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

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,...

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Published in:Biochemistry (Easton) 1999-11, Vol.38 (48), p.15893-15902
Main Authors: Bossard, Mary J, Tomaszek, Thaddeus A, Levy, Mark A, Ijames, Carl F, Huddleston, Michael J, Briand, Jacques, Thompson, Scott, Halpert, Stacie, Veber, Daniel F, Carr, Steven A, Meek, Thomas D, Tew, David G
Format: Article
Language:English
Subjects:
Binding Sites
Cathepsin K
Cathepsins - antagonists & inhibitors
Chromatography, High Pressure Liquid
Enzyme Reactivators
Hydrazines - chemistry
Hydrazines - pharmacology
Kinetics
Leucine - analogs & derivatives
Leucine - chemistry
Leucine - pharmacology
Magnetic Resonance Spectroscopy
Mass Spectrometry
Papain - antagonists & inhibitors
Protease Inhibitors - pharmacology
Spectrophotometry
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cited_by cdi_FETCH-LOGICAL-a412t-746a8cd03968fed95ea53bbbfaffe0de3cdc8b0e5463ab8dc315d4fdc5673f573
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container_end_page 15902
container_issue 48
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container_title Biochemistry (Easton)
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creator Bossard, Mary J
Tomaszek, Thaddeus A
Levy, Mark A
Ijames, Carl F
Huddleston, Michael J
Briand, Jacques
Thompson, Scott
Halpert, Stacie
Veber, Daniel F
Carr, Steven A
Meek, Thomas D
Tew, David G
description 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 × 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.
doi_str_mv 10.1021/bi991193+
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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 × 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. 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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 × 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.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>10625455</pmid><doi>10.1021/bi991193+</doi><tpages>10</tpages></addata></record>
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source American Chemical Society:Jisc Collections:American Chemical Society Read & Publish Agreement 2022-2024 (Reading list)
subjects Binding Sites
Cathepsin K
Cathepsins - antagonists & inhibitors
Chromatography, High Pressure Liquid
Enzyme Reactivators
Hydrazines - chemistry
Hydrazines - pharmacology
Kinetics
Leucine - analogs & derivatives
Leucine - chemistry
Leucine - pharmacology
Magnetic Resonance Spectroscopy
Mass Spectrometry
Papain - antagonists & inhibitors
Protease Inhibitors - pharmacology
Spectrophotometry
title 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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