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PubMed1985

Histamine release from human basophils by pepstatin A.

Pepstatin A, a pentapeptide isolated from cultures of actinomycetes, induced histamine secretion from human basophils in the concentration range of 3 X 10(-7) to 10(-4) M. The characteristics of this reaction were similar to those of f-met-peptide-induced histamine release: pepstatin A-induced release required Ca2+ and the release reaction was complete within 2 min at 22 or 37 degrees C, but did not occur at 4 degrees C. Release by both pepstatin A and f-met-peptide was reversibly inhibited by two non-releasing analogs of f-met-peptide, CBZ-Phe-Met and BOC-Met-Leu-Phe. Further, there was complete cross-desensitization between pepstatin A and f-met-peptide, while cells desensitized to pepstatin A released normally with anti-IgE and vice versa. A variety of pharmacological agents had similar effects on both pepstatin A and f-met-peptide-induced release (e.g., no enhancement with D2O; marked enhancement with cytochalasin B). We suggest that pepstatin A induces histamine release from human basophils by activating a cell surface receptor(s), also activated by the synthetic tripeptide f-met-peptide.

Basophils↗
PubMed2000

Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy.

Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathological progressions during tumor formation and oxidation stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity with an IC50 of 10 nM. The nature of its binding to cathepsin D was further characterized using a fluorescence polarization measurement. Results showed that BODIPY FL-pepstatin A selectively binds to cathepsin D at pH 4.5. In fixed cells, BODIPY FL-pepstatin A stained lysosomes, where it co-localized with cathepsin D. This staining was depleted when cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysosomes. The staining in the lysosomes can be competed with unlabeled pepstatin A. These properties, along with the good photostability of the BODIPY FL fluorophore, make this probe a novel tool for the study of the secretion and trafficking of cathepsin D.

Animals↗
PubMed2001

Aspartyl protease inhibitor pepstatin binds to the presenilins of Alzheimer's disease.

Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.

Alzheimer Disease↗
PubMed1984

Non-specific inhibition of pressor agents in vivo by the renin inhibitor pepstatin A.

The specificity of pepstatin A as an inhibitor of the cardiovascular actions of renin injected into anaesthetized rats has been investigated. Pepstatin A 70 micrograms/kg/min partially inhibited the pressor response to injected renin without affecting the pressor responses to injected angiotensin II, phenylephrine or vasopressin. Pepstatin A 150 micrograms/kg/min also produced partial inhibition of injected renin, but in addition caused significant inhibition of the other pressor agents. This was in contrast to the effects of the angiotensin converting enzyme inhibitor captopril, 100 micrograms/kg i.v., which caused greater inhibition of the renin pressor response than pepstatin A without affecting the pressor response to injected angiotensin II, phenylephrine or vasopressin. Finally the direct acting vasodilator hydralazine was found to have a similar non-specific inhibitory effect to pepstatin A on the pressor responses to injected pressor agents. It is concluded that pepstatin A reduces the pressor responsiveness to injected pressor agents and that this non-specific cardiovascular activity limits the usefulness of pepstatin A as a pharmacological tool to inhibit renal renin in vivo.

Animals↗
PubMed1977

Pepstatin inhibition mechanism.

Pepstatin is a low molecular weight, potent inhibitor specific for acid proteases with a Ki value of about 10(-10)M for pepsin. The chemical structure of pepstatin is essentially a hexapeptide which contains two residues of an unusual amino acid, 4-amino-3-hydroxy-6-methylheptanoic acid (statine). The complete structure of pepstatin is isovaleryl-L-valyl-L-valyl-statyl-L-alanyl-statine. To study its mode of inhibition, we prepared several derivatives and measured their kinetics of inhibition. Both N-acetyl-statine and N-acetyl-alanyl-statine are competitive inhibitors for pepsin with Ki values of 1.2 x 10(-4)M and 5.65 x 10(-6)M, respectively. The Ki value for N-acetyl-valyl-statine is 4.8 x 10(-6)M. These statyl derivatives, therefore, are very strong inhibitors. The Ki value for N-acetyl-statine is 600-fold smaller than that of its structural analog N-acetyl-leucine. The derivative which contains two statyl residues in a tetrapeptide exhibits inhibitory properties which approach those of pepstatin itself. Other acid proteases, human pepsin, human gastricsin, renin, cathepsin D, the acid protease from R. chinensis and bovine chymosin, also are inhibited by pepstatin and its derivatives. We suggest that the statyl residue is responsible for the unusual inhibitory capability of pepstatin and that statine is an analog of the previously proposed transition state for catalysis by pepsin and other acid proteases.

Animals↗
PubMed1979

Further studies of the effect of pepstatin on ascites accumulation in tumor-bearing mice.

Studies were done of the effect of pepstatin on ascites accumulation in mice bearing MM46, Ehrlich, CCM, SN36, L1210, and NTF ascites tumors. When pepstatin was injected subcutaneously at 80 mg/kg body wt before ascites accumulation, it inhibited the accumulation in all strains of the tumors tested. In MM46, CCM and NTF tumor strains there was also a decrease in the tumor cell numbers following pepstatin treatment. Kinetic studies on ascites accumulation with tumor strain MM46 demonstrated that even when pepstatin was injected after ascites accumulation it reduced the ascites volume. A dose-dependent effect was observed in this tumor strain when pepstatin was injected both before and after ascites accumulation. The results confirm previous studies of pepstatin's ability to retard ascites in L1210 and P-815Y ascites tumors and also broaden the concept of the mechanisms by which petstatin may be acting.

Animals↗
PubMed2003

Comparative pepstatin inhibition studies on individual human pepsins and pepsinogens 1,3 and 5(gastricsin) and pig pepsin A.

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.

Animals↗
PubMed1985

[Effects of pepstatin A on neutrophils; cross-deactivation with FMLP].

The ability of pepstatin A, a protease inhibitor produced by Streptomyces testaceus, to elicit a number of responses by the human PMN has been studied. In lysozyme and beta-glucuronidase release, pepstatin A 10(-5)M is equivalent to the synthetic oligopeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) 10(-7)M. In superoxide release, pepstatin A 10(-5)M produces 80% of that originated by FMLP 10(-7). After two minutes of incubation the superoxide release is important, there being no further increase after 10 minutes. Preincubation of the cells with cytochalasin B before stimulation with pepstatin A elicits a noticeable increase in O2- release. In chemotaxis, pepstatin A 10(-6) originates the same cell motility as FMLP 10(-9). Pepstatin A produces a cross deactivation with FMLP which adds further evidence to the hypothesis that both stimuli compete for the same receptor in the PMN.

Binding, Competitive↗
PubMed1982

Effects of pepstatin on reducing hypoxia-induced injury in the isolated guniea pig heart.

Intracellular cathepsin D is thought to play a role in myocardial injury produced by ischemia and hypoxia. Pepstatin, a known inhibitor of cathepsin D, was infused into isolated guniea pig hearts (Langendorff preparation) in order to observe if such an administration of pepstatin would protect against the effects of a two minute exposure to hypoxia. Hypoxia was produced by exposing the hearts to perfusion fluid aerated with 20% 02/5% CO2/75% N2 and containing 0.5 microgram/ml of norepinephrine. Contractile force, heart rate, coronary flow and ECG were monitored. Samples of heart tissue were assayed for cathepsin D activity. Infusion of 0.06 mg/min of pepstatin for 30 minutes produced no significant alterations in the parameters of cardiac function studied. However, this amount of pepstatin inhibited 97% of the cathepsin D activity of the hearts. The characteristics ECG alterations produced by hypoxia were significantly reduced after infusion of pepstatin. These data indicate that pepstatin may protect the heart against hypoxia-induced injury.

Animals↗
PubMed1991

Effect of pepstatin A on structure and polymerization of intermediate filament subunit proteins in vitro.

Pepstatin A, a pentapeptide aspartyl protease inhibitor, can interact with intermediate filament (IF) subunit proteins and induce their polymerization in the absence of salt into long filaments with a rough surface and a diameter of 15-17 nm. This polymerization appears to be driven primarily by non-ionic interactions between pepstatin A and polymerization-competent forms of IF proteins, resulting in a composite filament. Proteolytic fragments of vimentin, lacking portions of only the head domain or of both the head and tail domains, failed to copolymerize with pepstatin A into long filaments under these conditions. Rather, these peptides, as well as control proteins like bovine serum albumin, were found to decorate pepstatin A polymers (filaments, ribbons, and sheets) by sticking to their surfaces. In addition to the electron microscopy experiments, UV difference spectra, ultracentrifugation, and SDS-PAGE analysis of in vitro cleavage products of vimentin obtained with HIV-1 protease all provided independent evidence for a direct association of pepstatin A with IF subunit proteins, with subsequent alterations in the IF subunit protein conformation. These data show that non-ionic interactions can substitute for the effect of salt and effectively drive the higher-order polymerization of IF subunit proteins.

Amino Acid Sequence↗
PubMed2003

Crystal structures of Aspergillus oryzae aspartic proteinase and its complex with an inhibitor pepstatin at 1.9A resolution.

The X-ray structures of Aspergillus oryzae aspartic proteinase (AOAP) and its complex with inhibitor pepstatin have been determined at 1.9A resolution. AOAP was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=49.4A, b=79.4A, and c=93.6A. By the soaking of pepstatin, crystals are transformed into a monoclinic system with the space group C2 and cell dimensions of a=106.8A, b=38.6A, c=78.7A, and beta=120.3 degrees. The structures of AOAP and AOAP/pepstatin complex were refined to an R-factor of 0.177 (R(free)=0.213) and of 0.185 (0.221), respectively. AOAP has a crescent-shaped structure with two lobes (N-lobe and C-lobe) and the deep active site cleft is constructed between them. At the center of the active site cleft, two Asp residues (Asp33 and Asp214) form the active dyad with a hydrogen bonding solvent molecule between them. Pepstatin binds to the active site cleft via hydrogen bonds and hydrophobic interactions with the enzyme. The structures of AOAP and AOAP/pepstatin complex including interactions between the enzyme and pepstatin are very similar to those of other structure-solved aspartic proteinases and their complexes with pepstatin. Generally, aspartic proteinases cleave a peptide bond between hydrophobic amino acid residues, but AOAP can also recognize the Lys/Arg residue as well as hydrophobic amino acid residues, leading to the activation of trypsinogen and chymotrypsinogen. The X-ray structure of AOAP/pepstatin complex and preliminary modeling show two possible sites of recognition for the positively charged groups of Lys/Arg residues around the active site of AOAP.

Arginine↗
PubMed1990

Modulation of experimental systemic murine candidosis by intravenous pepstatin.

The effect of intravenous pepstatin-A on systemic candidosis in NWNI mice was investigated. True solutions of the inhibitor proved ineffective due to a very fast clearance. Pepstatin was effective as a crystal suspension (0.69 mg in 0.1 ml saline) which produced serum inhibitory activity for greater than 29 h. From the intravenously applied suspension, pepstatin was taken up predominantly into the liver, no inhibitor being taken up by the kidneys. The suspension was protective if it was injected once before the mice were infected and repeatedly following infection. It was also effective if it was administered concomitantly with the infecting agent and thereafter. The suspension was ineffective if it was only given once before infection, and it proved to be detrimental if it was given only after infection. The results support previous findings (2), suggesting a role of fungal proteinase early in the adherence of Candida to host epithelia. Our results also suggest an inhibition of lysosomal cathepsin-D in vivo by pepstatin, which prohibits a parenteral therapeutic use of nonmodified pepstatin A.

Animals↗
PubMed1983

Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D.

1. N-Pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane, a potential active-site-directed localization reagent for cathepsin D, was found to bind non-specifically to immuno-precipitates containing cathepsin D. 2. Three new water-soluble localization reagents were synthesized, by using NN'-bis-(3-aminopropyl)piperazine, 3-oxa-1,5-diamino-pentane or 3,6-dioxa-1,8-diamino-octane, as spacer arms between the pepstatin and dinitrophenyl moieties. 3. The hydrophilic dinitrophenyl-pepstatins were all tight-binding inhibitors of cathepsin D at pH 3.5, but showed little or no binding to immuno-precipitates containing the inactive enzyme at pH 7.4. 4. Gel-chromatographic experiments showed that, at pH 5.0, all the dinitrophenyl-pepstatins were bifunctional reagents able to bind cathepsin D and anti-dinitrophenyl antibody at the same time. Enzyme-inhibitor-antibody complexes were not formed at pH 7.4, thus confirming that the reagents were active-site-directed. 5. Cultured human synovial cells were fixed and incubated with the dinitrophenyl-pepstatins at pH 5.0 or pH 7.4. After washing briefly, the cells were incubated at the appropriate pH value with anti-dinitrophenyl antibody labelled with fluorescein. When examined by fluorescence microscopy the cells stained at pH 5.0 showed fluorescent perinuclear granules, which were not seen in the cells treated at pH 7.4. The distribution of cathepsin D, determined by indirect immuno-fluorescence at pH 7.4, closely resembled that revealed by the dinitrophenyl-pepstatins at pH 5.0. 7. NN'-(3-Pepstatinylaminopropyl-3'-dinitrophenylaminopropyl)piperazine gave the most intense lysosomal staining and showed no non-specific binding. We conclude that this reagent is suitable for the subcellular localization of the active conformation of cathepsin D.

Binding Sites↗
PubMed1989

Isolation of procathepsin D from mature cathepsin D by pepstatin affinity chromatography. Autocatalytic proteolysis of the zymogen form of the enzyme.

Procathepsin D is a rapidly processed precursor form of the lysosomal proteinase cathepsin D. The enzymic properties of procathepsin D have been studied by examining the pepstatin-binding characteristics of both the precursor and the mature enzyme. Procathepsin D bound to immobilized pepstatin at 4 degrees C in pH 3.5 buffer but not in pH 5.3 buffer, whereas mature forms of cathepsin D bound to immobilized pepstatin at both pH values. These characteristics of procathepsin D were exploited to isolate the proenzyme from mature forms and to determine whether activation of the proenzyme is an autocatalytic process. After incubation at 37 degrees C in pH 3.5 buffer, the proenzyme underwent pepstatin-inhibitable proteolysis resulting in a dramatically increased affinity of purified procathepsin D for pepstatin at pH 5.3. The low concentration of enzyme used in these studies suggests that procathepsin D cleavage to single-chain cathepsin D may occur via a unimolecular mechanism.

Acetylglucosaminidase↗
PubMed2004

Inhibition of 1,4-beta-D-xylan xylanohydrolase by the specific aspartic protease inhibitor pepstatin: probing the two-step inhibition mechanism.

This is the first report that describes the inhibition mechanism of xylanase from Thermomonospora sp. by pepstatin A, a specific inhibitor toward aspartic proteases. The kinetic analysis revealed competitive inhibition of xylanase by pepstatin A with an IC50 value 3.6 +/- 0.5 microm. The progress curves were time-depended, consistent with a two-step slow tight binding inhibition. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI* and the dissociation of EI* were 15 +/- 1 x 10(-5) and 3.0 +/- 1 x 10(-8) s(-1), respectively. The Ki value for the formation of EI complex was 1.5 +/- 0.5 microm, whereas the overall inhibition constant Ki* was 28.0 +/- 1 nm. The conformational changes induced in Xyl I by pepstatin A were monitored by fluorescence spectroscopy, and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. Pepstatin A binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde-labeled xylanase. Our results revealed that the inactivation of xylanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis, and a model depicting the probable interaction between pepstatin A with xylanase has been proposed.

Actinobacteria↗
PubMed1977

Pepstatin, an ascites retardant of L1210 tumor-bearing mice.

The effect of pepstatin on the kinetics of ascitic fluid accumulation in L1210 tumor-bearing mice (DBA/2) was observed. Following inoculation of 1.5x10(6) tumor cells, untreated mice reached a peak of fluid accumulation on day 6 and remained at this level until death on day 9. A "lag" phase of 4 days occurred before fluid accumulation was seen. Pepstatin administered SC in a single dose of 80 mg/kg during the lag phase, significantly retarded fluid accumulation as compared to untreated animals. Pepstatin administered following fluid accumulation was much less effective. We concluded that pepstatin prevents fluid accumulation rather than acts as a diuretic agent. The term "ascites retardant" is suggested for the pharmacologic actions of pepstatin, since it prevents fluid accumulation without diminishing the cell count.

Animals↗
PubMed1988

Prevention of acute gastric erosions in the rat by novel semi-synthetic amphipathic analogues of pepstatin.

Pepstatin is a potent aspartyl proteinase inhibitor which is virtually insoluble in physiological media. Five semi-synthetic amphipathic analogues of pepstatin, prepared by N terminal substitution of native pepstatin with hydrophilic oligopeptides, have been assessed for their ability to protect the mucosa in two animal models of acute gastric erosions. Concentrations of approximately 90 pmol/mg were achieved in the rat gastric mucosa after oral administration of a 20 mmol solution. These levels are theoretically adequate to inhibit all pepsin like proteinase activity (including zymogens). Each pepstatin analogue was tested by intragastric administration in a controlled hypotension/luminal acid animal model of acute gastric erosions in a group of six animals. All the inhibitors tested produced marked mucosal protection, as measured by a mucosal damage index, compared with control animals (control mean 241, range 100-420; Pepstatinyl-Gly-Orn-Orn-Cys (10 mmol) mean 3, range 0-8, p less than 0.01; Pepstatinyl-Gly-Cysteic acid-Cysteic acid (10 mmol) mean 5, range 0-21, p less than 0.01; Pepstatinyl-Gly-Lys-Lys (10 mmol) mean 18, range 0-60, p less than 0.01; Pepstatinyl-Gly-Cysteic acid-Cysteic acid (1 mmol) mean 24, range 0-86, p less than 0.01; Pepstatinyl-Gly-Orn-Orn-Cys (1 mmol) mean 57, range 0-116, p less than 0.01; Pepstatinyl-Gly-Asp-Asp (1 mg/ml suspension) mean 68, range 19-126, p less than 0.01; Pepstatinyl-Arg-OMe (1 mmol) mean 93, range 4-142, p less than 0.05, Pepstatinyl-Gly-Lys-Lys (1 mmol) mean 157, range 70-286, NS). In a platelet activating factor/20% luminal ethanol model of erosions the pepstatin analogues again provided mucosal protection although this only reached statistical significance for one of three compounds tested.

Animals↗
PubMedDate not supplied

Pepstatin A is inducing contractile effects on isolated rat aorta rings.

In a series of experiments dealing with the effects of angiotensin I (AI) and angiotensinogen on isolated rat aorta we observed that pepstatin A was able to induce contractile effects by itself. The endothelin pathway was excluded by the inhibitory effects of captopril, chymostatin and amastatin. In addition, few preliminary experiments showed that the contractile effects of pepstatin A were inhibited by the pretreatment with losartan, an antagonist of AT1 angiotensin receptors. Pepstatin A-induced contractile effects on isolated rat aorta were inhibited with high potency by captopril, chymostatin and amastatin and were totally blocked by captopril + amastatin and captopril + chymostatin. Finally, we concluded that the pepstatin A-induced contractile effects on isolated rat aorta rings are dependent on an angiotensinogen vascular pool, compulsory involve an angiotensin-converting enzyme-1 (ACE-1) mediated pathway and one or more non-classical pathways for the generation of angiotensin peptides. Further experiments are necessary to elucidate the mechanisms associated to pepstatin A-induced effects.

Angiotensin I↗