Identification of Kazal Inhibitor Scaffolds with Identical Canonical Binding Loops and Their Effects on Binding Properties

[Image: see text] Kazal inhibitors hold high potential as scaffolds for therapeutic molecules, taking advantage of the easily exchangeable canonical binding loop. Different Kazal inhibitor backbones have been suggested to be therapeutically useful, but the impact of different Kazal-like scaffolds on binding properties is still largely unknown. Here, we identified trypsin-targeting human serine protease inhibitor Kazal type 1 (SPINK1) homologues in different mammalian species that cluster in two P2–P1 combinations, implying the coevolution of these residues. We generated loop exchange variants of human SPINK1 for comparison with Kazal inhibitors from related species. Using comprehensive biophysical characterization of the inhibitor–enzyme interactions, we found not only affinity but also pH resistance to be highly backbone-dependent. Differences are mostly observed in complex stability, which varies by over one order of magnitude. We provide clear evidence for high backbone dependency within the Kazal family. Hence, when designing Kazal inhibitor-based therapeutic molecules, testing different backbones after optimizing the canonical binding loop can be beneficial and may result in increased affinity, complex stability, specificity, and pH resistance.