Characterizing degradation kinetics and cellular mechanisms of PROTAC compounds. Kristin M. Riching, Ph.D. Protein Degradation Therapeutics 2018

Transcription

1 Characterizing degradation kinetics and cellular mechanisms of PROTAC compounds Kristin M. Riching, Ph.D. Protein Degradation Therapeutics 28

2 Understanding the Dynamics and Mechanisms of Protein Degradation Challenges in understanding degradation and PROTAC efficacy: Protein degradation is a dynamic and complex process. How do we assess whether a PROTAC degrades its target? If no degradation is observed, what happened? How do we know what steps in the pathway to optimize or improve? Is degradation efficacy correlated to specific mechanisms? Solutions Develop technologies to understand the key steps for efficient and targeted degradation. Use assays to advance research and help development of degradation/protac compounds. Clin. Cancer Res. 8(), AACR 2

3 Comprehensive Approach for Cellular Characterization PROTAC Target Protein Degradation PROTAC permeability/binding PROTAC induced interactions Target Protein Degradation Phenotype Target + PROTAC PROTAC Ternary Complex VHL E3 Ligase HaloTag Fusion Cl Ubiquitin Ub Ub Ub Target Binary Complexes E3 Recruiter Ub Proteasome Proprietary Information. Not for further distribution.

4 Technologies for Monitoring Events within the Degradation Pathway Luminescence Protein Levels: Degradation and recovery NanoLuc Luciferase NanoLuc Fusion HiBiT Technology Binary Complementation of NanoBiT Enzyme HiBiT Fusion + LgBiT NanoBiT Fusion NanoLuc aa HiBiT 7kDa LgBiT NanoBiT NanoBRET: Bioluminescence Resonance Energy Transfer Fluorescent Acceptor Donor:Acceptor Complex Mechanism: PROTAC binding and protein:protein interactions Luminescent Donor OR + HaloTag Fusion Energy Transfer Energy Transfer NanoBiT or NanoLuc Fusion Tracer Compound 4

5 Luminescent imaging capturing endogenously tagged BRD4 degradation Brightfield CRISPR HiBiT-BRD4 Treatment with MZ shows uniform loss of BRD4 over 2 hours Imaging performed on Olympus LV2 How do we look at degradation quantitatively? Degradation rate Degradation IC5 Proprietary Information. Not for further distribution.

6 BET family comparisons of protein degradation and recovery HiBiT (CRISPR) BRD2, BRD3, or BRD4 Endogenously tagged protein + MZ or dbet.c M M Z nm MZ BRD2 BRD3 BRD4 B R D 2 c B R D 3 B R D 4. cm d B e t nm dbet BRD2 BRD3 BRD4 B R D 2 B R D 3 B R D Each family member reveals both unique degradation and recovery response Where should dose response curves be performed to understand degradation IC 5 s? Time component is critical, what about drug concentration treatment? Proprietary Information. Not for further distribution.

7 Live Cell Degradation Profiles Across Concentration Series B R D 2 B R D 3 B R D 4 M d B E T B R D 2 B R D 3 B R D 4 M M Z Target and concentration-specific degradation and recovery response to MZ and dbet 7

8 Using Profiles to Calculate PROTAC Degradation Parameters D max Degradation Profile Plateau +.(D max ) Plateau Time at D max Quantitative Parameters Rate of degradation Dmax/DC 5 Time at Dmax y = (y Plateau)e ƛt + Plateau ƛ = Degradation Rate Can calculate multiple parameters in a single degradation profile. B R D 2 Dmax B R D 3 M M Z B R D 2 Rate M M Z M M Z

9 D egrada tion R ate C onstant, (h r - ) F r a c t i o n a l R L U T im e (h o u rs) Graphical Representations of Calculated Profile Degradation Parameters D egrada tion R ate D e g r a d a t i o n M a x i m u m T im e a t D m a x, M M P R O T A C M P R O T A C DC 5 (nm) 5 B R D 2 B R D 3 B R D 4 B R D 2 dbe T B R D 3 dbe T B R D 4 dbe T B R D 2 M Z B R D 3 M Z B R D 4 M Z B R D 2 d B E T B R D 3 d B E T B R D 4 d B E T B R D 2 M Z B R D 3 M Z B R D 4 M Z C R B N / dbet V HL / M Z Can rank the order of PROTAC and family member responses across various quantitative parameters. 9

10 % D e g r a d a tio n Comparing Degradation from Endogenous and Ectopic Expression Endogenous B RHiBiT-BRD4 D 4 M M Z Transient NanoLuc-BRD4 B R D 4 M M Z Ectopic expression differs from endogenous by: Degradation rate Extent Protein recovery R a te v s C o n c. D m a x Ectopic expression recommendations: 2.5 E n d o g e n o u s E n d o g e n o u s Express at low levels T ra n s ie n t 5. T ra n s ie n t MZ DC 5 Endogenous = 8nM Transient = 85nM Use only qualitatively (yes or no for degradation). Stable cell line if possible for more uniform expression. M M Z M M Z

11 Monitoring PROTAC permeability and target engagement Target Engagement PROTAC NanoBRET Target engagement Drug Tracer Nano Luc Target Nano Luc Target Intracellular binding Intracellular affinity Target Binary Complexes E3 Recruiter Bioluminescence Resonance Energy Transfer to monitor protein:small molecule interactions Highly specific, biophysical measurement Monitor binding affinity, residence time and rank order compounds NanoBRET target engagement assays can be performed using: Transient, stable or CRISPR endogenously tagged NanoLuc or HiBiT fusions Target specific or E3 ligase fluorescent tracers Compatible with lytic or live-cell formats to assess cellular permeability.

12 N o r m a liz e d B R E T N o r m a liz e d B R E T Comparing lytic versus live cell to assess permeability NanoBRET TE E3 Competitive Displacement PROTAC V H L, L y s a te V H L, L iv e C e ll V H V H M Z M Z OR E3 Ligase Tracer NanoLuc-E3 Ligase Parental M C o m p o u n d M C o m p o u n d Compound Target IC 5 (nm, Lysate) IC 5 (nm, Live Cell) VH298 VHL MZ VHL No difference in VHL binding affinities observed in lytic format between parental (VH298) and MZ PROTAC. NanoBRET target engagement in live cells shows binding affinity shift, indicating reduced permeability of MZ. 2

13 N o r m a liz e d B R E T IC 5 ( M ) Measuring intracellular binding to target BRD4 protein NanoBRET TE Target Competitive Displacement PROTAC B R D 4, L iv e C e ll J Q B R D 4 E n g a g e m e n t d B E T d B E T J Q - BET Tracer OR M Z M Z NanoLuc-BRD4 Parental M C o m p o u n d T im e (m in.) Compound Target IC 5 (nm, Live Cell) MZ BRD4 432 dbet BRD4 747 JQ BRD4 54 Significant shift in intracellular binding affinities for BRD4 by MZ and dbet as compared to parental JQ. Kinetic binding analysis showed slow intracellular engagement, suggesting reduced permeability of PROTACs. 3

14 Monitoring Interactions within the Degradation Pathway by NanoBRET NanoBRET Ternary Complex Assay HaloTag -Ub Acceptor Two NanoBRET Ubiquitination Assays Alexa594-Antibody Acceptor NanoBRET Proteasome Assay HaloTag-E3 Ligase Ub Ub Ub Ub HaloTag- Ubiquitin Ub Ub Ub Ub Alexa594 Ab HaloTag- Proteasome Subunit NanoLuc or HiBiT Fusion Protein NanoLuc or HiBiT Fusion Protein NanoLuc or HiBiT Fusion Protein NanoLuc or HiBiT Fusion Protein NanoBRET protein:protein interaction assays can be performed using: Transient, stable or CRISPR endogenously tagged NanoLuc or HiBiT fusions as energy donor. Transient or stable HaloTag fusions as energy acceptors 4

15 F o ld in c re a s e in B R E T F o ld in c re a s e in B R E T F o ld in c re a s e in B R E T Kinetic monitoring of ternary complexes NanoBRET Assay HiBiT BET-PROTAC-E3 B R D 2 B R D 3 B R D 4 5 C R B N / d B e t ( u M ) 8 V H L / M Z ( u M ) T im e (h r s ) T im e (h r s ) T im e (h r s ) Kinetic patterns are different with each PROTAC. Faster complex formation with dbet/crbn, but more stable formation with MZ/VHL 5

16 F o ld in c re a s e in B R E T F o ld in c re a s e in B R E T F o ld in c re a s e in B R E T Kinetic monitoring of BET family ubiquitination NanoBRET Assay HiBiT BET-HaloTag -Ub Ub Ub Ub B R D B R D B R D 4 D M S O d B e t ( u M ) Ub M Z ( u M ).5 2. T im e (h r).5 2. T im e (h r).5 2. T im e (h r) Robust ubiquitination of BRD2 and BRD4 treated with MZ The different E3 ternary complexes recruited by dbet and MZ show differential ubiquitination on respective targets. 6

17 F o ld in c re a s e in B R E T D e g ra d a tio n R a te Endogenous ubiquitination patterns and correlation with degradation rate NanoBRET Assay HiBiT BET-Polyclonal Ub A n ti-u b iq u itin B R D 2 d B E T 2.5 C o r r e la tio n o f D e g r a d a tio n R a te w ith U b iq u itin a tio n B R D 2 d B E T Ub Ub Ub B R D 3 d B E T B R D 4 d B E T 2. B R D 3 d B E T B R D 4 d B E T Ub 5 B R D 2 M Z B R D 3 M Z B R D 4 M Z.5 B R D 2 M Z B R D 3 M Z B R D 4 M Z T im e (h r s ) U b iq u itin a tio n F o ld In c re a s e in B R E T Relative intensity differences and patterns similar to HaloTag -Ub NanoBRET Assay. Anti-Ubiquitin measurements are done using lytic, endpoint measurements. Level of ubiquitination shows linear relationship, high correlation with calculated degradation rate. 7

18 B R E T R a tio (m B U ) B R E T R a tio (m B U ) Kinetic trafficking of BRD4 to the Proteasome N L -B R D 4 & H T -P S M D 3 NanoLuc-BRD4:HaloTag-PSMD N L -B R D 4 & H T -P S M D 3 V e h ic le C o n tro l M Z V ( e huic M le ) C/ Vo nh tro 2 9l 8 (. 2 u M ) 4 M Z ( ( u M ) / V H (..327 u M )) 3 MZ Z ( ( u M ) / V H ( (.3.7 u M )) 3 2 M Z Z ( ( u M ) / V H ( ( u M ) ) 2 M Z ( u M ) / V H (3.3 3 u M ) M Z ( u M ) / V H ( ) M Z ( u M ) / V H ( u M ) T im e (m in ) 5 2 T im e (m in ) Detect increase in trafficking of BRD4 to proteasome in the presence of PROTAC. Parental compounds can be used as controls. Can simultaneously monitor loss of target (NanoLuc-BRD4) in proteasome assay. 8

19 Targeted degradation phenotype using HaloTag PROTACs HaloPROTAC Study phenotype of degraded targets HaloPROTAC targets HaloTag fusion proteins for degradation through VHL recruitment ACS Chem Biol, 25,, 83 Help define targets important for PROTAC development VHL E3 Ligase HaloTag Fusion Cl Degradation Goals: Generate endogenously tagged HaloTag fusions Pair with HiBiT (HiBiT-HaloTag) for easy detection Characterize kinetics of degradation for variety of proteins Proprietary Information. Not for further distribution.

20 Kinetic degradation profiles of HiBiT-HaloTag-BRD4 with HaloPROTAC H ib it -H a lo T a g -B R D 4 H T P R O T A C.5 u M u M. u M u M u M 4 5 u M 2 3 T im e (h r) u M u M Efficient loss of endogenous HiBiT-HaloTag-BRD4 using HaloPROTAC, even at nm concentration HaloPROTAC also demonstrates burst/rapid degradation loss HiBiT could be appended to any fusion tag PROTAC to enable live-cell kinetic detection Proprietary Information. Not for further distribution.

21 Summary Differentiating cellular technologies are available to study key processes in PROTAC-mediated degradation. HiBiT and NanoLuc technology Measure kinetics of degradation in live cells. Can be used with CRISPR to study endogenous proteins. Able to quantitate key degradation parameters. NanoBRET technology HiBiT- Fusion HiBiT or NanoLuc technology aa HiBiT + LgBiT LgBiT OR NanoLuc Fusion NanoLuc NanoBRET Bioluminescence Resonance Energy Transfer Monitor dynamic pathway interactions and signaling mechanisms in live cells. Luminescent Donor Fluorescent Acceptor Donor:Acceptor Complex Can assess PROTAC cellular permeability. Follow induced interactions with E3 ligase components, Ubiquitin and subsequent trafficking to proteasome. OR + NanoBiT or NanoLuc Fusion HaloTag Fusion Energy Transfer 2