BROMOscan Quantitative Ligand Binding Assays

Transcription

1 BROMOscan Quantitative Ligand Binding Assays Identification of Best In Class Bromodomain Inhibitors Elizabeth Quinn, PhD Director, LeadHunter Discovery Services

2 Outline Introduction Challenges to Epigenetic Drug Discovery Solution BROMOscan Technology Platform LeadHunter Service Portfolio 2

3 What is Epigenetics? Gene regulation independent of DNA sequence determines whether genes are turned on or off in a cell, in disease, in response to stimuli Our DNA is impacted by several environmental & genetic factors Addition of methyl groups and histone tail modifications Combination of marks determine gene expression Leads to health issues cancer, diabetes, autoimmunity Arrowsmith, et al. Nature Rev Drug Disc. 11: ,

4 Histone Code Histones globular proteins wrapped in DNA Histone tails subject to a wide range of covalent interactions Epigenetic marks addition & removal of chemical groups Acetylation of lysine residues (Kac) most frequently occurring posttranslational modifications (PTMs) which control gene transcription 4

5 Epigenetic Proteins A Maxmen, Nature 488: , Modification Write Erase Read Acetyl HAT HDAC Bromo Methyl HMT HDM Chromo, PHD, Tudor, MBT Enzyme Enzyme Binding 5

6 Bromodomains Are Druggable Evidence for small molecule inhibition of readers, writers, erasers Mainly HDAC inhibitors in clinic until recently Diazapines are selective BET inhibitors GSK OTX105 RVX 208 Oncology Inflammation DeWoskin and Million, NDD, 12: ,

7 Therapeutic Potential of Bromodomains Basic understanding of transcription Novel approaches to cancer biology AML, NUT midline carcinoma, myeloma, glioblastoma Inflammation & viral infection 7

8 Challenges for Screening Bromodomains Assay development difficult Available technologies have drawbacks Difficult to run as a large assay panel Chemical Probes/drugs Assay Output Sensitivity Throughput Ligand Needed? Limitation AlphaScreen Low IC50 High High Yes High false positive rate Tm Shift High C Medium High No Indirect Octet BLI Low Kd Medium Medium No Biotinylated protein ITC Low Kd Low Low No Protein consumption Need for a large menu of assays, HTP screening tool 8

9 KINOMEscan Technology The Solution KINOMEscan applied to BRDs World s Largest Kinase Panel Cover >80% human kinome 456 assays lipid, TK, mutants Enables new drug discovery paradigms Our Goal for Bromodomains Leverage our expertise Build a first in class panel of bromodomain binding assays 9

10 BROMOscan Technology: How it Works Competition Test Compound + Test Compound No Competition Measure amount of bromodomain bound to immobilized ligand in the presence and absence of test compound Ultrasensitive qpcr readout (6 log range) 10

11 BROMOscan Menu 40 Human Bromodomain Assays 34 of 57 distinct bromodomains (~60% coverage) Multiple putative therapeutic targets: BET, ATAD2, CREBBP/EP300, TRIM24(PHD,Bromo.) Ability to perform K d FUP studies on single vs tandem domains Human Bromodomain Dendrogram Bromodomain Targets ATAD2A BRD3(1,2) BRDT(2) PBRM1(5) ATAD2B BRD4(1) BRDT(1,2) PCAF BAZ2A BRD4(2) BRPF1 SMARCA2 BAZ2B BRD4(1,2) BRPF3 SMARCA4 BRD1 BRD4(full length, short iso) CECR2 TAF1(2) BRD2(1) BRD7 CREBBP TAF1L(2) BRD2(2) BRD8(1) EP300 TRIM24(Bromo.) BRD2(1,2) BRD8(2) FALZ TRIM24(PHD,Bromo.) BRD3(1) BRD9 GCN5L2 TRIM33(PHD,Bromo.) BRD3(2) BRDT(1) PBRM1(2) WDR9(2) * BRD2, BRD3, BRDT tandem assays also available ǂ BRD4(full length, short iso) assay also available Roman numerals indicate SGC defined families 11

12 BROMOscan Assay Validation Small Molecules Measure K d s consistent with known ITC values (when available) Inhibitors: JQ1 (active enantiomer) I BET (active and inactive enantiomers) PFI 1 & Bromosporine (SGC) and other known BRD inhibitors Acetylated Histone Tail Peptide Ligands Measure K d s consistent with published values (when available) Demonstrate selectivity for acetylated over nonacetylated peptides 12

13 Global Validation of BROMOscan Panel Comparison of BROMOscan and SGC T m Shift Data for the Promiscuous Inhibitor Bromosporine T m Shift (100 um Bromosporine) 24/25 BROMOscan assays analyzed Each square represents a unique bromodomain Outstanding agreement between BROMOscan and T m Shift data Cross validates the formats Further quantifies Bromosporine promiscuity SGC T m shift data provided by S. Knapp, PhD in collaboration with DiscoveRx Bromosporine K d (nm) Striking agreement between two very different assay readouts 13

14 Assay Validation: Family II (BET) Family II (BET) assays BRD2(1) BRD2(2) BRD3(1) BRD3(2) BRD4(1) BRD4(2) BRDT(1) BRDT(2) 14

15 BET Family Assay Validation JQ1 Kds Correlate with Literature Values Data Comparison Similar Kd values measured with BROMOscan & ITC Domain affinity rank order identical Optimized conditions ensure true thermodynamic Kd measurements *Nature (2010) 408:

16 BET Family Validation I BET Nature (2010) 468: BROMOscan data consistent with published ITC data Potency Rank order Lack of potent activity for inactive I BET enantiomer 16

17 Family IV Validation: BRD1 Selective Binding to Acetylated Histone Tail Peptide Ligand Affinity Measurements Bromodomain 1 internally discovered acetylated peptide ligand 2 corresponding non acetylated peptide Histone Tail Peptide BROMOscan K d (nm) BRD1 H4(Ac) 1 15,000 BRD1 H4 2 >100,000 Assay Signal K d = 5,000 nm Also determine K d Values of 1 50 um for other known BRD inhibitors [Ligand], nm 17

18 Bromodomain Inhibitor Profiles I BET PFI 1 Bromosporine BET Selective Semi BET Selective Promiscuous 18

19 Dual Kinase/Bromodomain Inhibitors Rationally Designed Polypharmacology Question: Do kinase inhibitors cross react with bromodomain epigenetic reader proteins? Experiment: Screen diverse panel of mature kinase inhibitors across internal bromodomain assay panel (BROMOscan) Implications: Outstanding leads for BRD inhibitor discovery Proof of principle for development of dual kinase/brd inhibitors New paradigm for oncology drug discovery 19

20 BROMOscan Bromodomain Potency and Selectivity Result: Potent (Kd = M), diverse BRD profiles for 4/68 (5.9%) kinase inhibitors tested Best in class in vitro and cellular BRD4 potency 20

21 BROMOscan Summary Specific Sensitive Quantitative HTS Compatible Generic Assay Rigorous assay validation with optimized bait ligands Heterogeneous screen to solubility limit of compound No fluorescent compound interference Ultrasensitive qpcr method Accurate Kd values over a broad dynamic range <100 pm to >10 um Optimized conditions for true thermodynamic affinity (Kd) measurements Detect small amts of protein, low amts of kinase Screen more than 100,000 wells/day No interference from compound fluorescence Unique, semi automated set up Generic, standardized assay conditions Industrial scale screening & profiling Fast time to results 2 week data delivery 21

22 Flexible Service Menu Service Number of Assays Description Growing panel of 32 human bromodomains validated for single point testing Choose only the targets you need for personalized or custom panels Quantitative binding constants on any of the 40 bromodomain assays 40 Kd profile of entire BROMOscan menu LIBRARY 1-32 Compound library screening against a single target or against the full panel 22

23 DiscoveRx Solution Portfolio for Epigenetics 23

24 >80% Coverage of Druggable Targets + Primary Cell Phenotypic Assays 0% GPCRs Kinases NHRs Pathways Bromodomain HDAC/HMT Depth of Coverage % Primary Cell Profiling 1,120 assays covering 751 druggable targets All major drug target classes covered Multiple assays for single target 24

25 Acknowledgements Dan Treiber Jeremy Hunt Lisa Wodicka Pietro Ciceri Lisa Ramos Mark Floyd Gabe Pallares KINOMEscan Team Jay Bradner Jun Qi Stefan Knapp Susanne Muller Knapp 25