NanoBRET: A Quantitative Technique to Measure Kinase Target Engagement in Live Cells Matthew Robers
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1 anobret: A Quantitative Technique to Measure Kinase Target Engagement in Live Cells Matthew Robers Sr. Research Scientist & Group Leader
2 Premise: Targets may behave differently in cells than in isolation Target Engagement Analysis Target Inhibition Biochemical K i Cellular IC 5 Target [Inhibitor] Commonly observe potency offset in cells 2
3 Enabling mechanistic analysis of kinase pharmacology Engagement in isolation Intracellular Affinity Cellular Effect RTKs PI3K JAK SRC Target MAPK AKT Target Phenotype Biochemical Assays Target Engagement Phosphorylation Gene expression Mechanistic cell health 3
4 Tracking drug targets with anoluc (Luc) plophorus Luciferase anoluc Luciferase DDR1-Luc (PM) Directed evolution CDK2-Luc (nuclear) Multimeric enzyme Unstable 19 kda, monomeric > 1 6 brighter Thermostable Ectopic Expression CRISPR/Cas9 IRE1α-Luc (ER) 4
5 Measuring molecular proximity Bioluminescence Resonance Energy Transfer (BRET) BRET B D A D A anoluc anobret 59 ~ 3 nm BRET Ratio (A/D) Increasing ccupancy (agonism) 5
6 Bioluminescence Resonance Energy Transfer (BRET): A Quantitative Target The anobret Engagement Assay Target Engagement Assay ormat iphone 5 picture BRET Tracer Tracer Add Tracer Add Test Drugs D e m o n s tra te D y n a m ic E q u ilib riu m 5 Luc Target B R E T R a tio (m B U ) [Unlabeled Drug] Robers et al. ature Communications. 215 [Tracer] (µ M) 6
7 The anobret Target Engagement (TE) Assay uses both genetic and chemical reporters to query test compounds Chemical Reporter: Broad Spectrum Provides Quantitation! Kinase Test Compound Luc Genetic Reporter: Target-specific Provides Specificity! 7
8 anobret is a quantitative readout for target engagement VS Kinase Luc Determine Tracer Affinity Measure TE at Tracer K d -apparent 8
9 Example of quantitative capabilities for measuring intracellular affinity for full-length kinases DDR1-Luc (PM) Generate a K d -apparent: Use [Tracer] that yields a constant IC 5 value 9
10 Example Data: Profiling of BTK Kinase Inhibitors in EK293 Cells Dasatinib Type I Bosutinib Type I IC 5 Profiling using anobret Tracer K-5 Cl S Ibrutinib Covalent Cl oretinib Type II Cl BRET Ratio (mbu) BTK-anoLuc Live EK293 Cells B osutinib oretinib Dasatinib Ibrutinib EC5 [Test Compound], µ M Bosutinib.65 oretinib.88 Dasatinib.12 Ibrutinib.13 1
11 Creating a kinome-wide TE capability for full-length kinases anobret Pan-Kinase Tracer Examples anoluc/kinase Library 178 Live Cell TE Assays 1 2 Cl Cl B B 3 S B 4 B 5 Cl B 6 Cl S B - Vasta et al. Cell Chemical Biology
12 Creating a kinome-wide TE capability for full-length kinases anobret Pan-Kinase Tracer Examples Cl Cl S B B 4 B B EPA5 EPB2 anoluc/kinase TRK2 GR1 Library GR4 178 Live Cell TRK1 TE Assays EPB3 EPA3 EPA4 EPA6EPB4 EPA7 EPA8 MuSK DDR2 DDR1 IG1R EPA2 EPA1 EPB6 GR2 ISR MET ALK LTK GR3 LT1 CS1R AXL MER RET LT3 KIT TYR3 PDGRβ TIE1 TIE2 5 Cl B 6 Cl S B - Vasta et al. Cell Chemical Biology
13 Enabling live cell kinase profiling Type I, Type II, Allosteric Probes PIK 3CA BTK BRET Ratio (mbu) m ipalisib Taselisib P I-13 A pitolosib BRET Ratio (mbu) B osutinib oretinib Dasatinib Ibrutinib [Compound], µ M [Compound], µ M R IPKK 1 BRET Ratio (mbu) 1 1 Ponatinib ecrostatin II 8 8 KW-2449 APY [Com pound], µ M [Compound], µ M A B L BRET BRET Ratio Ratio (mbu) (mbu) ABL-1 ABL-1 Dasatinib Ponatinib 2 G 2 Im a tin ib [Com pound], µ [Com pound], µ M 13
14 Common observation for kinases: Right shifted IC 5 in live cells vs biochemical assay LCK 1 B o s u tin ib Dasatinib 1 L C K In h ib ito r P o te n c ie s Expected BRET Ratio (mbu) Staurosporine Saracatinib pki (anobret) Lysed Cells (observed) L iv e C e lls L iv e C e lls (observed) (observed) 3 lysed cells pki (Biochem ical Analysis) [Com pound] (µ M ) 14
15 Literature analysis: Cellular Potency ffsets May Vary Depending n The Kinase Published Biochemical K d 2 Cl Cl Published Cellular IC 5 RTKs Kinase Crizotinib: Biochemical vs p-elisa B iochem ical, Log K d, nm ALK T IE 2 Abl TrkA TrkB AXL Perfect Correlation I S R LCK MET P ABL P SRC P MAPK P LCK AKT P P Cellular pelisa, Log IC 5 nm - Davis et al. at Biotechnology Cui et al. J. Med. Chem. 211 or LCK: Cell-free K d < 2 nm Cellular p-elisa IC 5 > 3 nm 15
16 Why would results differ in cells vs biochemical assays? Kinase Plasma Membrane Protein Complexes Activated State Auto-inhibited State Unpredictable [ATP] P P Kinase 16
17 anobret Assay Can Predict Cellular Potency ormalized BRET Crizotinib Target Engagement M ET- Luc ALK-Luc TrkB- Luc AXL-Luc TrkA- Luc Luc-ABL TIE2-Luc ISR-Luc LCK-Luc Target Engagem ent, Log IC 5 (n M ) ABL1 LCK I S R T IE 2 TRKA AXL TRKB ALK MET R 2 = [C rizotinib] (µ M) Phoshpo-ELISA, Log IC 5 (n M ) - Vasta et al. Cell Chemical Biology
18 uture Direction: Kinome-wide selectivity profiling in live cells Compound Diversity Set Target Diversity Set 18
19 Crizotinib: Model for kinome-wide live cell profiling 1 µm (C max ) Dose Cl 2 Selectivity predicted by Biochemistry (72) Cl MET ALK EML-ALK ERK AKT PRLIERATI / SURVIVAL Molecular targets of crizotinib in live cells? - Cui et al. J. Med Chem Davis et al. at. Biotech
20 Profiling crizotinib against 178 kinases in one experiment Kinase diversity set % ccupancy Arrayed Transfection + Tracers M ET -32 M AP4K2-31 LTK-32 CASK-31 AXL-32 M ERTK-32 M USK-32 ALK-32 EPA6-32 STK1-31 TRKA-32 TRK2-32 T EK-32 TRK1v1-32 TRK1v2-32 TYRR3-32 TIE2-32 EPA1-32 EPA7-32 IG1R-32 M AP3K13-31 TIE1-32 AURKA-32 IRAK3-31 LIM K1-32 CDK7-32 M APK7-31 LIM K2-32 M AP3K12-31 RIPK1-31 AURKB-32 PTK2B-32 abl2-31 AURKC-32 JAK2-32 SLK-31 M AP4K5-32 ISR-32 ABL1-32 RIPK2-31 TTK-32 BM P2K-31 PRKA-31 IKBKE-31 UAK1-31 EPA2-32 PTK2-31 CSK2A1-32 GR2-32 LRRK2-32 M AP4K3-31 EPA4-32 CS1R-32 SIK3-31 IRAK4-32 STK32A-31 EPB6-32 M AP3K9-31 TESK1-31 ULK1-31 AAK1-31 STK33-31 M APK6-31 PAK4-32 PLK4-31 LCK % ccupancy Expt 1 Reproducibility % ccupancy Expt 2 2
21 Improved intracellular selectivity for crizotinib 178 kinases analyzed in EK-293 Biochemical ccupancy at 1 µm (Ambit) 72 hits at >5% ccupancy Live Cell ccupancy at 1 µm (BRET) 16 its at >5% ccupancy - Davis et al. at. Biotech
22 Improved intracellular selectivity for crizotinib 178 kinases analyzed in EK-293 Biochemical ccupancy at 1 µm (Ambit) 72 hits at >5% ccupancy Live Cell ccupancy at 1 µm (BRET) 16 its at >5% ccupancy MET and ALK - Davis et al. at. Biotech
23 Improved intracellular selectivity for clinically-relevant inhibitors Biochemical (Ambit) Crizotinib Dasatinib ilotinib Crizotinib Intracellular (anobret) Dasatinib ilotinib 23
24 Measuring Compound Residence Time In Live Cells Closed System: Drug concentration static pen System: Drug concentration dynamic 24
25 BRET to monitor in-cell residence time (τ) ypothetical Data BRET Signal Short Residence Long Residence Super long residence or covalent Time 25
26 Tuning for durable residence time at BTK with reversible covalent inhibitors Inverted Cyanoacrylamide Electrophile Kinase Recognition Cys BTK Ibrutinib (irreversible, covalent) - Bradshaw et al., at Chem Bio.,
27 Tunable engagement at BTK (Cys481) BRET reveals a wide range of τ despite similar K d GDC-834 (reversible, non-covalent) Varying branch-chain alkyl capping groups Ibrutinib (irreversible, covalent) BTK-Luc BTK Live Cell Residence Time Analysis ormalized BRET [Compound], µ M -4 h equilibration 4 24 GDC-834 BRET Ratio (mbu) Tim e (m in) Vehicle 4 24 GDC-834 Ibrutinib Washout 27
28 Striking discordance between τ and K d 9 demonstrates durable binding despite weaker affinity BLK anoluc-blk Live Cell Analysis of Residence Tim e ormalized BRET [Compound], um BRET Ratio (mbu) V ehicle 4 9 Ibrutinib Tim e (m in) 28
29 Kinetic selectivity at BTK over TEC, BLK Com pound 4 Residence Tim e In Cell ormalized BRET Compound 4 E quilibrium A nalysis [Compound 4], um BLK BTK TEC ormalized BRET BLK TEC BTK Ibrutinib (BTK) Kinase τ (hrs) BTK >1 TEC 9.1 BLK T im e (m in ) 29
30 Summary Biochemical analysis may not always predict cellular activity Engagement in isolation Intracellular Affinity Cellular Effect RTKs PI3K JAK SRC Kinase MAPK AKT Kinase Selectivity may vary between open and closed system conditions Phenotype uture Directions: Expanding anobret TE assays over the kinome Creating a Selectivity Profiling Capability 3
31 Assay optimization data are provided online BTK-anoLuc Tracer Affinity Live EK293 Cells BRET Ratio (mbu) Tracer nly Tracer + excess unlabeled EC 5 =.53µ M [anobret Tracer K-5], µ M BTK-anoLuc Compound Affinity Live EK293 Cells BRET Ratio (mbu) [Test Compound], µ M [Tracer] 4 µ M 2 µ M 1 µ M.5µ M.25µ M.13µ M IC5 4µM.31 2µM.18 1µM.18.5µM.17.25µM.12.13µM.18 31
32 Thank you! Questions? 32
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