C. Palacios, C. Cespón, C. Martín de la Vega, G. Roy, A. Serrano, M. Salinas, P. Gónzalez-Porqué
The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu.Na, Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.
{"title":"Lauryl Gallate Inhibits the Activity of Protein Tyrosine Kinase c-Src Purified from Human Platelets","authors":"C. Palacios, C. Cespón, C. Martín de la Vega, G. Roy, A. Serrano, M. Salinas, P. Gónzalez-Porqué","doi":"10.1080/14756360127567","DOIUrl":"https://doi.org/10.1080/14756360127567","url":null,"abstract":"The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu.Na, Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.","PeriodicalId":15776,"journal":{"name":"Journal of enzyme inhibition","volume":"47 1","pages":"527 - 533"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76010638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1080/14756360109162358
J. Stojan
An analysis of sigmoid-shaped progress curves in the reaction between Electric Eel acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7, AChE) and its substrate acetylthiocholine in low concentrations at pH 7 is presented. In order to be able to explain an initial apparent inhibition of the enzyme-substrate reaction, the rate of detection reaction had to be taken into account. The theoretical curves obtained by the fitting of differential equations for the reaction mechanism to the data of six progress curves simultaneously, exactly reproduce the course of the experimental curves. The measurements performed with various concentrations of detection reagent confirm the proposed cause of sigmoidity.
{"title":"Slow Detection Reaction can Mimic Initial Inhibition of an Enzymic Reaction","authors":"J. Stojan","doi":"10.1080/14756360109162358","DOIUrl":"https://doi.org/10.1080/14756360109162358","url":null,"abstract":"An analysis of sigmoid-shaped progress curves in the reaction between Electric Eel acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7, AChE) and its substrate acetylthiocholine in low concentrations at pH 7 is presented. In order to be able to explain an initial apparent inhibition of the enzyme-substrate reaction, the rate of detection reaction had to be taken into account. The theoretical curves obtained by the fitting of differential equations for the reaction mechanism to the data of six progress curves simultaneously, exactly reproduce the course of the experimental curves. The measurements performed with various concentrations of detection reagent confirm the proposed cause of sigmoidity.","PeriodicalId":15776,"journal":{"name":"Journal of enzyme inhibition","volume":"9 1","pages":"89 - 94"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78922092","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1080/14756360109162359
Nikolaos P. Rodis, G. Digenis
A series of novel low molecular weight thiocarbamate esters (1e-6e) were synthesized and evaluated as inhibitors of human leukocyte elastase (HLE). The thiocarbamate esters studied consist of a substituted primary or secondary aliphatic or aromatic amine and a 1-phenyl-1H-tetrazole-5-thiol (Table I). The HLE catalyzed hydrolysis of N-methoxysuccinyl- L-Ala-L-Ala-L-Pro-L-Val-p-nitroanilide substrate was utilized as the measure of inhibition. N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (1e) exhibited the highest inhibitory activity (kobs/[I] = 2.1 × 105 M−1 · min−1) and N-allyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (2e) (Kobs/[I] = 6.1×104 M−1 · min−1) exhibited the second highest inhibitory activity of all the thiocarbamates. The aromatic N-phenyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (4e) showed the lowest inhibitory activity (Kobs/[I] = 1.9 × 102 M−1 · min−1 among the N-monosubstituted derivatives, similar to that of N-ethyl-N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (5e) (Kobs/[I] = 1.8 × 102 M−1 min−1). The N-isopropyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (3e) (Kobs/[I] = 3.3 × 103 M−1 · min−1) was about 10 fold more active than (4e) and N, N-diisopropyl, 1-phenyl-1H-tetrazole- 5-thiocarbamate (6e) showed no inhibitory activity against HLE. In the present work less than 3% of HLE specific activity was regained after 24 hours incubation with each of the tested N-monosubstituted thiocarbamates (1e-4e). The time-dependent inhibition of HLE by the thiocarbamate compounds (1e-5e) seems to involve the interaction and possible chemical modification of one enzyme residue. Straight chain nonpolar aliphatic substituents on the nitrogen of the thiocarbamate functionality may be essential for high inhibitory activity. As the degree of substitution (branching) on the nitrogen of the thiocarbamate functionality increases the inhibitory activity of the compounds decreases. The time-dependent inhibition of HLE and the slow deacylation rates by the N-monosubstituted thiocarbamates are consistent with irreversible inhibition.
{"title":"Synthesis and in-vitro Evaluation of Novel Low Molecular Weight Thiocarbamates as Inhibitors of Human Leukocyte Elastase","authors":"Nikolaos P. Rodis, G. Digenis","doi":"10.1080/14756360109162359","DOIUrl":"https://doi.org/10.1080/14756360109162359","url":null,"abstract":"A series of novel low molecular weight thiocarbamate esters (1e-6e) were synthesized and evaluated as inhibitors of human leukocyte elastase (HLE). The thiocarbamate esters studied consist of a substituted primary or secondary aliphatic or aromatic amine and a 1-phenyl-1H-tetrazole-5-thiol (Table I). The HLE catalyzed hydrolysis of N-methoxysuccinyl- L-Ala-L-Ala-L-Pro-L-Val-p-nitroanilide substrate was utilized as the measure of inhibition. N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (1e) exhibited the highest inhibitory activity (kobs/[I] = 2.1 × 105 M−1 · min−1) and N-allyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (2e) (Kobs/[I] = 6.1×104 M−1 · min−1) exhibited the second highest inhibitory activity of all the thiocarbamates. The aromatic N-phenyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (4e) showed the lowest inhibitory activity (Kobs/[I] = 1.9 × 102 M−1 · min−1 among the N-monosubstituted derivatives, similar to that of N-ethyl-N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (5e) (Kobs/[I] = 1.8 × 102 M−1 min−1). The N-isopropyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (3e) (Kobs/[I] = 3.3 × 103 M−1 · min−1) was about 10 fold more active than (4e) and N, N-diisopropyl, 1-phenyl-1H-tetrazole- 5-thiocarbamate (6e) showed no inhibitory activity against HLE. In the present work less than 3% of HLE specific activity was regained after 24 hours incubation with each of the tested N-monosubstituted thiocarbamates (1e-4e). The time-dependent inhibition of HLE by the thiocarbamate compounds (1e-5e) seems to involve the interaction and possible chemical modification of one enzyme residue. Straight chain nonpolar aliphatic substituents on the nitrogen of the thiocarbamate functionality may be essential for high inhibitory activity. As the degree of substitution (branching) on the nitrogen of the thiocarbamate functionality increases the inhibitory activity of the compounds decreases. The time-dependent inhibition of HLE and the slow deacylation rates by the N-monosubstituted thiocarbamates are consistent with irreversible inhibition.","PeriodicalId":15776,"journal":{"name":"Journal of enzyme inhibition","volume":"31 1","pages":"105 - 95"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77051797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1080/14756360109162356
R. Olmo, M. Blanco, JesÚS M. Socorro, Juan A. Martín, J. Teijón
Structural variations of lysozyme as a consequence of its interaction with CdAc2, as well as the implications on the protein functionality have been studied. Variations in the conformation of the macromo-lecule are seen, however these changes are not reflected on the secondary structure. The interaction of the salt with the polypeptide chain is weak and thermodynamically unfavourable. Molecular aggregates (dimer forms) are observed at the highest salt concentrations. This interaction causes an inhibitory effect on lysozyme, the activity loss being 50% at the highest salt concentration studied. The inhibition is of mixed type with an uncompetitive component. Thus cadmium does not bind to the active site of the enzyme which is in accordance with the not very large activity loss observed. The substrate inhibition of lysozyme is favoured in the presence of the salt, so interaction with the macromolecule is at low affinity sites.
{"title":"Effect of Cadmium Acetate on the Conformation of Lysozyme: Functional Implications","authors":"R. Olmo, M. Blanco, JesÚS M. Socorro, Juan A. Martín, J. Teijón","doi":"10.1080/14756360109162356","DOIUrl":"https://doi.org/10.1080/14756360109162356","url":null,"abstract":"Structural variations of lysozyme as a consequence of its interaction with CdAc2, as well as the implications on the protein functionality have been studied. Variations in the conformation of the macromo-lecule are seen, however these changes are not reflected on the secondary structure. The interaction of the salt with the polypeptide chain is weak and thermodynamically unfavourable. Molecular aggregates (dimer forms) are observed at the highest salt concentrations. This interaction causes an inhibitory effect on lysozyme, the activity loss being 50% at the highest salt concentration studied. The inhibition is of mixed type with an uncompetitive component. Thus cadmium does not bind to the active site of the enzyme which is in accordance with the not very large activity loss observed. The substrate inhibition of lysozyme is favoured in the presence of the salt, so interaction with the macromolecule is at low affinity sites.","PeriodicalId":15776,"journal":{"name":"Journal of enzyme inhibition","volume":"12 1","pages":"65 - 80"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87175850","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1080/14756360109162370
Christian G. Castro, B. M. Britt
Binding of the transition state analogue coformycin and the ground state analogue 1-deaazadenosine to bovine adenosine deaminase have been thermody-namically characterized. The heat capacity changes for coformycin and 1-deazaadenosine binding are - 4.7 × 0.8 kJ/mole-K and -1.2 × 0.1 kJ/mole-K, respectively. Since the predominant source of heat capacity change in enzyme interactions are changes in the extent of exposure of nonpolar amino acid side chains to the aqueous environment and the hydrophobic effect is the predominant factor in native structure stabilization, we propose that the binding of either class of ligand is associated with a stabilizing enzyme conformational change with coformycin producing the far greater effect Analysis of the T dependence of the second order rate constant for formation of the enzyme/coformycin complex further reveals that the conformational change is not rate limiting. We propose that the enzyme may facilitate catalysis via the formation of a stabilizing conformation at the reaction transition state.
{"title":"Binding Thermodynamics of the Transition State Analogue Coformycin and of the Ground State Analogue 1-Deazaadenosine to Bovine Adenosine Deaminase","authors":"Christian G. Castro, B. M. Britt","doi":"10.1080/14756360109162370","DOIUrl":"https://doi.org/10.1080/14756360109162370","url":null,"abstract":"Binding of the transition state analogue coformycin and the ground state analogue 1-deaazadenosine to bovine adenosine deaminase have been thermody-namically characterized. The heat capacity changes for coformycin and 1-deazaadenosine binding are - 4.7 × 0.8 kJ/mole-K and -1.2 × 0.1 kJ/mole-K, respectively. Since the predominant source of heat capacity change in enzyme interactions are changes in the extent of exposure of nonpolar amino acid side chains to the aqueous environment and the hydrophobic effect is the predominant factor in native structure stabilization, we propose that the binding of either class of ligand is associated with a stabilizing enzyme conformational change with coformycin producing the far greater effect Analysis of the T dependence of the second order rate constant for formation of the enzyme/coformycin complex further reveals that the conformational change is not rate limiting. We propose that the enzyme may facilitate catalysis via the formation of a stabilizing conformation at the reaction transition state.","PeriodicalId":15776,"journal":{"name":"Journal of enzyme inhibition","volume":"16 1","pages":"217 - 232"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90898372","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1080/14756360109162353
Regis Le Lain, P. Nicholls, H. Smith, F. Maharlouie
In a screening programme for inhibitors of human testis 17β-hydroxysteroid dehydrogenase (17β-HSD type 3), as potential agents for the treatment of hormone-dependent prostatic cancer, we have used crude human testis microsomal 17β-hydroxysteroid dehydrogenase as a convenient source of the enzyme. Crude human enzyme was shown to have a similar substrate profile to recombinant Type 3 17β-HSD from the same source as determined by the low Km/Vmax ratio for the reduction of androste-nedione compared to the oxidation of testosterone, and a low level of activity in reduction of oestrone. Screening of a wide range of compounds of different structural types as potential inhibitors of the microsomal enzyme in the reduction step revealed that certain p-benzoquinones and flavones/isoflavones were potent inhibitors of the enzyme, diphenyl-p-benzoquinone (2.7 μM), phenyl-p-benzoquinone (5.7 μM), 7-hydroxyflavone (9.0 μM), baicalein (9.3 μM) and biochanin A (10.8 μM). Some structure-activity relationships within the flavone/isoflavone series are discussed. Studies with rat testis microsomal 17β-HSD showed that it differed from the human enzyme mainly in its greater ability to accept oestrone as substrate and the pH-optimum for oxidation of testosterone. It was found to be much less sensitive to inhibition by the compounds studied so negating it use as a more readily available tissue for the screening of potential inhibitors.
{"title":"Inhibitors of Human and Rat Testes Microsomal 17 β-Hydroxysteroid Dehydrogenase (17β-HSD) as Potential Agents for Prostatic Cancer","authors":"Regis Le Lain, P. Nicholls, H. Smith, F. Maharlouie","doi":"10.1080/14756360109162353","DOIUrl":"https://doi.org/10.1080/14756360109162353","url":null,"abstract":"In a screening programme for inhibitors of human testis 17β-hydroxysteroid dehydrogenase (17β-HSD type 3), as potential agents for the treatment of hormone-dependent prostatic cancer, we have used crude human testis microsomal 17β-hydroxysteroid dehydrogenase as a convenient source of the enzyme. Crude human enzyme was shown to have a similar substrate profile to recombinant Type 3 17β-HSD from the same source as determined by the low Km/Vmax ratio for the reduction of androste-nedione compared to the oxidation of testosterone, and a low level of activity in reduction of oestrone. Screening of a wide range of compounds of different structural types as potential inhibitors of the microsomal enzyme in the reduction step revealed that certain p-benzoquinones and flavones/isoflavones were potent inhibitors of the enzyme, diphenyl-p-benzoquinone (2.7 μM), phenyl-p-benzoquinone (5.7 μM), 7-hydroxyflavone (9.0 μM), baicalein (9.3 μM) and biochanin A (10.8 μM). Some structure-activity relationships within the flavone/isoflavone series are discussed. Studies with rat testis microsomal 17β-HSD showed that it differed from the human enzyme mainly in its greater ability to accept oestrone as substrate and the pH-optimum for oxidation of testosterone. It was found to be much less sensitive to inhibition by the compounds studied so negating it use as a more readily available tissue for the screening of potential inhibitors.","PeriodicalId":15776,"journal":{"name":"Journal of enzyme inhibition","volume":"8 1","pages":"35 - 45"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79246283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1080/14756360109162357
J. Vioque, R. Sánchez‐Vioque, A. Clemente, J. Pedroche, M. M. Yust, F. Millán
Extensive rapeseed protein hydrolysate obtained sequentially with Alcalase and Flavourzyme showed inhibitory activity towards Alcalase. Inhibitory activity decreased as the hydrolytic process progressed probably by heat denaturation and/or partial protease degradation. Alcalase rapeseed inhibitors were purified by gel filtration and subsequent ion exchange chromatography. They are composed of peptides of 8.4 and 6.1 kDa linked by interchain disulphide bonds, as observed by reducing SDS-PAGE, with a native molecular weight of 18 kDa. Aminoacid composition of the inhibitors was characterized by the high proportion of methionine (4.2%) and cysteine (4.6%). Alcalase inhibitors were partially resistant to heat treatment; after heating at 70 °C for 45 minutes more than 50% of the original inhibitory activity remained in the purified protein but after heating at 90 °C for 5 minutes, inhibitory activity decreased very fast to a basal level. The possible relation of these protease inhibitors with the 2S albumin storage proteins is discussed.
{"title":"Alcalase Rapeseed Inhibitors: Purification and Partial Characterization","authors":"J. Vioque, R. Sánchez‐Vioque, A. Clemente, J. Pedroche, M. M. Yust, F. Millán","doi":"10.1080/14756360109162357","DOIUrl":"https://doi.org/10.1080/14756360109162357","url":null,"abstract":"Extensive rapeseed protein hydrolysate obtained sequentially with Alcalase and Flavourzyme showed inhibitory activity towards Alcalase. Inhibitory activity decreased as the hydrolytic process progressed probably by heat denaturation and/or partial protease degradation. Alcalase rapeseed inhibitors were purified by gel filtration and subsequent ion exchange chromatography. They are composed of peptides of 8.4 and 6.1 kDa linked by interchain disulphide bonds, as observed by reducing SDS-PAGE, with a native molecular weight of 18 kDa. Aminoacid composition of the inhibitors was characterized by the high proportion of methionine (4.2%) and cysteine (4.6%). Alcalase inhibitors were partially resistant to heat treatment; after heating at 70 °C for 45 minutes more than 50% of the original inhibitory activity remained in the purified protein but after heating at 90 °C for 5 minutes, inhibitory activity decreased very fast to a basal level. The possible relation of these protease inhibitors with the 2S albumin storage proteins is discussed.","PeriodicalId":15776,"journal":{"name":"Journal of enzyme inhibition","volume":"68 1","pages":"81 - 87"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74113246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1080/14756360109162354
W. Reichert, R. Hartmann, J. Jose
A eucaryotic cell assay was established to identify novel, dual and selective inhibitors of human 5α-reductase. For this purpose the cDNAs encoding 5α-reductase type I and type II were inserted into a pRcCMV vector and expressed in human embryonic kidney (HEK293) cells. Single cell clones with substantially high enzymatic activity were selected and established as permanent cell lines. KM values were determined for both isozymes. The inhibitory potency of several steroidal and non-steroidal compounds synthesized in our group, as well as finasteride and 4MA as controls, were tested by measuring the conversion of [3H]androstenedione. Reaction products were quantified by a HPLC reversed phase technique. Using the new cell assays, selective as well as novel dual 5α-reductase inhibitors with IC50 values between 1.0 and 2.5 μM were identified.
{"title":"Stable Expression of the Human 5α-Reductase Isoenzymes Type I and Type II in HEK293 Cells to Identify Dual and Selective Inhibitors","authors":"W. Reichert, R. Hartmann, J. Jose","doi":"10.1080/14756360109162354","DOIUrl":"https://doi.org/10.1080/14756360109162354","url":null,"abstract":"A eucaryotic cell assay was established to identify novel, dual and selective inhibitors of human 5α-reductase. For this purpose the cDNAs encoding 5α-reductase type I and type II were inserted into a pRcCMV vector and expressed in human embryonic kidney (HEK293) cells. Single cell clones with substantially high enzymatic activity were selected and established as permanent cell lines. KM values were determined for both isozymes. The inhibitory potency of several steroidal and non-steroidal compounds synthesized in our group, as well as finasteride and 4MA as controls, were tested by measuring the conversion of [3H]androstenedione. Reaction products were quantified by a HPLC reversed phase technique. Using the new cell assays, selective as well as novel dual 5α-reductase inhibitors with IC50 values between 1.0 and 2.5 μM were identified.","PeriodicalId":15776,"journal":{"name":"Journal of enzyme inhibition","volume":"20 1","pages":"47 - 53"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73148259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1080/14756360109162382
K. Peters, G. Jahreis, Eva-Maria Kötters
A number of substrate analogous peptides containing a phosphoramidate, phosphonate ester, hydro-xamate, carboxylate or sulfhydryl group are known to be inhibitors of thermolysin and other metallo-proteinases. According to the specificity, most of the inhibitors mimic the prime site of the active center. Hitherto, peptidyl derivatives with a thiol group at the C-terminus have not been described. We have synthesized the protected cysteamides Ac-Ala-Ala-CA-SH and Z-Aa1-Aa2-CA-SH (Aa1: Ala, Pro; Aa2: Ala, Leu). The binding of these thiol peptide inhibitors to the metalloproteinases is characterized first by the coordination of the thiolate group of the inhibitor to the catalytic zinc ion and second by the subsite interaction of the peptide ligand in the active site of the enzyme. All peptide derivatives were competitive inhibitors of the zinc metalloproteinase thermolysin. The strongest inhibition was found with Z-Pro-Leu-CA-SH (Ki = 30 μM). Substitution of the N-protecting benzylox-ycarbonyl residue towards the acetyl group in the peptide inhibitor, the inhibition constant decreased about 25 times.
许多含有磷酰胺、膦酸酯、羟基、羧酸或巯基的底物类似肽被认为是热溶酶和其他金属蛋白酶的抑制剂。根据特异性，大多数抑制剂模拟活性中心的主要位点。迄今为止，在c端有巯基的肽基衍生物还没有被描述过。我们合成了受保护的半胱胺类化合物Ac-Ala-Ala-CA-SH和Z-Aa1-Aa2-CA-SH (Aa1: Ala, Pro;Aa2: Ala, Leu)。这些硫醇肽抑制剂与金属蛋白酶结合的特征首先是抑制剂的硫酸基团与催化锌离子的配位，其次是肽配体在酶的活性位点的亚位相互作用。所有肽衍生物都是锌金属蛋白酶热溶酶的竞争性抑制剂。Z-Pro-Leu-CA-SH (Ki = 30 μM)的抑制作用最强。将保护n的苯氧基羰基残基置换为乙酰基后，抑制常数降低约25倍。
{"title":"A New Class of Potent Reversible Inhibitors of Metallo-proteinases: C-terminal Thiol-peptides as Zinc-coordinating Ligands","authors":"K. Peters, G. Jahreis, Eva-Maria Kötters","doi":"10.1080/14756360109162382","DOIUrl":"https://doi.org/10.1080/14756360109162382","url":null,"abstract":"A number of substrate analogous peptides containing a phosphoramidate, phosphonate ester, hydro-xamate, carboxylate or sulfhydryl group are known to be inhibitors of thermolysin and other metallo-proteinases. According to the specificity, most of the inhibitors mimic the prime site of the active center. Hitherto, peptidyl derivatives with a thiol group at the C-terminus have not been described. We have synthesized the protected cysteamides Ac-Ala-Ala-CA-SH and Z-Aa1-Aa2-CA-SH (Aa1: Ala, Pro; Aa2: Ala, Leu). The binding of these thiol peptide inhibitors to the metalloproteinases is characterized first by the coordination of the thiolate group of the inhibitor to the catalytic zinc ion and second by the subsite interaction of the peptide ligand in the active site of the enzyme. All peptide derivatives were competitive inhibitors of the zinc metalloproteinase thermolysin. The strongest inhibition was found with Z-Pro-Leu-CA-SH (Ki = 30 μM). Substitution of the N-protecting benzylox-ycarbonyl residue towards the acetyl group in the peptide inhibitor, the inhibition constant decreased about 25 times.","PeriodicalId":15776,"journal":{"name":"Journal of enzyme inhibition","volume":"54 1","pages":"339 - 350"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90407279","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1080/14756360109162369
Sabine Gritsch, S. Guccione, R. Hoffmann, A. Cambria, G. Raciti, Thierry Langer
A molecular modelling study was performed using the CATALYST software package on a dataset of 100 thiosemicarbazide and thiazole derivatives acting as MAO-B irreversible inhibitors in order to, (i) better elucidate the possible role of the ligand features which are significant for binding and (ii) generate chemical features based pharmacophore models which were subsequently used as 3D queries for database searching. Based on known MAO-B inhibitors, pharmacophore hypotheses were created in order to find similarities between the thiazoles and thiosemicarbazides and identify the key sub-structures most likely to be significant for high MAO-B inhibitory activity.
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