Monitoring Protein:Protein Interactions in Living Cells Using NanoBRET Technology Danette L. Daniels, Ph.D. Group Leader, Functional Proteomics
Proteomics and studying dynamic interactions AP-MS Proteomics Affinity purifications (AP) Mass spectrometry (MS) Long lists of data Direct or indirect interactors? Strong or transient? Static or biased picture??? Strategy for studying dynamic interactions in living cells Monitor interactions in cellular context Develop live cell assay for drug screening of protein interactions Analyze both protein:protein and protein:small molecule interactions Utilize Bioluminescence Resonance Energy Transfer (BRET)
Principles of Biolumuniscence Resonance Energy Transfer BRET BRET Proximity D A D A Geometry Spectral Overlap Donor Acceptor Protein:protein interaction Emission Current BRET technologies have limitations: Luciferase Protein Protein A Protein B Fluorescent Protein Noise BRET Poor spectral separation Requires high donor expression Poor Signal-to noise
Improving BRET with NanoLuc donor and HaloTag acceptor BRET BRET Proximity D A D A Geometry Spectral Overlap Donor Acceptor NanoLuc Luciferase NanoBRET Assay HaloTag fusion In vivo fluorescent labeling NanoBRET Donor Acceptor 400 500 600 700 400 500 600 700 BRET NLuc signal = background Increased signal and decreased spectral overlap Improved Signal-to-noise
median RLU NanoLuc luciferase NanoLuc (Nluc) is a 19.1 kda, ATP-independent luciferase that utilizes a novel coelenterazine analog (furimazine) to produce high intensity, glow-type luminescence. NanoLuc (Nluc) Firefly (Fluc) Renilla (Rluc) 1.0 10 8 1.0 10 7 1.0 10 6 Amino acids M.W. Mol. Vol. Å 3 Nluc 171 19.1 14 Rluc 312 36.0 32 Fluc 550 60.6 44 1.0 10 5 NanoLuc (Nluc) NLuc RLuc8 RLuc Smallest, brightest luciferase Can be used as a reporter or fusion partner
Relative emission NanoLuc has excellent physical properties Thermal stable enzyme Retains activity following 30 min incubation at 55 C Melting temps: Nluc, 58 C; Fluc, 31 C Active over broad ph range Fully active between ph 7-9 Retains significant activity at ph 5-7 Fluc: sharp decrease in activity below ph = 8 Monomeric enzyme Facilitates use as transcriptional reporter or fusion partner No post-translational modifications detected in mammalian cells No disulfide bonds Supports high levels of activity inside living cells Uniform distribution in cells No apparent compartmental bias in the absence of targeting sequences 400 500 600 700 (nm) 6 100 80 60 40 20 0 460 nm 480 nm U2OS cells 565 nm Unfused NLuc Immunofluorescence NanoLuc Renilla Firefly
HaloTag is a Genetically Engineered Protein Fusion Tag Protein of Interest HT + Cl O O Functional group Protein of Interest HT O O Functional group A monomeric, 34 kda, modified bacterial dehalogenase genetically engineered to covalently bind specific, synthetic HaloTag ligands Irreversible, covalent attachment of chemical functionalities Suitable as either N- or C- terminal fusion
Ligands Impart Multi-functionality HaloTag protein HaloTag ligand HaloTag surfaces Nonmagnetic resin Magnetic resin Glass slides R HaloTag fluorescent ligands Many different colors Cell permeable ligands Cell non-permeable ligands NanoBRET Ligand HaloTag reactive ligands Attach functional group of choice i.e. Quantum dots, PET ligands Selectable functionalties: a single fusion construct may be attached to a broad range of functional properties
Developed and custom NanoBRET assays available Available Assays (~100) Epigenetic Readers and Enzymes Transcription Factors (p53/mdm2) Kinases (Ras/Raf) Membrane Receptors GPCRs Custom Design NanoBRET Assays Inhibitor Screening BRD4:Histone H3.3 + JQ1 IC50 = 65.4 nm NanoBRET Protein:Protein 0.010 0.005 0.000 cmyc:max HDAC1:HDAC2 0.025 0.020 0.015 0.010 0.005 0.000 P53:MDM2 + Nutlin-3
Strategy for developing NanoBRET assay 1. Append donor and acceptor tags to proteins 2. Find combination gives best energy transfer and follows proper physiology Protein A Protein B NL HT NL Each N or C terminus for a total of 8 possible constructs NL A + B HT NL A + HT B A NL + B HT HT A + B HT A + NL B NL A HT + B NL HT A NL + HT B A HT + NL B 3. Determine optimal donor to acceptor DNA ratio to minimize free/unbound donor to maximize dynamic range NL
Empirical Optimization of BRET Pairs for FKBP/Frb FKBP D FKBP D Y F P -F rb / F K B P -R L Y F P -F rb / R L -F K B P FRB A A FRB F rb -Y F P / F K B P -R L 8 F rb -Y F P / R L 8 -F K B P Y F P -F K B P / F rb -R L 8 D FKBP FRB A D A FKBP FRB Y F P -F K B P / R L 8 -F rb F K B P -Y F P / F rb -R L 8 F K B P -Y F P / R L 8 -F rb FKBP FRB A D A FKBP FRB D H T -F rb / F K B P -N L H T -F rb / N L -F K B P F rb -H T / F K B P -N L F rb -H T / N L -F K B P FKBP A A FKBP H T -F K B P / F rb -N L H T -F K B P / N L -F rb D FRB D FRB F K B P -H T / F rb -N L F K B P -H T / N L -F rb 0 1 2 3 4 5 6 7 8 9 1 0 S /B r a tio
S /B R a tio Comparison of NanoBRET with conventional BRET Protein interaction model: FKBP / Frb FKBP + HT 1 0 8 N L u c : H T R L u c 8 : Y F P Frb Nluc 6 Rapamycin 4 FKBP Frb HT Nluc 2 0 0.0 0 0 1 0.0 1 1 1 0 0 1 0 0 0 0 R a p a m y c in [n M ] Improved signal:background as compared to other BRET systems Greater light output with NanoLuc enables BRET at low levels of expression
m B R E T u n its m B R E T u n its NanoBRET Enables PPI Analysis at Low Expression Levels NanoBRET Frb-NLuc / FKBP-HT BRET1 Frb-RLuc8 / FKBP-YFP 1 0 Z value 0.61 0.40-1.39 1 0 Z value 0.47 0.11-0.22 1 1 2 0 6.6 2.2 0.7 4 0.2 4 6 0.0 8 2 0.0 2 7 0.0 0 9 0.0 0 3 2 0 6.6 2.2 0.7 4 0.2 4 6 0.0 8 2 0.0 2 7 0.0 0 9 0.0 0 3 D N A [n g /w e ll] D N A [n g /w e ll] FKBP-HT FKBP-YFP
Applications of NanoBRET technology for drug screening NLuc HT Protein A Protein B Identify interactions NLuc Protein A HT Protein B Monitor modulations Protein:Protein NLuc Protein A Measure compound binding NLuc Protein A Rank order compounds Protein:Small Molecule
Use of NanoBRET in epigenetics research Epigenetic reader proteins Bromodomain family Implicated in numerous diseases Promising initial compounds Current assays utilize purified domain and histone fragments Complexity of chromatin difficult to recapitulate in in vitro assays NanoBRET bromodomain-histone interactions in live cells Arrowsmith, et. al (2012) Nature Reviews Drug Discovery 11, 384-400
NanoBRET for monitoring interactions with chromatin H2A-HaloTag H3.3-HaloTag HaloTag fusion HT Histone TMR H2B-HaloTag H4-HaloTag Confocal Imaging Observed chromatin incorporation of HaloTag fusions: H2A, H2B, H3.3, and H4 Other Histone variants available (H3.1, H3.2, H2A.X, H2A.Z, MacroH2A) Scale bars = 10µm
Corrected BRET Ratio Average 610/450 BRET Ratio NanoBRET for monitoring BRD4 binding to histone H3 or H4 HaloTag fusion H3 or H4 NanoLuc fusion NanoLuc HT BRD4 0.012 0.01 H3.3 H4 0.008 0.006 0.004 0.002 0 NL-BRD4 NL-BRD4 NL-BRD4 / No HaloTag Detected specific interaction of BRD4 with Histones H3 and H4.
mbret BRET panel of full-length bromodomains with H3.3 55 50 45 40 35 30 25 20 15 10 5 0 NanoLuc Fusions Optimization for placement of NanoLuc and transfection ratios of bromodomains Absolute BRET ratio dependent on numerous factors: Proximity, kd, and occupancy Optimized conditions also for Histone H4. 18
Measuring Effect of BET Inhibitor on Histone-Bromodomain Interactions BRD4-Histone H3.3 CBP-Histone H3.3 IC50 = 65.4 nm BRD4-Histone H4 CBP-Histone H4 Measured potency differences amongst bromodomain proteins and histones IC50 = 222.8 nm
Corrected mbret ratios Corrected mbret ratios Assay compatibility for screening in 384 and 96 well formats BRD4-Histone H3.3 384 format BRD4-Histone H3.3 96 format 60 60 50 40 Z'=0.72 45% Inhibition 50 40 Z'=0.83 49% Inhibition 30 30 20 20 10 10 0 Untreated 10µM IBET151 0 Untreated 10µM IBET151
Relative viability Domains alone can be used and cell toxicity measured BD1 of BRD4 Cell-Titer Glo Effect of IBET-151 on viability HEK293T cells Z =0.7 120% 100% 80% 60% 40% 20% 0% 0 0.001 0.01 0.05 0.1 0.5 1 5 10 µm IBET-151 Assay possible with the bromodomain alone or larger fragments Cell toxicity can be directly measured using Cell Titer Glo 21
Comparing inhibition of full length protein to domains BRD4 BD1 BD2 BRD4 Constructs Full-length BD1 BRD4 Full-length /H3 BRD4 (BD1)/H3 In this case, similar IC50s obtained for domain alone as compared to full-length protein
mbret Inhibition of BAZ2A bromodomain with GSK2801 BAZ2A Full-length/H3 + GSK2801 BrD of BAZ2A /H3 + GSK2801 Observe no detectable inhibition of the full-length BAZA with GSK2801 Excellent inhibition of BAZ2A bromodomain (BrD) alone
% BRET remaining after 10μM treatment Breaking down the contributions of the BAZ2A domains MBD PHD BD BAZ2A Full-length MBD+PHD+BD PHD+BD BD 100 90 80 70 60 50 40 30 20 10 0 Full-length MBD+BRD+PHD BRD+PHD BRD Alone Addition of PHD domain significantly reduces inhibition by GSK2801
Differential inhibition of CBP full length vs domains of CBP CBP KIX BD HAT CREBBD Full-length BD-HAT BD CBP Full-length /H3 CBP (BD-HAT) /H3 CBP (BD) /H3 IC50=N/A 0% Inhibition IC50=1.03μM 14% Inhibition IC50=1.0μM 70% Inhibition Observe no inhibition of the full-length CBP with SGC CBP-30 Similar IC50s for BD-HAT and BD domain fragments, yet greater % inhibition with BD alone
Monitoring small molecule binding to target proteins with NanoBRET Drug Tracer NLuc Target protein NLuc Target Express Nluc fusion transiently or stably Measure BRET Via Endpoint Or In Real Time Add tracer +/- compounds Add furimazine 26
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 NanoBRET for the study of compound binding to various targets Monitor small molecule binding NLuc BRD Bromodomain NLuc Bromodomain Protein B Protein B 3 0 0 2 0 0 1 0 0 N L u c -B R D F u s io n s 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 L iv e H e L a C e lls Increasing BRET Ratio 0 1 0-4 1 0-2 1 0 0 1 0 2 B E T T ra c e r (u M ) BET Tracer binds specifically to BET family members or BET bromodomains alone.
N o rm a liz e d B R E T R e s p o n s e Competition profiles for various BET inhibitors Competition Experiments NLuc Bromodomain NLuc Bromodomain Decreasing BRET Ratio N L u c -B R D 4 (F u ll L e n g th ) C o m p e titiv e D is p la c e m e n t 1 1 0 1 0 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0-1 0 1 0-4 1 0-3 1 0-2 1 0-1 1 0 0 1 0 1 1 0 2 [In h ib ito r], u M IB E T IB E T -1 5 1 (+ )-J Q 1 C P I2 0 3 P F I-1 Competition studies of BET inhibitors reveal rank order potencies of the different compounds for binding to BRD4.
Tracer + Cold Tracer Nuclear Localization of BRET HeLa Cells transfected with Nluc-BRD4 2uM IBET Tracer +/- 10uM Cold IBET Donor Acceptor Bright Field
Summary NanoBRET technology Improved BRET protein interaction system using NanoLuc and HaloTag Increased signal:background and dynamic range Monitor intracellular protein:protein and protein:small molecule interactions Assays are compatible for screening in 96 and 384 well formats Rank order drug targets and determine specificity of compound binding Rank order compounds against a specific target NanoBRET is compatible for imaging experiments
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