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

Abstract

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 nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602 . 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 Gü2602 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.

Nitriles: an attractive approach to the development of covalent inhibitors

“…25. 122 In the same work, the authors developed a structurally distinct azanitrile-based compound ( 56 ) to inhibit CatK with a K i of 0.91 nM. Rankovic and colleagues employed a 2-cyanopyrimidine-based compound to obtain highly selective and potent compounds for CatK.…”

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans

“…All protease substrates were purchased from Bachem (Bubendorf, Switzerland) with the exception of Abz-Phe-Arg-Phe(NO 2 )-OH from MP Biomedicals (Irvine, CA, USA). Human cathepsins L, K, and V were produced in the Pichia pastoris expression system as described previously [12,56,57]. Bovine cathepsins C and H were prepared as described in [58,59]; human cathepsin D was prepared as described in [60].…”

Emerging and Re-emerging Warheads for Targeted Covalent Inhibitors: An Update

“…Their use in covalent inhibitors has been pioneered by the Gütschow group, who developed the first aza­dipeptide nitriles (e.g., 139c , Figure A) as extremely potent inhibitors of cysteine proteases. This strategy has been successfully employed to human cysteine proteases like cathepsins (e.g., compounds 139a , 139b , and 139c , and Gü2602 , (( 140 ), all Figure A), and viral proteases such as SARS-CoV-2 M pro (e.g., compound 141 , Figure A) . The mechanism of cysteine addition to cyano­hydrazides is depicted in Figure B.…”