Degrading the Barriers to Drug Discovery in Ubiquitin E3 Ligase Pathways

Transcription

1 Degrading the Barriers to Drug Discovery in iquitin E3 Ligase Pathways Blaine N. Armbruster, PhD. Sr. Manager - Discovery & Development Solutions EMD Millipore 1 Oct, 2012

2 Outline Introduction ti to EMD Millipore Drug Discovery & Development group iquitin pathway potential for drug discovery and challenges Assay development for EMD Millipore s ubiquitin pathway portfolio Sample data generated through iquibitinprofiler service assays 2

3 Drug Discovery & Development Solutions Trusted expertise through every phase. Biomarker Solutions Screening & Profiling Services In Vitro Toxicity Studies Cell-Based Assay Services Biopharmaceutical Analysis Services Pharmacokinetic Services Immunogenicity Services 3

4 What is the relevance of the ubiquitination pathway for drug discovery? iquitination regulates numerous biological processes, including: Protein degradation Gene transcription Cell cycle progression DNA repair Apoptosis Receptor endocytosis Dysregulation of the ubiquitinproteasome pathway is linked to a number of diseases, including: Cancer Neurodegeneration Inflammation Diabetes Viral infection Proof of clinical relevance for the ubiquitin/proteosome pathway being relevant in drug discovery Velcade proteasome inhibitor in clinic (treatment of multiple myeloma) 4

5 iquitination iti three step process AMP + PPi ATP E1 E1 Proteosome degradation E2 Substrate E2 Substrate E3 E3 Substrate Substrate Localization & Signaling Substrate 5

6 iquitin iti cascade hierarchy, h diversity it & specificity it E1 E1 1 Number of enzymes E2 E2 E2 E2 >30 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 >600 Specificity ye3 >600 >1000 Substrates 6

7 Barriers to Drug Discovery in iquitin E3 Ligase Pathways iquitin pathway is complex Process involves three sets of enzymes E3 ligase can be composed of multiple subunits to direct proper substrate recognition E3 ligases can be regulated by post-translational translational modifications (e.g., neddylation) The substrate often needs to be phosphorylated for ubiquitination to occur Gap in commercial support for highly qualified reagents Very few E3 ligases available in the market Less have been tested for functional activity 7

8 Assay Development Case study: RING E3 ligases - SCF Complexes

9 Considerations for assay development Goal: To develop assays to measure the activity of E3 ligases by detecting the covalent addition of ubiquitin to a substrate Components & component optimization: iquitin (biotinylated) ATP (for E1 enzyme activity) E1 activating enzyme E2 conjugating enzyme E3 ligase enzyme (this may consist of multiple proteins) Substrate (with appropriate modification, e.g., phosphorylation) 9

10 E3 ligase cascade assay reaction: example - SCF RING E3 ligase complex Cdk2 Cks 1 p27 Cyclin E B B B B B B Tag E2 E1 ATP Skp1 Cul1 Rbx1 Assay reaction components: iquitin (biotinylated) ATP (for E1 enzyme activity) E1 activating enzyme (yellow) E2 conjugating enzyme (gray) E3 ligase enzyme (this may consist of multiple proteins) (green) Substrate (epitoped tag and with appropriate modification, e.g., phosphorylation) (red) 10

11 Protein Production For the SCFSkp2 complex, 10 different proteins are required to enable the assay The substrate is expressed with an epitope tag for assay purposes E3 complex expressed as a tetramer in insect cells Additional accessory yproteins are required Analogous complexes for βtrcp and Fbw7 Lane 1: Molecular weight markers Lane 2: UBE1 (E1) Lane 3: ch3 (E2) Lane 4: Cks1 Lane 5: SCF Skp2 complex (E3) (blue - cul1, red - Skp2, black - Skp1, green - Rbx1)

12 Detection and quantification of ubiquitination Readout utilizes a electrochemiluminescence format that is analogous to an ELISA After reaction assay incubation is stopped, the assay contents are transferred to a detection plate The epitope tagged substrate from the assay reaction is captured on the detection plate A ruthenium conjugated streptavidin molecule binds to biotinylated ubiquitin that was added to the substrate during the assay reaction Excitation of ruthenium ion by an electrical charge results in the generation of a luminescent signal B B Substrate Tag Plate Surface B Biotin B Ru Streptavidin 12

13 Assay Development Assay development process follows a common profile for each target Initial preparatory stages ensure that the substrate is phosphorylated and that up-stream activity (E1 activation and E2 ubiquitin charging) is present β-catenin phosphorylated by GSK3α 13

14 Assay Development βtrcp1 - Construct Screen +/- Substrate Standard Plate + Substrate - Substrate Signal D10HP009N D10HP010N βtrcp Screen Available Protein Constructs & TAG Options Screen for Optimum Buffer and Capture Conditions Check Signal Specificity by Individual Assay Component Knockouts Optimise Assay Component Concentrations (E1, E2, ATP etc.) Compound Work e.g. Known Inhibitors DMSO Tolerance Assay Reproducibility Z Assay Timecourse 14

15 Assay Development ch3 - IKBa Signal vs. 'Knockout' Controls Standard Plate Signal All No iquit in No E1 No E2 No E3 No Subst rat e No ATP Screen Available Protein Constructs & TAG Options Screen for Optimum Buffer and Capture Conditions Check Signal Specificity by Individual Assay Component Knockouts Optimise Assay Component Concentrations (E1, E2, ATP etc.) Compound Work e.g. Known Inhibitors DMSO Tolerance Assay Reproducibility Z Assay Timecourse 15

16 Assay Development Signal βtrcp Mediated IΚBα iquitination (ch3) - e1 Titration [e1] (nm) Signal βtrcp Mediated IΚBα iquitination (ch3) - ATP Titration [ATP] (µm) Signal βtrcp Mediated IΚBα iquitination (ch3) - ch3 Titration [ch3] (nm) Screen Available Protein Constructs & TAG Options Screen for Optimum Buffer and Capture Conditions Check Signal Specificity by Individual Assay Component Knockouts Optimise Assay Component Concentrations (E1, E2, ATP etc.) t Compound Work e.g. Known Inhibitors DMSO Tolerance Assay Reproducibility Z Assay Timecourse 16

17 Assay Development btrcp iquitination of IKBa - Timecourse Signal Time (Minutes) Screen Available Protein Constructs & TAG Options Screen for Optimum Buffer and Capture Conditions Check Signal Specificity by Individual Assay Component Knockouts Optimise Assay Component Concentrations (E1, E2, ATP etc.) Compound Work e.g. Known Inhibitors DMSO Tolerance Assay Reproducibility Z Assay Timecourse 17

18 Assay Development btrcp Assay with IKBa and ch3 - DMSO Tolerance Signal % DMSO Screen Available Protein Constructs & TAG Options Screen for Optimum Buffer and Capture Conditions Check Signal Specificity by Individual Assay Component Knockouts Optimise Assay Component Concentrations (E1, E2, ATP etc.) Compound p Work e.g. Known Inhibitors DMSO Tolerance Assay Reproducibility Z Assay Timecourse 18

19 iquitinprofiler service ubiquitin cascades with biologically-relevant substrates 19 E1 E2 E3 Substrate Therapeutic area e1 ch3 SCF Skp2/Cks1 * p27 e1 ch3 SCF Fbw7 * Cyclin E1 Cancer e1 ch5a SCF Fbw7* Cyclin E1 e1 ch3 SCF btrcp1* IκBα e1 ch4 SCF btrcp1 * IκBα Cancer, Inflammation e1 ch3 SCF btrcp1 * β-catenin e1 ch4 SCF btrcp1 * β-catenin e1 ch4 MDM2/CK1δ* p53 e1 ch5c MDM2/CK1δ* p53 e1 ch4 MDM2* MDM2 (auto) Cancer e1 ch5c MDM2* MDM2 (auto) e1 ch4 c-cbl* Src e1 ch4 c-cbl* Kit e1 ch4 VHL* HIF-1α e1 ch5a VHL* HIF-1α Cancer, Inflammation e1 ch5c VHL* HIF-1α e1 ch7 Parkin* Parkin (auto) Neurodegeneration e1 ch7 Parkin* p38/jtv-1 (Parkinson s disease) e1 ch13/uev1a CHIP** p53 Cancer & Neurodegeneration e1 ch13/uev1a CHIP** Hsp70 (Parkinson s disease) e1 ch13/uev1a CHIP** CHIP (auto) e1 ch5c MuRF1* Cardiac Troponin I Cardiovascular *RING type, **U-box, ***HECT type

20 Example of compound selectivity it profiling against SCF E3 ligase cascades Compounds were profiled at a final concentration of 100 µm PYR41, which inhibits e1 (E1) activity, was included as a control. Cascades 20 E1 E2 E3 Substrate e1 ch3 SCF βtrcp1 IκBα e1 ch3 SCF βtrcp1 β-catenin e1 ch3 SCF Fbw7 Cyclin E1 e1 ch3 SCF Skp2/Cks1 p27

21 Example of follow up dose response studies: SCF Skp2/Cks1 (substrate: p27) Example of dose response curves from hits derived from a small library screen (138 compounds) screened at 20 and 200 µm 21

22 Compound screening against Parkin E3 ligase Parkin Auto-iquitination Parkin - p38/jtv-1 iquitination Activity (% Contro ols) 100 PYR-41 Ro SMER3 60 RITA log 10 conc (M) Activity (% Contro ols) 100 PYR-41 Ro SMER3 60 RITA log 10 conc (M) Complex Compound potency (um) E3 Substrate PYR41 Ro SMER3 RITA Parkin Parkin (auto) Parkin p38/jtv

23 Pharmacological comparison of MDM2-p53 inhibition by binding vs. functional activity assays MDM2-p53 TR-FRET Binding Assay MDM2-p53 Functional Assay trols) Binding (% Cont RITA Nutlin (+) HLI-373 Nutlin (-) trols) Activity (% Cont RITA Nutlin (+) HLI-373 Nutlin (-) log 10 conc (M) log 10 conc (M) Assay Complex Compound potency (um) Type E3 Substrate RITA HLI373 Nutlin (+) Nutlin (-) Functional MDM2 p >300 >300 Binding MDM2 p53 >100 >

24 Major takeaways Yes, the ubiquitin pathway is complex Yes,, E3 ligases can be complex targets But, with appropriate expertise, theses complexities can be overcome, thus E3 ligases are permissible targets for drug discovery 24

25 Acknowledgments 25 Phil Adams Andrew Plater Jonathan Clark Jennifer Hill Simon Hawdon Anna Woodward Clare Hadden Lynn Byers Steve Davies Dundee, Scotland How to find out more information: