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Proximity Binding Assay for PROTAC Ternary Complex Analysis
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DOI：10.1021/acssensors.5c04944.png)
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Abstract
En 中文
The engineered formation of ternary complexes, in which two proteins are bridged by small molecules such as PROTACs or molecular glues, is a prerequisite for the targeted enzymatic degradation of pathogenic proteins; however, the combined analysis of these ternary interactions during the drug discovery process remains challenging. Here, we introduce a proximity binding assay for the simultaneous measurement of binary and ternary interaction kinetics on a biosensor surface. Target proteins and the substrate binding subunit of ubiquitin E3 ligase are tethered to mobile swivel arms of a Y-shaped DNA scaffold. The Y-structure induces spatial proximity between the proteins and presents them to PROTAC analytes flown across the sensor. PROTAC-induced ternary complex formation is measured by fluorescence energy transfer (FRET), while binary interactions are detected by fluorescence quenching. The assay is applied to cereblon (CRBN) and von Hippel-Lindau (VHL) as E3 ligase substrate receptors, a range of compounds including AT1, MZ1, dBETs, and ARV-825 as PROTACs, and the two bromodomains of BRD2, BRD3, BRD4, and BRDT proteins as targets. Automated workflows enable the measurement of 384 real-time sensorgrams in a single run using picomole sample quantities. The insights into proximity-mediated binding kinetics can enable the development of PROTACs and molecular glues with improved properties for targeted protein degradation.
Keywords:
Assays
Degradation
Kinetic parameters
Kinetics
Sensors
targeted protein degradation
PROTAC
molecular glue
ternary complex
kinetics
affinity
avidity
Y-structure
switchSENSE
CRBN
VHL
BRD
BET
Journal
IF:
9.1
Papers:
1.0K
Citations:
2.6W
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