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LifeSensors271 Great Valley Parkway
Malvern PA 19355
Phone: 610-644-8845
www.lifesensors.com
PROTAC® Drug Discovery
Proteolysis-targeting Chimeric Molecules
PROTAC is a registered trademark of Arvinas Operations, Inc. and is used under license.
LifeSensors
Leading Biotech in UPS Drug Discovery and Diagnostic R&D
~500 Products, Proteins, Ubiquitin Affinity Reagents (TUBEs), Inhibitors, Assays, Kits and Proprietary Protein Expression Systems (SUMO)
Drug Discovery, UPS and PROTAC Screening Services
Profiling Compounds Against Ubiquitin Ligases and DUBs
Custom Assay Development and Collaborative Research
Ubiquitin Proteasome System
E1 – Ubiquitin activating enzyme
Requires ATP to attach Ub to E1
E2 – Ubiquitin conjugating enzyme
Transfers Ub from E1 to E3
E3 – Ubiquitin ligases
Transfers Ub to self or substrate
Forms mono-Ub or poly-Ub chains
DUB – Deubiquitinase
Removes mono-Ub or poly-Ub chains
Proteasome – Degrades ubiquitylated proteins
• Natural ubiquitin-binding domains (UBDs)
• Designed and engineered for high affinity and selectivity for polyubiquitin chains
• Superior to antibodies with regards to selectivity and versatile applications
• Labeling with tags (i.e., 6xHis, GST, Biotin, Magnetic, fluorophores, etc.)
• Pan-selective and polyubiquitin chain linkage-selective TUBEs
• Variety of applications, mass spec proteomics, imaging, HTS and biomarkers
TUBE Properties
Current State of PROTAC Drug Discovery
Current PROTAC methods are time consuming and irreproducible
Blots are time consuming, non-quantitative, prone to artifacts
Reporter genes cell lines don’t correlate with true function
Link between ubiquitylation and degradation, chicken or the egg!
Not all degradation in cells is ubiquitin mediated
Speed up PROTAC drug discovery
Microtiter plate based, SAR in one day vs two weeks, rank order potency
6
Degraders are Essential Catalytic Activators
Challenges for PROTAC Discovery
Challenges with Current Cell-based Strategies
Reporter Gene Assays
Nonnative
Expensive
Requires expert user
Requires a validated cell line
Multiplexing is challenging
Western Blot assays
Unreliable
Low throughput
No absolute quantitation
Time consuming
No Multiplexed detection
Enables accurate determination of substrate (POI) ubiquitylation for monitoring the effects of PROTAC treatments on patterns of cellular substrate ubiquitylation
PROTAC® Assay Plate (PA950)
ComponentsCoated plates: One pre-coated and blocked 96-well strip plate (Store at -80°C)Blocking concentrate (BC): 12 mL of 5X blocking agent for antibody dilution (Store at 4°C)Plastic plate seals: Two providedDetection reagent 1 (DR1): 1.0 mL vial of DR1 reagent (Store at 4°C)Detection reagent 2 (DR2): 1.0 mL vial of DR2 reagent (Store at 4°C)MG132: 25 µL vial (10 mM in DMSO; Store at -20°C)PR-619: 25 µL vial (22 mM in DMSO; Store at -20°C)1,10-phenanthroline: 100 µL vial (500 mM in DMSO; Store at -20°C)
Components required but not supplied
Phosphate buffered saline (PBS) and PBS containing 0.1% Tween 20 (PBST)
Disposable gloves, pipette tips, reagent reservoirs
Multi-channel pipettors
Automatic plate washer (optional)
A luminescence microplate reader
Schematic of PROTAC® Assay Plate
Example of Cell-based PROTAC Assay
Dose response of dBET1 PROTAC on ubiquitylation and degradation of BRD3
Establish both ubiquitylation and degradation rate
Absolute quantitation
Adaptable to high throughput screening
Example of Cell-based PROTAC Assay
Time course of dBET1 PROTAC on ubiquitylation and degradation of BRD3
Example of Cell-based PROTAC Assay
Time course of cereblon-based pan kinase PROTAC on ubiquitylation and
degradation of selected kinases Aurora Kinase A and BTK
In vitro PROTAC High-throughput Screening Service
Plate-based in vitro assay
Purified E1, E2, Ub, ATP
Add E3 ligase, substrate, PROTAC
Use UbiQuant S ULTRA plate
Measure substrate ubiquitylation
Detection by chemiluminescence
Help customer discover potent and selective PROTACs
Express biologically active E3 ligase and substrate
Develop and optimize in vitro HTS assay for PROTAC
Screen custom libraries at LifeSensors
Confirmation and counter screening to eliminate off-target compounds
Biophysical and biochemical assay development
Cell-based assays to determine target engagement by PROTAC
TUBE-based mass spec to identify target engagement
Work performed under CDA and Master Service Agreement
Fee for service model, defined milestone-based agreement
PROTAC Drug Discovery Service
Contact Information
We are your partner for PROTAC Drug Discovery
Contact Us!
Research & Product Inquiries R&D [email protected] 610-644-8845 (ext 339)
Custom Service & Assays BD [email protected] 610-644-8845 (ext 310)
