Next Generation Chemoproteomic Platforms for Functional Diagnostics and Small Molecule Drug Discovery

Targeting the proteome to treat diseases has been a long-standing goal in biomedical research. Yet less than 3% of the human proteome have FDA approved drugs against them. This is largely because over 35% of the proteome remains poorly understood. Moreover, we need to identify those proteins within the proteome that directly drive disease. To uncover new therapeutic targets, we must assess the activity of the proteins under relevant backgrounds—not just their abundance. Furthermore, many disease-driving proteins are still considered "undruggable," underscoring the need for innovative strategies to therapeutically target these challenging proteins. Here we report single cell Activity Dependent Proximity Ligation (scADPL) which integrates automated microfluidic single-cell capture and nanoliter-volume handling to translate activity of proteins into multiplexed, amplifiable oligonucleotide barcodes. Activity-based probes targeting serine hydrolases and cysteine proteases were used to profile a panel of six enzymes known to be associated with cancer aggressiveness. Using the single-cell ADPL (scADPL) workflow on cancer cell lines and primary breast cancer organoids, we identified an activity signature that correlates with metastatic progression in breast cancer. This platform enables measurement of activity signatures at the single-cell level, allowing us to distinguish signals from cancer cells versus normal cells—signatures that would be masked in bulk analyses. Additionally, the low sample input requirement makes it possible to profile patient-derived cells under physiologically relevant conditions, helping to identify true disease drivers. To enable efficient targeting of traditionally undruggable proteins, we developed a high-throughput chemoproteomics screening platform called In-situ Proximity Amplified Rapid Chemoproteomics (SPARC). SPARC employs activity-based probes to convert protein engagement into a barcoded DNA signal that is amplified and quantified. Drug binding to the target protein leads to signal loss or signal gain, indicating inhibition or activation of target protein. Using SPARC, we identified inhibitors and activators for a panel of five full-length kinase-GFP fusion proteins in their native, intracellular context. In addition to detecting drug–protein interactions, SPARC provides multiple layers of information that help reduce false discovery rates and accelerate drug development. It captures protein localisation and abundance, allowing us to distinguish true target engagement from changes in protein levels. SPARC is also coupled with a viability assay to assess whether observed effects are due to specific interactions or general cytotoxicity from off-target effects. Overall, these chemoproteomic platforms advance drug discovery by enabling the identification of disease-relevant targets through scADPL and the rapid discovery of modulators—activators or inhibitors—of these targets using the SPARC screening pipeline.