Mechanism-based Irreversible Inhibitor Research Articles

Active site mapping of affinity-labeled rat oxidosqualene cyclase.

Rat liver oxidosqualene cyclase (OSC), a 78-kDa membrane-bound enzyme, was purified and labeled with the mechanism-based irreversible inhibitor, [3H]29-methylidene-2,3-oxidosqualene (Abe, I., Bai, M., Xiao, X.-Y., and Prestwich, G. D. (1992) Biochem. Biophys. Res. Commun. 187, 32-38). A 6-kDa CNBr peptide was separated by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to a polyvinylidene difluoride membrane. The sequence of the first 30 amino acids of this peptide were determined by Edman degradation and showed unexpectedly high similarity to the fungal OSC from Candida albicans (50% identity with Arg413-Val442) and to the bacterial squalene cyclase from Alicyclobacillus (formerly Bacillus) acidocaldarius (37% identity with Lys356-Leu385). Further, radioanalysis clearly established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala or DDTAEA) were equally labeled by the irreversible inhibitor. This result provides the first information on the structural details of the active site of OSC and shows for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of sesquiterpene cyclases and prenyl transferases.

5-Ethynyluracil (776C85): a potent modulator of the pharmacokinetics and antitumor efficacy of 5-fluorouracil.

5-Ethynyluracil (5-EU, 776C85) is a mechanism-based irreversible inhibitor of dihydropyrimidine dehydrogenase (EC 1.3.1.2), the rate-determining enzyme in 5-fluorouracil (5-FU) catabolism. In the present study, 5-EU was found to be a potent modulator of 5-FU catabolism in mice and rats. Liver extracts prepared up to 6 hr after a 5-EU dose (2 mg/kg) were > 96% inhibited in their ability to catalyze 5-FU degradation. 5-EU treatment increased the elimination t1/2 and the area under the plasma concentration-time curve of 5-FU. 5-FU oral bioavailability was approximately 100% in rats pretreated with 5-EU. Consequently, 5-EU induced a linear relationship between the area under the plasma concentration-time curve and the oral dose of 5-FU. As expected from the preservation of plasma 5-FU, 5-EU potentiated the antitumor activity and the toxicity of 5-FU in two mouse tumor models (Colon 38 and MOPC-315). However, 5-EU potentiated the antitumor activity to a greater degree and thereby increased the therapeutic index of 5-FU 2- to 4-fold.

Affinity labeling of vertebrate oxidosqualene cyclases with a tritiated suicide substrate

Pig and rat liver oxidosqualene cyclase (OSC) enzymes were purified to homogeneity and showed single bands on SDS-polyacrylamide gel electrophoresis with molecular masses of 75 kDa (pig) and 78 kDa (rat). Pig liver OSC was purified for the first time (441-fold with a yield of 39%). Chemical affinity labeling of pure or crude preparations of the liver cyclases using the mechanism-based irreversible inhibitor of OSC, [3H]29-methylidene-2,3-oxidosqualene ([3H]29-MOS), showed a single radioactive band at 75 kDa (pig) and 78 kDa (rat). Affinity labeling experiments were also performed with dog and human microsomal preparations and with yeast and plant cyclases. All of the vertebrate OSC enzymes were specifically labeled with [3H]29-MOS and gave a single band with molecular masses ranging from 70 to 80 kDa (rat, 78 kDa; dog, 73 kDa; pig, 75 kDa; and human, 73 kDa). In contrast, yeast lanosterol cyclase and plant cycloartenol cyclase were not labeled, demonstrating subtle differences in the active sites of animal, plant, and fungal enzymes.

Antiretroviral activity of mechanism-based irreversible inhibitors of S-Adenosylhomocysteine hydrolase

S-Adenosylhomocysteine hydrolase (AdoHcy-nase) is a key enzyme in transmethylation reactions. The objective of the present study was to examine the potential antiretroviral activities of novel mechanism-based irreversible AdoHcy-nase inhibitors. (Z)-4′,5′-didehydro-5′-deoxy-5′-fluoroadenosine (ZDDFA), (E)-4′,5′-didehydro-5′-deoxy-5′-fluoroadenosine (EDDFA), (Z)-4′,5′-didehydro-5′-deoxy-5′-chloroadenosine (ZDDCA) and 5′-deoxy-5′-acetylenic adenosine (DAA) inhibited AdoHcy-nase activity with Ki values of 0.55, 1.04, > 10.0 and 3.30 μM, respectively. These four compounds were tested for antiviral activity in vitro against Moloney leukemia virus (MoLV) in the XC-plaque assay. MoLV replication in murine fibroblasts (SC-1) was inhibited by ZDDFA, EDDFA and DAA with IC 50 values of 0.05, 0.25 and 3.30 μg/ml, respectively. ZDDCA did not inhibit MoLV infection at the concentrations tested. Antiviral activity correlated with the ability of the individual compounds to maintain sustained elevations in intracellular S-adenosylhomocysteine (AdoHcy) concentrations in the SC-1 cells. ZDDFA, the most potent inhibitor of AdoHcy-nase and MoLV was also the most active in maintaining sustained elevations in intracellular AdoHcy levels. The antiviral activity of ZDDFA was also examined in murine C3H1OT 1 2 fibroblasts which constitutively produce MoLV. Pretreatment with ZDDFA (1.0 μg/ml) for 24 hr inhibited virus production by 88%. Similar to the SC-1 cells, and concomitant with enzyme inhibition, there was a 300-fold increase in AdoHcy levels in ZDDFA (1.0 μg/ml) treated C3H1OT 1 2 cells. Incorporation of a [ 3H]methyl group from tritiated S-adenosylmethionine into total RNA in C3H1OT 1 2 cells was inhibited by ZDDFA without affecting cell viability. These results suggest that mechanism-based inhibitors of AdoHcy-nase, such as ZDDFA, may have potential as antiretroviral agents.

Inhibition of monoamine oxidase by the R and S enantiomers of N[3-(2,4-dichlorophenoxy)propyl]-N-methyl-3-butyn-2-amine

The chemical synthesis of the R-(+)- and S-(−)-enantiomers of N[3-(2,4-dichlorophenoxy)propyl]- N-methyl-3-butyn-2-amine is described. These compounds are derivatives of the mechanism-based irreversible monoamine oxidase inhibitor clorgyline in which a methyl group is substituted for a hydrogen atom on the propargyl methylene carbon. The enantiomeric clorgyline derivatives were both found to be reversible, linearly-competitive inhibitors of monoamine oxidase-A and -B. Thus the methyl-substitution does not prevent non-covalent binding of the inhibitors to the active sites, but it does prevent these compounds from reacting within that complex to form covalent adducts with the enzymes. The substitution of the methyl group resulted in a decreased affinity, as compared to clorgyline, for non-covalent binding to monoamine oxidase-A, with the S-enantiomer having the lower affinity. In contrast, the S-enantiomer showed increased affinity for monoamine oxidase-B. These effects resulted in the high selectivity of the parent compound, clorgyline, towards monoamine oxidase-A being lost. The R-enantiomer showed a small degree of selectivity towards the A-form of the enzyme, whereas the S-enantiomer had a higher affinity for the B-form. These results are discussed in terms of the chemical mechanisms proposed for the interactions of the monoamine oxidases with substrates and inhibitors.

Irreversible inhibition of rat hepatic transacetylase activity by N-Arylhydroxamic acids

Both N-hydroxy-2-acetamidofluorene ( N-OH-AAF) and the heterocyclic analogue, 2-( N-hydroxyacetamido)carbazole ( N-OH-AAC), were shown to be mechanism-based irreversible inhibitors (suicide inhibitors) of partially purified rat hepatic N-acetyltransferase (NAT) activity. Although N-OH-AAC exhibited an apparent first-order inactivation rate constant ( k i ) that was 7-fold lower than that of N-OH-AAF, their relative k i / K D values indicate that N-OH-AAC was the more potent and efficient inactivator of transacetylase activity. Inactivation of NAT activity by these N-arylhydroxamic acids appeared to involve contributions by electrophiles that react with the enzyme subsequent to release from the active site and by electrophiles that remain complexed with the active site. The irreversible nature of the enzyme inactivation was demonstrated by the failure to recover transacetylase activity upon either extensive dialysis or gel filtration of preparations that had been subjected to incubation with N-OH-AAF or N-OH-AAC. The use of the nucleophile N-acetylmethionine to trap the electrophilic reactants formed in the transacetylase-catalyzed bioactivation process resulted in a lower rate and extent of formation of methylthio adducts with N-OH-AAC than with N-OH-AAF. The results of this study indicate that N-OH-AAF and N-OH-AAC have been potential for use as tools in the investigation of rat hepatic transacetylases.

Effects of 10-(2-propynyl)-estr-4-ene-3, 17-dione (MDL 18,962)—a mechanism-based irreversible inhibitor of aromatase—In cultured human foreskin fibroblasts

In male subjects, peripheral aromatization of androgens accounts for most of the estrogen production, and skin is an important site of such enzymatic activity. We have studied the effects of a mechanism-based, irreversible aromatase inhibitor, 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962) on androgen action and metabolism in cultured human foreskin fibroblasts. Cells were incubated simultaneously in the presence of substrate, androstenedione, and inhibitor, MDL 18,962. Aromatase activity was linear with time up to 3 h of incubation at 37°C in the absence and presence of 1.0–10nM inhibitor. The IC 50 for four different cell strains ranged from 4.0 to 8.6 nM MDL 18,962. Kinetic analysis of competitive inhibition by the Eadie-Hofstee method yielded an apparent K i of 2.75 nM for the inhibitor Preincubation of cells with MDL 18,962 resulted in irreversible inhibition of aromatase activity which was time- and concentration-dependent. We calculated a K i of 7.6 nM for MDL 18,962. Preincubation of cells with 25 nM MDL 18,962 suppressed enzyme activity for up to 6 h following removal of the inhibitor, before a return of enzyme activity due to synthesis of new enzyme. MDL 18,962 (0.2–20μM) did not influence the 5α -reduction of testosterone (200 nM). In addition, binding of dihydrotestosterone (2nM) to androgen receptors was not affected by MDL 18,962 (25–1000 nM). In summary, MDL 18,962 is a specific, high potency inhibitor of aromatase. By virtue of its high binding affinity to the enzyme active site, it competes very effectively with substrate, resulting in irreversible inactivation of aromatase.

Human myeloma cells acquire resistance to difluoromethylornithine by amplification of ornithine decarboxylase gene.

Stepwise increments of the concentration of 2-difluoromethylornithine (DFMO), a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (ODC), resulted in a selection of cultured human IgG-myeloma cells (Sultan cell line) capable of growing in the presence of up to 3 mM-DFMO. This capacity was associated with 10-fold increase in ODC activity in the dialysed extracts of drug-resistant myeloma cells, markedly enhanced synthesis rate for ODC enzyme molecules, as revealed by a 20 min [35S]methionine labelling of cellular proteins, followed by specific immunoprecipitation and SDS/polyacrylamide-gel electrophoresis, dose-dependently increased expression of ODC mRNA in resistant cells (effective dose causing 50% inhibition), dose-dependent amplification of ODC gene sequences in a 9-kilobase-pairs EcoRI genomic DNA fragment, and (v) a 10-fold increase in the ED50 (effective dose causing 50% inhibition) for the anti-proliferative action of DFMO in these myeloma cells. These results represent one of the few gene amplifications described in cultured human cells.

Dissociation of cytochrome P-450 inactivation and induction

Polycyclic aromatic hydrocarbons bind to a cytosolic receptor that translocates into the nucleus and induces the synthesis of mRNA for a subset of cytochrome P-450 isozymes. The failure to detect a “phenobarbital” receptor, however, suggests that the induction of P-450 b e , the isozymes induced by phenobarbital, is mediated by a less direct mechanism. One attractive alternative is that a receptor exists for an endogenous substance that is specifically turned over by the trace amounts of P-450 b e present in uninduced liver. Changes in the concentration of this substance caused by agents that inhibit P-450 b e would then trigger the induction response. We report here that suppressing the catalytic activities of P-450 b e for prolonged periods with 1-aminobenzotriazole, a mechanism-based irreversible inhibitor, neither induces P-450 b e nor interferes with induction of these isozymes at the level of transcription by phenobarbital. The results argue against mechanisms for the induction of P-450 b e keyed to the effective catalytic availability of these isozymes.

Difluoromethylornithine-induced amplification of ornithine decarboxylase genes in Ehrlich ascites carcinoma cells

Stepwise increments of the concentration of 2-difluoromethylornithine, a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (EC 4.1.1.17), resulted in a selection of cultured Ehrlich ascites carcinoma cells capable of growing in the presence of up to 50 mM difluoromethylornithine. Dialyzed extracts of drug-resistant tumor cells exhibited a very high ornithine decarboxylase activity and contained large excess of immunoreactive ornithine decarboxylase protein. Hybridization analyses with cloned complementary DNA revealed that the difluoromethylornithine-resistant tumor cells also expressed mRNA of the enzyme at greatly enhanced rate. The overproduction of ornithine decarboxylase by the tumor cells grown under the pressure of difluoromethylornithine was at least partly attributable to a 10 to 20-fold increase in the total gene dosage of ornithine decarboxylase involving an amplification of several genes of the gene family. The gene amplification developed appeared to be stable, as the gene dosage only slowly (during a period of several months) returned towards the normal level upon the removal of difluoromethylornithine. The overproduction of ornithine decarboxylase was accompanied by an enhanced resistance of the enzyme towards difluoromethylornithine in vitro.

Beta-substituted ethylamine derivatives as suicide inhibitors of lysyl oxidase.

Lysyl oxidase initiates the covalent cross-linking of elastin and collagen, converting lysyl residues in these proteins to peptidyl aldehyde residues. The present study explored structural and electron withdrawing features required to generate mechanism-based inhibitors of this enzyme with antifibrotic potential. It was found that the electron withdrawing nitrile moiety of beta-aminopropionitrile (BAPN), a naturally occurring syncatalytic inhibitor of lysyl oxidase, can be replaced by chlorine, bromine, or the nitro function to yield 2-haloamines or nitroethylamine compounds which also act as mechanism-based irreversible inhibitors of this enzyme. BAPN and 2-bromo- and 2-chloroethylamine exhibit similar KI values of 6-10 microM. However, the enzyme becomes irreversibly inactivated significantly faster by either of the 2-haloamines than by BAPN. 2-Nitroethylamine has by far the poorest affinity for the enzyme and inactivates much more slowly than the other amines of this series, consistent with interference with optimal enzyme-inhibitor interactions by the anionic nitro group. Unlike BAPN, 2-bromoethylamine is processed to a detectable aldehyde product upon incubation with enzyme, showing a partition ratio of 1.2 mol of acetaldehyde formed per mol of 2-bromo-ethylamine which becomes covalently incorporated in the enzyme. The results are consistent with the processing of 2-bromo-ethylamine to an enzyme-ethyleneamine Schiff base subject to hydrolysis to acetaldehyde or to covalent attack at carbon 2 by an enzyme nucleophile. Thus, beta-haloamines represent a new series of suicide inhibitors of lysyl oxidase which can inactivate the enzyme faster than BAPN and hence may have antifibrotic potential.

Enzyme-activated inhibitors, alternate substrates, and a dead end inhibitor of the general acyl-CoA dehydrogenase.

Aspects of the binding and dehydrogenation of acyl-CoA thiol esters by the general acyl-CoA dehydrogenase from pig liver were investigated using a dead-end inhibitor, S-octyl-CoA, several alternate substrates, and three active site-directed inhibitors. Experiments with S-octyl-CoA indicate that the carbonyl group of acyl-CoA thiol esters is not absolutely required for binding to the enzyme. However, the mode of binding of the 8-carbon thiol ether can be distinguished from the mode of binding of the enoyl-CoA product, octenoyl-CoA. Octanoyl pantetheine, octanoyl-etheno-CoA, and octanoyl-3'-dephospho-CoA are alternate substrates of the dehydrogenase. Steady state kinetic constants obtained with these alternate substrates indicate that the adenosine 5'-diphosphate, but not the 3'-phosphate, of the nucleotide moiety of acyl-CoA substrates contribute to the tight binding of the substrates. The substrate analogs 3'-butynoyl-CoA and 3-octynoyl-CoA are active site-directed, mechanism-based irreversible inhibitors of the dehydrogenase. These inhibitors covalently modify the apoprotein rather than the flavin. This finding and the fact that 2,3-octadienoyl-CoA also completely and irreversibly inhibits the enzyme indicate that th 3-acetylenic thiol esters inhibit the enzyme by a mechanism involving: (1) base-catalyzed abstraction of a protein at C-2 followed by isomerization to the allene carbanion, (2) protonation of the carbanion, and (3) attack of a nucleophile in the enzyme-active site on C-3 of the 2,3-dienoyl-CoA. The data show that the alkynoyl-CoA's are activated and bound at the active site of the enzyme. The results suggest that abstraction of a proton at C-2 of acyl-CoA substrates is the initial step in the catalytic pathway of dehydrogenation of substrates by the enzyme.

3] Mechanism-based irreversible enzyme inhibitors

There are two extreme forms of mechanism for specific irreversible enzyme inhibitors. One comprises the classical affinity labeling agents, which are substrate analogs containing chemically reactive functional groups. The specific binding of these inhibitors to the active-site regions of the enzyme ensures a greater probability of chemical reaction with a residue within this region as opposed to nonspecific chemical reactions. This chapter explains the irreversible enzyme inhibitors. It defines the conditions on which the successful design of mechanism-based irreversible enzyme inhibition is dependent. It cites specific reagents like β-γ – Unsaturated Molecules, diazoesters and ketones, Allyl chloride and sulfates, vinyl chlorides and other reagents. It also explains the synthsis of Vinyl Glycine and p-Nitrophenyl Diazoacetate. The chapter concludes that mechanism-based irreversible enzyme inhibitors are more than an enzymological curiosity and can be useful as general selective irreversible enzyme inhibitors.