Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrite Gu1303 and a 3-cyano-3-aza-beta-amino acid Gu2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gu2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

Automated summary

This study investigates the binding mechanism of two model cyanohydrazide inhibitors with Cathepsin K (CatK) and reveals that the extraordinary potency of Gu2602 is favored by its conformational flexibility. The study also demonstrates the effectiveness of cyanohydrazides in targeting mature CatK in osteosarcoma cells and inhibiting the autoactivation of CatK. These findings provide important structural insights for the design of cyanohydrazide inhibitors as potential drugs.