Function-based OBOC screening for the direct discovery of potent protein-protein interaction inhibitors.

Protein-protein interactions (PPIs) represent an attractive class of therapeutic targets, yet the discovery of PPI inhibitors remains challenging because PPI interfaces are typically shallow and lack well-defined binding pockets. Moreover, target binding alone does not necessarily translate into functional inhibition, as compounds can engage a target protein without disrupting its interaction with a partner, and in some cases may even stabilize the complex. Combinatorial library technologies such as one-bead-one-compound (OBOC) screening enable the exploration of vast chemical diversity, but because conventional OBOC screening selects hits based on target binding rather than biological function, it is susceptible to nonfunctional or false-positive hits. These include compounds that bind outside the functional PPI interface and therefore lack inhibitory activity, compounds that stabilize rather than disrupt the PPI, and artifacts arising from avidity, nonspecific adsorption, or physical entrapment of the target protein within the polymer bead matrix. Here, we report a function-based OBOC screening platform that establishes PPI disruption, rather than target binding, as the primary selection criterion. By integrating a FRET-based competition assay with OBOC combinatorial libraries, bead-displayed compounds are selected only when they competitively and potently displace a quencher-tagged binding partner from a preassembled target complex, ensuring that fluorescence-positive beads directly report potent, functional PPI disruption. To validate this strategy, we applied the platform to the SRC-1/STAT6 interaction by screening a stapled peptide OBOC library, directly identifying highly potent functional inhibitors. Proof-of-concept experiments using DNA-encoded beads further confirmed the compatibility of this selection strategy with DNA-encoded library technologies, which enable access to substantially larger and more diverse combinatorial libraries. Together, this work establishes a broadly applicable framework for transforming affinity-based OBOC screening into function-based screening, enabling the direct discovery of biologically active PPI inhibitors.