Stopped-flow kinetic investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the allosteric site

Herein, we report on the role of the allosteric site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The K(D) for the activation was determined to be 25.9 +/- 2.3 microM and the rate constant for the oxidation of the iron cofactor, k(2), to be 182 +/- 4 s(-1). Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a K(i) of 13.7 +/- 1.3 microM for the catalytic site and a K(i)' of 140 +/- 9 microM for the allosteric site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a K(i) of 17.5 +/- 3.8 microM. These results suggest that OS binds to an allosteric site that is separate from the catalytic iron site. We further observed that the allosteric site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS.     

Inhibition of bacteriophage lambda protein phosphatase by organic and oxoanion inhibitors.

Bacteriophage lambda protein phosphatase (lambdaPP) with Mn(2+) as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of lambdaPP, albeit with inhibition constants (K(i)) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (K(i) = 5.1 +/- 1.6 microM) whereas phosphonoacetic acid (K(i) = 380 +/- 45 microM) and acetohydroxamic acid (K(i) > 75 mM) modestly inhibited lambdaPP. Low-temperature EPR spectra of Mn(2+)-reconstituted lambdaPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn(2+) ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn(2+))(2) dimer is most prominent upon the addition of orthovanadate (K(i) = 0.70 +/- 0.20 microM) and PhAH, indicating that these inhibitors tightly interact with the (Mn(2+))(2) form of lambdaPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by lambdaPP and other serine/threonine protein phosphatases.     

Effect of natural phenols on the catalytic activity of cytochrome P450 2E1

The effect of protocatechuic acid, tannic acid and trans-resveratrol on the activity of p-nitrophenol hydroxylase (PNPH), an enzymatic marker of CYP2E1, was examined in liver microsomes from acetone induced mice. trans-Resveratrol was found to be the most potent inhibitor (IC(50) = 18.5 +/- 0.4 microM) of PNPH, while protocatechuic acid had no effect on the enzyme activity. Tannic acid with IC(50) = 29.6 +/- 3.3 microM showed mixed- and trans-resveratrol competitive inhibition kinetics (K(i) = 1 microM and 2.1 microM, respectively). Moreover, trans-resveratrol produced a NADPH-dependent loss of PNPH activity, suggesting mechanism-based CYP2E1 inactivation. These results indicate that trans-resveratrol and tannic acid may modulate cytochrome P450 2E1 and influence the metabolic activation of xenobiotics mediated by this P450 isoform.     

Biochemical characterization of a phosphinate inhibitor of Escherichia coli MurC

The bacterial UDP-N-acetylmuramyl-L-alanine ligase (MurC) from Escherichia coli, an essential, cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent ligation of L-alanine (Ala) and UDP-N-acetylmuramic acid (UNAM) to form UDP-N-acetylmuramyl-L-alanine (UNAM-Ala). The phosphinate inhibitor 1 was designed and prepared as a multisubstrate/transition state analogue. The compound exhibits mixed-type inhibition with respect to all three enzyme substrates (ATP, UNAM, Ala), suggesting that this compound forms dead-end complexes with multiple enzyme states. Results from isothermal titration calorimetry (ITC) studies supported these findings as exothermic binding was observed under conditions with free enzyme (K(d) = 1.80-2.79 microM, 95% CI), enzyme saturated with ATP (K(d) = 0.097-0.108 microM, 95% CI), and enzyme saturated with the reaction product ADP (K(d) = 0.371-0.751 microM, 95% CI). Titrations run under conditions of saturating UNAM or the product UNAM-Ala did not show heat effects consistent with competitive compound binding to the active site. The potent binding affinity observed in the presence of ATP is consistent with the inhibitor design and the proposed Ordered Ter-Ter mechanism for this enzyme; however, the additional binding pathways suggest that the inhibitor can also serve as a product analogue.     

Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid

Structural and genetic studies indicate that the antibacterial compound triclosan, an additive in many personal care products, is an inhibitor of EnvM, the enoyl reductase from Escherichia coli. Here we show that triclosan specifically inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis and a target for the antitubercular drug isoniazid. Binding of triclosan to wild-type InhA is uncompetitive with respect to both NADH and trans-2-dodecenoyl-CoA, with K(i)' values of 0.22+/-0.02 and 0.21+/-0.01 microM, respectively. Replacement of Y158, the catalytic tyrosine residue, with Phe, reduces the affinity of triclosan for the enzyme and results in noncompetitive inhibition, with K(i) and K(i)' values of 36+/-5 and 47+/-5 microM, respectively. Consequently, the Y158 hydroxyl group is important for triclosan binding, suggesting that triclosan binds in similar ways to both InhA and EnvM. In addition, the M161V and A124V InhA mutants, which result in resistance of Mycobacterium smegmatis to triclosan, show significantly reduced affinity for triclosan. Inhibition of M161V is noncompetitive with K(i)' = 4.3+/-0.5 microM and K(i) = 4.4+/-0.9 microM, while inhibition of A124V is uncompetitive with K(i)' = 0. 81 +/- 0.11 microM. These data support the hypothesis that the mycobacterial enoyl reductases are targets for triclosan. The M161V and A124V enzymes are also much less sensitive to isoniazid compared to the wild-type enzyme, indicating that triclosan can stimulate the emergence of isoniazid-resistant enoyl reductases. In contrast, I47T and I21V, two InhA mutations that occur in isoniazid-resistant clinical isolates of M. tuberculosis, show unimpaired inhibition by triclosan, with uncompetitive inhibition constants (K(i)') of 0.18+/-0.01 and 0.12+/- 0.01 microM, respectively. The latter result indicates that InhA inhibitors targeted at the enoyl substrate binding site may be effective against existing isoniazid-resistant strains of M. tuberculosis.     

Discovery of Helicobacter pylori shikimate kinase inhibitors: bioassay and molecular modeling

Shikimate kinase (SK) is the fifth enzyme in the shikimate pathway and catalyzes the phosphate transfer from ATP to shikimate in generating shikimate 3-phosphate and ADP. SK has been developed as a promising target for the discovery of antibacterial agents. In this report, two small molecular inhibitors (compound 1, 3-methoxy-4-{[2-({2-methoxy-4-[(4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]phenoxy}methyl)benzyl]oxy}benzaldehyde; compound 2, 5-bromo-2-(5-{[1-(3,4-dichlorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}-2-furyl)benzoic acid) against Helicobacter pylori SK (HpSK) were successfully identified with IC(50) values of 5.5+/-1.2 and 6.4+/-0.4 microM, respectively. The inhibition kinetics shows that compound 1 is a noncompetitive inhibitor with respect to both shikimate and MgATP, and compound 2 is a competitive inhibitor toward shikimate and noncompetitive inhibitor with respect to MgATP. The surface plasmon resonance (SPR) technology based analysis reveals that the equilibrium dissociation constants (K(D)s) of compounds 1 and 2 with HpSK enzyme are 4.39 and 3.74 microM, respectively. The molecular modeling and docking of two inhibitors with HpSK reveals that the active site of HpSK is rather roomy and deep, forming an L-shape channel on the surface of the protein, and compound 1 prefers the corner area of L-shape channel, while compound 2 binds the short arm of the channel of SK in the binding interactions. It is expected that our current work might supply useful information for the development of novel SK inhibitors.     

A bisubstrate analog inhibitor of the carboxyltransferase component of acetyl-CoA carboxylase

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report, the synthesis of a bisubstrate analog inhibitor of carboxyltransferase is described. The inhibitor was synthesized by covalently linking biotin to coenzyme A via an acyl bridge between the sulfur of coenzyme A and the 1'-N of biotin. The steady-state kinetics of carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The inhibitor exhibited competitive inhibition versus malonyl-CoA and noncompetitive inhibition versus biocytin, with a slope inhibition constant (K(is)) of 23 +/- 2 microM. The bisubstrate analog has an affinity for carboxyltransferase 350 times higher than biotin. This suggests the inhibitor will be useful in structural studies, as well as aid in the search for chemotherapeutic agents that target acetyl-CoA carboxylase.     

Inactivation of dopamine beta-hydroxylase by beta-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition kinetics of carba- and C-fucosyl analogues of GDP-fucose against fucosyltransferase V: implication for the reaction mechanism

Inhibition kinetics of two isosteric analogues of GDP-fucose (GDP-Fuc) were investigated against fucosyltransferase V using electrospray ionization mass spectrometry coupled to multiple reaction monitoring. The carba-Fuc analogue was found to be a competitive inhibitor with a K(i) value of 67.1+/-9.8 microM, similar to the K(m) value for GDP-Fuc (50.4+/-5.5 microM), while the C-Fuc analogue exhibited significantly weak competitive inhibition with a K(i) value of 889+/-93 microM.     

Synthesis of photoaffinity probes [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside and [(4(')-benzoyl)phenoxy]-D-glucopyranoside for the identification of sugar-binding and phlorizin-binding sites in the sodium/D-glucose cotransporter protein

In this paper we describe the synthesis and photochemical and biochemical properties of two new photoaffinity probes designed for studies on the structure-function relationship of the sodium D-glucose cotransporter (SGLT1). The two probes are [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside (TIPDG), a mimic for the phenyl glucopyranoside arbutin which is transported by SGLT1 with a very high affinity, and [(4(')-benzoyl)phenoxy]-D-glucopyranoside (BzG), a model compound for phlorizin, the most potent competitive inhibitor of sugar translocation by SGLT1. Both photoaffinity probes TIPDG (lambda(max)=358 nm) and BzG (lambda(max)=293 nm) can be activated at 350-360 nm, avoiding protein-damaging wavelengths. In inhibitor studies on sodium-dependent D-glucose uptake into rabbit intestinal brush border membrane vesicles TIPDG and BzG showed a fully competitive inhibition with regard to the sugar with respective K(i) values of 22+/-5 microM for TIPDG and 12+/-2 microM for BzG. These K(i) values are comparable to those of their parent compounds arbutin (25+/-6 microM) and phlorizin (8+/-1 microM). To further test the potential of TIPDG and BzG as photoaffinity probes, truncated loop 13 protein, supposed to be part of the substrate recognition site of SGLT1, was exposed to TIPDG and BzG in solution. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis demonstrated that TIPDG and BzG successfully labeled the protein. These preliminary results suggest that both photoaffinity probes are promising tools for the study of the structure-function relationship of SGLT1 and other SGLT1 family transporter proteins.     

Time-dependent inhibition of protein farnesyltransferase by a benzodiazepine peptide mimetic

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) catalyze the prenylation of proteins with a carboxy-terminal tetrapeptide sequence called a CaaX box, where C refers to cysteine, "a" refers to an aliphatic residue, and X typically refers to methionine, serine, or glutamine (FTase), or to leucine (GGTase-I). Marsters and co-workers [(1994) Bioorg. Med. Chem. 2, 949--957] developed inhibitors of FTase with cysteine and methionine attached to an inner hydrophobic benzodiazepine scaffold. We found that the most potent of these compounds (BZA-2B) resulted in the time-dependent inhibition of FTase. The K(i) of BZA-2B for FTase, which is the dissociation constant of the initial complex, was 79 +/- 13 nM, and the K(i)*, which is the overall dissociation of inhibitor for all enzyme forms, was 0.91 +/- 0.12 nM. The first-order rate constant for the conversion of the initial complex to the final complex was 1.4 +/- 0.2 min(-1), and that for the reverse process was 0.016 +/- 0.002 min(-1). The latter rate constant corresponds to a half-life of the final complex of 45 min. Our experiments favor the notion that the inhibitor binds to the FTase--farnesyl diphosphate complex which then undergoes an isomerization to form a tighter FTase*--farnesyl diphosphate--BZA2-B complex. Diazepam, a compound with a benzodiazepine nucleus but lacking amino acid extensions, was a weak (K(i) = 870 microM) but not time-dependent inhibitor of FTase. Cys-Val-Phe-Met and Cys-4-aminobenzoyl-Met were instantaneous and not time-dependent inhibitors of FTase. Furthermore, BZA-4B, with a leucine specificity determinant, was a classical competitive inhibitor of GGTase-I and not a time-dependent inhibitor.     

Inhibition of dipeptidyl peptidase-IV (DPP-IV) by atorvastatin

Dipeptidyl peptidase-IV (DPP-IV) is an enzyme responsible for the inactivation of the glucoregulatory incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). In this report, we show that the hypolipidemic agent atorvastatin is a competitive inhibitor of porcine DPP-IV in vitro, with K(i)=57.8+/-2.3 microM. These results may have implications in the development of novel DPP-IV inhibitors based on the use of atorvastatin as a lead compound for the treatment of type 2 diabetes.     

Resveratrol prevents apoptosis in K562 cells by inhibiting lipoxygenase and cyclooxygenase activity.

The natural polyphenolic compound resveratrol (trans-3,4', 5-trihydroxystilbene) is shown to prevent apoptosis (programmed cell death) induced in human erythroleukemia K562 cells by hydrogen peroxide and other unrelated stimuli. Resveratrol reversed the elevation of leukotriene B4 (from 6.40 +/- 0.65 to 2.92 +/- 0.30 pmol.mg protein-1) and prostaglandin E2 (from 11.46 +/- 1.15 to 8.02 +/- 0.80 nmol.mg protein-1), induced by H2O2 challenge in K562 cells. The reduction of leukotriene B4 and prostaglandin E2 correlated with the inhibition of the 5-lipoxygenase activity, and the cyclooxygenase and peroxidase activity of prostaglandin H synthase, respectively. Resveratrol also blocked lipoperoxidation induced by hydrogen peroxide in K562 cell membranes. Resveratrol was found to act as a competitive inhibitor of purified 5-lipoxygenase and 15-lipoxygenase and prostaglandin H synthase, with inhibition constants of 4.5 +/- 0.5 microM (5-lipoxygenase), 40 +/- 5.0 microM (15-lipoxygenase), 35 +/- 4.0 microM (cyclooxygenase activity of prostaglandin H synthase) and 30 +/- 3.0 microM (peroxidase activity of prostaglandin H synthase). Altogether, the results reported here suggest that the anti-apoptotic activity of resveratrol depends on the direct inhibition of the main arachidonate-metabolizing enzymes.     

Exosite occupation by heparin enhances the reactivity of blood coagulation factor IXa

Blood coagulation factor IXa (fIXa) is a trypsin-like serine protease with low inherent activity that is greatly enhanced in the factor X activation complex. Molecular details of the conversion of fIXa from an inactive enzyme into a fully functional procoagulant are unclear. Recent studies have identified a heparin-binding exosite in the protease domain of fIXa. Effects of exosite occupation on fIXa activity are unclear. We used the Kunitz-type inhibitor bovine pancreatic trypsin inhibitor (BPTI) to probe fIXa reactivity in the absence and in the presence of heparin. While fIXa alone was poorly reactive with BPTI (K(i) approximately 0.7 mM), this reactivity was increased roughly 20-fold (K(i) = 37 +/- 6 microM) by heparin. This was reproducible with low-molecular-weight heparin (enoxaparin; K(i) = 70 +/- 12 microM). Surface plasmon resonance studies of the interaction between heparin and BPTI indicated an unstable interaction with very low affinity (K(d) = 172 microM). In contrast, kinetic studies revealed a high-affinity interaction between heparin and fIXa (K(d) = 128 +/- 26 nM) and showed that the enhancement of BPTI inhibition of fIXa by heparin was well described by a competitive inhibition model where heparin acts as an affecter of fIXa reactivity with inhibitor. Fluorescence studies with dansyl-EGR-fIXa supported the high-affinity interaction between heparin and fIXa and suggested an altered environment in the fIXa active-site region upon heparin binding. This modulating effect of heparin was supported by the observation of a heparin-induced increase in reactivity of fIXa toward a pentapeptide substrate. When viewed together, the results imply that specific physiological exosite interactions with heparin can induce alterations in the environment of the extended fIXa active site that can result in increased reactivity.     

Characterization of novel imidazole derivative, JM-8686, a potent inhibitor of allene oxide synthase

The inhibitory properties of a first synthetic jasmonic acid biosynthesis inhibitor, JM-8686, were investigated. Steady-state kinetic analysis indicates that the compound is a competitive inhibitor of allene oxide synthase (AOS) with a K(i) value of approximate 0.62+/-0.15 microM. Dialysis experiment indicates that AOS inactivation by JM-8686 is reversible. The optical difference spectroscopy analysis of JM-8686 and AOS interaction indicates that JM-8686 induced type II binding spectra with a K(d) value of approximate 1.6+/-0.2 microM, suggesting that JM-8686 binds to the prosthetic heme iron of AOS. Comparison of the inhibitory potency of the compound against HPL (CYP74B) from tomato revealed that JM-8686 was a highly selective inhibitor for AOS.     

Benzodiazepine compounds as inhibitors of the src protein tyrosine kinase: screening of a combinatorial library of 1,4-benzodiazepines.

We screened 1680 spatially separated compounds of a diverse combinatorial library of 1,4-benzodiazepines for their ability to inhibit the kinase activity of protein tyrosine kinases Src, Yes, Abl, Lck, Csk, and fibroblast growth factor receptor. This screening yielded novel ligands for the protein tyrosine kinase Src. In the 1, 4-benzodiazepine-2-one scaffold, the preferred substituent at position R(1) was 4-hydroxyphenylmethyl or a 3-indolemethyl derived from a tyrosine or tyrptophan used in building the benzodiazepine, while the substituent at R(2), introduced by alkylating agents, was preferably aromatic in nature. The preferred ring structure introduced on the bicyclic ring of the scaffold by acid chlorides was a p-hydroxy phenyl group. The lead compound, designated as N-L-Yaa, has a L-4-hydroxyphenylmethyl ring at R(1) and a biphenylmethyl substituent at R(2). The compound has an IC(50) of 73 microM against Src, 2- to 6-fold lower than against other protein tyrosine kinases and >10-fold lower than against other nucleotide-utilizing enzymes. The mechanism of binding of N-L-Yaa to Src is mixed against the peptidic substrate with a K(i) of 35 microM and noncompetitive against ATP-Mg with a K(i) of 17 microM. Multiple inhibition analysis of the lead compound in the presence of other competitive inhibitors demonstrated that the binding of the lead compound is nonexclusive to the other competitive inhibitor. The inhibitor was found to be nontoxic to the AFB-13-human fibroblasts cells and inhibited the colony formation of HT-29 colon adenocarcinoma cells that are dependent on Src activity.     

Oleyl sulfate reveals allosteric inhibition of soybean lipoxygenase-1 and human 15-lipoxygenase

Inhibition of lipoxygenase (LO) is currently an important goal of biomedical research due to its critical role in asthma, atherosclerosis, and cancer regulation. Steady-state kinetic data indicate that oleic acid (OA) is a simple competitive inhibitor for soybean lipoxygenase; however, kinetic isotope effect (KIE) data suggest a more complicated inhibitory mechanism. To investigate the inhibitory effects of fatty acids on lipoxygenase more thoroughly, we have synthesized a novel inhibitor to lipoxygenase, (Z)-9-octadecenyl sulfate (oleyl sulfate, OS), which imparts kinetic properties that are inconsistent with simple competitive inhibition for both SLO-1 and 15-HLO. The KIE exhibits a hyperbolic rise with addition of OS, indicating the formation of a catalytically active ternary complex with K(D) values of 0.6 +/- 0.2 and 0.4 +/- 0.05 microM for SLO-1 and 15-HLO, respectively. The steady-state kinetics show that SLO-1 proceeds through a hyperbolic mixed-type inhibition pathway, where OS binding (K(i) = 0.7 +/- 0.3 microM) causes an approximate 4-fold increase in the K(m)(app) (alpha = 4.6 +/- 0.5) and a decrease in the k(cat) by approximately 15% (beta = 0.85 +/- 0.1). 15-HLO also exhibits a hyperbolic saturation of k(cat)/K(m) consistent with the observed rise in its KIE. Taken together, these findings indicate the presence of an allosteric site in both SLO-1 and 15-HLO and suggest broad implications regarding the inhibition of LO and the treatment of LO-related diseases.     

Slow-binding inhibition of human prostaglandin endoperoxide synthase-2 with darbufelone, an isoform-selective antiinflammatory di-tert-butyl phenol.

The antiinflammatory agent darbufelone, ((Z)-5-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl] methylene]-2-imino-4-thiazolidinone, methanesulfonate salt), was discovered as a dual inhibitor of cellular prostaglandin and leukotriene production. To study the mechanism of action of this drug, we expressed human prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2 and purified the recombinant enzymes using buffers that contain octylglucoside. In cyclooxygenase assays following a 15-min incubation of enzyme with inhibitor, darbufelone potently inhibits PGHS-2 (IC(50) = 0.19 microM) but is much less potent with PGHS-1 (IC(50) = 20 microM). Interestingly, when the assay buffer contains traces of Tween 20 (0.0001%), darbufelone appears inactive with PGHS-2 due to a detergent interaction that is detectable by absorption spectroscopy. We therefore used octylglucoside, which does not affect darbufelone in this way, in place of Tween 20 in our PGHS buffers. Inhibition of PGHS-2 with darbufelone is time dependent: with no preincubation, darbufelone is a weak inhibitor (IC(50) = 14 microM), but after a 30-min incubation it is 20-fold more potent. Plots of PGHS-2 activity vs preincubation time at various darbufelone concentrations reach a plateau. This finding is inconsistent with irreversible or one-step slow-binding inhibition. A two-step slow-binding inhibition model is proposed in which the E.I complex (K(i) = 6.2 +/- 1.9 to 14 +/- 1 microM) slowly transforms (k(5) = 0.015-0.030 s(-)(1)) to a tightly bound E.I form with K(i) = 0.63 +/- 0.07 microM and k(6) = 0.0034 s(-)(1). In steady-state kinetics inhibition experiments performed with no preincubation, we find that darbufelone is a noncompetitive inhibitor of PGHS-2 (K(i) = 10 +/- 5 microM). Darbufelone quenches the fluorescence of PGHS-2 at 325 nm (lambda(ex) = 280 nm) with K(d) = 0.98 +/- 0.03 microM. The PGHS substrate, arachidonate, and various cyclooxygenase inhibitors do not alter this binding affinity of darbufelone but a structural analogue of darbufelone competes directly for binding to PGHS-2. Di-tert-butyl phenols such as darbufelone may inhibit PGHS-2 by exploiting a previously unrecognized binding site on the enzyme.     

Purification and characterization of Streptomyces griseus catechol O-methyltransferase

A soluble (100,000 x g supernatant) methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to catechols was present in cell extracts of Streptomyces griseus. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7. 5 and 35 degrees C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the K(m) for 6,7-dihydroxycoumarin was 500 +/- 21.5 microM, and that for S-adenosyl-L-methionine was 600 +/- 32.5 microM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of S-adenosyl-L-methionine, with a K(i) of 224 +/- 20.6 microM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg(2+), p-chloromercuribenzoic acid, and N-ethylmaleimide.     

A lead compound for the development of ABA 8'-hydroxylase inhibitors

(1'S*,2'S*)-(+/-)-6-Nor-2',3'-dihydro-4'-deoxo-ABA (2) was designed and synthesized as a candidate lead compound for developing a potent and specific inhibitor of ABA 8'-hydroxylase. This compound acted as an effective competitive inhibitor of the enzyme, with a K(I) value of 0.40microM, without exhibiting ABA activity. However, compound 2 also functioned as an enzyme substrate, making it a short-lived inhibitor. The 8'-difluorinated derivative of 2 (4) was synthesized as a long-lasting alternative. Compound 4 resisted 8'-hydroxylation, but inhibited ABA 8'-hydroxylation as effectively as 2. These results suggest that compound 2 is a useful lead compound for the future design and development of an ideal ABA 8'-hydroxylase inhibitor.