ABSTRACT
1,4-Disubstituted imidazole inhibitors of Staphylococcus aureus and Escherichia coli enoyl acyl carrier protein reductase (FabI) have been identified. Crystal structure data shows the inhibitor 1 bound in the enzyme active site of E. coli FabI.
Subject(s)
Anti-Bacterial Agents/pharmacology , Imidazoles/pharmacology , Oxidoreductases/antagonists & inhibitors , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/drug effects , Escherichia coli Proteins , Fatty Acid Synthase, Type II , Imidazoles/chemical synthesis , Imidazoles/chemistry , Microbial Sensitivity Tests , Staphylococcus aureus/drug effectsABSTRACT
An SAR study of a screening lead has led to the identification of 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as inhibitors of Staphylococcus aureus enoyl acyl carrier protein reductase (FabI).
Subject(s)
Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Oxidoreductases/antagonists & inhibitors , Anti-Bacterial Agents/chemistry , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) , Escherichia coli/drug effects , Escherichia coli Proteins , Fatty Acid Synthase, Type II , Inhibitory Concentration 50 , Microbial Sensitivity Tests , Staphylococcus aureus/drug effects , Structure-Activity Relationship , Triclosan/pharmacologyABSTRACT
5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.
Subject(s)
Caspase Inhibitors , Cysteine Proteinase Inhibitors/chemical synthesis , Isatin/analogs & derivatives , Isatin/chemical synthesis , Sulfonamides/chemical synthesis , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Caspase 3 , Caspase 7 , Cell Line , Cell Survival/drug effects , Chondrocytes/cytology , Chondrocytes/drug effects , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Drug Design , Humans , Isatin/chemistry , Isatin/pharmacology , Jurkat Cells , Kinetics , Mice , Models, Molecular , Molecular Conformation , Neutrophils/cytology , Neutrophils/drug effects , Neutrophils/physiology , Recombinant Proteins/antagonists & inhibitors , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
The protease granzyme B (GrB) plays a key role in the cytocidal activity during cytotoxic T lymphocyte (CTL)-mediated programmed cell death. Multiple caspases have been identified as direct substrates for GrB, suggesting that the activation of caspases constitutes an important event during CTL-induced cell death. However, recent studies have provided evidence for caspase-independent pathway(s) during CTL-mediated apoptosis. In this study, we demonstrate caspase-independent and direct cleavage of the 45 kDa unit of DNA fragmentation factor (DFF45) by GrB both in vitro and in vivo. Using a novel and selective caspase-3 inhibitor, we show the ability of GrB to process DFF45 directly and mediate DNA fragmentation in the absence of caspase-3 activity. Furthermore, studies with DFF45 mutants reveal that both caspase-3 and GrB share a common cleavage site, which is necessary and sufficient to induce DNA fragmentation in target cells during apoptosis. Together, our data suggest that CTLs possess alternative mechanism(s) for inducing DNA fragmentation without the requirement for caspases.
Subject(s)
Caspases/metabolism , DNA Fragmentation , Deoxyribonucleases/metabolism , Nuclear Proteins/metabolism , Proteins/metabolism , Serine Endopeptidases/metabolism , Apoptosis , Apoptosis Regulatory Proteins , Binding Sites , Caspase 3 , Caspase Inhibitors , Enzyme Activation , Granzymes , Humans , Jurkat Cells , Molecular Structure , Nuclear Proteins/chemistry , Proteins/chemistry , Substrate SpecificityABSTRACT
Triclosan, a widely used antibacterial agent, possesses potent activity against Staphylococcus aureus. This study reports on an investigation of the antibacterial target of triclosan in this pathogen. A strain of S. aureus overexpressing the enoyl-[acyl-carrier-protein] reductase (FabI), demonstrated by Western immunoblotting, gave rise to an increase in the MIC of triclosan, while susceptibilities to a range of unrelated antibacterials were unaffected. There are approximately 12 000 molecules of FabI per cell in mid-log phase growth. This number increased by approximately three- to four-fold in the S. aureus FabI overexpressor. Triclosan selectively inhibited the incorporation of [(14)C]acetate into TCA-precipitable product, an indicator of fatty acid biosynthesis. Furthermore, it inhibited de novo fatty acid biosynthesis in this organism. In vitro, triclosan inhibited recombinant, purified S. aureus FabI with an IC(50) of approximately 1 microM. The combination of these biochemical and genetic data provide further evidence that the mode of action of triclosan in S. aureus is via inhibition of FabI.
Subject(s)
Anti-Infective Agents, Local/pharmacology , Staphylococcus aureus/drug effects , Triclosan/pharmacology , DNA Replication/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolismABSTRACT
In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.
Subject(s)
Apoptosis , Caspases/physiology , Kidney Tubules, Proximal/cytology , Trans-Activators , Amino Acid Chloromethyl Ketones/pharmacology , Cytoskeletal Proteins/metabolism , Humans , Mitogen-Activated Protein Kinases/physiology , Okadaic Acid/pharmacology , Oligopeptides/pharmacology , beta Catenin , p38 Mitogen-Activated Protein KinasesABSTRACT
Structure-based design of a combinatorial array was carried out in order to identify non-peptidic thiomethylketone inhibitors of caspases 3 and 8. Five compounds from the designed array were active against caspase 3, and two were active against caspase 8. A 2.5-A resolution co-crystal structure of caspase 3 and a thiomethylketone array member is reported. The structure-based design strategy has proved useful for identifying caspase inhibitors.
Subject(s)
Caspase Inhibitors , Combinatorial Chemistry Techniques , Cysteine Proteinase Inhibitors/chemistry , Drug Design , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/chemistry , Catalytic Domain , Computer-Aided Design , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemical synthesis , Models, Molecular , Molecular Structure , Protein ConformationABSTRACT
Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.
Subject(s)
Apoptosis , Caspases/physiology , Chondrocytes/physiology , Amino Acid Chloromethyl Ketones/pharmacology , Caspase 3 , Caspase Inhibitors , Cell Line , Collagen/genetics , Humans , Oligopeptides/pharmacology , Promoter Regions, Genetic , Up-RegulationABSTRACT
Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.
Subject(s)
Apoptosis , Caspase Inhibitors , Enzyme Inhibitors/chemistry , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Binding Sites , Camptothecin/pharmacology , Caspase 3 , Caspase 7 , Chondrocytes/drug effects , Collagen/genetics , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Humans , Isatin/analogs & derivatives , Mice , Models, Molecular , Molecular Structure , Neutrophils/drug effects , Neutrophils/enzymology , Osteoarthritis/drug therapy , Promoter Regions, Genetic , Recombinant Proteins/chemistry , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
Polycystic kidney disease (PKD) is characterized by the development of large renal cysts and progressive loss of renal function. Although the cause of the development of renal cysts is unknown, recent evidence suggests that excessive apoptosis occurs in PKD. With the use of terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, we have confirmed the presence of apoptotic bodies in cystic kidneys of congenital polycystic kidney (cpk) disease mice carrying a homozygous mutation at 3 wk of age. Apoptosis was localized primarily to the interstitium with little evidence of cell death in cyst epithelium or noncystic tubules. In addition, we observed that the expression of various caspases, bax and bcl-2, was upregulated in cystic kidneys. With the use of various substrates in enzyme activity assays, we have demonstrated a greater than sevenfold increase in caspase 4 activity and a sixfold increase in caspase 3 activity. These data suggest that there is a caspase-dependent apoptosis pathway associated with PKD and support the hypothesis that apoptotic cell death contributes to cyst formation in PKD.
Subject(s)
Apoptosis , Caspases/metabolism , Polycystic Kidney Diseases/pathology , Animals , Mice , Mice, Mutant Strains , Polycystic Kidney Diseases/enzymology , Polycystic Kidney Diseases/genetics , Signal TransductionABSTRACT
An efficient strategy for the synthesis of a tetrapeptidyl substrate combinatorial array directed toward the caspases is described. Testing of this array with caspases 1 and 4 gave substrate hydrolytic profiles characteristic of each caspase, and permitted the identification of efficiently processed substrates. A comparison of this approach to that using a positional scanning library is presented.
Subject(s)
Caspases/metabolism , Oligopeptides/metabolism , Caspase 1/metabolism , Caspases, Initiator , Catalysis , Kinetics , Oligopeptides/chemical synthesis , Substrate SpecificityABSTRACT
TL1 is a recently discovered novel member of the tumor necrosis factor (TNF) cytokine family. TL1 is abundantly expressed in endothelial cells, but its function is not known. The present study was undertaken to explore whether TL1 induces apoptosis in endothelial cells and, if so, to explore its mechanism of action. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to TL1 showed morphological (including ultrastructural) and biochemical features characteristic of apoptosis. TL1-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 72 ng/ml). The effect of TL1 was not inhibited by soluble TNF receptors 1 or 2. TL1 up-regulated Fas expression in BPAEC at 8 and 24 h after treatment, and significantly activated stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (p38 MAPK). The peak activities of SAPK and p38 MAPK in TL1-treated BPAEC were increased by 9- and 4-fold, respectively. TL1-induced apoptosis in the BPAEC was reduced by expression of a dominant-interfering mutant of c-Jun (62.8%, p < 0.05) or by a specific p38 inhibitor, SB203580 (1-10 microM) dose-dependently. TL1 also activated caspases in BPAEC, and TL1-induced apoptosis in BPAEC was significantly attenuated by the caspase inhibitor, ZVAD-fluromethyl-ketone. The major component activated by TL1 in BPAEC was caspase-3, which was based on substrate specificity and immunocytochemical analysis. These findings suggest that TL1 may act as an autocrine factor to induce apoptosis in endothelial cells via activation of multiple signaling pathways, including stress protein kinases as well as certain caspases.
Subject(s)
Apoptosis , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Caspases/metabolism , Endothelium, Vascular/physiology , Mitogen-Activated Protein Kinases , Receptors, Tumor Necrosis Factor/physiology , Animals , Antigens, CD/metabolism , Caspase 3 , Cattle , Cells, Cultured , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , JNK Mitogen-Activated Protein Kinases , Molecular Sequence Data , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Pulmonary Artery , Pyridines/pharmacology , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor, Type I , Receptors, Tumor Necrosis Factor, Type II , Up-Regulation , fas Receptor/biosynthesis , p38 Mitogen-Activated Protein KinasesABSTRACT
Cardiomyocyte apoptosis has been demonstrated in animal models of cardiac injury as well as in patients with congestive heart failure or acute myocardial infarction. Therefore, apoptosis has been proposed as an important process in cardiac remodeling and progression of myocardial dysfunction. However, the mechanisms underlying cardiac apoptosis are poorly understood. The present study was designed to determine whether the family of caspase proteases and stress-activated protein kinase (SAPK/JNK) are involved in cardiac apoptosis. Cultured rat neonatal cardiac myocytes were treated with staurosporine to induce apoptosis as evidenced by the morphological (including ultrastructural) characteristics of cell shrinkage, cytoplasmic and nuclear condensation, and fragmentation. Nucleosomal DNA fragmentation in myocytes was further identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end-labeling (TUNEL). Staurosporine-induced apoptosis in myocytes was a time- and concentration-(0.25-1 micro M)-dependent process. Staurosporine-induced apoptosis in myocytes was reduced by a cell-permeable, irreversible tripeptide inhibitor of caspases, ZVAD-fmk, but not by the ICE-specific inhibitor, Ac-YVAD-CHO. At 10, 50 and 100 muM of ZVAD-fmk, staurosporine-induced myocyte apoptosis was reduced by 5.8, 39.1 (P<0.01) and 53.8% (P<0.01), respectively. Staurosporine, at 0.25-1 micro M, increased caspase activity in cardiomyocytes by five- to eight-fold, peaking at 4-8 h after stimulation. Based on substrate specificity analysis, the major component of caspases activated in myocytes was consistent with caspase-3 (CPP32). Moreover, the appearance of the 17-kD subunit of active caspase-3 in staurosporine-treated myocytes was demonstrated by immunocytochemical analysis. In contrast, staurosporine induced a rapid and transient inhibition of SAPK/JNK in myocytes. The SAPK activity in myocytes was reduced by 68.3 and 58.3% (P<0.01 v basal) at 10 and 30 min after treatment with 1 micro M of staurosporine, respectively. Our results suggest that staurosporine-induced cardiac myocyte apoptosis involves activation of caspases, mainly caspase-3, but not activation of the SAPK signaling pathway.
Subject(s)
Apoptosis/drug effects , Caspases , Cysteine Endopeptidases/metabolism , Mitogen-Activated Protein Kinases , Myocardium/cytology , Myocardium/enzymology , Staurosporine/pharmacology , Amino Acid Sequence , Animals , Apoptosis/physiology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Caspase 3 , Cells, Cultured , Enzyme Activation/drug effects , Heart/drug effects , JNK Mitogen-Activated Protein Kinases , Oligopeptides/chemistry , Rats , Signal Transduction , Substrate SpecificityABSTRACT
Evaluation of a variety of PDE4 inhibitors in a series of cellular and in vivo assays suggested a strategy to improve the therapeutic index of PDE4 inhibitors by increasing their selectivity for the ability to inhibit PDE4 catalytic activity versus the ability to compete for high affinity [3H]rolipram-binding sites in the central nervous system. Use of this strategy led ultimately to the identification of cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxyl ic acid (1, SB 207499, Ariflo), a potent second-generation inhibitor of PDE4 with a decreased potential for side effects versus the archetypic first generation inhibitor, (R)-rolipram.
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Anti-Asthmatic Agents/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cyclohexanecarboxylic Acids/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Animals , Anti-Asthmatic Agents/chemical synthesis , Anti-Asthmatic Agents/metabolism , Anti-Asthmatic Agents/toxicity , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Binding, Competitive , Body Temperature/drug effects , Brain/metabolism , Bronchoconstriction/drug effects , Cyclic Nucleotide Phosphodiesterases, Type 4 , Cyclohexanecarboxylic Acids/chemical synthesis , Cyclohexanecarboxylic Acids/metabolism , Cyclohexanecarboxylic Acids/toxicity , Dogs , Gastric Acid/metabolism , Guinea Pigs , Humans , Mice , Monocytes/drug effects , Monocytes/metabolism , Nitriles , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/metabolism , Phosphodiesterase Inhibitors/toxicity , Pyrrolidinones/chemical synthesis , Pyrrolidinones/metabolism , Pyrrolidinones/pharmacology , Pyrrolidinones/toxicity , Rabbits , Recombinant Proteins/antagonists & inhibitors , Rolipram , Stereoisomerism , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Vomiting/chemically inducedABSTRACT
To identify critical amino acids within the central conserved region of recombinant human cAMP-specific phosphodiesterase 4 subtype A (rhPDE4A), we engineered the expression of point mutants in a fully active rhPDE4A/Met201-886. When histidine residues at positions 433, 437, 473, and 477, which are highly conserved among all PDE families, were changed independently to serine residues, cAMP hydrolyzing activities were substantially reduced or abolished. The ability of these mutants to bind prototypical PDE4 inhibitors [3H]-(R)-rolipram or [3H]RP 73401 was also decreased in parallel with the loss of catalytic activity. The parallel loss of catalytic activity and inhibitor binding suggests that these changes resulted from non-localized perturbations in the structure of the enzyme. More interesting results were obtained when histidine residues at positions 505 and 506 were changed independently to aspar agines. The K(m) value for cAMP increased 3-fold in H505N (K(m) = 11 +/- 3 microM) and 11-fold in H506N (K(m) = 44 +/- 6 microM) compared with the wild-type protein (K(m) = 4 +/- 1 microM). These mutant proteins bound [3H]-(R)-rolipram and [3H]RP 73401 with K(d) values of 1.8 +/- 0.4 and 0.3 +/- 0.1 nM, respectively, for H505N, and 3.9 +/- 0.9 and 0.5 +/- 0.1 nM, respectively, for H506N. These values are nearly identical to those obtained with the wild-type rhPDE4A/Met201-886. In contrast, the IC50 values for cAMP competition with either [3H]-(R)-rolipram or [3H]RP 73401 binding increased approximately 2-fold in H505N and approximately 13-fold in H506N compared with the wild type protein. These increases are virtually identical to the changes in the K(m) value for cAMP in these mutants. We conclude that His506 and, perhaps, His505 are involved in binding of cAMP to PDE4A/Met201-886 but not in binding of PDE4-selective inhibitors.
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases , Histidine/metabolism , Isoenzymes/metabolism , Phosphodiesterase Inhibitors/metabolism , Phosphoric Diester Hydrolases/metabolism , Amino Acid Sequence , Amino Acids/metabolism , Binding Sites , Catalysis , Cyclic Nucleotide Phosphodiesterases, Type 4 , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphodiesterase Inhibitors/pharmacology , Protein Binding , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacologyABSTRACT
Endothelin converting enzyme was partially purified by detergent extraction and ion exchange chromatography from porcine aortic endothelial cells. This kinetically homogeneous preparation catalyzes the hydrolysis of porcine big endothelin-1 to endothelin-1 with a pH optimum of 7. Human big endothelins-1, -2, and -3 are also hydrolyzed, but at progressively lower rates. Fragments of big porcine endothelin-1 comprising residues 16-39 and 16-29 are good substrates, but additional C-terminal truncations are devoid of substrate activity. Endothelin converting enzyme is characteristically inhibited by phosphoramidon and other metalloproteinase inhibitors including EDTA, o-phenanthroline, and diethylpyrocarbonate, but not by inhibitors of other classes of proteases or thiorphan. The inhibition by phosphoramidon is competitive with big porcine endothelin-1 suggestive of a common binding site for substrate and inhibitor. A number of novel analogs of phosphoramidon were synthesized by modifying various regions of the molecule and tested for inhibitory activity. The most potent of these, a methylphosphonic acid, has an IC50 of 0.05 microM.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Endothelium, Vascular/enzymology , Enzyme Inhibitors/pharmacology , Glycopeptides/pharmacology , Protease Inhibitors/pharmacology , Animals , Aorta , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/isolation & purification , Endothelin-Converting Enzymes , Endothelins/metabolism , Humans , Kinetics , Metalloendopeptidases , Molecular Structure , Stereoisomerism , Structure-Activity Relationship , Substrate Specificity , SwineABSTRACT
Considerable interest has been generated in the potential utility of phosphodiesterase (PDE) IV inhibitors as a novel class of anti-asthmatic agents. Because a detailed understanding of the molecular and biochemical characteristics of any molecular target of interest provides a key ingredient for rational drug design, we cloned a cDNA encoding a PDE IV (hPDE IV) from a human monocyte library and expressed, purified and characterized the recombinant gene product. Purified hPDE IV has kinetic characteristics consistent with native PDE IV isolated from tissue sources. In addition, it is inhibited by rolipram (Ki = 60 nM) and other archetypical PDE IV-selective inhibitors. Purified hPDE IV also contains a high affinity binding site for rolipram (Kd = 2 nM), although the precise relationship between this site and the cAMP catalytic site is not clear. In other studies in which the regulation of PDE IV expression was examined in U937 cells, a human monocytic cell line, prolonged treatment with salbutamol was shown to induce an increase in the activity of PDE IV. This up-regulation of PDE IV activity appears to be mediated by cAMP and occurs at the transcriptional or pretranscriptional level. As a consequence of PDE IV up-regulation, the sensitivity of U937 cells to the inhibitory effects of adenylyl cyclase activators on cell function is greatly diminished. If such regulation of PDE IV occurs in inflammatory cells in vivo, it could have implications for the therapeutic use of beta-adrenoceptor agonists. Specifically, induction of PDE IV activity in asthmatics being treated with beta-adrenoceptor agonists could result in a heterologous desensitization of inflammatory cells to endogenous anti-inflammatory agents (e.g., epinephrine, prostaglandin E2).
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases , Phosphoric Diester Hydrolases/chemistry , Animals , Binding Sites/physiology , Cell Line , Cyclic Nucleotide Phosphodiesterases, Type 4 , Humans , Kinetics , Phosphodiesterase Inhibitors/metabolism , Phosphoric Diester Hydrolases/isolation & purification , Phosphoric Diester Hydrolases/physiology , Pyrrolidinones/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Rolipram , Up-Regulation/physiologyABSTRACT
Transient transfection of simian kidney (COS) cells with a recombinant plasmid encoding human preproendothelin-1 resulted in the production of biologically active endothelin-1. Conditioned medium from human preproendothelin-1 transfected cells demonstrated a significant increase in immunoreactive endothelin and big endothelin which co-eluted, when analyzed by reverse phase HPLC, with synthetic endothelin-1 and big endothelin-1, respectively. In addition, biological activity was confirmed by both inhibition of [125I]endothelin-1 binding to rat cerebellar and renal medullary membrane endothelin receptors and in vitro vasoconstriction of rabbit aorta. This is the first demonstration that human preproendothelin-1 is capable of being processed to a vasoactive form in a heterologous system and suggests that human preproendothelin-1 transfected COS cells may provide a useful model system for the study of endothelin biosynthesis.
Subject(s)
Endothelins/genetics , Animals , Biological Assay , Chlorocebus aethiops , Cloning, Molecular , DNA/genetics , Genetic Vectors , Humans , In Vitro Techniques , Rabbits , Recombinant Proteins , Transfection , VasoconstrictionABSTRACT
A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.
Subject(s)
Dopamine beta-Hydroxylase/genetics , Adrenal Glands/enzymology , Amino Acid Sequence , Animals , Base Sequence , Cattle , DNA/genetics , Glycosylation , Humans , Mass Spectrometry , Molecular Sequence Data , Peptide Mapping , Protein Processing, Post-Translational , Sequence Homology, Nucleic AcidABSTRACT
p-Cresol is a mechanism-based inhibitor of bovine dopamine beta-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate: oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) (DBH) which covalently modifies a tyrosine at position 216 during inactivation (DeWolf, W.E., Jr., Carr, S.A., Varrichio, A., Goodhart, P.J., Mentzer, M.A., Roberts, G.D., Southan, C., Dolle, R.E. and Kruse, L.I. (1988) Biochemistry 27, 9093-9101). Here we report the recovery and characterization of additional minor peptides that are produced during the inactivation of DBH with p-[3H]cresol. Sequence and structural analysis of these peptides indicates tyrosine 357 as a second, minor site of modification.