Mechanism of Inhibition of Cathepsin K by Potent, Selective 1,5-Diacylcarbohydrazides: A New Class of Mechanism-Based Inhibitors of Thiol Proteases

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• 作者：Mary J. Bossard ; Thaddeus A. Tomaszek ; Mark A. Levy ; Carl F. Ijames ; Michael J. Huddleston ; Jacques Briand ; Scott Thompson ; Stacie Halpert ; Daniel F. Veber ; Steven A. Carr ; Thomas D. Meek ; and David G
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：November 30, 1999
• 年：1999
• 卷：38
• 期：48
• 页码：15893 - 15902
• 全文大小：182K
• 年卷期：v.38,no.48(November 30, 1999)
• ISSN：1520-4995

文摘

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 includecompounds 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 resultingfrom 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 asreflected by 2-3 orders of magnitude differences in K_i and k_obs/I, but all eventually form the same stablecovalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 × 10^-4 s^-1), indicatinghydrolysis 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 generatea 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 stableinhibited form of the enzyme is the thioester complex. 1,5-Diacylcarbohydrazides represent a new classof unreactive cysteine protease inhibitors that share a common mechanism of action across members ofthe papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity andpotency.