Effect of pepstatin, a specific protease inhibitor, on the experimental peptic ulcers.
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Acetates↗
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Pepstatin inhibition of human renin. Kinetic studies and estimation of enzyme purity.
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Humans↗
Renin inhibition by pepstatin in experimental hypertension.
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Acute Disease↗
Biosynthetic studies on pepstatin. Biosynthesis of (3s, 4s)-4-amino-3-hydroxy-6-methylheptanoic acid moiety.
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Acetates↗
Effects of proteinase inhibitors on the cutaneous lesion of Sporothrix schenckii inoculated hairless mice.
Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin. On the other hand, proteinase II is an aspartic proteinase, inhibited by pepstatin. The addition of either pepstatin or chymostatin to the culture medium did not inhibit cell growth, however the addition of both inhibitors strongly inhibited fungal growth. Accordingly, this suggested that extracellular proteinases play an important role in cell growth and that such cell growth may be suppressed if these proteinases are inhibited. In order to substantiate this speculation in sporotrichosis, the effects of proteinase inhibitors on the cutaneous lesions of mice were studied. Ointments containing 0.1% chymostatin, 0.1% pepstatin and 0.1% chymostatin-0.1% pepstatin were applied twice daily on the inoculation sites of hairless mouse skin, and the time courses of the lesions examined. The inhibitory effect in vivo on S. schenckii was similar to that demonstrated in our previous in vitro study. Compared to the control, the time course curve of the number of nodules present after the application of either pepstatin or chymostatin was slightly suppressed. The application of both pepstatin and chymostatin, however, strongly suppressed nodule formation. This study not only confirmed the role of 2 proteinases of S, schenckii for fungal growth in vivo, but also may lead to their use as new topical therapeutic agents.
Animals↗
Essential role of pepsin in pathogenesis of acid reflux esophagitis in rats.
Pepsin, a protease activated by gastric acid, is a component of the refluxate, yet the role of pepsin in the pathogenesis of reflux esophagitis has not been well studied. In the present study, we examined the effect of pepstatin, a specific inhibitor of pepsin, on acid reflux esophagitis. Acid reflux esophagitis was induced in rats by ligating both the pylorus and the forestomach for 3 or 4 hr. Pepstatin, ecabet Na (the anti-ulcer drug), and L-glutamine were administered intragastrically after the ligation. Pepstatin or ecabet Na, given intragastrically, significantly prevented esophageal lesions, even though they did not affect basal acid secretion in pylorus-ligated rats. Pepstatin significantly inhibited pepsin activity in vivo and in vitro, while ecabet Na inhibited this activity in vitro. By contrast, L-glutamine given intragastrically aggravated the lesions in a dose-dependent manner, but even in the presence of L-glutamine the development of esophageal lesions was totally prevented by coadministration of pepstatin or ecabet Na. L-Glutamine increased the pH of gastric contents to approximately 2.0, the optimal pH for the proteolytic activity of pepsin in vitro. In addition, intragastric administration of exogenous pepsin worsened the severity of esophageal damage. These results suggest that pepstatin is highly effective against acid reflux esophagitis, without influencing acid secretion, while L-glutamine aggravated these lesions by increasing the pepsin activity by shifting the intraluminal pH to the optimal pH range for proteolytic action. It is assumed that pepsin plays a major pathogenic role in the development of acid reflux esophagitis.
Animals↗
Kinetic and mechanistic analysis of the association and dissociation of inhibitors interacting with secreted aspartic acid proteases 1 and 2 from Candida albicans.
In order to elucidate the characteristics of different aspartic proteases (Sap) secreted by Candida albicans, the kinetics of the interaction (k(on), k(off)) between Sap1 and Sap2 with acetyl-pepstatin and pepstatin A was determined at different pH by biosensor technology. The enzymes were biotinylated and coupled to a streptavidin-coated sensor chip, whereupon acetyl-pepstatin or pepstatin A was injected and the interaction was measured in real time. Sap2 showed a faster k(on) and a higher affinity for acetyl-pepstatin than Sap1, regardless of pH. The values for both k(on) and k(off) decreased with increased pH from 3.8 to 5.0, except for the k(off) for Sap1, which was only influenced by the pH change from 3.8 to 4.4. Binding of acetyl-pepstatin to Sap1 or Sap2 obviously proceeds by a different mechanism than dissociation of the inhibitor. Association appears to be coupled to protonation of a catalytic aspartic acid residue, consistent with reduced k(on) values at higher pH. In contrast, the stability of the complex is reduced at lower pH due to reduced hydrogen bonding capacity of aspartic acid residues acting as hydrogen bond acceptors. Differences in the number and distribution of charged nonactive site residues in Sap1 and Sap2 evidently result in different electrostatic properties of the binding sites, primarily influencing the association step.
Amino Acid Sequence↗
A specific inhibitor of keratinolytic proteinase from Candida albicans could inhibit the cell growth of C. albicans.
The authors investigated the influence of culture medium pH and various kinds of protease inhibitors on the growth of Candida albicans when cultivated in liquid medium containing human stratum corneum (HSC) as the nitrogen source. Rapid growth of C. albicans was observed with weakly acidic media, particularly at pH 4.0. From among the various kinds of protease inhibitors added to the media at pH 4.0, pepstatin, a carboxyl protease inhibitor, most strongly inhibited the growth of C. albicans dependent upon its concentration. The antifungal effect of pepstatin was not fungicidal, but was nevertheless effective even at a very low concentration of 0.01 microgram/ml. This inhibitory effect of pepstatin was considerably stronger than that of the well-known antifungal agent, clotrimazole. Pepstatin is a specific inhibitor of keratinolytic proteinase (KPase) from C. albicans; it belongs to the carboxyl proteinases group and has an optimum pH at 4.0. Pepstatin showed a strong antifungal effect, possibly through KPase inhibition, in biologic (HSC) medium that was similar to that encountered in vivo. Our results suggest that KPase may play an important role in the growth of C. albicans and that pepstatin has the possibility of being used as a new type of antifungal agent.
Antifungal Agents↗
Role of aspartic proteases in disseminated Candida albicans infection in mice.
A murine model of disseminated candidiasis involving intranasal challenge with Candida albicans was developed and used to explore the role of C. albicans aspartic proteases as virulence factors during early dissemination. Pretreatment of neutropenic mice with the aspartic protease inhibitor pepstatin A by intraperitoneal injection afforded strong dose-dependent protection against a subsequent lethal intranasal dose of an aspartic protease-producing strain (ATCC 32354) of C. albicans. Administration of 0.6 mg of pepstatin A kg of body weight(-1) prior to challenge and on days 1 to 4 postchallenge resulted in 100% survival at day 15 postchallenge, whereas 100% of animals receiving saline had died by day 6. This effect was comparable to the dose-dependent protection obtained with amphotericin B, which resulted in 100% survival when administered at 0.1 mg kg(-1). The reduction in mortality afforded by pepstatin A correlated with its dose-dependent blockade of C. albicans numbers in the lungs, liver, and kidneys. By sharp contrast, no protection by pepstatin A was observed in mice challenged intravenously, and protection was markedly attenuated in mice given pepstatin A after intranasal challenge only. These data show the utility of pepstatin A in the prophylaxis of disseminated Candida infections and suggest that Candida aspartic proteases play an essential role early in dissemination.
Administration, Intranasal↗
Biochemical mechanisms that interact with membrane-associated IL-1 RII (60-kDa decoy) receptors in populations of adherent macrophages and vascular endothelium.
The aim of this investigation was to identify the potential biochemical mechanisms that alter the integrity of membrane-associated IL-1 RII (decoy) receptor complexes expressed by populations of adherent macrophages and vascular endothelium. The initial research strategy utilized to achieve this objective involved delineating the ability of macrophage activation or exposure of macrophages and vascular endothelium to a spectrum of enzyme proteases to influence the expression of membrane-associated IL-1 RII (decoy) or generate soluble fragments of this receptor complex. Results from these investigations revealed that stimulated macrophages displayed proportional increases in both the expression of membrane-associated IL-1 RII (decoy) and release of soluble receptor fragments. Exposure of macrophages and vascular endothelium to the reference proteases discovered the ability of cathepsin-D to biochemically deplete membrane-associated IL-1 RII (decoy) in addition to generating soluble fragments of this receptor complex. Complementary investigations isolated a carboxyl/aspartate protease from activated macrophages utilizing pepstatin-A affinity chromatography. Exposure of vascular endothelium to pepstatin-A binding proteins resulted in a detectable depletion of membrane-associated IL-1 RII (decoy) and generation of soluble receptor fragments. Evaluation of pepstatin-A binding proteins by SDS-PAGE identified a primary protein fraction with a molecular mass of 47-52 kDa that closely correlates with the known molecular size of leukocyte cathepsin-D fractions. Macrophage pepstatin-A binding protein fractions evaluated by nondenaturing haemoglobin-substrate PAGE (Hb-PAGE) analysis detected a lucent proteolytic band at 47-52 kDa. Macrophage pepstatin-A binding proteins also hydrolyzed a synthetic enzyme-specific substrate that selectively recognizes cathepsin-D biochemical activity. In conclusion, the leukocyte carboxyl/aspartate protease cathepsin-D can biochemically alter the integrity and generate soluble fragments of membrane-associated IL-1 RII (60-kDa decoy) receptor complexes expressed by macrophages and vascular endothelium.
Animals↗
Dissection of the pH dependence of inhibitor binding energetics for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues.
The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (DeltaG, DeltaH, DeltaCp, and DeltaS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKa values of ionizable residues whose pKa values change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKa of 7.5, based on the three-dimensional structure of the pepstatin-plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is -1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.
Animals↗
Inhibitors of human immunodeficiency virus-1 protease.
We have shown that the interaction of pepstatin A with human immunodeficiency virus-1 protease (HIV-1 protease) can be characterized by a high-affinity mode (Ki = 478 +/- 27 nM), resulting in pure competitive inhibition of the hydrolytic activity of HIV-1 protease toward the fluorogenic substrate. Binding of pepstatin in this mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 10 microM pepstatin. Haloperidol, in contrast, interacts with HIV-1 protease with weaker affinity (Ki = 19 +/- 1 microM) in a mode which results in pure noncompetitive inhibition of the hydrolytic activity of HIV-1 protease. Binding of haloperidol in this mode induces a red shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 200 microM haloperidol. Addition of both pepstatin and haloperidol at concentrations in the range of their Ki values results in additive inhibition of the hydrolytic activity of HIV-1 protease, as well as an additive effect on the tryptophan fluorescence of protease. However, at saturating concentrations of pepstatin and haloperidol, the effect of haloperidol was predominant, as measured by the changes in the intrinsic fluorescence of HIV-1 protease.
Amino Acid Sequence↗
Chymosin-mediated proteolysis, calcium solubilization, and texture development during the ripening of cheddar cheese.
Full fat, milled-curd Cheddar cheeses (2 kg) were manufactured with 0.0 (control), 0.1, 1.0, or 10.0 micromol of pepstatin (a potent competitive inhibitor of chymosin) added per liter of curds/whey mixture at the start of cooking to obtain residual chymosin levels that were 100, 89, 55, and 16% of the activity in the control cheese, respectively. The cheeses were ripened at 8 degrees C for 180 d. There were no significant differences in the pH values of the cheeses; however, the moisture content of the cheeses decreased with increasing level of pepstatin addition. The levels of pH 4.6-soluble nitrogen in the 3 cheeses with added pepstatin were significantly lower than that of the control cheese at 1 d and throughout ripening. Densitometric analysis of urea-PAGE electro-phoretograms of the pH 4.6-insoluble fractions of the cheese made with 10.0 micromol/L of pepstatin showed complete inhibition of hydrolysis of alpha(S1)-casein (CN) at Phe23-Phe24 at all stages of ripening. The level of insoluble calcium in each of 4 cheeses decreased significantly during the first 21 d of ripening, irrespective of the level of pepstatin addition. Concurrently, there was a significant reduction in hardness in each of the 4 cheeses during the first 21 d of ripening. The softening of texture was more highly correlated with the level of insoluble calcium than with the level of intact alpha(S1)-CN in each of the 4 cheeses early in ripening. It is concluded that hydrolysis of alpha(S1)-CN at Phe23-Phe24 is not a prerequisite for softening of Cheddar cheese during the early stages of ripening. We propose that this softening of texture is principally due to the partial solubilization of colloidal calcium phosphate associated with the para-CN matrix of the curd.
Calcium↗
Biochemical entities that influence membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complex expression and receptor fragment production in adherent vascular endothelium.
The research aim of the present investigation was to identify leukocyte enzyme-proteases that have the capacity to biochemically recruit the passive participation of vascular endothelium in cytokine receptor 'shedding' phenomenon involving membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complexes. Achieving this research objective involved the design of a laboratory approach that delineated to what extent enzyme-proteases released by activated macrophages directly interact with, and liberate soluble fragments of membrane-associated cytokine receptor complexes. Results from this segment of the investigation revealed that cathepsin-D, a leukocyte carboxyl/aspartate protease, altered the integrity and generated soluble fragments of TNF RII (80-kDa) and IL-1 RI (80-kDa) receptor complexes expressed by vascular endothelium. Furthermore, laboratory findings also suggested that cathepsin-D possessed the ability to variably deplete biologically functional membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complexes. Complementary investigations isolated a carboxyl/aspartate protease from activated macrophages utilizing pepstatin-A affinity chromatography. Exposure of vascular endothelium to pepstatin-A binding proteins resulted in a detectable depletion of membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) in addition to the generation of soluble receptor fragments. Analysis of macrophage pepstatin-A binding proteins by SDS-PAGE identified a primary fraction with a molecular mass of 47-52-kDa that closely correlated with the known molecular mass of leukocyte cathepsin-D. Evaluation of macrophage pepstatin-A binding-protein fractions by non-denaturing Hb-PAGE detected a lucent proteolytic band at 47-52-kDa compatible with the known molecular mass of leukocyte cathepsin-D. Macrophage pepstatin-A binding proteins also hydrolyzed a synthetic enzyme-specific substrate that selectively recognizes cathepsin-D biochemical activity. In conclusion, the leukocyte carboxyl/aspartate protease, cathepsin-D can biochemically alter the integrity and generate soluble fragments of membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) receptor complexes expressed by vascular endothelium. The relevance of this concept is in part based on investigations that have discovered that genetic 'knock-out' mice incapable of expressing IL-1 RI (80-kDa) or TNF RI (55-kDa) receptor complexes are highly resistant to developing the pathophysiological alterations classically associated with conditions of endotoxic-shock.
Journal Article↗
Exocrine secretion and processing of pro-xenopsin in rat gastric lumen.
This study examines the effects of acid, acid secretagogues, and pepstatin on gastric luminal concentrations of xenopsin-like immunoreactivity (XPLI) by in situ luminal perfusion of the stomach in anesthetized rats. During perfusion with saline over a 2-h period, the concentration of XPLI fell in parallel with acid output. Levels of XPLI fell more rapidly when the saline contained 20 micrograms/ml pepstatin A and when phosphate-buffered saline (pH 7.0) was used. These treatments did not, however, alter acid output. After a basal condition was established at 90 min, intravenous injection of carbachol, pentagastrin, or histamine stimulated acid and pepsin secretion and also led to an increase in XPLI concentration, which was pepstatin sensitive. Acid itself was also a stimulus for pepsin and XPLI output, which were correlated at various levels of acidity. Although pepstatin blocked the effect of acid on XPLI output, it did not lead to an accumulation of xenopsin (XP) precursor in the luminal fluid. However, the decrement in acid-stimulated XPLI output seen in the presence of pepstatin was matched by an increment in XP precursor associated with the mucosal surface. During high-pressure liquid chromatography, approximately 70% of the acid-generated XPLI eluted at the position of mammalian XP. These data support the notion that, during heightened acid output, XP is secreted by a pepsin-dependent process or generated by the action of pepsin on XP precursor present on the mucosal surface of the rat stomach.
Animals↗
Structure-based thermodynamic design of peptide ligands: application to peptide inhibitors of the aspartic protease endothiapepsin.
The prediction of binding affinities from structure is a necessary requirement in the development of structure-based molecular design strategies. In this paper, a structural parameterization of the energetics previously developed in this laboratory has been incorporated into a molecular design algorithm aimed at identifying peptide conformations that minimize the Gibbs energy. This approach has been employed in the design of mutants of the aspartic protease inhibitor pepstatin A. The simplest design strategy involves mutation and/or chain length modification of the wild-type peptide inhibitor. The structural parameterization allows evaluation of the contribution of different amino acids to the Gibbs energy in the wild-type structure, and therefore the identification of potential targets for mutation in the original peptide. The structure of the wild-type complex is used as a template to generate families of conformational structures in which specific residues have been mutated. The most probable conformations of the mutated peptides are identified by systematically rotating around the side-chain and backbone torsional angles and calculating the Gibbs potential function of each conformation according to the structural parametrization. The accuracy of this approach has been tested by chemically synthesizing two different mutants of pepstatin A. In one mutant, the alanine at position five has been replaced by a phenylalanine, and in the second one a glutamate has been added at the carboxy terminus of pepstatin A. The thermodynamics of association of pepstatin A and the two mutants have been measured experimentally and the results compared with the predictions. The difference between experimental and predicted Gibbs energies for pepstatin A and the two mutants is 0.23 +/- 0.06 kcal/mol. The excellent agreement between experimental and predicted values demonstrates that this approach can be used in the optimization of peptide ligands.
Aspartic Acid Endopeptidases↗
Protease activity associated with loss of adhesiveness in mouse teratocarcinoma.
We have observed the spontaneous conversion of an embryoid body (multicellular) form of 129/J mouse ascites teratocarcinoma to a single cell form. Concomitant with the conversion, a rapid increase in growth rate and ascites fluid accumulation have been observed. This report presents data on protease activity in the ascites fluid after the conversion and evidence that the protease causes decreased tumor cell adhesiveness. Ascites protease has a pH optimum of 2.0-3.0 and is inhibited by both DAN (diazoacetyl-DL-norleucine methyl ester) and Pepstatin A. Using denatured bovine hemoglobin as a substrate, the low pH optimum and inhibition by DAN and Pepstatin A allow tentative identification of the enzyme as the carboxypeptidase Cathepsin D. Approx. 0.036 X 10(-2) micrograms of Cathepsin D per mg of protein was found in the ascites fluid of the single cell form of the mouse teratocarcinoma. We show that Pepstatin A-derivatized agarose beads bind the single ascites cells, causing them to display increased cell-cell adhesion, a phenomenon not observed with control beads. The results suggest that ascites protease may play a role in transformation of a slow growing, clustered tumor into a rapidly growing, non-adhesive, single cell form. We found that an embryoid-like tissue culture line of mouse teratocarcinoma, that we established, disaggregated into single cells, upon addition of ascites fluid from the single cell tumor, to the culture medium. Pepstatin A prevented disaggregation of the cell clusters. These results further support the contention that specific ascites protease plays a role in the transformation of a clustered tumor into a single cell form.
Animals↗
Cockroach allergen Bla g 2: an unusual aspartic proteinase.
BACKGROUND: Enzymatic activity of mite, fungal, and bee venom allergens is thought to potentiate their allergenicity. Bla g 2 is a potent cockroach allergen, but despite sharing sequence homology with aspartic proteinases, it contains critical amino acid substitutions that impair proteolytic activity. The biologic function of Bla g 2 remains unclear. OBJECTIVE: We sought to investigate the effects of specific amino acid substitutions on enzymatic activity, and the peptide-binding capability of Bla g 2. METHODS: Site-directed mutagenesis was used to produce a recombinant Bla g 2 mutant (Mut) with corrected canonical triads and a flap region. Another mutant (MutF - ) was expressed after additional mutations in the flap region of Mut. Bla g 2 wild-type (Wt), Mut, and MutF - were assayed for aspartic proteinase activity, and Bla g 2 Wt was tested for pepstatin binding. RESULTS: Recombinant Bla g 2 Wt and Mut did not show enzymatic activity in a milk-clotting and hemoglobin assay. By using a modified hemoglobin assay, residual activity inhibited by pepstatin was detected for MutF - and Wt at 20 microg/mL, whereas pepsin was active at a 1000-fold lower concentration. Most of Bla g 2 binding to pepstatin-agarose was nonspecific. CONCLUSION: Residual proteolytic activity was found for Bla g 2 at concentrations of approximately 4 mM. This weak activity suggests that proteolysis is not the primary function of this allergen and that it is unlikely to contribute to the allergenicity of Bla g 2. Bla g 2 has a cleft that might specifically bind ligands other than pepstatin.
Amino Acid Sequence↗