Targeting Proteins for Degradation: Characterizing PROTAC Kinetics and Mode of Action using Live-Cell Assays. Kristin M. Riching, Ph.

Transcription

1 Targeting Proteins for Degradation: Characterizing PROTAC Kinetics and Mode of Action using Live-Cell Assays Kristin M. Riching, Ph.D

2 PROTACs: An Attractive Drug Discovery Strategy PROTACs, or Proteolysis Targeting Chimeras, induce selective degradation of target proteins by simultaneously binding to target proteins and E3 ubiquitin ligases. Basic PROTAC Structure Ligase Recognition Domain Target Protein Ligand E3 iquitin Ligase Target Protein Proteosome Degraded Target Why PROTACs over classical inhibitors? Improved potency due to catalytic mode of action (MOA). Prolonged pharmacodynamics due to need for protein resynthesis. Potential for targeting undruggables with ligands that can bind anywhere on a protein surface. 2

3 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. 18(1), AACR 3

4 PROTAC Cellular Permeability Target Engagement Five Key Steps for Monitoring PROTAC Activity Target Binary E3 Recruiter Active E2/E3 Complex Target iquitination Proteasome Recruitment Target Degradation E2 Ternary E3 iquitination Proteasome Engagement Protein Level Ternary Complex Formation 4

5 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 11aa HiBiT 17kDa 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 5

6 BET Family Bromodomain Inhibitors and PROTACs Significant therapeutic promise for variety of leukemia, lymphomas and other hematologic cancers First generation: Protein interaction inhibitors Next generation: Heterobifunctional degraders/protacs MZ1 dbet1 ACS Chem. Biol., 215, 1, 177. Science, 215, 348, Nature 21 December 23, Nature , VHL System CRBN System Goals: Can we understand the key steps for efficient and targeted degradation? Does this insight help development of therapeutic degradation compounds? E2 Rbx1 (E3) Cullin2 BET VHL EloC EloB E2 Rbx1 (E3) Cullin4A BET BET CRBN 6

7 Approach for Studying the BET Family Members and Degradation 7

8 Assay #1: Monitoring Target Protein Degradation Degradation Assay Target + PROTAC Target Degradation NanoLuc luciferases for protein level detection NanoLuc Luciferase NanoLuc Fusion HiBiT Technology Binary Complementation of NanoBiT Enzyme HiBiT Fusion + LgBiT NanoBiT Fusion NanoLuc 11aa HiBiT 17kDa LgBiT NanoBiT Target NanoLuc and HiBiT fusion proteins can be generated using: Transiently or stably expressing fusion vectors. CRISPR endogenous tagging RECOMMENDED for all quantitative calculations (rate, DC 5, and Dmax). Degradation assays can be performed: As lytic or in live cells. As long-term kinetic analysis (24 48 hours) with Endurazine or Vivazine extended substrate. 8

9 HiBiT CRISPR of BET Family Members and Initial Testing Luminescent Blot Luminescent Imaging of HiBiT-BRD4 + MZ1 PROTAC HiBiT CRISPR BRD2, 3 or 4 kda BRD2 BRD3 BRD4 26 Time (minutes) HiBiT- BRD JQ1 16 Endogenously tagged protein 11 Endogenously tagged BET family members Bright Field Endpoint Luminescent (RLU) Analysis with dbet1 or MZ1 RLU hours BRD2 BRD3 BRD4 DMSO 1 µ M dbet1 RLU hours BRD2 BRD3 BRD4 DMSO 1 µ M MZ1 MZ1 ACS Chem. Biol., 215, 1, 177. dbet1 Science, 215, 348,

10 Live Cell Kinetic Degradation Analysis at Fixed Concentration for 24 Hours HiBiT CRISPR BRD2, 3 or 4 Endogenously tagged protein + MZ1 or dbet1 Fractional RLU µ M MZ c 1nM MZ1 Time (hours) BRD2 BRD3 BRD4 c BRD2 BRD3 BRD4 Fractional RLU 1..5 c.1µ M db et1 1nM dbet Time (hours) BRD2 BRD4 BRD3 BRD2 BRD3 BRD4 Each family member revealed both unique degradation and recovery response to MZ1 and dbet1. Demonstrated how degradation measurements at fixed points in time can differ drastically. Used the extended furimazine substrate, Endurazine, and read continuously on a GloMax Discover. 1

11 Live Cell Degradation Profiles Across Concentration Series BRD2 BRD3 BRD4 µ M dbet1 1 Fractional RLU 1..5 Fractional RLU 1..5 Fractional RLU Time (hours) Time (hours) Time (hours) BRD2 BRD3 BRD4 µ M MZ1 1 Fractional RLU 1..5 Fractional RLU 1..5 Fractional RLU Time (hours) Time (hours) Time (hours) Target and concentration-specific degradation and recovery response to MZ1 and dbet1 11

12 Using Profiles to Calculate PROTAC Degradation Parameters D max Degradation Profile 1 Plateau +.1(D max ) Plateau Time at D max Quantitative Parameters Rate of degradation Dmax/DC 5 Time at Dmax yy = (yy PPPPPPPPPPPPPP)ee ƛtt + PPPPPPPPPPPPPP ƛ = DDDDDDDDDDDDDDDDDDDDDD RRRRRRRR Time (hours) Can calculate multiple parameters in a single degradation profile. Fractional RLU 1..5 Fractional RLU 1..5 BRD2 Dmax BRD3 Fractional RLU µ M MZ Time (hours) BRD2 Rate Time (hours) µ M MZ µ M MZ

13 Graphical Representations of Calculated Profile Degradation Parameters Degradation Rate Degradation M axim um Tim e at D max, 1 µ M Degradation Rate Constant, λ (hr -1 ) µ M PROTAC Fractional R LU µ M PROTAC DC 5 (nm) Time (hours) BRD2 BRD3 BRD4 BRD2 dbet1 BRD3 dbet1 BRD4 dbet1 BRD2 MZ1 BRD3 MZ1 BRD4 MZ1 BRD2 dbet1 BRD3 dbet1 BRD4 dbet1 B R D 2 M Z 1 B R D 3 M Z 1 B R D 4 M Z CRBN / dbet1 VHL / MZ1 Can rank the order of PROTAC and family member responses across various quantitative parameters. 13

14 Comparing Degradation from Endogenous and Ectopic Expression Fractional RLU 1..5 Endogenous BRD4 HiBiT-BRD4 µ M MZ Fractional RLU Transient NanoLuc-BRD4 µ M MZ Ectopic expression differs from endogenous by: Degradation rate Extent Protein recovery Time (hours) Rate vs Conc. Time (hours) Dmax Ectopic expression recommendations: 2.5 Endogenous 1 Endogenous Express at low levels. Fractional RLU Transient % Degradation Transient MZ1 DC 5 Endogenous = 8nM Transient = 85nM Use only qualitatively (yes or no for degradation). Stable cell line if possible for more uniform expression. µ M MZ1 µ M MZ1 14

15 Assay #2: Monitoring PROTAC Permeability and Target Engagement Target Engagement PROTAC NanoBRET Target engagement Drug Tracer Target Target NanoLuc Intracellular binding NanoLuc 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. 15

16 Comparing Lytic versus Live Cell to Assess Permeability NanoBRET TE E3 Competitive Displacement PROTAC VHL, Lysate V H L, Live C ell E3 Ligase Tracer NanoLuc-E3 Ligase OR Parental Normalized BRET 1..5 VH298 MZ1 Normalized BRET 1..5 VH298 MZ µ M Compound µ M Compound Compound Target IC 5 (nm, Lysate) IC 5 (nm, Live Cell) VH298 VHL MZ1 VHL No difference in VHL binding affinities observed in lytic format between parental (VH298) and MZ1 PROTAC. NanoBRET target engagement in live cells shows binding affinity shift, indicating reduced permeability of MZ1. 16

17 Measuring Intracellular Binding to Target BRD4 Protein NanoBRET TE Target Competitive Displacement BRD4, Live Cell BRD4 Engagement PROTAC JQ1 1 dbet NanoLuc-BRD4 BET Tracer OR Parental Normalized BRET 1..5 dbet1 MZ1 IC 5 (µ M) 1.1 JQ -1 MZ µ M Compound Tim e (m in.) Compound Target IC 5 (nm, Live Cell) MZ1 BRD4 432 dbet1 BRD4 747 JQ1 BRD4 54 Significant shift in intracellular binding affinities for BRD4 by MZ1 and dbet1 as compared to parental JQ1. Kinetic binding analysis showed slow intracellular engagement, suggesting reduced permeability of PROTACs. 17

18 Assay #3: Monitoring E3 Ternary Complex Formation NanoBRET protein:protein interactions Target PROTAC E3 Component Target-PROTAC-E3 Luminescent Donor Fluorescent Acceptor Donor:Acceptor Complex OR + NanoBiT or NanoLuc Fusion HaloTag Fusion Energy Transfer 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 E3 ligases or components as energy acceptors. Compatible with lytic or live-cell formats. Long-term kinetic analysis also possible using stabilized Vivazine substrate. 18

19 Ternary Complex Detection and Controls NanoLuc- BRD4 HaloTag- VHL PROTAC +/- MG132 NanoBRET Assay Target-PROTAC-E3 BRET Ratio (mbu) NL-BRD4 & HT-VHL No PROTAC MZ1 (1uM) Luminescense (RLU) Relative Levels of NL-BRD4 No PROTAC MZ1 (1uM) No Proteasome Inhibitor M G 132 (1uM ) Pre-Treatment No Proteasome Inhibitor M G 132 (1uM ) Pre-Treatment Monitor ternary complex formation and degradation simultaneously. Using MG132 can increase signal window. Using irrelevant E3 ligase components demonstrate signal specificity. BRET Ratio (mbu) BRD4:VHL Drug (um) DMSO MZ1 dbet1 19

20 Kinetic Monitoring of Ternary Complexes NanoBRET Assay HiBiT BET-PROTAC-E3 BRD2 BRD3 BRD4 Fold increase in BRET Fold increase in BRET 1 5 Fold increase in BRET CRBN / dbet1 (1uM) VHL / MZ1 (1uM) Time (hrs) Time (hrs) Time (hrs) Kinetic patterns are different for each family member and each PROTAC. Analysis in the presence of MG132 enables study of cellular stability of ternary complex. Studies greater than 1 hour should use stabilized Vivazine substrate. 2

21 Monitoring Ternary Complexes in Lysates HiBiT-BRD4 HiBiT-BRD4 HaloTag-E3 Lyse Cells +/- PROTAC Lytic NanoBRET Assay Target-PROTAC-E3 BRET Ratio (mbu) CRBN / dbet1 V H L / M Z1. Untreated 1 µ M PROTAC Untreated 1 µ M PROTAC PROTACs are added after cell lysis with digitonin. Can screen for compounds that induce ternary complexes without eliminating hits due to low cell permeability. 21

22 Assay #4: Monitoring iquitination Active E3 Complex E3 Target iquitination Two NanoBRET iquitination Assays HaloTag - Acceptor Alexa594-Antibody Acceptor NanoBRET protein:protein and protein:antibody ubiquitination assays can use the following: Transient, stable or CRISPR endogenously tagged NanoLuc or HiBiT fusions as energy donors. Transient HaloTag -iquitin as energy acceptor (live-cell assay). Primary or secondary fluorescently labelled antibody as energy acceptor (lytic assay). Long-term kinetic analysis possible using stabilized Vivazine substrate. 22

23 Kinetic Monitoring of BET Family iquitination NanoBRET Assay HiBiT BET-HaloTag - Fold increase in BRET BRD2 Fold increase in BRET BRD3 Fold increase in BRET BRD4 DMSO db et1 (1uM ) MZ1 (1uM) Time (hr) Time (hr) Time (hr) Live-cell kinetic patterns are different for each family member and each PROTAC. The different E3 ternary complexes recruited by dbet1 and MZ1 show differential ubiquitination on respective targets. Studies greater than 1 hour should use stabilized Vivazine substrate. 23

24 Endogenous iquitination Patterns and Correlation with Degradation Rate NanoBRET Assay HiBiT BET-Polyclonal Fold increase in BRET 1 5 A nti-u biquitin BRD2 dbet1 BRD3 dbet1 BRD4 dbet1 BRD2 MZ1 BRD3 MZ1 BRD4 MZ1 Degradation Rate Correlation of Degradation Rate w ith U biquitination BRD2 dbet1 BRD3 dbet1 BRD4 dbet1 BRD2 MZ1 BRD3 MZ1 BRD4 MZ Time (hrs) iquitination Fold Increase in BRET Relative intensity differences and patterns similar to HaloTag - NanoBRET Assay. Anti-iquitin measurements are done using lytic, endpoint measurements. Level of ubiquitination shows linear relationship, high correlation with calculated degradation rate. 24

25 Assay #5: Monitoring Translocation to Proteasome Target iquitination Proteasome Recruitment NanoBRET Proteasome Assay NanoBRET protein:protein proteasomal assays can use the following: Transient, stable or CRISPR endogenously tagged NanoLuc or HiBiT fusions as energy donors. Transient HaloTag -PSMD3 proteasomal subunit as the energy acceptor. Long-term kinetic analysis possible using stabilized Vivazine substrate. 25

26 Kinetic Trafficking of BRD4 to the Proteasome NL-BRD4 & HT-PSMD3 NanoLuc-BRD4:HaloTag-PSMD3 BRET Ratio (mbu) Vehicle Control MZ1 (1uM) / VH298 (.12uM) MZ1 (1uM) / VH298 (.37uM) MZ1 (1uM) / VH298 (1.11uM) MZ1 (1uM) / VH298 (3.33uM) MZ1 (1uM) / VH298 (1uM) Tim e (m in) Detect increase in trafficking of BRD4 to proteasome in the presence of PROTAC. Parental compounds can be used as controls. We do not recommend using proteasome inhibitors for the assay. Can simultaneously monitor loss of target (NanoLuc-BRD4) in proteasome assay. 26

27 Mechanistic Contributions to Degradation Phases Degradation Initiation Degradation Maximum Recovery PROTAC Binding Permeability Affinity Degradation Rate iquitination Dmax and Time at Dmax Compound potency Ternary complex stability Protein Recovery New protein synthesis Compound stability 27

28 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 11aa 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, iquitin and subsequent trafficking to proteasome. OR + NanoBiT or NanoLuc Fusion HaloTag Fusion Energy Transfer 28

29 Thank You! Questions? 29