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In drug discovery, efficient screening of protein-drug interactions (PDIs) is hampered by the limitations of current biophysical approaches. Here, we develop a biological nanopore sensor for single-molecule detection of proteins and PDIs using the pore-forming toxin YaxAB. Using this YaxAB nanopore, we demonstrate label-free, single-molecule detection of interactions between the anticancer Bcl-xL protein and small-molecule drugs as well as the Bak-BH3 peptide. The long funnel-shaped structure and nanofluidic characteristics of the YaxAB nanopore enable the electro-osmotic trapping of diverse folded proteins and high-resolution monitoring of PDIs. Distinctive nanopore event distributions observed in the two-dimensional (ΔI/Io-versus-IN) plot illustrate the ability of the YaxAB nanopore to discriminate individual small-molecule drugs bound to Bcl-xL from non-binders. Taken together, our results present the YaxAB nanopore as a robust platform for label-free, ultrasensitive, single-molecule detection of PDIs, opening up a possibility for low-cost, highly efficient drug discovery against diverse drug targets.

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application.

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