Time-dependent inactivation

Baer, B. R. Wienkers, L. C. Rock, D. A. Time-dependent inactivation of P450 3A4 by raloxifene identification of Cys239 as the site of apoprotein alkylation. Chem. Res. Toxicol. 2007, 20, 954-964. 

True enough, treatment of PAP with FMPP resulted in a time-dependent inactivation of the enzyme. Competitive inhibitors of PAP protected against inactivation. The authors suggest that FMPP represents a useful basic structure which can be incorporated into the design of more specific phosphatase inhibitors for example, the modified tyrosine 77 could be incorporated into a particular peptide to give a suicide substrate that is selective for a protein phosphatase which preferentially hydrolyses that peptide. 

An attractive strategy for predicting the clinical significance of irreversible inhibition is to use human hepatocytes wherein the natural turnover of enzymes might be preserved and in vivo cellular concentrations of inhibitors and metabolites would be achieved. Zhao et al. demonstrated time-dependent inactivation of CYP3A in cryopreserved hepatocytes for amprenavir, diltiazem, erythromycin, raloxifene, and TAO (126). Except for TAO, significant differences in inactivation efficiency potency between hepatocytes and HLMs were... 

Zhao P, Kunze KL, Lee CA. Evaluation of time-dependent inactivation of CYP3A in cryopreserved human hepatocytes. Drug Metab Dispos 2005 33(6) 853-861. 

One of the second type of inhibitor analogs which cause a time-dependent inactivation of alanine racemase is (l-aminoethyl)phosphonic acid, the phosphonate analog of alanine (Ala-P). Ala-P effectively and specifically inactivates alanine racemases from Grampositive bacteria (Bacillus, Staphylococcus, Streptococcus), and serves as a reversible inhibitor of Gram-negative bacterial (Escherichia, Salmonella) alanine racemase.47 53 The mechanism of inhibition was studied with B. stearothermophihis alanine racemase.47 The d- or L-Ala-P leads to an E-I complex with a Ki value of 1 mM, then is slowly isomerized (A nac<=6-9 min-1) to a stoichiometric enzyme complex (E-I ). The Ed dissociates extremely slowly with the... 

Based on this premise, y-acetylenic GABA (IV) waB synthesized (11) and found to be an irreversible inhibitor of GABA-T, in vitro and in vivo (12). Thus, when GABA-T, partially purified from pig brain, is incubated for varying time periods with y-acetylenic GABA, a time-dependent inactivation process is observed which follows pseudo first-order kinetics. Enzyme half lives range from 28 minutes to 9 minutes with concentrations of inhibitor between 0.029 mM and 0.29 mM. Time dependent inactivation is... 

Aminocephalosporanic acid (15, Scheme 9) is an important intermediate in the production of many semisynthetic cephalosporin antibiotics (66, 67). However, direct deacylation of cephalosporin C (13) to 15 by cephalosporin C acy-lase is unfavorable, so an enzymatic process is used involving D-amino acid oxidase (DAAO) oxidation of 13 to A-glutaryl-7-aminocephalosporanic acid (14, GL-7-ACA) followed by deacylation to 15 and glutaric acid, catalyzed by GL-7-ACA acylase from Pseudomonas sp. 130 (Scheme 9) (68, 69). GL-7-ACA acylase underwent pseudo first-order time-dependent inactivation by 7 3-bromoacetyl aminocephalos-poranic acid (16) (70). Dialysis did not regenerate enzyme activity, indicating irreversible inhibition. The rate of inactivation was lowered by the presence of either glutaric acid or 15,... 

Figure 16 (a) A plot showing time-dependent inactivation by affinity labeling agents and mechanism-based inactivators used for determination of kinetic constants, (b) Replot of the half-lives of inactivation from Fig. 16a versus the inverse of the inactivator concentration to determine the K and values for affinity labeling agents and mechanism-based inactivators. 

The impetus for the newer work was the observation that benzyl clavulanate (11c) is a time-dependent inactivator of HLE (ICjq = 5 //M) whereas clavulanic acid (11b) is inactive [211, 212]. This finding led to the hypothesis that, since HLE is an endopeptidase whereas the bacterial serine proteinases are carboxypeptidases, quench(ing) the negative charge that the -lactam antibiotics normally require might yield HLE inhibitors. For synthetic reasons the group at Merck decided to use 7-aminocephalospo-ranic acids (1 Id) for most of their initial SAR studies. In contrast to the result with clavulanic acid, conversion of the cephalosporin 2 -carboxyl group in (1 Id) to an ester (He) was insufificent to transform the compound to an HLE inhibitor. 

The normal branched chain isovaleryl CoA (149) is then desaturated to the a,)3-enonyl CoA (150) by a flavin-linked branched chain acyl CoA dehydrogenase (equation 24). When the methylenecyclopropane acyl CoA (151) is exposed to acyl CoA dehydrogenase, time-dependent inactivation ensues with covalent modification of the bound FAD coenzyme (equation 25), possibly via a 6,5-adduct although the structure is unproven. ... 

A second new class of MAO mechanism-based inactivators, (aminoalkyl)tri-methylsilanes, have been reported by Silverman and Banik (114). The idea for this class of MAO inactivators is based on the known activation of the carbon-silicon bond toward homolytic cleavage reaction when the silicon atom is /3 to a radical cation (115, 116). The aminomethyl-, aminoethyl-, and (amino-propyl)trimethylsilanes are all pseudo-first-order time-dependent inactivators of beef liver MAO that reduce the flavin cofactor during the inactivation reaction. Since denaturation of the inactivated enzyme allows flavin leoxidation, covalent bond formation might be to an amino acid residue (114). The stabilities of the enzyme adducts from the (aminoalkyl)trimethylsilanes were found to be differ-... 

The inhibition of pyruvate formate-lyase by hypophosphite was first observed by Novelli in work on the CoA-independent carboxylate exchange reaction between pyruvate and formate (186). In a more detailed study by Knappe et al., time-dependent inactivation is observed to occur with concomitant loss of the enzyme free radical EPR signal (180). The inactivation kinetics are first order and the rate of inactivation is accelerated when the enzyme is in the acetylated form. Furthermore, inactivation by [ HJhypophosphite leads to the stoichiometric release of tritium to H2O, and treatment of PFL with [ P]hypophosphite produces an alkali-labile radiolabel that is covalently bound to the inactive enzyme (180). 

The inhibition studies were first performed with the E. coli, and later with the M. tuberculosis enzymes, since it was recently reported that bioA, the gene encoding for DAPA AT, was implicated in the long-term survival of mycobacteria. With the E. coli protein, a strong time-dependent inactivation was observed with the cis isomer, whereas the trans one was less potent. It was established that an aromatic PLP-inhibitor adduct was formed, the structure of which (13) was established by comparison with an authentic sample." Although not covalently bound to the enzyme, its affinity makes the inactivation quasi-irreversible. The X-ray structure of the inactivated enzyme reveals that the aromatic moiety of 13 is located in the AOP binding site, constituted of aromatic residues (Figure 12). 

The time-dependent inactivation of aromatase by 10-hydroperoxy-4-estrene-3,17-dione, reportedly a mechanism-based inactivator of aromatase -is inhibited by NADPH or an alternative substrate and is partially reversed by dithiothreitol. Other high affinity 10-substituted analogs, such as the mechanism-based inhibitor 10-p-mercaptoestr-... 

Flynn, G.A., J.O. Johnston, C.L. Wright, and B.W. Metcalf (1981). The time-dependent inactivation of aromatase by 17-fi-hydroxy-lO-methylthioestra-1,4-dien-3-one. Biochem. Biophys. Res. Commun. 103,913-918. 

Numazawa, M., A. Mutsumi, N. Asano, and Y. Ito (1993). A time-dependent inactivation of aromatase by 19-substituted androst-4-ene-3,6,17-diones. Steroids 58, 40-46. 

Inhibition of cyclooxygenase by eicosatetraynoic acid occurred through a dual mechanism immediate inhibition was followed by a time-dependent inactivation of the enzyme. The latter could be blocked by a-naphthols, indicating a chain reaction involving a free radical . The second stage in cyclooxygenase inhibition by eicosatetraynoic acid is thus mechanism-based. 

The cytochrome P-450-catalyzed oxidation of N.N-bis-carbethoxy-2,3-diazabicyclo[2,2,0]hex-5-ene (, R = C02Et), a relatively stable derivative, results in time-dependent inactivation of the enzyme.The inactivation is paralleled by the accumulation of a heme adduct Identified as N-(2-cyclobutenyl)protoporphyrin IX, a finding that points to a catalytically unmasked cyclobutadlenoid species as the heme-alkylating agent. 

As previously described, irreversible enzyme inhibition is defined as time-dependent inactivation of the enzyme, which implies that the enzyme has, in some way or form, been permanently modified, because it can no longer carry out its function. This modification is the result of a covalent bond being formed with the inhibitor and some amino acid residue in the protein. Furthermore, this bond is extremely stable and, for all practical purposes, is not hydrolyzed fo give back the enzyme in its original state or structure. In most examples of irreversible inhibition, a new enzyme must be generated through gene transcription and translation for the enzyme to continue its normal catalytic action. Basically, there are two types of irreversible enzyme inhibitors, the affinity labels or active site-directed irreversible inhibitors and the mechanism-based irreversible enzyme inactivators. 

Figure 4. Time-dependent inactivation of IMPase by S6,721-l. 50 pi of inhibitor at 10 pM were preincubated in 50 mM Tris-HCl, pH 7.5, at 25°C. After 0, 10, or 30 minutes 50 pi IMPase were added to about 1 pM final enzyme concentration and the incubation was continued. At the indicated time points, 5 pi aliquots were added to 450 pi assay mix (legend to Fig. 1), but without substrate and kept on ice. Residual enzyme activities were determined by starting the reaction with 50 pi substrate and monitoring the absorbance at 4 time points as described in the legend to Fig. 1.

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