Inhibition of papain by nitriles: Mechanistic studies using NMR and kinetic measurements

Liang, Tzyy-Chyau; Abeles, Robert H.

doi:10.1016/0003-9861(87)90068-3

Cited by 49 publications

(32 citation statements)

References 25 publications

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“…A similar tetrahedral complex was found to develop during crystal soaking of a related enzyme, arginine deiminase (66, 67). Analogous reversible covalent inhibition has been reported of other thiol-dependent enzymes by nitriles, cyanamides and aldehydes, which form reversible thioimidate, thioamidine and hemithioacetal bonds, respectively (68-70). Also, structural studies of other reversible tetrahedral adducts have been reported, such as carbinolamine formation between pyruvate and Lys133 of aldolase (71).…”

Section: Resultsmentioning

confidence: 62%

Developing Dual and Specific Inhibitors of Dimethylarginine Dimethylaminohydrolase-1 and Nitric Oxide Synthase: Toward a Targeted Polypharmacology To Control Nitric Oxide

Wang

Monzingo²,

Shi-sheng

et al. 2009

Biochemistry

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Molecules that block nitric oxide's (NO) biosynthesis are of significant interest. For example, nitric oxide synthase (NOS) inhibitors have been suggested as anti-tumor therapeutics, as have inhibitors of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catabolizes endogenous NOS inhibitors. Dual-targeted inhibitors hold promise as more effective reagents to block NO biosynthesis than single-targeted compounds. In this study, a small set of known NOS inhibitors are surveyed as inhibitors of recombinant human DDAH-1. From these, an alkylamidine scaffold is selected for homologation. Stepwise lengthening of one substituent converts an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor and then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N5-(1-iminohexyl)-l-ornithine for neuronal NOS (1.7, 3, 20, >1,900 μM, respectively) and DDAH-1 (990, 52, 7.5, 110 μM, respectively). A 1.9Å X-ray crystal structure of the N5-(1-iminopropyl)-l-ornithine : DDAH-1 complex indicates covalent bond formation between the inhibitor's amidino carbon and the active-site Cys274, and solution studies show reversible competitive inhibition, consistent with a reversible covalent mode of DDAH inhibition by alkylamidine inhibitors. These represent a versatile scaffold for the development of a targeted polypharmacological approach to control NO biosynthesis.

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Section: Resultsmentioning

confidence: 62%

Developing Dual and Specific Inhibitors of Dimethylarginine Dimethylaminohydrolase-1 and Nitric Oxide Synthase: Toward a Targeted Polypharmacology To Control Nitric Oxide

Wang

Monzingo²,

Shi-sheng

et al. 2009

Biochemistry

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“…[18] These inhibitors make a covalent thioimidate bond with the active site Cys, [19] mimicking the covalent acyl-intermediate observed during catalytic turnover, yet have only a short lifetime that leads to rapid reversibility of inhibition. [20] The C -alkyl amidines appear to use a similar mechanism, reacting with the active-site Cys of DDAH-1 to form a tetrahedral adduct similar to that found on the normal catalytic pathway. However, the potency of these inhibitors is moderate, with values only 24-fold lower than the K M for substrate ( 1 , 170 μM).…”

Section: Resultsmentioning

confidence: 95%

Characterization of C‐Alkyl Amidines as Bioavailable Covalent Reversible Inhibitors of Human DDAH‐1

Lluis¹,

Wang²,

Monzingo³

et al. 2010

ChemMedChem

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C-alkyl amidine analogs of asymmetric Nω,Nω -dimethyl-L-arginine are dual-targeted inhibitors of both human DDAH-1 and nitric oxide (NO) synthase, and provide a promising scaffold for developing therapeutics to control NO overproduction in a variety of pathologies including septic shock and some cancers. Using a two-part clickchemistry-mediated activity probe, a homologated series of C-alkyl amidines are ranked for their ability to inhibit DDAH-1 within cultured HEK 293T cells. N5-(1-iminopentyl)-L-ornithine was determined to be the most potent compound in vitro (Kd = 7 μM) as well as in cultured cells, and the binding conformation and covalent reversible mode of inhibition was investigated by comparison of interactions made with DDAH-1 and a catalytically inactive C274S variant as gauged by Xray crystallography and isothermal titration calorimetry. By interrupting the ability of the inhibitor to form a covalent bond, the contribution of this interaction can be estimated. These results suggest further stabilization of the covalent adduct as a promising strategy for lead optimization in the design of effective reagents to block NO synthesis.

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“…The functional role of Asn-74 in AS-B glutaminase activity therefore appears strikingly similar to that of Gln-19 in the active site of papain (Menard et al, 1995;Dufour et al, 1995b). In the thiol proteases, it is likely that the side chain of this conserved glutamine residue defines an oxyanion hole, stabilizing either tetrahedral intermediate 14 and/or the thioester 15 in the mechanism of thiolate-catalyzed peptide bond hydrolysis (Scheme 4) based on experiments employing aldehyde (Lewis and Wolfenden, 1977;Mackenzie et al, 1986), and nitrile (Hanzlik et al, 1990;Moon et al, 1986;Brisson, et al, 1986;Liang and Abeles, 1987) inhibitors of papain. Further evidence for the catalytic equivalence of Gln-19 in papain and Asn-74 in AS-B has been obtained using nitrile 18, which is a weak, noncompetitive inhibitor of AS-B glutaminase activity at pH 6.5 (Boehlein et al, 1997b).…”

Section: B Mechanistic Comparison Of As-b and Papainmentioning

confidence: 99%

Mechanistic Issues in Asparagine Synthetase Catalysis

Richards

Schuster

1998

Advances in Enzymology - And Related Areas of Molecular Biology

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Inhibition of papain by nitriles: Mechanistic studies using NMR and kinetic measurements

Cited by 49 publications

References 25 publications

Developing Dual and Specific Inhibitors of Dimethylarginine Dimethylaminohydrolase-1 and Nitric Oxide Synthase: Toward a Targeted Polypharmacology To Control Nitric Oxide

Developing Dual and Specific Inhibitors of Dimethylarginine Dimethylaminohydrolase-1 and Nitric Oxide Synthase: Toward a Targeted Polypharmacology To Control Nitric Oxide

Characterization of C‐Alkyl Amidines as Bioavailable Covalent Reversible Inhibitors of Human DDAH‐1

Mechanistic Issues in Asparagine Synthetase Catalysis

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