Unbiased, Target-Agnostic Platform for the Synthesis and Screening of PROTAC Libraries

Abstract Most approaches to PROTAC discovery require prior knowledge of the protein target, limiting the scope of targetable biology. Here we report an unbiased, direct-to-biology platform for the high-throughput synthesis and phenotypic screening of PROTAC libraries. The platform employs the Ugi multicomponent reaction to couple diverse carboxylic acid-containing ligands with VHL-recruiting E3 ligase ligand−linker conjugates in a single well plate step, followed by solid-phase extraction and cell-based phenotypic screening without predetermined target selection. A pilot library derived from seven structurally diverse protein ligands was screened in a cell viability assay against six cancer cell lines, yielding potent and selective hits. Global proteomics identified BRD4 as the primary target of lead compound B5, a BI2536-derived VHL-recruiting degrader that exhibited single-digit nanomolar degradation potency (DC50 = 4.9 nM, Dmax = 96%) and complete selectivity for BRD4 over PLK1. Target engagement and ternary complex formation were confirmed by NanoBRET, Western blot, and Boltz-2 computational modeling. The modularity of the Ugi reaction further enabled direct synthesis of two photoaffinity labeling probes, PALTAC-1 and PALTAC-2, which confirmed BRD4 as a direct binding target via photocrosslinking and pull-down experiments. This platform enables the unbiased discovery of degrader drugs and their targets from phenotypic screens and provides a generalizable framework for converting existing compound libraries into functional PROTAC collections.