Proximity Binding Assay for PROTAC Ternary Complex Analysis

TY - JOUR

T1 - Proximity Binding Assay for PROTAC Ternary Complex Analysis

AU - Ponzo, Irene

AU - Soldà, Alice

AU - Crowe, Charlotte

AU - Dahl, Göran

AU - Jahodová, Tereza

AU - Heerwig, Andreas

AU - Geschwindner, Stefan

AU - Ciulli, Alessio

AU - Rant, Ulrich

N1 - Copyright: © 2026 The Authors. Published by American Chemical Society

PY - 2026/5/22

Y1 - 2026/5/22

N2 - 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.

AB - 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.

KW - affinity

KW - avidity

KW - BET

KW - BRD

KW - CRBN

KW - kinetics

KW - molecular glue

KW - PROTAC

KW - switchSENSE

KW - targeted protein degradation

KW - ternary complex

KW - VHL

KW - Y-structure

UR - https://www.scopus.com/pages/publications/105036420502

U2 - 10.1021/acssensors.5c04944

DO - 10.1021/acssensors.5c04944

M3 - Article

C2 - 42011828

AN - SCOPUS:105036420502

SN - 2379-3694

VL - 11

SP - 3876

EP - 3888

JO - ACS Sensors

JF - ACS Sensors

IS - 5

ER -