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PubMed2006

Antimalarial effects of human immunodeficiency virus type 1 protease inhibitors differ from those of the aspartic protease inhibitor pepstatin.

Human immunodeficiency virus type 1 protease inhibitors (HIVPIs) and pepstatin are aspartic protease inhibitors with antimalarial activity. In contrast to pepstatin, HIVPIs were not synergistic with a cysteine protease inhibitor or more active against parasites with the cysteine protease falcipain-2 knocked out than against wild-type parasites. As with pepstatin, HIVPIs were equally active against wild-type parasites and against parasites with the food vacuole plasmepsin aspartic proteases knocked out. The antimalarial mechanism of HIVPIs differs from that of pepstatin.

Animals↗
PubMed1982

Electron microscopic visualization of cathepsin D using mercury-labeled pepstatin as an enzyme inhibitor.

Pepstatin, a specific inhibitor of pepsin, cathepsin D (E), renin, etc., was used in a new method to demonstrate the sites of enzymes. Pepstatin (Pst) was covalently attached to glutathione (GSH), using a dicyclohexylcarbodiimide, and CH3Hg+ was then put in the residue of SH for electron microscopic observation. Pst-GS-HgCH3 thus obtained was nonisotopical, had a very low molecular weight (about 1,200 daltons), and still almost completely retained its inhibitory activity. Sections of rat liver (less than 1 mm3 thick), prefixed by glutaraldehyde, were incubated in the prepared reaction medium. Cathepsin D, located in the lysosomes, was demonstrated by this compound, but when the sections were preincubated with pepstatin, this was not the case. These results demonstrate that mercury-labeled pepstatin is a useful reagent for the histochemical staining of acid proteases for electron microscope.

Animals↗
PubMed1979

Inhibition of dog renin activity by pepstatin A.

The inhibitory effect of pepstatin A, an isovaleryl pentapeptide isolated from streptmyces, was studied. Pepstatin A inhibited the action of dog renin on homologus substrate. The Ki value was 7.7 X 10(-7) M and the mode of the inhibition was non-competitive. Pestatin A suppressed angiotensin I generation in dog plasma. Dose-dependent hypotensive effect of pepstatin A was observed in rats with sustained increase in blood pressure during continuous infusion of dog renin. It is concluded that pepstatin A inhibits the dog renin activity in vitro and in vivo.

Angiotensin I↗
PubMed1990

[Effect of pepstatin A on Candida albicans infection in the mouse].

The intravenous injection of 10(6) to 10(7) Candida albicans cells revealed to be a reliable model in mice produce infections of the kidney. Higher germ contents could be yielded in the kidney after the application of protease positive Candida-strains as compared to protease negative ones. Additionally, after the infection with protease positive strains a proteolytic activity could be found in the kidney homogenates in vitro on casein plates. The modulation of this Candida infection by pepstatin A, an inhibitor of extracellular yeast proteases in vitro, has also been studied in the same mice model in vivo. The growth rate of Candida albicans has been measured in the left kidney by counting the germ content as described by Haenel. Infections could be reduced after single doses of 120 micrograms pepstatin A contrary to 60 micrograms pepstatin A. The same was with three doses of 180 micrograms at 24 h intervals. This protease inhibitory effect could also be found in kidney homogenates in vitro on casein plates and lasted until 48 h post injection. On the basis of this positive effects on the Candida infection in mice pepstatin A should be considered as an adjuvans in the therapy of severe yeast infections.

Animals↗
PubMed1979

Gastric ulcer therapy with a pepsin-inactivating peptide, pepstatin: a double-blind randomized clinical trial.

Sixty consecutive patients with gastric ulcer were studied at random after treatment with 100 mg pepstatin or 100 mg placebo seven times a day at fixed hours and controlled 2, 4, 8, and 12 weeks after initial diagnosis. Examinations comprised clinical symptoms and endoscopic evaluation of ulcer healing. Eight patients were withdrawn from the study. Ulcer healing and symptoms were better with placebo than with pepstatin, but a type-2 error of a 10% effect in favour of pepstatin may be overlooked. In gastric ulcer treatment, pepstatin seems to be of only fringe benefit, if of any benefit at all.

Clinical Trials as Topic↗
PubMed1981

Biological activity of aspartic proteinase inhibitors related to pepstatin.

We have synthesized eight tripeptide analogs of pepstatin in which both the side-chain and stereochemistry of the novel amino acid statine have been altered. They have been compared to pepstatin for inhibition of pepsin and cathepsin D activity, inhibition of autolysis at pH 4, and inhibition of protein degradation in cultured cells. Effective inhibition of aspartic proteinase activity appears to require the novel amino acid to have a bulky hydrophobic side-chain and the S-configuration at both chiral centers. However, the Cbz-Val-Val-(3S4S)-statine peptide was more effective than pepstatin in cultured cells, and inhibition was also achieved, and in some cases enhanced relative to pepstatin, by its stereoisomers and by tripeptides containing valyl and alanyl analogs of statine.

Amino Acids↗
PubMed1983

Host cathepsin D response to tumor in the normal and pepstatin-treated mouse.

In view of the postulated role of cathepsin D in cachexia, investigations have been pursued on the host tissue response of cathepsin D activity in DBA/2 mice inoculated with 5 X 10(5) L1210 tumor cells. The results confirmed previous investigators' findings of the increase in cathepsin D activity (specific activity) in liver and muscle of tumor bearers. In addition, it was found that this increase was a general response of the host since heart, kidney, lung, and spleen cathepsin D specific activity were also enhanced in tumor bearers. These increases ranged from an average of 10% for spleen to 100% for gastrocnemius muscle. This effect was age related in heart and kidney. As a working hypothesis, we propose the concept that tumor bearers release protease-enhancing factor(s) which trigger increase or enhancement of cathepsin D activity in host tissues by yet unknown mechanisms. Pepstatin (60 mg/kg), a known inhibitor of cathepsin D in vitro, was shown to provide long-lasting inhibition (3 to 6 days) of cathepsin D in vivo in non-tumor bearers particularly in spleen, liver, kidney, lung, and heart. Evidence is provided from assays of cell fractions that this inhibition takes place at or in the lysosome. The duration of the effectiveness of pepstatin was altered in tumor bearers in that cathepsin D activity of heart, lung, and spleen had returned to near normal values in 48 hr following pepstatin injection. However, in muscle, liver, and kidney, significant inhibition (90%) still persisted in tumor bearers as it did in non-tumor bearers. Pepstatin or related antiproteases may prove useful as "anticachexia" agents by decreasing proteolysis in muscle and other tissues.

Age Factors↗
PubMedDate not supplied

Effects of E-64 (cysteine-proteinase inhibitor) and pepstatin (aspartyl-proteinase inhibitor) on metastasis formation in mice with mammary and ovarian tumors.

The effects of E-64 (Cathepsin B and L inhibitor) and Pepstatin A (Cathepsin D inhibitor) on spontaneous and experimental metastasis formation were investigated in mice with MCa mammary carcinoma, M5076 ovarian sarcoma and L1210 leukemia. Pepstatin induced a marked decrease in the number of spontaneous metastasis in MCa or M5076 tumor bearing mice. This phenomenon was also noted with E-64 but only in M5076 tumor bearing mice. On the other hand, both these agents were unable to prevent the formation of experimental metastasis in mice injected i.v. with L1210, MCa or M5076 tumor cells or with tumor cells in which Cathepsin B, L and D activities were inhibited by a 24 hour continuous exposure to high non-cytotoxic concentrations of E-64 and/or Pepstatin. These data suggest that Cathepsin B, L and D seem to be involved in the early steps of the metastatic process rather than in the hematogenous spread of tumor cells. However, other pharmacological activities which may account for the discrepant effects of E-64 or Pepstatin on experimental and spontaneous metastasis cannot be ruled out.

Animals↗
PubMed1976

Mode of inhibition of acid proteases by pepstatin.

Four derivatives of pepstatin, each of which contains the unusual amino acid 4-amino-3-hydroxy-6-methylheptanoic acid (statine) have been prepared. All four are porcine pepsin inhibitors. Both N-acetylstatine 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 values 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 Rhizopus chinensis and bovine chymosin, also are inhibited by pepstatin and its derivatives. It is suggested 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.

Amino Acid Sequence↗
PubMed1975

Pepstatin, an inhibitor of leukokinin formation and ascitic fluid accumulation.

Ascites fluid accumulation accompanying a mastocytoma or L1210 murine tumor is significantly retarded following the i.p. or s.c. injection of moderate quantities of pepstatin, a known acid protease inhibitor. No effect on cell count was noted by pepstatin treatment. The probable mechanism by which pepstatin acts is by inhigiting the enzymatic formation of chemical mediators known as leukokinins. These are pharmoacologically active peptiedes having potent permeability characteristics previously described by this laboratory. Leukokinins are formed by cathepsin D-like enzymes present in the invading cells and in the ascites fluid acting on a protein substrate, leukokininogen. present in the ascites fluid. Pestatin inhibits the action of these leukokinin-forming enzymes invitro but has no effect on kallikreins (bradykinin-forming enzymes) in vitro. Human ascites fluid from a patient with ovarian carcioma was found to have a paepstatin-inhibited, leukokinin-generating system, as does the mouse. A 'chemical mediator' theory is proposed for ascites fromation which broadens the previously held theory of lymphatic blockage (Holm-Nielsen) and may explain the recent findings of Hirabayashi and Graham of increased plasma-ascites exchange in peritoneal carcionmatosis. Pepstatin inhibition of chemical mediator formation may represent a new therapeutic approach to ascites fluid accumulation in neoplastic disease.

Animals↗
PubMed2004

Calculating proton uptake/release and binding free energy taking into account ionization and conformation changes induced by protein-inhibitor association: application to plasmepsin, cathepsin D and endothiapepsin-pepstatin complexes.

The protein-inhibitor binding energies of enzymes are often pH dependent, and binding induces either proton uptake or proton release. The proton uptake/release and the binding energy for three complexes with available experimental data were numerically studied: pepstatin-cathepsin D, pepstatin-plasmepsin II and pepstatin-endothiapepsin. Very good agreement with the experimental data was achieved when conformational changes were taken into account. The role of the desolvation energy and the conformational changes was revealed by modeling the complex, the separated molecules in the complex conformation and the free molecules. It was shown that the conformational changes induced by the complex formation are as important for the proton transfer as the loss of solvation energy caused by the burial of interface residues. The residues responsible for the proton transfer were identified and their contribution to the proton uptake/release calculated. These residues were found to be scattered along the whole protein rather than being localized only at the active site. In the case of cathepsin D, these residues were found to be highly conserved among the cathepsin D sequences of other species. It was shown that conformation and ionization changes induced by the complex formation are critical for the correct calculation of the binding energy. Taking into account the electrostatics and the van der Waals (vdW) energies within the Boltzmann distribution of energies and allowing ionization and conformation changes to occur makes the calculated binding energy more realistic and closer to the experimental value. The interplay between electrostatic and vdW forces makes the pH dependence of the binding energy smoother, because the vdW force acts in reaction to the changes of the electrostatic energy. It was found that a small fraction of the ionizable groups remain uncharged in both the free and complexed molecules. The sequence and structural position of these groups aligns well within the three proteases, suggesting that these may have specific role.

Amino Acid Sequence↗
PubMed2001

Molecular model for the binary complex of uropepsin and pepstatin.

The three-dimensional structure of human uropepsin complexed with pepstatin has been modelled using human pepsin as a template. Uropepsin is an aspartic proteinase from the urine, produced in the form of pepsinogen A in the gastric mucosa. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo twofold axis. A structural comparison between binary complexes of pepsin:pepstatin and uropepsin:pepstatin is discussed.

Binding Sites↗
PubMed1998

No role for pepstatin-A-sensitive acidic proteinases in reovirus infections of L or MDCK cells.

Strong evidence indicates that virions of mammalian reoviruses undergo proteolytic processing by acid-dependent cellular proteinases as an essential step in productive infection. Proteolytic processing takes the form of a series of cleavages of outer-capsid proteins final sigma3 and mu1/mu1C. Previous studies showed an effect of both NH4Cl and E-64 on these cleavages, indicating that one or more of the acid-dependent cysteine proteinases in mammalian cells (cathepsins B and L, for example) is required; however, these studies did not address whether acid-dependent aspartic proteinases in those cells (cathepsin D, for example) may also be required. To determine the role of aspartic proteinases in reovirus entry, studies with pepstatin A, a specific inhibitor of aspartic proteinases, were performed. The results showed that pepstatin A neither blocks nor slows reovirus infection of L or MDCK cells. Experiments using ribonuclease A and other proteins as cleavable substrates showed that cathepsin-D-like proteinases from these cells are inhibited within the tested range of pepstatin A concentrations both in vitro and within living cells. In other experiments, virion-bound final sigma3 protein was shown to be a poor substrate for cleavage by cathepsin D in vitro, consistent with the findings with inhibitors. In sum, the data indicate that cathepsin-D-like aspartic proteinases provide little or no activity toward proteolytic events required for infection of L or MDCK cells with reovirus virions.

Animals↗
PubMed1994

Preparation and preliminary characterization of poly(ethylene glycol)-pepstatin conjugate.

The carboxyl function of pepstatin has been coupled, through an amide bond, to methoxypoly(ethylene glycol) (5 kDa), to which an amino function had been previously grafted. The mPEG-pepstatin conjugate inhibits hog pepsin (aspartic proteinase) in vitro as pepstatin itself, however, with a 400 times higher apparent Ki. The conjugate apparently does not inhibit proteinases belonging to other proteinase families such as serine (trypsin, carboxypeptidase Y), cysteine (Papaya proteinase III), or metallo (collagenase) proteinases.

Carboxypeptidases↗
PubMed2006

Effect of pepstatin A on the virulence factors of Candida albicans strains isolated from vaginal environment of patients in three different clinical conditions.

The aspartate proteinase inhibitor pepstatin A was used to study a possible correlation among proteinase activity and other virulence factors of Candida albicans strains isolated from the vaginal environment of patients in three different clinical conditions: asympthomatic, vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). The addition of 1.0 muM pepstatin A did not have any significant effect on hyphae formation, biofilm production and in the cell surface hydrofobicity of isolates in the three different clinical conditions. However, pepstatin A reduced the adherence of C. albicans to vaginal mucosa epithelial cells (53.1, 48.7 and 59.9%, respectively to isolates from asymptomatic, VVC and RVVC patients). This result suggests that the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles in cellular adhesion.

Adolescent↗
PubMed1984

The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture.

The production of [14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl-L-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 +/- 1.4% to 10.5 +/- 2.4% with 10 microM TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 microM TLCK. The incorporation of [14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 micrograms/ml leupeptin or 60 micrograms/ml pepstatin A. However, the production of collagen was reduced to 10.9 +/- 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 microM TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.

Amino Acid Chloromethyl Ketones↗
PubMed1990

Affinity purification of HIV-1 and HIV-2 proteases from recombinant E. coli strains using pepstatin-agarose.

A procedure is described which employs pepstatin-agarose for the affinity purification of either HIV-1 or HIV-2 protease from two similar recombinant E. coli constructs that were developed for the expression of these enzymes. HIV-2 protease was routinely expressed at much higher levels than the HIV-1 enzyme and pepstatin-agarose was the only chromatography step required to isolate pure HIV-2 protease from crude bacterial lysates. A Mono S ionic exchange step following pepstatin-agarose chromatography was sufficient to bring the HIV-1 protease to homogeneity. Purification of either enzyme can be completed in several days yielding homogeneous preparations suitable for crystallization and other physical characterization.

Chromatography, Affinity↗
PubMed1988

The effect of pepstatin A, an inhibitor of the pro-opiomelanocortin (POMC)-converting enzyme, on POMC processing in mouse intermediate pituitary.

In our previous studies, we have purified a unique, paired basic residue-specific, prohormone-converting enzyme from pituitary intermediate lobe secretory vesicles. This enzyme, an aspartyl protease, was shown to cleave the intermediate lobe prohormone, pro-opiomelanocortin (POMC), to adrenocorticotropin, beta-endorphin and a 16 kDa NH2-terminal glycopeptide, in vitro [(1985) J. Biol. Chem. 260, 7194-7205]. To provide some evidence that this enzyme plays a role in prohormone conversion in the intact cell, the ability of pepstatin A, an aspartyl protease inhibitor, to block POMC processing in the mouse intermediate pituitary was investigated. By the use of a radioactive pulse-chase paradigm, [3H]POMC processing was found to be inhibited by 36.4% in pepstatin A-treated intermediate lobes. This result is consistent with the inactivation of pro-opiomelanocortin-converting enzyme by pepstatin A in the intact pituitary and further supports a role of this enzyme in POMC processing in vivo.

Animals↗