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PubMed1989

Inhibition of HIV replication in cell culture by the specific aspartic protease inhibitor pepstatin A.

After incubation of H9 cells infected with human immunodeficiency virus (HIV) with pepstatin A at 10(-4) M for 2, 4, or 11 days, the culture medium contained significantly less HIV core antigen (p24) than controls without pepstatin A and no or only borderline activity of reverse transcriptase was detected. In addition, after pepstatin A treatment no infectious HIV at 2 or 4 days and only minimal amounts at 11 days were detectable in the culture medium.

Aspartic Acid Endopeptidases↗
PubMed1992

Crystallization and initial crystallographic results for pepstatin A inhibited bovine cathepsin D.

Cathepsin D was purified from bovine liver by a method using two pepstatin A affinity columns. The eluted protein was combined with pepstatin A and the complex crystallized from 15% polyethylene glycol 8000 at pH 5.9. The crystals diffract to a resolution of 3.0 A and have space group P2(1)2(1)2(1) with unit cell dimensions a = 74.8 A, b = 76.0 A, c = 157.7 A. There are two molecules in the asymmetric unit. The structure was solved by molecular replacement using a pepsin search model and both molecules showed clearly interpretable density in the position expected for pepstatin A in a preliminary difference map. The refined model has r.m.s. deviations from ideal bond lengths and angles of 0.014 A and 3.2 degrees, respectively, and a crystallographic R factor of 17%.

Animals↗
PubMed2006

Correction of the mineralization defect in hyp mice treated with protease inhibitors CA074 and pepstatin.

Increased expression of several osteoblastic proteases and MEPE (a bone matrix protein) occurs in X-linked hypophosphatemic rickets (hyp). This is associated with an increased release of a protease-resistant MEPE peptide (ASARM peptide), a potent inhibitor of mineralization. Cathepsin B cleaves MEPE releasing ASARM peptide and hyp osteoblast/osteocyte cells hypersecrete cathepsin D, an activator of cathepsin B. Our aims were to determine whether cathepsin inhibitors correct the mineralization defect in vivo and whether hyp-bone ASARM peptide levels are reduced after protease treatment. Normal littermates and hyp mice (n = 6) were injected intraperitoneally once a day for 4 weeks with pepstatin, CAO74 or vehicle. Animals were then sacrificed and bones plus serum removed for comprehensive analysis. All hyp mice groups (treated and untreated) remained hypophosphatemic with serum 1,25 vitamin D3 inappropriately normal. Serum PTH was significantly elevated in all hyp mice groups relative to normal mice (P = 0.0017). Untreated hyp mice had six-fold elevated levels of serum alkaline-phosphatase and two-fold elevated levels of ASARM peptides relative to normal mice (P < 0.001). In contrast, serum alkaline phosphatase and serum ASARM peptides were significantly reduced (normalized) in hyp mice treated with CA074 or pepstatin. Serum FGF23 levels remained high in all hyp animal groups (P < 0.0001). Hyp mice treated with protease inhibitors showed dramatic reductions in unmineralized osteoid (femurs) compared to control hyp mice (Goldner staining). Also, hyp animals treated with protease inhibitors showed marked and significant improvements in growth plate width (42%), osteoid thickness (40%) and cortical area (40%) (P < 0.002). The mineralization apposition rate, bone formation rate and mineralization surface were normalized by protease-treatment. High-resolution pQCT mineral histomorphometry measurements and uCT also confirmed a marked mineralization improvement. Finally, the growth plate and cortical bone of hyp femurs contained a massive accumulation of osteoblast-derived ASARM peptide(s) that was reduced in hyp animals treated with CA074 or pepstatin. This study confirms in vivo administration of cathepsin inhibitors improves bone mineralization in hyp mice. This may be due to a protease inhibitor mediated decrease in proteolytic degradation of the extracellular matrix and a reduced release of ASARM peptides (potent mineralization inhibitors).

Animals↗
PubMed2002

Pepstatin A attenuates the inhibitory effect of N-acetyl-L-cysteine on proliferation of hepatic myofibroblasts (stellate cells).

The pharmacological interaction between pepstatin A, a specific inhibitor of cathepsin D, and N-acetyl-L-cysteine was analyzed using hepatic stellate cells in primary culture. Isolated rat stellate cells were cultured on plastic dishes in Dulbecco's modified Eagle's medium (DMEM). Proteins and phospho-proteins were detected by Western blot. DNA synthesis was determined by [3H]thymidine uptake. Pepstatin A restored DNA synthesis of stellate cells stimulated by either platelet-derived growth factor-BB (PDGF-BB) or insulin-like growth factor-I (IGF-I), an effect that was attenuated by N-acetyl-L-cysteine. This agent induced the recovery of both the expression of PDGF receptor beta and IGF-I receptor beta and the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) and Akt under stimulation with either PDGF-BB or IGF-I, which were downregulated by N-acetyl-L-cysteine. Expression of cathepsin D was induced in activated stellate cells. These results indicate that pepstatin A hampers the inhibitory effect of N-acetyl-L-cysteine on stellate cell growth by ameliorating growth factor receptor downregulation.

Acetylcysteine↗
PubMed1983

Role of cellular proteinases in acute myocardial infarction. I. Proteolysis in nonischemic and ischemic rat myocardium and the effects of antipain, leupeptin, pepstatin and chymostatin administered in vivo.

To test the hypothesis that cellular proteinases contribute to ischemic myocellular death, measurements were made of tyrosine release (an index of overall proteolysis) from incubated slices of nonischemic and ischemic myocardium obtained at various times after coronary artery occlusion in rats. Proteolysis failed to increase in ischemic myocardium throughout the first 24 hours of occlusion, when irreversible damage develops, indicating that cellular proteinases do not undergo generalized activation in this phase. These data represent the first assessment of myocardial proteolysis throughout the development of ischemic death, and suggest that cellular proteinases do not play a causal role in this process. However, the possibility remains that ischemia selectively accelerates the breakdown of vital proteins, a phenomenon that may not be detected by measuring overall proteolysis. To determine whether future studies on the effects of proteolytic inhibition on infarct size are feasible, the ability of the proteinase inhibitors antipain, leupeptin, pepstatin and chymostatin, given in vivo, to interfere with proteolysis in ischemic myocardium was also evaluated. Leupeptin (10 or 40 mg/kg) inhibited proteolysis in a dose-related fashion (-49 and -72%, respectively, p less than 0.001). Antipain (20 mg/kg) decreased protein breakdown by 60% (p less than 0.001). The combination of antipain (20 mg/kg), leupeptin (40 mg/kg) and pepstatin (5 mg/kg) suppressed proteolysis almost completely at both 15 minutes (-88%, p less than 0.001) and at 6 hours (-72%, p less than 0.05) of ischemia, that is, throughout the development of irreversible injury. These results demonstrate that whatever proteolysis is occurring during acute myocardial infarction is largely mediated by cathepsins A, B, D, L and H and by calcium-activated neutral protease (that is, the enzymes sensitive to the inhibitors used). Because antipain, leupeptin and pepstatin significantly suppress such proteolysis, these agents might be useful in further assessing any potential contribution of cellular proteinases to the production of ischemic myocellular death. In addition, this study provides a new experimental model that affords serial assessments of regional myocardial proteolysis during the evolution of myocardial infarction.

Animals↗
PubMed2001

Ab initio molecular dynamics-based assignment of the protonation state of pepstatin A/HIV-1 protease cleavage site.

A recent 13C NMR experiment (Smith et al. Nature Struct. Biol. 1996, 3, 946-950) on the Asp 25-Asp25' dyad in pepstatin A/HIV-1 protease measured two separate resonance lines, which were interpreted as being a singly protonated dyad. We address this issue by performing ab initio molecular dynamics calculations on models for this site accompanied by calculations of 13C NMR chemical shifts and isotopic shifts. We find that already on the picosecond time-scale the model proposed by Smith et al. is not stable and evolves toward a different monoprotonated form whose NMR pattern differs from the experimental one. We suggest, instead, a different protonation state in which both aspartic groups are protonated. Despite the symmetric protonation state, the calculated 13C NMR properties are in good agreement with the experiment. We rationalize this result using a simple valence bond model, which explains the chemical inequality of the two C sites. The model calculations, together with our calculations on the complex, allow also the rationalization of 13C NMR properties on other HIV-1 PR/inhibitor complexes. Both putative binding of the substrate to the free enzyme, which has the dyad singly protonated (Piana, S.; Carloni, P. Proteins: Struct., Funct., Genet. 2000, 39, 26-36), and pepstatin A binding to the diprotonated form are consistent with the inverse solvent isotope effect on the onset of inhibition of pepsin by pepstatin and the kinetic iso-mechanism proposed for aspartic proteases (Cho, T.-K.; Rebholz, K.; Northrop, D.B. Biochemistry 1994, 33, 9637-9642).

Aspartic Acid↗
PubMed1999

Simplified pepstatins: synthesis and evaluation of N-terminally modified analogues.

The promising strategy of gastric ulcer healing with perorally administered epidermal growth factor (EGF) is so far strongly limited by the pepsinic degradation of this therapeutic polypeptide in the stomach. The incorporation of EGF in a bioadhesive polymer-pepsin inhibitor conjugate used as drug carrier matrix, however, might provide sufficient protection toward pepsinic degradation. The synthesis of appropriate pepsin inhibitors represents a prerequisite for the development of such polymer-inhibitor conjugates. The presented study demonstrates that modifications at the N-terminus of simplified analogues of pepstatin which can be synthesized in a simple and straight way result only in slight variations of the inhibitory activity. These analogues display only 10-fold reduced inhibitory activity, compared to pepstatin A, when bearing a greater N-terminal group like isovaleryl, Boc, or Cbz. Compounds which are substituted at the N-terminus by a shorter N-acyl group like propionyl or cyclopropylcarbonyl show further reduced activity (0.01, compared to pepstatin A). The presence of an amide or a urethane moiety at the N-terminus has no considerable effect on enzyme inhibition. Therefore, the N-terminus of these analogues is able to be modified forming a covalent bond to various bioadhesive polymers via a suitable functionality.

Anti-Ulcer Agents↗
PubMed1981

Bimane-labelled pepstatin, a fluorescent probe for the subcellular location of cathepsin D.

1. Pepstatinyl-cystamine was synthesized. The disulphide bond was cleaved and the pepstatin-bound thiol was made to react with monobromobimane. The fluorescent N-pepstatinyl-S-bimanyl-2-aminoethanethiol was purified. 2. Human cathepsin D showed tight binding of the bimane-labelled pepstatin at pH 3.5. The titration curves were used to determine the apparent dissociation constant, KD; values of approx. 1 x 10(-10) M were obtained. 3. Gel-chromatographic experiments showed that, like that of pepstatin, the binding of N-pepstatinyl-S-bimanyl-1-aminoethanethiol to cathepsin D was strongly pH-dependent. Binding was seen at pH 5.0, but could not be demonstrated at pH 7.4. 4. Cultured human synovial cells were fixed and incubated with the fluorescent inhibitor at pH 5.0 or pH 7.4. When examined by fluorescence microscopy the cells stained at pH 5.0 showed a punctate perinuclear distribution of bimane fluorescence. By contrast, the cells stained at pH 7.4 showed no fluorescence. 5. The distribution of cathepsin D, determined by indirect immunofluorescence at pH 7.4, closely resembled that of the fluorescent inhibitor seen at pH 5.0. 6. We conclude that N-pepstatinyl-S-bimanyl-2-aminoethanethiol is a fluorescent probe selective for the active conformation of cathepsin D.

Cathepsin D↗
PubMed2002

Linear non-competitive inhibition of solubilized human gamma-secretase by pepstatin A methylester, L685458, sulfonamides, and benzodiazepines.

Cerebral deposition of amyloid beta-protein (A beta) is believed to play a key role in the pathogenesis of Alzheimer's disease. Because A beta is produced from the processing of amyloid beta-protein precursor (APP) by beta- and gamma-secretases, these enzymes are considered important therapeutic targets for identification of drugs to treat Alzheimer's disease. Unlike beta-secretase, which is a monomeric aspartyl protease, gamma-secretase activity resides as part of a membrane-bound, high molecular weight, macromolecular complex. Pepstatin and L685458 are among several structural classes of gamma-secretase inhibitors identified so far. These compounds possess a hydroxyethylene dipeptide isostere of aspartyl protease transition state analogs, suggesting gamma-secretase may be an aspartyl protease. However, the mechanism of inhibition of gamma-secretase by pepstatin and L685458 has not been elucidated. In this study, we report that pepstatin A methylester and L685458 unexpectedly displayed linear non-competitive inhibition of gamma-secretase. Sulfonamides and benzodiazepines, which do not resemble transition state analogs of aspartyl proteases, also displayed potent, non-competitive inhibition of gamma-secretase. Models to rationalize how transition state analogs inhibit their targets by non-competitive inhibition are discussed.

Amyloid Precursor Protein Secretases↗
PubMed1987

Conformational analysis of pepstatin and related renin inhibitors by 400 MHz 1H n.m.r. spectroscopy.

The conformational behaviour of pepstatin (Iva-Val-Val-Sta-Ala-Sta) and of two derived renin inhibitors, Boc-Phe-Nle-Sta-Ala-Sta-OMe, 1, and Boc-Phe-Nle-X-Ala-Sta-OMe, 2 (X = -NH-CH(iPr)-CHOH-CH2-CO-) was assessed in DMSO-d6 at various temperatures and in deuteriopyridine at -35 degrees. Complete assignment of almost all proton signals was achieved by 2D COSY, 2D NOESY and selective NOE experiments. The three compounds show similar extended conformations in both solvents, with the hydrophobic lateral chains extending away from the peptide backbone. In the case of pepstatin the solvated conformation is closely related to the structure found in the crystal of the pepstatin-Rhizopus chinensis complex. Strong NOE effects and precise determination of vicinal coupling constants show the lack of large structural differences between 1 and 2 at the level of the internal Sta or X residues, which are assumed to interact with the aspartyl residues of the renin active-site. This suggests that the 100-fold lower inhibitory potency of 2 is mainly due to unfavorable close contacts of the beta-branched residue X with constituent amino acids of the enzyme.

Dimethyl Sulfoxide↗
PubMed1979

Cytotaxin receptors of neutrophils: evidence that F-methionyl peptides and pepstatin share a common receptor.

Pepstatin, a chemotactic microbial pentapeptide, competes with f-Met-Leu-[3H]Phe for binding to human neutrophils. Furthermore, porcine neutrophils, which neither specifically bind nor respond chemotactically to the synthetic f-methionyl peptides, also fail to respond chemotactically to pepstatin. These results suggest that pepstatin shares a receptor on the neutrophil with f-methionyl peptides, despite their completely different amino acid compositions. The specificity of this cytotaxin receptor may therefore be broader than expected and depend on ligand characteristics distinct from primary structure.

Animals↗
PubMed1976

Inhibition of human plasma renin activity by pepstatin.

Pepstatin, a pentapeptide isolated from streptomyces, is a powerful inhibitor of several acid proteases. Its ability to inhibit the renin-angiotensinogen reaction was studied in vitro, in various human plasma. A 50% inhibition of plasma renin activity was obtained with 10(-6)M pepstatin at pH 5.7 and 10(-5)M at pH 7.4. The inhibition of plasma renin activity by pepstatin was studied in hypertensive patients with various plasma renin levels. The inhibitory effect could be demonstrated in patients with low, normal and high renin activity at both pH's. The type of inhibition and the inhibitory constant were investigated by Dixon plot and Lineweaver-Burk plot in three separate experiments. On both representations, a competitive type of inhibition was found with an inhibitory constant of about 1.2 x 10(-6)M.

Humans↗
PubMed1984

Depressor activity of intracerebroventricularly administered pepstatin in young spontaneously hypertensive rats.

The effect of prolonged intracerebroventricular (i.c.v.) infusion of N-acetyl-pepstatin in young and adult spontaneously hypertensive rats was studied. In young animals, pepstatin infusion resulted in a decrease in blood pressure and heart rate. Water intake and body weight were not affected. The depressor effect was accompanied by a slight increase in plasma renin activity and decreases in plasma vasopressin and plasma catecholamines. The blood pressure of adult rats with already established hypertension was not significantly affected. In addition, changes in plasma renin or catecholamines were not observed in these animals while vasopressin levels were slightly increased. The involvement of a possibly decreased sympathetic activity in the depressor effect of pepstatin is suggested. It is concluded that increased brain renin activity contributes to the development of hypertension of spontaneously hypertensive rats.

Aging↗
PubMed1979

Pepstatin, an inhibitor of acid kininogenases and ascites retardant in neoplastic disease.

Evidence is reviewed that pepstatin, an inhibitor of acid kininogenases such as cathepsin D, may be an effective therapeutic agent in retarding ascites accumulation in certain cancers. The evidence for this conclusion is based on the actions of pepstatin in retarding ascites in six different tumor strains inoculated into various species of mice, as well as the demonstration that cathepsin D activity is reduced in vivo in several organs following pepstatin administration. The latter is significant since we have postulated that ascites formation, in good part, is due to leukokinin formation, which is catalyzed by cellular-released cathepsin D.

Animals↗
PubMed1973

Pentapeptide (pepstatin) inhibition of brain acid proteinase.

The pentapeptide pepstatin obtained from culture filtrates of actinomycetes completely inhibited brain acid proteinase (cathepsin D) at exceedingly low concentrations. Among the brain enzymes tested, the effect is specific for acid proteinase because addition of 1000-fold higher concentrations was without effect on neutral proteinase, aminopeptidase, and arylamidases. Pepstatin also inhibits pepsin as tested with hemoglobin or with N-acetylphenylalanyl-L-diiodotyrosine as substrate. Pepstatin must be regarded as the most powerful agent yet described that inhibits intracellular acidic proteolytic enzyme in brain.

Actinomyces↗
PubMed1973

Inhibition of cathepsin D-type proteinase of macrophages by pepstatin, a specific pepsin inhibitor, and other substances.

The macrophage is the main cell participating in chronic inflammation. It contains an acid-acting, cathepsin D-type proteinase with the specificity of pepsin, which may release mediators of the inflammatory process. To find new pharmaceutical inhibitors of this proteinase, we tested a variety of chemical compounds in vitro. For this survey, the possible inhibitor (at a concentration of 0.4 mg/ml) was assayed with partially purified cathepsin D-type proteinase from beef lung (a macrophage-rich tissue) and hemoglobin as the substrate. Diazophenylbutanone, three acetophenones, two barbiturates, a gold salt, a copper chelate of a substituted nicotinic acid, a hexapeptide containing a d-amino acid, and Pepstatin inhibited this enzyme; over 200 other potential inhibitors did not. By far the most active and specific inhibitor found to date is Pepstatin, a pentapeptide with two gamma-NH linkages, two beta-OH groups, and five branched aliphatic side chains. Banyu Pharmaceutical Co., Tokyo, Japan, produces this nontoxic compound for the treatment of peptic ulcers. In vitro, as little as 4 ng of Pepstatin inhibits the acid-acting cathepsin D-type proteinase purified from beef and rabbit lung as well as the similar proteinase of rabbit peritoneal and pulmonary macrophages.

Acetophenones↗
PubMed1992

Structures of complexes of rhizopuspepsin with pepstatin and other statine-containing inhibitors.

The three-dimensional structures of the complexes of the aspartic proteinase from Rhizopus chinensis (Rhizopuspepsin, EC 3.4.23.6) with pepstatin and two pepstatin-like peptide inhibitors of renin have been determined by X-ray diffraction methods and refined by restrained least-squares procedures. The inhibitors adopt an extended conformation and lie in the deep groove located between the two domains of the enzyme. Inhibitor binding is accompanied by a conformational change at the "flap," a beta-hairpin loop region, that projects over the binding cleft and closes down over the inhibitor, excluding water molecules from the vicinity of the scissile bond. The hydroxyl group of the central statyl residue of the inhibitors replaces the water molecule found between the two active aspartates, Asp-35 and Asp-218, in the native structure. The refined structures provide additional data to define the specific subsites of the enzyme and also show a system of hydrogen bonding to the inhibitor backbone similar to that observed for a reduced inhibitor.

Aspartic Acid Endopeptidases↗
PubMed2000

Pepstatin A-sensitive aspartic proteases in lysosome are involved in degradation of the invariant chain and antigen-processing in antigen presenting cells of mice infected with Leishmania major.

We previously reported that CA074, a specific inhibitor of cathepsin B, significantly deviated immune responses from the disease-promoting Th2 type to the protective Th1 type in BALB/c mice infected with Leishmania major. Herein, we found that pepstatin A-sensitive aspartic proteases (PSAP) in lysosomes seem to play a different role from that of cathepsin B in antigen-processing and Ii-degradation. That is, cathepsin B appears to digest 16-, 28-, and 31-kDa peptides of soluble leishmania antigen (SLA), whereas PSAP seems to process mainly 28-kDa peptides. Furthermore, the latter protease contributed to the degradation of Ii but cathepsin B did not. Following treatment with pepstatin A, both Th1 and Th2 responses were profoundly suppressed in resistant DBA/2 mice (H-2(d)) and in susceptible BALB/c mice (H-2(d)), and both strains of mice became markedly susceptible compared with the untreated groups, probably owing to failure in degradation of Ii and partly to failure in digestion of 28-kDa peptide.

Animals↗