Cellular DNA-Encoded Library (DEL) Screening Coupled with MICRO-TAG Real-Time Cellular Target Engagement Enables Discovery of Novel Therapeutics

Transcription factors are challenging drug targets that require the native cellular environment for interrogation of direct engagement with potential therapeutic candidates. The transcription factor beta-catenin is a therapeutically valuable yet challenging drug target, serving as signaling hub of the complex and highly interconnected WNT signaling network. Its central role and extensive pathway crosstalk have complicated efforts to distinguish effectors that bind beta-catenin directly from those acting indirectly through upstream WNT components. This mechanistic distinction is critical for guiding rational drug discovery. We applied the MICRO-TAG® Cell Target Engagement technology to interrogate 25 reported beta-catenin/WNT effectors. Using a novel direct target engagement system employing real-time temperature series, we quantified ligand-dependent stabilization of beta-catenin across multiple doses, profiling their cellular target engagement potencies, under cellular conditions. Parallel functional assays, including TOPFlash reporter activity and WNT-responsive surface markers, integrated direct binding with downstream signaling readouts. This integrated approach distinguished direct beta-catenin binders from effectors acting indirectly through upstream pathway components. By coupling cellular target engagement with functional readout, the MICRO-TAG® system enables scalable, mechanism-resolved drug discovery system for complex signaling pathways. This approach establishes a new paradigm for functionally interrogating therapeutic candidates through integrated target engagement and functional readout, within a unified, quantitative, and biologically relevant framework that is readily translatable to other challenging drug targets.