How to Interrogate Epigenetic Events in Live Cells

Transcription

1 How to Interrogate Epigenetic Events in Live Cells Danette L. Daniels, PhD Group Leader, Functional Proteomics Proprietary Information. Not for further distribution.

2 What is Epigenetics? Change in phenotype without change in genotype - Waddington 1942 Players: DNA, RNA, histones, proteins (readers, writers and erasers) Events: Regulation of chromatin structure and transcription Nature Reviews Drug Discovery (2014) 13: Proprietary Information. Not for further distribution. 2

3 Epigenetic Modifications and Chromatin Context Chromatin landscape Epigenetic regulation is a compilation of numerous marks or signals: Histone modifications DNA modifications RNA (non-coding) modification or processing Pulmonary Circulation (2011) 1: Regulation of transcription Epigenetic marks promote different outcomes: Chromatin compaction or opening Transcriptional repression or activation Subsequent protein or RNA recruitment Proprietary Information. Not for further distribution. 3

4 Readers, Writers & Erasers The epigenetics field refers to the three major classes of epigenetic proteins as: 1. Writers: Place the marks 2. Readers: Bind the marks 3. Erasers: Remove the marks Proprietary Information. Not for further distribution. 4

5 Epigenetics and Link to Disease Numerous epigenetic proteins implicated in numerous diseases Alterations of histone modifications Aberrant DNA modifications Changes in expression of epigenetic proteins Increased activity of epigenetic enzymes Loss or inappropriate expression of non-coding RNAs (ncrna) Nature (2008) 454: What makes epigenetics such an exciting field for drug development? Plasticity, dynamics, and potential reversibility! Proprietary Information. Not for further distribution. 5

6 Promise of Epigenetic Inhibitors Epigenetic compounds treat numerous diseases and conditions Most advanced compounds are enzymatic inhibitors Emerging inhibitors developed against protein:protein interactions Nature Reviews Genetics (2016) 17: Proprietary Information. Not for further distribution. 6

7 BRD4, BET Family Members, and Functional Roles BET members contain tandem bromdomains (BD1 and BD2) and extra-terminal region BD1 BD2 mb ET BRDT BRD4 BRD3 BRD-NUT translocations observed in NUT midline carcinomas BRD2 BRD4 Roles: Nature Reviews Drug Discovery (2014) 13: BRD4 regulates transcription through interactions with Mediator, SEC, and ptefb In cancers, BRD4 drives expression of oncogenic proteins such as Myc Proprietary Information. Not for further distribution. 7

8 Mechanism of Action of BET Inhibitors BRD4 Chromatin Displacement Displaces BRD4 from chromatin BET inhibitors are acetyl-lysine mimics which block BRD4:histone interaction BRD4 displacement results in transcriptional reduction of oncogenic proteins BRD4 involved in numerous complexes and may interact with chromatin through different interactions Nature (2011) 478: BRD4 and bromodomain family members are complex, multi-domain proteins. Could we study them and chromatin interactions in live cells with full-length proteins? Proprietary Information. Not for further distribution. 8

9 Monitoring Protein:Protein Interactions in Live Cells and Application to Chromatin Proprietary Information. Not for further distribution.

10 Two Technologies Enabling Cellular Studies of Protein Interactions NanoLuc Luciferase Small size, 19kDa Monomeric enzyme Thermally stable enzyme Fusion tag Extremely bright, 150X increase HaloTag Technology Monomeric protein, 34kDa Fusion tag Extremely fast association rate Irreversibly binds chloroalkane ligands Ligands include fluorophores and surfaces Histone H2A-HaloTag in HEK293 cells We can apply NanoLuc and HaloTag technologies to studies of protein:protein interactions Proprietary Information. Not for further distribution. 10

11 S /B R a tio Monitoring Protein Interactions in Living Cells Using NanoBRET NanoBRET BRET = Bioluminescence Resonance Energy Transfer Luminescent Donor Protein A Fluorescent Acceptor Protein B A:B interaction FKBP:FRB Comparison with BRET2 N L u c : H T 1 0 R L u c 8 : Y F P NanoLuc HaloTag Energy Transfer R a p a m y c in [n M ] Key characteristics Improved signal: background, decreased spectral overlap Greater assay window as compared to other BRET systems NanoLuc enables BRET at low expression levels Monitor modulation or changes of interactions NanoBRET is ratiometric Acceptor signal Donor signal with ligand Acceptor signal Donor signal no ligand Corrected BRET Ratio 11

12 Monitoring Specific Displacement of BRD4 with BET Inhibitors BRD4-Histone H3.3 CBP-Histone H3.3 IC50 = 65.4 nm BRD4-Histone H4 CBP-Histone H4 Measuring potency differences amongst bromodomain proteins and histones IC50 = nm ACS Chemical Biology (2015) 10: Proprietary Information. Not for further distribution. 12

13 Tale of a Bivalent Bromodomain Inhibitor Proprietary Information. Not for further distribution. 13

14 Targeting Both Bromodomains of BRD4 with a Single Drug? BRD4 BD1 BD2 Full-length (WT) Current inhibitors are monovalent: 1:1 Drug to BD region BRD4 expect 2:1 Drug to Protein (2BDs) Would a bivalent drug that targets two bromodomains at the same time be more potent? IBET (Monovalent) Bi-BET (Bivalent) Nature (2010) 468: See the paper co-authored by AstraZeneca and Promega research teams: Potent and selective bivalent inhibitors of BET bromodomains Nature Chem. Biol. (2016) 12: for more information Proprietary Information. Not for further distribution. 14

15 What is the Binding Mode of a Bivalent BET Inhibitor? Possible mechanisms (Drug:BRD4) Bi-BET (Bivalent) 1. Cis binding (2:1) Separate engagement into BDs of same protein. Similar to monovalent. BRD4 BD1 BD2 Full-length (WT) 2. Cis binding (1:1) Engagement of both BDs in same protein. Distinct from monovalent. BRD4 BD1 BD2 Full-length (WT) Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 15

16 What is the Binding Mode of a Bivalent BET Inhibitor? Third possible mechanism (Drug:BRD4) Bi-BET (Bivalent) 3. Trans binding: (1:2) or (2:2) Dimerization of BRD4 through BDs BRD4 BD1 BD2 Full-length (WT) BRD4 Full-length (WT) BRD4 BD1 BD2 Full-length (WT) BRD4 Full-length (WT) Proprietary Information. Not for further distribution. 16

17 B R E T r a t i o ( m B U ) B R E T r a t i o ( m B U ) Chromatin Binding Assays with Bivalent Inhibitor BRD4 BD1 BD2 X BRD4 Constructs Full-length (WT) N433F BD2 Mutant BRD4 B R D 4 WTW T B R D 4 N F M u t a n t BRD4 N433F Mutant IBET Or Bi-BET = Bivalent compound 1 = IBET762 (monovalent) A Z I B E T = Bivalent compound 1 = IBET762 (monovalent) A Z I B E T C o m p o u n d [ µ M ] C o m p o u n d [ µ M ] Bivalent BET inhibitor orders of magnitude (pm) more potent than monovalent Point mutation into BD2 shifted the bivalent inhibitor to looking like a monovalent Suggest both domains important for inhibition Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 17

18 B R E T R a t i o / n m Bivalent Compound Able to Induce Dimerization of Isolated BD1 Domain BRD4 BD1 BD2 X-Ray Crystal Structure BRD4 BD1-BD1 Dimer + Bi-BET BD1-BD1 + BiBET interaction :Full-length (WT) :BD1 NanoBRET Data BRD4 BD1-BD1 Dimer + Bi-BET 0. 3 Bivalent B i d e n t a t e M o n o d e n t a t e Monovalent µ C o m p o u n d [ u M ] Both sides of bivalent compound able to engage a bromodomain simultaneously Possible to dimerize BRD4 through trans binding mechanism Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 18

19 mbu Bivalent Compound Unable to Induce Dimerization of Full-Length BRD4 in Cells BRD4 BD1 BD2 Full-length (WT) BRD4 + BRD4 + BiBET NanoBRET Data Nl-BRD4 +HT-BRD4 Dimer + Bi-BET + Bi-BET NL-BRD4 HT-BRD4 X Bivalent Inhibitor (Bi-BET) D/A: 1:100 D/A: 1:25 D/A: 1:6.25 D/A: 1:1.562 D/A: 1:0.390 D/A: 1:0.097 D/A: 1:0.024 D/A: 1:0.006 No induction of full-length BRD4 dimer observed in cells across various concentrations Proprietary Information. Not for further distribution. 19

20 Alterations in Sedimentation and SAXS of BD1BD2 Construct with Monovalent and Bivalent Inhibitors Analytical ultracentrifugation analysis BD1 BD2 Small-angle X-ray scattering BD1BD2 Tandem Domain Monovalent Bivalent Bivalent Bivalent Sedimentation analysis indicates dispersed Apo-BRD4 BD1BD2 is monomer of highly extended conformation Additional of Bivalent compound results in more compact shape Similar results observed using SAXS Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 20

21 Development of a Conformation Biosensor for Cellular Studies NanoBRET tandem BD1BD2 biosensor to detect conformational/distance changes Increase BRET Basal BRET Bivalent Engagement HT NL BD1 BD2 HT BD1 BRD4 BD2 NL Basal BRET Monovalent Engagement HT BD1 BRD4 BD2 NL BD1 BD2 BRD4 X X BD1BD2 Tandem Domain BD1N140F BD1 Mutant BD1BD2N433F BD2 Mutant Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 21

22 N o r m a l i z e d B R E T r e s p o n s e NanoBRET BRD4 Biosensor Supports Cis Binding Mechanism in Cells NanoBRET BRD4 BD1BD2 Biosensor BRD4 BD1 BD2 BD1BD2 Tandem Domain X BD1N140F BD2 Mutant B R D 4 B D 1 B D B R D 4 B D 1 B D 2 N F 6 B R D 4 B D 1 B D 2 6 B R D 4 B D 1 B D 2 N F Cis binding (1:1) within a BRD l o g 1 0 ( [ c o m p o u n d ] ) / M Bivalent compound (blue) shifts conformation and holds signal Point mutant in biosensor shifts bivalent inhibitor to monovalent Point mutant and shape of curve support Cis binding mechanism Targeting tandem reader domains within same protein is possible! Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 22

23 Monitoring Protein:Small Molecule Interactions in Live Cells Proprietary Information. Not for further distribution. 23

24 BRET BRET NanoBRET Target Engagement Enables Monitoring Protein:Small Molecule Interactions in Live Cells Drug Tracer NanoBRET Target Engagement Nano Luc Target Nano Luc Target Express target as fusion to luciferase (energy donor) Intracellular binding [BRD Tracer] Intracellular affinity [unlabeled BET inhibitor] Provide permeable fluorescent tracer for target (energy acceptor) Estimate binding affinity by competitive displacement of the tracer with the compound Highly specific, Intracellular, Biophysical measurements Target verification Optimization of chemical series Affinity Residence time See the article by Promega R&D: Target engagement and drug residence time can be observed in living cells with BRET. Nature Communications (2015) 6: Proprietary Information. Not for further distribution. 24

25 B R E T R a tio (m B U ) B a c k g ro u n d C o rr e c te d BET Family Target Engagement Assays NLuc-Bromodomain Fusions BET Tracer BRD2: BRD3: NLuc BETs NLuc BETs BRD4: N L u c -B R D F u s io n s BRD4 BD1: L iv e H e L a C e lls BRD4 BD2: BRDT: KAT2B: (non-bet control) B R D 2 B R D 3 B R D 4 B RD4 B D 1 B R D 4 B D 2 B R D T K A T2 B BET tracer specific for BET family proteins Binds each with different affinities B E T T ra c e r (u M ) Proprietary Information. Not for further distribution. 25

26 Target Engagement Studies of Inhibitor Binding to BRD4 in Living Cells BRD4 BD1 BD2 X BRD4 Constructs :Full-length (WT) :N433F BD2 Mutant :BD1 IBET 1 Bivalent BET inhibitor orders of magnitude (pm) more potent for binding than monovalent Point mutation shifts the bivalent inhibitor to looking like a monovalent or BD1 alone pic 50 for compound binding identical to pic 50 for chromatin displacement Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 26

27 Similar Potency Shifts in Binding to BET Family Members BRD2/IBET Tracer BRD3/IBET Tracer BRDT/IBET Tracer All BET family members show increased potency of binding to bivalent inhibitor as compared to monovalent Suggests similar binding mechanism Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 27

28 Measuring Cell Viability and c-myc Levels After Bivalent Treatment Cell viability assays (MM.1S) c-myc levels following compound washout Monovalent Bivalent Bivalent (6); Monovalent (1) Bivalent inhibitor orders of magnitude more potent for growth arrest in BRD4-sensitive cell line Bivalent compound shows loss of Myc for significantly longer times than monovalent compound Does binding of bivalent drug to BRD4 have increased affinity or residence time? Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 28

29 Target Engagement Measuring Residence Time Inside Cells 1. Saturate with Test Ligand 3. Add Tracer 2. Wash Luc Target Luc Target 4. Measure BRET (real time) Luc Drug Tracer Target Proprietary Information. Not for further distribution. 29

30 Residence Time Analysis of BRD4 with Bivalent and Monovalent Inhibitors BRD4 BRD4/IBET Tracer BRD4 BD1/IBET Tracer :Full-length (WT) :BD1 Monovalent Bivalent Bivalent compound has protracted residence time for binding to BRD4 as compared to monovalent Protracted residence time not observed with domain alone Nature Chem. Biol. (2016) 12: Proprietary Information. Not for further distribution. 30

31 Promega Offerings for Epigenetics Studies Proprietary Information. Not for further distribution. 31

32 Tools for Epigenetic Protein Studies & Drug Screening Epigenetic Histone Modulators Assays and Technologies Readers NanoBRET technology - Protein:protein and protein:small molecule interactions - Compound screening HaloTag technology - Imaging - Proteomics - HaloCHIP OGT Writers Methyltransferases (MTase-Glo Assay) Kinases (ADP-Glo Kinase Assays, Kinase-Glo Assays) OGT (UDP-Glo Assay) Erasers Deacetylases (HDAC-Glo and SIRT-Glo Assays) Demethylases (Succinate-Glo Assay - Jumonji class) For more information: Epigenetics on promega.com 32

33 Epigenetics Offerings for Nucleic Acids Assays Sample Preparation Instruments DNA MTase-Glo to detect DNMT activity ReliaPrep FFPE, Blood or Large Volume gdna purification gdna extraction from small volume QuantiFluor ssdna or dsdna systems Maxwell Quantus Fluorometer RNA pmirglo Dual Luciferase vectors MTase-Glo to detect RNMT activity SimplyRNA mirna Tissue purification kits ReliaPrep FFPE RNA purification ReliaPrep RNA Cell and Tissue Miniprep System SV Total RNA isolation QuantiFluor RNA system Maxwell Quantus Fluorometer Nature (2008) 454: For more information: Epigenetics on promega.com Proprietary Information. Not for further distribution. 33

34 Summary An exciting and rapidly growing area of basic science and drug discovery! Correlative biochemical and cellular studies of epigenetic proteins critical for understanding function and mechanism of action inside live cells. Promega has many technologies and reagents for epigenetics research: - Methyltransferase-Glo MT Assay - Protein:Protein Interaction & Target Engagement assays for Bromodomains - HDAC-Glo Assays & Screening Systems - Succinate-Glo Assay, and many more We also have options for collaborating with us, having access to these technologies and newer products. And we have a Custom Assay Services team to build a specific assay for you. 34

35 Thank You For specific webinar questions: Danette L. Daniels, PhD Or for questions in general, contact our Technical Services Scientists: For Custom Assay Services inquiries: Proprietary Information. Not for further distribution. 35