Showing papers in "Journal of Enzyme Inhibition in 1993"

Biochemical characterisation of a pancreatic elastase inhibitor from the leech Hirudinaria manillensis.

Abstract: The jawed leech, Hirudinaria manillensis is closely related to Hirudo medicinalis, both belonging to the same family Arhynchobdellida. From Hirudo, two potent peptide inhibitors, hirudin (a thrombin inhibitor) and eglin (an elastase/chymotrypsin inhibitor) have been characterised in detail. During our studies to isolate thrombin inhibitor from the leech Hirudinaria a potent inhibitor, analogous to eglin, was also detected. Results indicate that this inhibitor, which we have named 'GELIN', is significantly different from eglin. Gelin was isolated and purified to homogeneity by ion exchange chromatography and reverse phase HPLC. The isoelectric point of Gelin was estimated to be 4.55, in contrast to 6.45 for eglin. The molecular weight of Gelin was similar to eglin, as estimated by SDS-PAGE. Amino-terminal sequence analysis of the first 29 residues show no sequence homology with eglin or any other serine protease inhibitors. Circular dichroism studies showed that the secondary structure of Gelin has no helix, 58% beta sheets and 42% random structures compared to 19% helix, 56% beta sheets and 25% random structures in eglin. Like eglin, Gelin inhibits elastase, cathepsin G and chymotrypsin but has little or no activity towards plasmin, thrombin, pepsin and trypsin. These data suggest that the elastase inhibitors from these two species of leech are fundamentally different in structure, indicative of independent evolutionary origin.

Xanthine Oxidase Inhibitors from the Roots of Eggplant (Solanum Melongena L.)

Abstract: Stigmasterol 1, stigmasterol-β-D-glucoside 2, β-sitosterol-β-D-glucoside 3, dioscin 4, protodioscin 5 and methyl protodioscin 6 were isolated and characterized from the butanol soluble part of the ethanolic extract of eggplant roots (Solanum melongena L. Solanaceae). Except for stigmasterol, these compounds have not been previously isolated from Solanum melongena L.Since the roots of the eggplant have been used in folk medicine for rheumatism, inflammation and foot pain1,2, these compounds were tested for their inhibitory effect on xanthine oxidase. The results showed that the phytosterols 1,2 and 3 displayed a stronger inhibition on xanthine oxidase than the steroidal glycosides 4, 5 and 6. From the structural features, the active moiety seems to be the double bond for both phytosterols and steroidal glycosides or 22-OH group in furostanol glycoside 5 as regards xanthine oxidase inhibition based on this study.

Inhibition of aminopeptidases by phosphonic acid and phosphinic acid analogues of aspartic and glutamic acids.

Abstract: More than 30 phosphonic and phosphinic acid analogues of aspartic and glutamic acids were synthesized in order to probe how the structural differences of these molecules were reflected in their ability to inhibit cytosolic (LAP) and microsomal (APM) aminopeptidases. Although most of the compounds studied were found to exert only a modest inhibitory effect, the studies provide some information on the structural requirements of the binding subsites and catalytic centers of both enzymes.

Studies on Aldose Reductase Inhibitors from Fungi. II. Moniliformin and Small Ring Analogues

Abstract: The fungal metabolite moniliformin and several small ring analogues were evaluated for potential substrate and inhibitory activity in the rat lens aldose reductase (AR) assay. Even though all of these compounds possess carbonyl moieties and structural similarities to AR substrates, none were found to function as substrates over a concentration range of 1.0 mM to 10 μM. All of the compounds did display inhibitory activity with IC50s ranging from 19–110 μM. The most inhibitory compounds were the four-membered ring moniliformin (ICI 19 μM), the five-membered analogue croconic acid (IC50 28 μM) and six-membered derivative tetrahydroxy p-benzoquinone (1C50 23 μM). Modification of moniliformin by methylation (methyl moniliformin) or hydroxylation (squaric acid) resulted in a significant decline in inhibitory activity. All of the compounds evaluated except moniliformin displayed uncompetitive, non-competitive or mixed-type kinetics relative to the substrate (glyceraldehyde) and cofactor (NADPH), kinetic ...

Peptidyl Ammonium Methyl Ketones as Substrate Analog Inhibitors of Proline-Specific Peptidases

Abstract: Prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DP IV) are serine enzymes cleaving highly specific prolyl peptide bonds. Both enzymes were found to be inhibited by newly designed peptidyl ammonium and pyridinium methyl ketones acting as slow binding inhibitors. The most potent inhibitor of PEP is Z-Pro-Pro-CH2N+C5H5 exhibiting a K*i value of 1.8 nM with a first-order rate constant of kon 0.0022 s−1 for the formation of the tight enzyme-inhibitor complex. DP IV and H-Pro-Pro-CH2N+(CH3)3 form an enzyme-inhibitor-complex with an apparent second order rate constant of 2713M−1s−1. In contrast to the very stable N-terminal protected Z-Pro-Pro-CH2N+ (CH3)3, the deblocked derivative decomposes rapidly in aqueous solution.

Synthesis and the Antiperoxidase Activity of Seleno Analogues of the Antithyroid Drug Propylthiouracil

Abstract: The synthesis of 6-n-propyl-2-selenouracil (PSeU, Ib) and its methyl derivative (MSeU, Ic) are described. Replacement of the sulfur atom at C2 by selenium increased with antiperoxidase activity of these analogues five fold when compared to the clinically used antithyroid drug propylthiouracil (PTU). The structure-activity relationships of these agents are discussed.

Further evidence for the importance of free carboxylate in epoxysuccinate inhibitors of thiol proteases.

Abstract: Analogs of Ep-475 (2a), designed to explore the role played by the carboxylate in epoxysuccinate thiol protease inhibitors, have been synthesized and tested as inhibitors of papain and cathepsin B. Papain and cathepsin B are rapidly inactivated by carboxylates 2a and 6a, but are inactivated much more slowly by 2b-2f, 6c, and 6f, in which the carboxylate is absent or replaced by an amide, ester, or ketone. This order of reactivity contrasts with the inherent reactivity of substituted epoxides toward a non-enzymatic thiolate, previously shown to decrease in the order: COCH3 < CO2CH3 < CONH2 < H < CO2H. The results suggest that electrostatic attraction between the carboxylate of the inhibitor and protonated His159 of papain facilitates docking of the inhibitor in the active site of the enzyme, a conclusion reached previously from X-ray crystallographic structures of epoxysuccinates bound to papain. The most reactive isoleucine analog, 6a, was significantly less reactive than leucine-containing Ep-475...

Inhibition of β-N-Acetylglucosaminidase by Glycon-Related Analogues of the Substrate

Abstract: Inhibition studies on β-N-acetylglucosaminidase (EC 32130) of widely differing origins (animal, plant, fungus) were carried out with N-acetylglucosaminono-1,5-lactone (1), N-acetylglucosaminono-1,5-lactam (2), 1,5-imino-JV-acetylglucosaminitol (3), and N-acetylglucosaminono-1,5-lactone oxime (4) The inhibition was competitive in all cases, and K, values were generally in the range of 015-2μM, except for the fungal enzyme (5-20μM) To assess the kinetics of enzyme-inhibitor complex formation, continuous enzyme activity monitoring was done with 3,4-dinitrophenyl-β-N-acetylglucosaminide as the substrate A slow approach to the binding-equilibrium in the time scale of minutes could not be observed with any of the inhibitors tested (1-4) The results are evaluated as to the bearing of the enzyme source on best performance of the test compounds, the sub-type of inhibition mechanism is discussed, and suggestions are made for further analogue syntheses as well as potential applications of 1-4 (partic

Some 1-, and 3-substituted 3-(4'-aminophenyl)pyrrolidine-2,5-diones as selective inhibitors of aromatase.

Abstract: l-Alky1-3-(4′aminophenyl)pyrrolidine-2,5-diones (7,R = C3H7-C7H15) are potent inhibitors of aromatase in vitro, the l-hexyl (K; = 62nM) being about 100-fold more potent than aminoglutethimide (AG), and more selective in their ratio of aromatase:CSCC inhibitory potency. The 1-pentyl, l-hexyl and 1-heptyl derivatives are more stable to liver microsomal metabolism in vitro than AG possibly due to inhibition of the liver cytochrome P450s.l,3-Dialkyl-3(4′-aminophenyl)pyrrolidine-2,5-diones (9) have been synthesised by a novel method. Although the higher homologues (di-pentyl and di-hexyl) are more potent in vitro as inhibitors of aromatase than AG. they are less active than their 1-alkyl counterparts with the same alkyl substituent.

Binding of bovine basic pancreatic trypsin inhibitor (kunitz) as well as bovine and porcine pancreatic secretory trypsin inhibitor (kazal) to human cathepsin g: a kinetic and thermodynamic study

Abstract: The effect of pH and temperature on kinetic and thermodynamic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor; BPTI) as well as bovine and porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor; bovine and porcine PSTI, respectively) to human cathepsin G (EC 3.4.21.20) has been investigated. The affinity of the macromolecular inhibitors examined for cathepsin G is characterized by an endothermic, entropy-driven, behaviour, and shows the following trend: BPTI

Modulation of 3 alpha-hydroxysteroid dehydrogenase activity by the redox state of glutathione.

Abstract: 3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50), purified to homogeneity from rat liver, was strongly inactivated by incubation with a disulfide such as GSSG, L-cystine or L-cystamine, as well as an SH-reagent such as DTNB (5,5'-dithiobis(2-nitrobenzoic acid)), NEM (N-ethylmaleimide) or iodoacetic acid. The inactivation advanced with incubation time. Coenzyme (NADP+) completely protected the enzyme from this inactivation by disulfides, but neither of the substrates (androsterone and benzenedihydrodiol) did. The activity of inactivated enzyme was restored by treatment with thiols such as DTT (dithiothreitol) or GSH. In the GSH/GSSG redox buffer, the enzyme existed in an equilibrium between active (reduced) and inactive (oxidized) forms.

Reactions of usual and atypical human serum cholinesterase phenotypes with progressive and reversible inhibitors

Abstract: The reaction of usual (U) and atypical (A) cholinesterase phenotypes was studied with six organophosphorus compounds, two pyridinium oximes (HI-6 and PAM-2) and with 4–4-bipyridine (4,4-BP) No difference in the inhibition rate constants for the two phenotypes was found with the progressive inhibitors tabun, sarin, paraoxon and soman The other two progressive inhibitors, VX and the positively charged phosphostigmine, inhibited the U phenotype more strongly than the A phenotypeThe positively charged reversible inhibitor HI-6 showed a higher affinity for the U than for the A phenotype, while PAM-2 and the non-charged 4,4′-BP did not show a significant difference in their affinity towards the two enzymesBoth phenotypes phosphylated by VX or sarin were reactivatable by HI-6 and PAM-2, and the A phenotype was always reactivated more slowly than the U phenotype The paraoxon-inhibited phenotypes were reactivated at equal rates with PAM-2 but were not reactivated with HI-6 The phosphylated phenotypes

Peptidyl Carbamates as Novel Elastase Inhibitors: Structure-Activity Relationship Studies

Abstract: Twenty-six novel peptidyl carbamates and thiocarbamates were synthesized and evaluated as elastase inhibitors. Eighteen compounds inhibited porcine pancreatic elastase, whereas only eleven of the newly synthesized compounds inhibited human leukocyte elastase. Neither of the other serine dependent proteases, trypsin or chymotrypsin, were affected by any of the active inhibitors. Structure-activity relationship studies indicated that inhibition was dependent on P1 and P'1 substitution as well as on the presence of the carbamate functionality. Placement of an isostere of valine at P1 and a 1-(phenyl mercaptotetrazole at P'1 resulted in the most active human leukocyte elastase inhibitor within this series of compounds (Ki - 3.0 x 10(-7) M).

Exploring the hexokinase glucose binding site through correlation analysis and molecular modeling of glucosamine inhibitors

Abstract: A series of N-substituted glucosamines has been designed, synthesized, and tested as inhibitors of yeast hexokinase All derivatives exhibited competitive inhibition kinetics with respect to glucose Quantitative structure-activity relationships were derived from the resulting inhibition data The most significant equation demonstrated the existence of highly specific steric effects for the seven mew-substituted benzoylglucosamines included in the relationship Molecular modeling of potential complexes between the inhibitors and the hexokinase substrate binding site strongly suggests that the steric effects arise from potential contacts with two amino acid residues lying in the region occupied by the amide substituents

Enzyme selectivity analyses of arylsulfonylamino acid aldose reductase inhibitors.

Abstract: Arylsulfonylamino acids, displaying a wide range of inhibitory activities versus rat lens aldose reductase (RLAR), were analyzed for enzyme selectivity in several test systems. These RLAR inhibitors were found not to produce significant inhibition of genetically-linked reductases (aldehyde reductase, ALR), catalytically similar reductases (Pachysolen tannophilus xylose reductase, PTXR), functionally distinct oxidoreductases (glutathione reductase, GR, lactate dehydrogenase, LDH, and gamma-transaminase, GABA-T), and thymidylate synthase (TS). These data suggest that aldose reductase differs significantly from other oxidoreductases in its inhibitor binding domain(s). Furthermore, the aldose reductase selectivity demonstrated by the arylsulfonylamino acids suggests that these compounds may not inhibit other key metabolic transformations in various cell types and that they may function as selective probes for studies of the relationship between aldose reductase mediated biochemical changes and the pathologies of chronic diabetes.

Additional peptidyl diazomethyl ketones, including biotinyl derivatives, which affinity-label calpain and related cysteinyl proteinases.

Abstract: Calpain, the calcium-activated cysteinyl proteinase, can be irreversibly inactivated by peptidyl diazomethyl ketones in which the peptide portion contains a penultimate leucine residue. Some new derivatives of this type have been synthesized and examined for their rates of inactivation of chicken gizzard and human platelet calpain. Two derivatives containing a C-terminal biotin residue, Biot-Aca-Leu-TyrCHN2 and Biot-Aca-Leu-Leu-TyrCHN2, have also been prepared in the expectation that their application to the study of the function of calpain and related proteases will prove fruitful.

Deoxyribonuclease inhibitors produced by streptomycetes.

Abstract: Streptomyces sp. strain No. A-5838 produces three types of inhibitors of DNase II. Two of them, DNI-2 and DNI-3, were distinguished from the previously reported DNase II inhibitors, 5838-DNI and 5923-DNI, by their inhibitory profiles towards phosphodiesterases. DNI-2 has Mr 654, and is considered to be a coproporphyrin. DNI-3 is an acidic substance with Mr about 60,000 as estimated by gel filtration. The inhibitory activities of both inhibitors were shown to be temperature-dependent whereas only that of DNI-2 was pH-dependent.

Cholinesterase studies with (R) (+)- and (S)(-)-5-(1,3,3-trimethylindolinyl)-N-(1-phenylethyl)carbamate.

Abstract: A limited number of carbamates have been found useful for treatment of cholinergic symptoms with pyridostigmine and physostigmine being the main focus. In recent years 5-(1,3,3-trimethylindolinyl)N,N-dimethylcarbamate (I) has received considerable attention in the Chinese literature for a similar role. We report on the first synthesis of stereoisomers of an analog of (I). The isomers prepared were (R)(+)-5-(1,3,3-trimethylindolinyl)-N-(1-phenylethyl)carbamate (II) and (S)(-)-5-(1,3,3-trimethylindolinyl)-N-(1-phenylethyl)carbamate (III). The pKa value for each isomer was 6.8. Eel acetylcholinesterase inhibition studies were carried out at 25.0 degrees C over the pH range of 6.0 to 9.0. They reflect the first pH profiles using enantiomorphs of a cholinesterase inhibitor. The inhibition potencies for (II) and (III) over the range examined were similar. At pH 7.60 the ki for II = 7.38 x 10(3) M-1 min-1 (SD = 398) and for (III) the ki = 6.67 x 10(3) M-1 min-1 (SD = 355). In accord with the findings of Wilson and Bergmann20 on physostigmine our results indicate that the protonated form of (II) and (III) is the more potent inhibitor.

Inhibition of Human Synovial β-Glucuronidase by Steroidal Compounds

Abstract: IntroductionIn our laboratory we have focused on several enzymes, among them β-glucuronidase, which have been shown to be released in abnormal quantities during chronic inflammatory diseases such as Rheumatoid Arthritis.1,2 This enzyme, which is involved in the catalysis of β-glucuronides, has been characterized in mammalian tissues.3 In previous studies4,5 we have demonstrated that it was inhibitied by a number of synthetic antiinflammatory gold (I) complexes, such as gold (I) thiomalate (Myochrisin) and gold (I) thiosulfate (Solganol). although the mechanism of inhibition has not been verified we have suggested from these and previous studies6 that a thiol group on the enzyme may be the primary site of binding to the gold complex.

Synthesis and antithyroid activity of fluorinated 2-thiouracil analogues

Abstract: The synthesis of four fluorinated-2 thiouracil derivatives, namely, 6-trifluoromethyl* -2-thiouracil, l-methyl-6-trifluoromethyl-2-thiouraci1, l-acetyl-6-trifluoromethyl-2-thiouracil and 6-n-heptafluoropropyl-2-thiou-racil was achieved. 6-n-Heptafluoropropyl-2-thiouracil displayed higher antithyroid activity followed by 6-trifluoromethyl derivative and 1-methyl-6-trifluoromethyl analogue when compared to 6-n-propyl-2-thiouracil. 1-Acetyl 6-trifluoromethyl-2-thiouracil exhibited no antithyroid activity. The structure-activity relationships (SAR) of this class of compounds are discussed.

Peptidyl Carbamates as Human Leukocyte Elastase Inhibitors: Design and Synthesis of Desmosine-Like Tetrapeptidyl Carbamate Inhibitors

Abstract: The rational design and synthesis of a series of peptidyl carbamates incorporating a derivatized ornithyl or lysyl residue at the P3 or P4 subsite is described. The derivatized residues were chosen as mimics of desmonsine cross-links ubiquitously found in mature elastin. The alteration of specific residues of the peptidyl carbamate, in addition to the realization of a stereospecific synthesis, required utilization of two convergent synthetic approaches. When tested for inhibitory activity against the serine dependent enzymes, human leukocyte elastase, porcine pancreatic elastase, trypsin, chymotrypsin, as well as acetyl cholinesterase, the compounds were found to be specific inhibitors of the elastases. Thus the series was found to exhibit inhibitor dissociation constants as low as 0.2 × 10−6M and 3.0 × 10−6M for human leukocyte and porcine pancreatic elastase, respectively. Michaelis-Menten kinetics demonstrated active site inhibition. Placement of Nδ-Bz-L-Orn at P4 withp-nitrophenol at P1’(23a) ...

A kinetic study of simultaneous suicide inactivation and irreversible inhibition of an enzyme. Application to 1-aminocyclopropane-1-carboxylate (ACC) synthase inactivation by its substrate S-adenosylmethionine.

Abstract: This paper deals with the development of an experimental method for the kinetic study of the inactivation of an enzyme by a racemic mixture of an inhibitor, whose isomers operate as suicide substrate and irreversible inhibitor respectively. The ratio between the isomer concentration in the biological or commercial source must be determined, but no separation of them is required. The method involves a kinetic analysis and an experimental design that enables the affinity (1/Km), rate of catalysis (kcat), rate of inactivation (lambda max), efficiency of catalysis (kcat/Km) and efficiency of inactivation (lambda max/Km) to be determined. The method has been applied to the kinetic characterization of the inactivation of 1-aminocyclopropane-1-carboxylate (ACC) synthase from tomato fruits by its substrate, S-adenosylmethionine (AdoMet). The ratio between AdoMet isomers with respect to its sulfonium centre, namely (-)-AdoMet and (+)-AdoMet, present in the commercial sample used, has been determined by 1H nuclear magnetic resonance.

Aminooxyphosphonates as slow binding inhibitors of aspartate and alanine aminotransferases from porcine heart

Abstract: Aminooxymethylphosphonic (AOMP), 1–aminooxyethylphosphonic (1-AOEP) and 2-aminooxyethyl-phosphonic (2-AOEP) acids have been synthesised and were found to be potent slow binding inhibitors of aspartate- and alanine-aminotransferases with Ki ranging from nanomolar to micromolar values. The half-life of the inhibited complexes varied from 8 min (AspAT-2-AOEP) to 11 h (AspAT-AOMP). Kinetic analysis of the interaction of both enzymes with AOMP suggested the formation of an E-I complex in a single slow binding process. In the case of other compounds, attempt to discriminate between a single- or a double-step mechanism, consistent with an E-I intermediate followed by a slow E-I to E-I* isomerisation process could not be clearly resolved. Spectral studies of the complex formed between PLP-bound enzyme and the aminooxy compound resulted in a shift from 362 nm, the absorption maximum of the native enzyme, to 380 nm, characteristic of the oxime produced. The kinetic parameters for aminooxyphosphonates were c...

Kinetic and Binding Studies on [125I]SDZ-283471, A Radiolabeled Inhibitor of Hiv-1 Proteinase

Abstract: Kinetic and binding studies on a novel type of potent inhibitors of HIV-1 proteinase containing a 2-aminobenzyl substituted statine moiety as dipeptide mimetic are reported. The compounds were characterized as fast-binding competitive inhibitors of the enzyme. Using the radioiodinated derivative [125I]SDZ-283471, monophasic association and dissociation curves were observed indicating a simple bimolecular reaction. While the association rate constant was similar to that of other inhibitors, the dissociation constant of SDZ-283471 was 20–500 times lower. Thus, the enzyme-inhibitor complex appears to be very stable in the case of the 2-aminobenzyl-statine compounds. Furthermore, we demonstrated that the inhibitors show appropriate specificity for HIV-1 and HIV-2 proteinases as compared to other aspartic proteinases. Using a competition assay, relative potencies of inhibitors modified in the P2 position were obtained and a preference for valine at this site was obseived.

1-Pentyl-3-(4-Aminophenyl)Pyrrolidine-2, 5-Dione, A Selective Aromatase Inhibitor: in vivo Studies

Abstract: 1-Pentyl, 1-hexyl and 1-heptyl-3–(4-aminophenyl)pyrrolidine-2, 5-dione, potent inhibitors of aromatase, lower oestrogen levels in PMSG-stimulated female rats in a comparable manner to the inhibitor aminoglutethimide (AG) used clinically for the treatment of breast cancer. Pharmacokinetic studies in the rat show t1/2 values for the 1-hexyl compound and AG of 1.8 and 5.5 h respectively. In 4 tests for CNS-depressant activity the overall order of activity was AG > I-heptyl=1-hexyl>>1-pentyl. The 1-pentyl compound has less tendency than AG to depress white cell and platelet counts in mice and overall is the drug candidate for further studies.

Adenosine Analogues as Inhibitors or Inactivators of S-Adenosylhomocysteine Hydrolase from Bovine Pancreas

Abstract: The ability of some substrate-analogues to inhibit or to inactivate S-adenosylhomocysteine hydrolase (SAHase) purified from bovine pancreas was investigated. Our results confirm that 3-deazaarysteromicin (DZAry) is a more potent competitive inhibitor than 3-deazaadenosine (DZA), while nebularine (purine riboside), contrary to previous reports, showed an uncompetitive inhibition. Moreover, 2-chloroadenosine and 2′-deoxyadenosine were found to be irreversible inactivators of SAHase with increasing potency, respectively. Ki values found for these drugs were of the same order of magnitude as those reported for SAHases from other mammalian tissues. The SAHase substrate-analogues studied are believed to act as antineoplastic and/or antiviral agents. It is conceivable to postulate that their therapeutic effects could be, at least in part, attributable to inhibition or even to inactivation of SAHase which, in turn, causes a reduction in S-adenosylmethionine-dependent methylation reactions.

Mechanistic Study of Hle Inhibition Using Dual Labeled Macromolecular Inhibitor

Abstract: The mechanism of inhibition of a specific and effective (Ki = 1–10 nM) macromolecular inhibitor of HLE was investigated. The inhibitor, polymer-bound peptidyl carbamate 1 was labeled with [3H] at its polymeric backbone (Mw = 27,000) and with [14C] in it peptidyl carbamate moiety. When the macromolecular inhibitor 1 was incubated with HLE to complete inhibition and then competitively displaced by an HLE substrate, only intact [3H/14C] polymer-bound inhibitor 1 was recovered. At the same time complete restoration of enzymatic activity was achieved. Gel permeation chromatography and HPLC were utilized to eliminate the possibility of the presence of low molecular weight fragments resulting from the interaction of 1 with HLE. It is concluded that 1 exerts its inhibitory action on HLE without the prior release of the low molecular weight inhibitor 3 (Mw = 570).

Screening and determination of kinetic parameters of aromatase inhibitors using human genital skin fibroblasts

Abstract: The screening of aromatase inhibitors usiing human genital skin fibroblasts (GSF) in culture is described. Using this system the selectivity in detecting inhibition compares well with that for human full-term placenta as a source of enzyme. IC50 and Ki values may be determined using GSF and the ranking of a group of known inhibitors tested compares with that using placental enzyme. The advantages of this method are that the tissue is readily available and the long term freezing of viable cells allows a ready supply of material for repeated experiments.

Affinity preparation of a protein inhibitor recognising a cell surface protease.

Abstract: Epithelial cell surfaces possess a trypsin-like protease, referred to as guanidinobenzoatase (GB). The cytoplasm of these cells contains an extractable protein (I) which recognises the cell surface GB by forming an enzyme-inhibitor complex (GB-I). Rhodamine-agmatine (Rh-Agm) was designed as a red fluorescent probe, directed to the active centre of GB, which can be used to locate cells with GB, employing fluorescence microscopy. Rh-Agm has a high affinity for GB and will displace I from GB-I on the surfaces of cells in frozen sections. Rh-Agm has been used to displace I from immobilised GB-I complexes on the surface of cultured colonic carcinoma cells in an affinity procedure aimed at purifying the inhibitors of GB obtained from cultured carcinoma cells. These inhibitors have been tested on protected frozen sections of normal colon and carcinoma of the colon, the formation of GB-I complexes being followed by a second yellow fluorescent probe which competes for the active centre of GB. The study of the protein-protein interactions to form GB-I has been facilitated by employing two synthetic fluorescent inhibitors of GB with differing affinities for GB and different fluorescent properties. The use of sections of tissue in this study has enabled a sequence of reactions to be carried out on the same cell surface GB, such that reversible inhibition reactions can be quickly demonstrated and recorded by fluorescence microscopy.

Experimental Method for the Kinetic Study of Unstable and Site-Directed Irreversible Inhibitors and its Application to the Inactivation of Chymotrypsin by Phenylmethylsulfonyl Fluorid

Abstract: The inactivation of enzymes by unstable irreversible inhibitors has only been experimentally characterized by means of discontinuous methods involving preincubation of the enzyme with the inhibitor and the removal of aliquots for further measurements of residual activity. A recent theoretical work proposed a continuous method for the kinetic study of these inhibitors in the presence of an auxiliary substrate. This method was based on approximate expressions for the evolution of the product concentration, which contained series expansions with five or more exponential terms, which severely complicates their use in practice. In this paper, a new experimental method has been developed for the kinetic study of unstable and site-directed irreversible inhibitors. This new method considers the operation of the enzymatic inactivation system in two different ranges of inhibitor concentration. Thus, at low inhibitor concentrations exact analytical equations describe the kinetic behaviour of the system from ...