Abstract 6408: Unlocking the unlockable: AS-MS for swift hit identification against challenging targets

Abstract Affinity selection-mass spectrometry (AS-MS) is a label-free, high-throughput platform that can be used to screen small-molecule ligands by incubating them with biomolecular targets, separating bound complexes via size-exclusion or ultrafiltration, and detecting binders using high-resolution mass spectrometry. This approach bypasses the need for chemical modifications, fluorescent tags, or functional assays, thereby preserving native binding interactions and minimizing artifacts. As a label-free method, it allows for the rapid evaluation of large libraries and ranking of binding affinities without requiring functional data. Additionally, the flexibility of AS-MS enables diverse hit identification strategies, including exploration of combinatorial libraries while remaining agnostic to binding sites or mechanisms, thus uncovering novel allosteric and orthosteric ligands.In particular, the versatility of AS-MS enables adaptation of affinity selection methods to target particularities by relying on SEC-based, filter-based, or pull-down techniques. These orthogonal selection modalities can be fine-tuned—by adjusting buffer conditions, SEC polymers and volume, incubation times, wash volumes, or competitive inhibitors—to modulate assay sensitivity and selectivity, thereby maximizing enrichment for ligands with desired on/off rates. In conjunction with chemically diverse libraries, it is a powerful and highly effective approach to identify agents with innovative mechanisms of action.As an illustration of how AS-MS accelerates drug discovery by accessing challenging or “undruggable” targets lacking enzymatic activity or favorable binding pockets, we present here several case studies. In these, we have utilized AS-MS screening technology in the search for novel molecular glues and targeted protein degraders (on the VHL-BRD3 pair and other undisclosed targets), and against a wide range of challenging targets, including transcription factors (such as TEAD), RNAs, and membrane proteins such as GPCRs or ion channels (P2X4 receptor), either as isolated targets or embedded in membrane preparations.In summary, AS-MS accelerates discovery against intractable targets, compressing timelines and delivering validated, mechanism-rich hits for optimization. Its integration with downstream biophysics (e.g., SPR, NMR) and structural biology further validates hits, paving the way for next-generation therapeutics against previously intractable disease drivers. Citation Format: Renaud Renaud. Unlocking the unlockable: AS-MS for swift hit identification against challenging targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6408.