Selective Identification of Allosteric Inhibitors and Co-Drug Combinations Targeting Kinases by Using a Nanopore Tweezer Approach.

Allosteric inhibitors represent a promising avenue for developing highly selective therapeutic drugs and overcoming drug resistance in kinase-targeted therapies. However, conventional screening methods are often limited by their reliance on known binding sites or by their low throughput. Here, we present a label-free nanopore tweezer approach that specifically detects allosteric binders to Abl kinase by monitoring ligand-induced ionic current fluctuations. In this method, an Abl kinase with a blocked active site is trapped within a cytolysin A (ClyA) nanopore, allowing real-time, single-molecule observation of co-binding of ATP-competitive and allosteric inhibitors to the Abl molecule. This strategy requires no prior structural knowledge of allosteric sites and is adaptable to high-throughput screening across the kinome and other enzyme targets. Our findings establish nanopore tweezers as a structure-based platform for identifying both allosteric modulators and co-drug combinations for a single target, thereby potentially accelerating the discovery of highly selective therapeutic agents.