title: Mechanism of inhibition of cathepsin K by potent, selective 1, 5-diacylcarbohydrazides: a new class of mechanism-based inhibitors of thiol proteases.
abstract: The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.
authors.affiliation: Departments of Molecular Recognition, Physical and Structural Chemistry, and Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, P.O. Box 1539, King of Prussia, Pennsylvania 19406, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Bossard
authors.forename: T A
authors.initials: TA
authors.lastname: Tomaszek
authors.forename: M A
authors.initials: MA
authors.lastname: Levy
authors.forename: C F
authors.initials: CF
authors.lastname: Ijames
authors.forename: M J
authors.initials: MJ
authors.lastname: Huddleston
authors.forename: J
authors.initials: J
authors.lastname: Briand
authors.forename: S
authors.initials: S
authors.lastname: Thompson
authors.forename: S
authors.initials: S
authors.lastname: Halpert
authors.forename: D F
authors.initials: DF
authors.lastname: Veber
authors.forename: S A
authors.initials: SA
authors.lastname: Carr
authors.forename: T D
authors.initials: TD
authors.lastname: Meek
authors.forename: D G
authors.initials: DG
authors.lastname: Tew
mesh_terms: D001665:Binding Sites; D056657:Cathepsin K; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D002851:Chromatography, High Pressure Liquid; D004793:Enzyme Reactivators; D006834:Hydrazines / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010206:Papain / Q000037:antagonists & inhibitors; D011480:Protease Inhibitors / Q000494:pharmacology*; D013053:Spectrophotometry
chemical_list: C403135:2,2'-N,N'-bis(benzyloxycarbonyl)-leucinylcarbohydrazide; D004793:Enzyme Reactivators; D006834:Hydrazines; D011480:Protease Inhibitors; D002403:Cathepsins; D010206:Papain; D056657:Cathepsin K; D007930:Leucine