anti c-Myc capture for p27 (Millipore 05-274, 1/500); 5 nM E1; 1 M E2 (HA); 3 to 100 nM E3/Cks1; 2 M Ub; 100 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Hyperbolic increase in signal with [E3/Cks1]. Optimum signal at 50 nM E3/Cks1. Re-plotting the data between 0 and 25 nM E3/Cks1 indicates a linear relationship. Final assay concentration set at 25 nM.

(28) FIG. 25: Live SCF.sup.Skp2/Cks1 AssayECL Cks1 titrationFIXED E3 tetramer. Used 60 minute incubation; 25 nM c-Myc p27; anti c-Myc capture for p27 (Millipore 05-274, 1/500); 5 nM E1; 1 M E2 (HA); 25 nM E3 tetramer; 3 to 100 nM Cks1; 2 M Ub; 100 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Linear increase in signal with [Cks1] up to 50 nM followed by plateau. Optimum signal at 50 nM Cks1. Final assay concentration set at 25 nM.

(29) FIG. 26: Live SCF.sup.Skp2/Cks1 AssayECL Skp1/Skp2 vs Cul1/Rbx1 titration. Live E1/E2/E3/p27/Ub/ATP (60 minute incubation) tested using ECL platform. Co-expressions of Skp1/Skp2 and Cul1/Rbx1 instead of tetramer. Used c-Myc and HA capture for p27 (Millipore 05-274 (c-Myc), 05-904 (HA), 1/500); 5 nM E1; 1 M E2 (HA or c-Myc), 25 mM Cks1; 25 nM p27; 2 M Ub; 100 M ATP; 50 to 1.5 nM Skp1/Skp2; 50 to 3.1 nM Cul1/Rbx1; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Optimum signal at 50 nM Cul1/Rbx1 and 25 nM Skp1/Skp2.

(30) FIG. 27: Comparison of the Skp1/Skp2 and Cul1/Rbx1 co-expressions with the Skp1/Skp2/Cul1/Rbx1 tetramer. Indicates very little difference in max signal strength.

(31) FIG. 28: Live SCF.sup.Skp2/Cks1 AssayECL time course. Used 10 to 100 minute incubation. 25 nM c-Myc p27; anti c-Myc capture for p27 (Millipore 05-274, 1/500); 5 nM E1; 1 M E2 (HA); 25 nM E3 tetramer; 25 nM Cks1; 2 M Ub; 100 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Linear increase in signal with time up to 100 minutes. Experiment repeated to check reproducibility. Comparable signal strength/profile seen for both experiments. High y-intercept may be due to stop method. Final reaction time set at 60 minutes.

(32) FIG. 29: Live SCF.sup.Skp2/Cks1 AssayECL Z. Used 60 minute incubation; 25 nM cMyc p27; anti c-Myc capture for p27 (Millipore 05-274, 1/500); 5 nM E1; 1 M E2 (HA); 25 nM E3 tetramer; 25 nM Cks 1; 2 M Ub; 100 M ATP; Streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). 1 sector of positive controls with one sector of negative (no Ubiquitin) controls. Total of 3 independent experiments. Signal strength, signal/background ratio, positive control % CV and Z all within acceptable limits. Some variation in signal strength.

(33) FIG. 30 (a): Live SCF.sup.Skp2/Cks1 AssayECL Inhibitor studyPYR-41 (E1 inhibitor). Used 60 minute incubation; 25 nM c-Myc p27 (phosphorylated in the absence of inhibitor); anti c-Myc capture for p27 (Millipore 05-274, 1/500); 5 nM E1; 1 M E2 (HA); 25 nM E3 tetramer; 25 nM Cks1; 2 M Ub; 100 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml; titration of PYR41 (Calbiochem 662105) from 300 M to 10 nM. IC.sub.50 of 55 Msignificantly less potent than quoted IC.sub.50 against the E1 assay only. BiogenNova quote IC.sub.50 value as 5 M vs. E1.

(34) FIG. 30 (b): E1/E2 AssayECL Inhibitor studyPYR-41. Used 60 minute incubation; 2 M Ub; 100 M ATP. For E1 assay (Anti-His capture) 50 nM E1. For E2 assay (Anti HA-capture) 5 nM E1; 50 nM E2. Used streptavidin sulfo tag (MSD R32AD-1, 1 g/ml); titration of PYR41 (Calbiochem 662105) from 300 M to 10 nM. IC.sub.50 values of 7.8 and 3.2 M for the E1 and E2 assays respectively. Significantly more potent than IC.sub.50 against the E3 assay.

(35) FIG. 31: Live SCF.sup.Skp2/Cks1 AssayECL R140 Kinase inhibitor panel (+PYR-41)

(36) FIG. 32: Live SCF.sup.Skp2/Cks1 AssayECL R140 Kinase inhibit or panel. Used 60 minute incubation; 25 nM c-Myc p27; anti-c-Myc capture for p27 (Millipore 05-274, 1/500); 5 nM E1; 1 M E2 (HA); 25 nM E3 tetramer; 25 nM Cks1; 2 M Ub; 100 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Several compounds show up as inhibitorsRottlerin, EGCG, K252c and AG538.

(37) FIG. 33: Live SCF.sup.Skp2/Cks1 AssayECL R140 Kinase inhibitor panel. Comparison of the SCF.sup.Skp2/Cks1 inhibition profile against the CDK2/CyclinE inhibition profile highlights very few similarities.

(38) FIG. 34: Live SCF.sup.Skp2/Cks1 AssayECL follow up IC.sub.50s. Used 60 minute incubation; 25 nM c-Myc p27; anti c-Myc capture for p27 (Millipore 05-274, 1/500); 5 nM E1; 1 M E2 (HA); 25 nM E3 tetramer; 25 nM Cks1; 2 M Ub; 100 M ATP; streptavidin sulfo tag (MSD, R32AD-1, 1 g/ml). Titration of PYR41 (Calbiochem 662105) from 300 M to 10 nM (control). Titration of Rottlerin, EGCG, AG538 and K252c from 300 M to 10 nM. PYR-41 IC.sub.50 of 84 MComparable to previous IC.sub.50 data.

(39) FIG. 35: E1, E2 and E3 assay non-specific binding. E1 assayanti His capture for specific binding and BSA block only for NSB. Clearly some NSB, however the relative portion of NSB drops as the concentration of E1 is reduced. NSB signal in the E3 assay is unlikely to be particularly high due to the low concentration of E1 present (5 nM).

(40) FIG. 36: E1, E2 and E3 assay non-specific binding. E2 assayAnti HA capture for specific binding and BSA block only for NSB. No indication of non-specific binding in the E2 assay.

(41) FIG. 37: E1, E2 and E3 assay non-specific binding. E3 assayanti c-Myc capture for specific binding and BSA block only for NSB. Clearly some NSB, however the relative portion of NSB drops as the concentration of p27 is reduced. Although there is some non-specific binding, it is unlikely to be NSB from the desired captured species.

(42) FIG. 38: E1, E2 and E3 assay non-specific binding. Multiple approaches were employed in order to minimise the E3 assay NSB signal including: Buffer pH; BSA; Salt; detergents; buffers; p27/E3 concentrations; blocking solutions. None were successful.

(43) FIG. 39: Schematic showing a current being passed through an electrode, which in turn causes stimulation of the labelled binding partner, in turn causing a signal to be emitted.

MODES FOR CARRYING OUT THE INVENTION

(44) Coding sequences used throughout this study were cloned and validated against appropriate GenBank entries (FIG. 1).

(45) Molecular weights of purified proteins were confirmed by SDS PAGE followed by Western blot analysis.

(46) Assay Development Pilot: Components Upstream of E3

(47) Initial experiments in the pilot phase focussed on establishing that the components upstream of the E3 are active, and then optimising the levels of each component required so that we limit the variables for the E3 assay.

(48) The majority of assays require the use of biotinylated ubiquitin in order to recruit streptavidin linked reporter molecules. Biotinylation reactions were carried out and assessed by ELISA to determine optimal biotin:ubiquitin ratios (FIG. 2).

(49) Results showed that using a labelling ratio of either 2:1 or 5:1 biotin:ubiquitin provided the best signal by ELISA. Higher ratios were detrimental, and lower ratios look to be inefficient.

(50) Positive Controls of Ubiquitinated E1 and/or E2

(51) Prolonged incubation of Ube1 (E1), UbcH3 (E2) and ubiquitin at ratios of 1:1:20 micromolar were set up to generate a positive control of ubiquitinated E1 and/or E2 which could then be used in establishing detection conditions for the HTRF and ECL platforms. Initial SDS polyacrylamide gel-based examination of ubiquitinated Eland E1/E2 showed that there was a ubiquitination event occurring, with an apparent shift in the molecular weight of E1 (FIG. 3). A decrease in the intensity of the ubiquitin band could also be seen in some samples. The assays were set up with stoichiometric amounts of E1 and E2 and a 20-fold excess of biotinylated ubiquitin. The band shifts were shown to be ATP dependent.

(52) The gels were also probed with an antibody against ubiquitinated species. The antibody does not recognise free ubiquitin. The Western blots for this experiment showed a clear band representing ubiquitinated E1. It was noted at this stage that the intensity of the band for E1 was markedly increased when E2 was present.

(53) Western blot analysis (Anti-mono and polyubiquitinated protein-HRP conjugate) revealed a positive response for Ube1 only in the presence of ATP. This indicates ubiquitination of Ube1. No positive response for UbcH3 was observed in the absence of Ube1. When Ube1 and UbcH3 were added together, we detected a positive response Ube1, which was time dependent.

(54) Developing the Detection MethodologyHTRF

(55) Samples were pre-incubated with equimolar amounts of the E1 and E2, and bio-ubiquitin and a dilution step was adopted to reduce the amount of E1 in the detect step to 10 nM. This resulted in encouraging ratios, indicating that the format was detecting the presence of ubiquitinated E1 and E2

(56) FIG. 4 shows the bell-shaped curve typical of a titration in this format of assay.

(57) The initial experiments were all carried out using prolonged incubation times to try to ensure that the reaction had progressed as far as it was likely to go in order to try to maximise the population of ubiquitinated E1 or E2 (FIG. 4).

(58) Subsequent assays brought the incubation times to a more realistic period. The 60 minute live assay (i.e. not pre-incubating) using a 2-way checkerboard for E1 and bio-ubiquitin showed that the HTRF ratio will form in this time-frame. Samples were again diluted to 10 nM E1 detection concentrations (FIG. 5).

(59) The amount of bio-ubiquitin excess over the E1 concentration is critical, else the signal gets lost in the background.

(60) All HTRF specific reagents were from Cisbio, including:

(61) Streptavidin-Xlent (611 SAXLB), Anti-6His-K (61 HISKLA), Anti-FLAG-K (61FG2KLA), and were used at concentrations specified by the manufacturer.

(62) Developing the MethodologyECL

(63) Unlike the HTRF format, the ECL assay is a wash format assay. The initial experiments using the pre-incubated material of 1:1:20 E1:E2:Bio-Ub at 2 mM ATP showed a signal response dependent on the amount of E1 or E2 present. The experiment enabled the selection of the type of plate (high bind vs low bind) and the selection of capture antibody concentrations for subsequent experiments (FIG. 6). It also indicated that the E2 was becoming ubiquitinated (FIG. 7).

(64) While the assay format is wash based, it has been shown that the concentration of bio-ubiquitin that can be used is not limitless, and will contribute to a significant background. Reducing the amount of bio-ubiquitin in the assay restores the S/B accordingly. An E1 titration gives a good response, and there are hints of sigmoidal behaviour (FIG. 8).

(65) The next steps for this format are the bio-ubiquitin and ATP titrations to establish the concentrations that will be considered non-rate limiting in downstream ubiquitination assays. The E2 also needs to be titrated into the system. There are 3 E2 molecules available, and the best performing one will be selected for the final stage of the pilot study.

(66) Continued Optimisation Using the ECL Assay

(67) Continuing with the E1 protein optimisation, ATP and ubiquitin titrations were performed to identify the conditions which are saturating, or non-rate limiting, for the E1 section of the cascade (FIG. 9).

(68) The results suggest that after 2 micromolar Ubiquitin and at 500 micromolar ATP, there is no additional signal generation. These concentrations have therefore been taken as the saturating conditions for the E1 protein.

(69) Following the identification of the ATP and ubiquitin concentrations to use, a time-course of signal generation was followed, to determine a linear part of the reaction velocity curve to use for subsequent inhibitor experiments (FIG. 10).

(70) What was found was that the reaction appears to happen very quickly, in fact within the dead time of setting up the assay. The fast nature will be an advantage in downstream events with the E2 and E3 assays.

(71) For the IC.sub.50 determination of Pyr-41, the data shown is that from a 30 minute pre-incubation and a 30 minute assay. The assay was performed in a separate plate and then transferred. For information purposes, another set of assays were performed within the capture/detect plate and stopped immediately by washing off the assay solution, or washed after 60 min. It was interesting to note that even with the wash at time 0 an IC.sub.50 for the compound that was in line with the data shown could still be determined.

(72) Reported values for the inhibitor are in the low micromolar range (FIG. 11).

(73) The detection of ubiquitinated E2 was explored, initially using material that had been pre-incubated for 24 hrs. Equimolar E1, E2 and ubiquitin were used for this preliminary experiment, and the concentration of capture antibody determined. The results below are representative, and all 3 E2 constructs were tested.

(74) This experiment was followed up with an E1 vs E2 checkerboard using 2 micromolar ubiquitin and 500 micromolar ATP, concentrations determined to be optimal for the E1 ubiquitination previously. The assay was this time set up in a live format rather than use material from a prolonged incubation (FIG. 12).

(75) Lower concentrations of E1 were tried in an attempt to bring down any background (FIG. 13). The lower range of E1 seemed to retain a decent E2 specific signal and also reduced non-specifics.

(76) The assay for the E3 ubiquitination of p27 was trialled, initially using conditions described in the Xu paper (Xu et al, 2007, JBC, 282 15462-470), and then using conditions identified in-house. The former also included a capture antibody titration for the FLAG-tagged p27 (FIG. 14).

(77) Both experiments demonstrate a signal for the ubiquitinated p27. The latter also indicates that the higher concentrations of p27 may be detrimental.

(78) Continued Optimisation Using the HTRF Format

(79) The progress with the E1 assay was continued by optimising the conditions for generation of signal.

(80) A time course for the assay was examined initially using equimolar concentrations of the E1, E2 and ubiquitin.

(81) Similar to the ECL format, the time-course in the HTRF assay indicates a very fast reaction (FIG. 15).

(82) E2 was titrated against fixed concentrations of Ub and E1. The detection of ubiquitination was configured in two ways, using Anti-6His-K and Anti-FLAG-K. The former should detect the combination of ubiquitinated E1 and E2 as both carry a 6His tag, while the latter will detect the E2 ubiquitination only.

(83) From the titration of E1 vs E2, concentrations of E1 and E2 were chosen for the ubiquitin titration, again detected with two configurations to arrive at conditions shown below for E1, E2, and Ub (100 nM E1, 1000 nM E2, 400 nM Ub) (FIG. 16).

(84) The samples require dilution to a lower concentration than used in the assay, and by using the dilution methodology, workable ratios have been obtained. The diluent itself has also proven to be important, as diluting in the Stop solution, which contains EDTA (120 mM), does reduce the signal window compared to stopping the assay and then diluting in reaction buffer.

(85) The various constructs of E2 and the two different E1 purifications were tested under the conditions derived above to see if some of the variables could be reduced.

(86) The bar chart on the next slide shows the different ratios observed for the E1/E2 and the specific E2 signals. A common dilution for detecting for both the E1 and E2 was identified (1/10 which gives 10 nM E1 in the detection), and it is this shown in FIG. 17.

(87) Using conditions identified previously, an IC.sub.50 determination for Pyr-41 using the E1 and E1/E2 assays was performed. The results show a marked difference in inhibitor potency compared to literature values and that determined in the ECL format. This may be a reflection of the higher enzyme concentrations used in the HTRF assay step (FIG. 18).

(88) Continued Optimisation Using Western Blots

(89) As well as the ECL and HTRF assay formats, material has been examined by using SDS-PAGE and Western blotting to verify the presence (or absence) of ubiquitinated or phosphorylated material (FIG. 19).

(90) The blots above show results from probing the membranes with an anti-phospho-p27 antibody. There is a clear indication that the p27 band has shifted, which could be interpreted as ubiquitination. The different loadings are 10, 20, 30 & 40 microliters of sample from right to left. It should be noted that there was not sufficient sample for the 40 microliter loading (estimated at 20 L), so the signal is reduced from what would be expected.

(91) On the right hand panel, a smear also appears in the No E1 lane. While this could mean that the E1 is not obligate, we have not seen this effect in either of the other assay formats, and so could also be a small contamination during the setup of this assay. Distinct banding can be seen in the lane with all components at 2 micromolar ubiquitin.

(92) FIG. 20 also shows anti-phospho-p27, this time from experiments designed to identify conditions to ensure a good level of phosphorylation which is required for the p27 to be ubiquitinated. It would appear that incubating the p27 with additional CDK2/Cyclin E and ATP for 20 minutes would suffice to ensure good phosphorylation.

(93) ECL FormatE3 Ubiquitin Ligase Assay

(94) Continuing with the E3 assay optimisation, p27 and E2 titrations were carried out using multiple tagging options on the E2 and p27 proteins.

(95) The results indicate that the optimum signal was achieved when pairing the HA-E2 with the c-Myc-p27. This combination was used for all further E3 assay optimisation experiments.

(96) Titrating the p27 against the E2 enzymes indicates an optimal signal strength at 1 M E2 and 50 nM p27 (FIG. 21).

(97) Following the identification of the optimum tag pairing, an E1 vs. E2 checkerboard titration was performed in order to identify the optimum conditions for these assay components (FIG. 22).

(98) E1Hyperbolic increase in signal with increasing E1 concentration with a signal plateau after 25 nM. However, due to the relatively high non-specific signal which originates from the E1 enzyme (LB1805p12-15) the signal to background (No E2 background) ratio was taken into consideration when identifying a final assay concentration for the E1 enzyme in the E3 assay. The maximum signal to background is seen between 3.125 and 12.5 nM E1. Final assay concentration of E1 set at 5 nM.

(99) E2Approximately linear increase in signal with increasing E2 concentration. Unlike the E1 enzyme, non-specific binding is not a problem (LB1805p35-37). Although it appears that the signal will continue to increase with increasing E2 concentration, in the interests of minimising the total amount of protein and the assay costs, the E2 concentration was set at 1 M.

(100) Continuing with the E3 assay component optimization, ubiquitin and ATP titrations were performed to identify saturating, or at least non-rate limiting, conditions (FIGS. 23 (a) and (b)).

(101) For each concentration of ATP tested, a hyperbolic increase in signal with increasing ubiquitin concentration was observed, with a signal plateau after approximately 2 M. Fitting the data to a hyperbolic function generated apparent K.sub.M values for ubiquitin binding at multiple ATP concentrations. All of the apparent K.sub.M values were between 0.46 and 0.58 M ubiquitin.

(102) The initial titration of ATP at multiple concentrations of ubiquitin demonstrated a high signal which was independent of ATP concentration (LB1805p80-83). A repeat of this experiment using lower concentrations of ATP(LB1805p87-89) again showed a signal largely independent of ATP concentration with only a moderate decrease at 7 M ATP. The apparent K.sub.M value was estimated at 4 M.

(103) Final assay concentrations were set at 2 M ubiquitin and 100 M ATP.

(104) Next, the E3 components Skp1/Skp2/Cul1/Rbx1 and Cks1 were titrated together (FIG. 24). The concentration of the E3 tetramer (Skp1/Skp2/Cul1/Rbx1) was set according to the concentration of the component which was least prevalent in the co-expression (Skp2). The Cks1 concentration was set as equimolar to the concentration of the component which was least prevalent in the co-expression (Skp2).

(105) A hyperbolic increase in signal with increasing E3/Cks1 concentration was observed, with a signal plateau after 50 nM. Re-plotting the data from 0 to 25 nM E3/Cks1 indicates a linear relationship between the signal and the E3/Cks1 concentration. Although the maximum signal is observed at 50 nM E3/Cks 1, a final assay concentration of 25 nM was chosen to ensure that the assay was within the linear region.

(106) Finally, the Cks1 component was titrated against a fixed concentration of the E3 tetramer (25 nM). A hyperbolic increase in signal with increasing Cks1 concentration was observed, with a signal plateau after 50 nM. Although the maximum signal is observed at 50 nM Cks1, a final assay concentration of 25 nM was chosen to ensure the assay is within the linear range (FIG. 25).

(107) Table 1 shows the final live SCF.sup.Skp2/Cks1 ECL assay concentrations.

(108) TABLE-US-00001 TABLE 1 Final live SCF.sup.Skp2/Cks1 ECL assay concentrations Final Assay Component Concentration Reference 6His-UBE1-(hu,FL) (E1) 5 nM LB1805p71-73 HA,6His-UbcH3-(hu,FL) (E2) 1 M LB1805p71-73 c-Myc Phospho-p27-(hu,FL) co- 25 nM LB1805p53-57 expressed with UT-CDK2-(hu,FL), 6His-Cyclin E1-(hu,FL), 6His-Skp2-(isoform 1, hu,FL), 25 nM LB1805p84-86 GST-Skp1-(hu,FL), 6His-Cul1- (hu,FL), UT-Rbx1-(hu,FL) (E3 tetramer) 6His-Cks1-(hu,FL) 25 nM LB1805p112-113 ATP 100 M LB1805p87-89 Biotinylated Ubiquitin 2 M LB1805p80-83
Trialling Different E3 Co-expressions

(109) The preceding E3 experiments were all carried out using an E3 comprised of a Skp1/Skp2/Cul1/Rbx1 tetramer alongside Cks1. Several other co-expressions were also available. Both a Skp1/Skp2 and a Cul1/Rbx1 co-expression were available with sufficient purity/yield to be used in an assay. A titration of these 2 components against each other was carried out in comparison to the tetramer co-expression. The concentration of the Skp1/Skp2 or Cul1/Rbx1 was set according to the concentration of the component which was least prevalent. The optimum conditions were seen at 25 nM Skp1/Skp2, 50 nM Cul1/Rbx1 in the presence of 25 nM Cks1. The comparison of the Skp1/Skp2 with Cul1/Rbx1 co-expressions against the Skp1/Skp2/Cul1/Rbx1 tetramer showed a largely similar max signal strength (FIG. 26). As all the previous experiments were carried out using the tetramer it was decided that all remaining assay development should be completed using the tetramer. Depending on the expression levels, yields and purities of the different co-expressions, this approach offers an alternative approach.

(110) Comparison of the Skp1/Skp2 and Cul1/Rbx1 co-expressions with the Skp1/Skp2/Cul1/Rbx1 tetramer indicates very little difference in max signal strength. The tetramer was used for future experiments for ease of use and to corroborate with previous data (FIG. 27).

(111) Time-course Assays

(112) Following the identification of optimal conditions for all of the assay components, a time-course (0-100 minutes) was carried out to determine a linear part of the reaction velocity curve to set for future inhibitor work (FIG. 28). The experiment was performed on 2 different days to check the reproducibility of the data. In both experiments, a comparable and an approximately linear relationship is observed between the reaction time and the signal strength. The reaction time for future experiments was set at 60 minutes. It is evident that the data does not pass through the origin as would be expected but intercepts the y-axis. This may be a result of the stop method. The assay stop contains EDTA which sequesters metal ions and hence prevents the Mg-ATP complex necessary for E1 function. However, the relatively large amount of E2 present means that even after the E1 has been halted, there may still be some ubiquitinated E2 present capable of p27 biotinylation leading to a lag period after the stop has been added and an artificially high signal at each time point.

(113) In order to assess the reproducibility of the final assay conditions, a Z assay was carried out (FIG. 29)64 wells (1 sector) of positive controls and 64 wells of negative (no ubiquitin) controls. The experiment was performed 3 different times to assess the reproducibility of the data. In each case the signal strength, signal/background ratio, positive control % CV and Z values were within acceptable limits (Z>0.6 (Zhang, Ji-Hu, Journal of Biomolecular Screening, Vol. 4, No. 2, 67-73 (1999)), % CV<15%).

(114) Inhibitor Studies

(115) Each of the assays in the p27 ubiquitination cascade (E1, E2 and E3) were tested against the E1 inhibitor PYR-41. No pre-charging of either the E1 or E2 was carried out prior to the introduction of the compound although in the E3 assay, p27 phosphorylation by CDK2/Cyclin E was carried out in the absence of inhibitor.

(116) IC.sub.50 values of 7.8 and 3.2 M were generated for the E1 and E2 assays respectivelyComparable to the reported value of 5 M (Calbiochem). However, a significantly less potent IC.sub.50 of 55 M was seen for the E3 assay (FIGS. 30 (a) and (b)).

(117) In order to test the feasibility of screening the E3 assay against a range of inhibitors, the assay was tested against a panel of 41 inhibitors (40 known kinase inhibitors and PYR-41) (FIG. 31). As expected, the PYR-41 showed a high degree of inhibition (>80%), and several other inhibitors also indicated moderate amounts of inhibition (Rottlerin, EGCG and K252c) (FIGS. 32 and 33). Using the same assay conditions, the E3 assay was tested against these inhibitors (FIG. 33). Both the Rottlerin and the EGCG inhibitors give IC.sub.50 values in the M range. The K252c inhibitor would appear to be a false positive result. One potential concern was that the inhibition was the result of inhibition of the CDK2/Cyclin E (and hence phosphorylation of the p27), despite the fact that phosphorylation was carried out in the absence of inhibitor. Comparison of the inhibition profiles for both the E3 assay and Cdk2/Cyclin E shows that this is not the case (FIG. 34).

(118) Non-specific Binding Assays

(119) Non-specific binding has previously been identified as a potential problem for both the biotinylated-ubiquitin on its own and for the E1 enzyme in the E2 assay. The NSB signal for the ubiquitin was minimized by limiting the amount of ubiquitin in the assay to 2 M. Similarly, limiting the concentration of E1 enzyme in both the E2 and E3 assays to 5 nM allows decent E2 specific signal whilst reducing the non-specific signal from the E1 (FIG. 35).

(120) Similar experiments have shown that non-specific binding signal is not a problem in the E2 assay. This indicates that under the E2 assay conditions neither the E2 (at any concentration), E1 (at 5 nM) or the ubiquitin (at 2 M) contributes to a non-specific signal (FIG. 36).

(121) Similar experiments have also been carried out for the E3 assay (FIG. 37). We again see a non-specific signal. However, our previous experiments have indicated that this signal is not the result of the E1 (at 5 nM), the ubiquitin (at 2 M) or the E2 at any concentration. We must infer therefore that the NSB signal is the result of NSB from either the E3 or from the p27 complex. Further experiments have indicated that it is likely to be NSB of the p27 substrate itself. As a result, the non-specific signal is not an issue when testing the E3 assay.

(122) Multiple approaches have been employed to minimize the proportion of non-specific signal (FIG. 38).