Structure-activity studies with high-affinity inhibitors of pyroglutamyl-peptidase II.

Inhibitors of PPII (pyroglutamyl-peptidase II) (EC 3.4.19.6) have potential applications as investigative and therapeutic agents. The rational design of inhibitors is hindered, however, by the lack of an experimental structure for PPII. Previous studies have demonstrated that replacement of histidine in TRH (thyrotropin-releasing hormone) with asparagine produces a competitive PPII inhibitor (Ki 17.5 microM). To gain further insight into which functional groups are significant for inhibitory activity, we investigated the effects on inhibition of structural modifications to Glp-Asn-ProNH2 (pyroglutamyl-asparaginyl-prolineamide). Synthesis and kinetic analysis of a diverse series of carboxamide and C-terminally extended Glp-Asn-ProNH2 analogues were undertaken. Extensive quantitative structure-activity relationships were generated, which indicated that key functionalities in the basic molecular structure of the inhibitors combine in a unique way to cause PPII inhibition. Data from kinetic and molecular modelling studies suggest that hydrogen bonding between the asparagine side chain and PPII may provide a basis for the inhibitory properties of the asparagine-containing peptides. Prolineamide appeared to be important for interaction with the S2' subsite, but some modifications were tolerated. Extension of Glp-Asn-ProNH2 with hydrophobic amino acids at the C-terminus led to a novel set of PPII inhibitors active in vitro at nanomolar concentrations. Such inhibitors were shown to enhance recovery of TRH released from rat brain slices. Glp-Asn-Pro-Tyr-Trp-Trp-7-amido-4-methylcoumarin displayed a Ki of 1 nM, making it the most potent competitive PPII inhibitor described to date. PPII inhibitors with this level of potency should find application in exploring the biological functions of TRH and PPII, and potentially provide a basis for development of novel therapeutics.

Inactivation of pyroglutamyl aminopeptidase by L-pyroglutamyl chloromethyl ketone.

A chloromethyl ketone derivative of pyroglutamic acid was newly synthesized and its reactivity with bacterial pyroglutamyl aminopeptidase (L-pyroglutamyl-peptide hydrolas, EC 3.4.11.8) as an affinity labelling reagent was examined. The compound was found to inactivate the enzyme markedly and rapidly at very low concentrations, though the enzyme was resistant to N-tosyl-phenylalanyl chloromethyl ketone. The rate of the enzyme inactivation by pyroglutamyl chloromethyl ketone was retarded in the presence of a poor substrate, pyroglutamyl valine. The enzyme inactivated by treating with p-chloromercuribenzoate failed to react with pyroglutamyl chloromethyl ketone. These results strongly suggest an active site-directed mechanism for the enzyme inactivation by pyroglutamyl chloromethyl ketone. This compound was shown to be useful as a titrant for the catalytically active protein of pyroglutamyl aminopeptidase.

N-alpha-carbobenzoxy pyroglutamyl diazomethyl ketone as active-site-directed inhibitor for pyroglutamyl peptidase.

Pyroglutamyl-peptidase (L-pyroglutamyl-peptide hydrolase, EC 3.4.19.3) from Bacillus amyloliquefaciens was covalently labeled with a newly synthesized N-carbobenzoxy-L-pyroglutamyl diazomethyl ketone (Z-PGDK) and was completely inactivated. The inactivation reaction proceeded in pseudo-first order. The kinetic studies demonstrated a rate-limiting step in the inhibition reaction, resulting in the formation of a reversible (enzyme.reagent) complex. The calculated KI,app is 0.12 mM at pH 7.58. The rate of inactivation was pH dependent with an extrapolated pK value of approx. 8.6. The enzyme could be protected against inactivation by a poor substrate, pyroglutamyl-valine. The PCMB-inactivated enzyme, that could be reversibly reactivated by mercaptoethanol, failed to react with Z-PGDK. The enzyme was insensitive toward the D-isomer of Z-PGDK and other diazomethyl ketone derivatives of carbobenzoxy amino acids such as Z-L-proline and Z-L-phenylalanine. These results strongly suggest that the Z-PGDK reacts as an affinity label, presumably with a cysteine residue as the site of alkylation in pyroglutamyl-peptidase, as was reported for chloromethyl ketone derivatives of pyroglutamic acid and its N-carbobenzoxy derivative.

A study of a synaptosomal thyrotropin releasing hormone-inactivating pyroglutamate aminopeptidase from bovine brain.

Pyroglutamate aminopeptidase type II is a highly specific membrane-bound neuropeptidase that has the ability to remove N-terminal pyroglutamate (Glp) from Thyrotropin Releasing Hormone (Glp-His-Pro-NH2) or very closely related tripeptides or tripeptide amides. In this paper we report on the purification and characterisation of a pyroglutamate aminopeptidase activity from the synaptosomal membranes of bovine brain. The Triton X-100 solubilised enzyme was purified nearly 600-fold by a combination of conventional column chromatography steps with a recovery/yield of 17.0%. Phase-partitioning experiments with Triton X-114 showed the activity to be an integral membrane protein. This detergent-solubilised pyroglutamate aminopeptidase activity was found to have a relative molecular mass of 240 kDa on a calibrated S-200 column. HPLC analysis on a C18 reverse-phase column showed that the purified activity displayed a very narrow substrate specificity cleaving only Thyrotropin Releasing Hormone (TRH) or the very closely related acid-TRH, LHRH (1-3) and the TRH-analogue (methyl-His)-TRH and had a Km of 100 microM for the fluorimetric substrate Glp-His-Pro-methyl-coumarin. The enzyme was inactivated by the metalchelator 1,10-ortho-phenanthroline but showed less sensitivity to EDTA. It also showed some inhibition by thiol protease inhibitors such as iodoacetate and n-ethyl-maleimide. In summary, we have purified a pyroglutamate aminopeptidase from the synaptosomal membrane of bovine brain. This enzyme displays characteristics consistent with it being classified as a PAP type II neuropeptidase with only minor differences from other proteases in this group.

Regulation of a thyrotropin-releasing hormone-degrading enzyme in GH3 cells: induction of pyroglutamyl peptidase I by 3,5,3'-triiodothyronine.

The effect of exposure of GH3 cells to T3 on the TRH-degrading enzymes pyroglutamyl peptidase I (EC 3.4.19.3) and prolyl endopeptidase (EC 3.4.21.26) was studied. T3 produced a dose-dependent increase in the specific activity of pyroglutamyl peptidase I after 3 days of exposure. The EC50 for T3 was 5 X 10(-10) M. The specific activity of prolyl endopeptidase was unaffected by exposure to T3. The increase in pyroglutamyl peptidase I activity was dependent upon the time of exposure of the cells to this hormone. A maximal effect occurred at 72 h. The stimulation of pyroglutamyl peptidase I by T3 was totally blocked by cycloheximide, indicating that this enzyme is induced in GH3 cells by T3. The effect of T3 on the two TRH-degrading enzymes was also studied in the ACTH-secreting cell line AtT20. T3 had no effect on these enzymes in the AtT20 cell, suggesting that the effect of T3 on pyroglutamyl peptidase I may be cell specific. These studies indicate that the induction of pyroglutamyl peptidase I by T3 may contribute to the negative feedback regulation of T3 levels.

Inhibition of pyroglutamyl peptidase II synthesis by phorbol ester in the Y-79 retinoblastoma cell.

Pyroglutamyl peptidase II (EC 3.4.19.-), a highly specific membrane-bound TRH-degrading enzyme, is inactivated in Y-79 human retinoblastoma cells by exposure to 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a biphasic manner. We have previously demonstrated a rapid decrease in pyroglutamyl peptidase II activity to 10% of the control level within 15 min, which returns to 70% of the control level by 1 h. This decrease results from enzyme phosphorylation by TPA-activated protein kinase-C. We now report a second phase of inactivation after longer exposure of cells to TPA. After 1 h, enzymatic activity slowly and progressively declined. By 7 h, only 15% of control activity remained. Cotreatment of cells with H-7, a protein kinase-C inhibitor, prevented this second phase of inactivation. Immunoblot experiments demonstrated a reduction in the amount of pyroglutamyl peptidase II in Y-79 membranes after long term exposure to TPA. Y-79 cells were labeled with [35S]methionine, and pyroglutamyl peptidase II was immunoprecipitated. A decreased incorporation of [35S]methionine paralleled the decrease in enzyme activity. These studies demonstrate that the second phase of inactivation after exposure to TPA is due to an inhibition of enzyme synthesis.

Rapid inactivation and phosphorylation of pyroglutamyl peptidase II in Y-79 human retinoblastoma cells after exposure to phorbol ester.

Pyroglutamyl peptidase II (EC 3.4.19.-), a membrane-bound metalloproteinase, is a highly specific TRH-degrading enzyme. Exposure of Y-79 human retinoblastoma cells to 12-0-tetradecanoyl phorbol 13-acetate (TPA) decreased the activity of this enzyme in a time- and concentration-dependent manner (IC50 5 x 10(-9) M). After 15 min of TPA treatment, only 10% of pyroglutamyl peptidase II activity remained. TPA treatment did not affect the activity of the cytosolic enzyme pyroglutamyl peptidase I (EC 3.4.19.3) or the membrane-bound enzyme dipeptidyl peptidase IV (EC 3.4.19.3). Pretreatment of the cells with the protein kinase C inhibitors H-7 or sphingosine prevented the inactivation of pyroglutamyl peptidase II by TPA. The time course of the TPA-mediated effect paralleled the time course of translocation and activation of protein kinase C in this cell line. Immunoblot analysis demonstrated that inactivation of pyroglutamyl peptidase II was not due to dissociation or internalization of this enzyme molecule. Incubation of TPA-activated Y-79 cell membranes with gamma-[32P]-ATP followed by immunoprecipitation revealed a time-dependent phosphorylation of a 48 kilodalton subunit of pyroglutamyl peptidase II. These studies indicate that the phorbol ester effect is mediated by protein kinase C, and reveal a mechanism of potentiation of the action of TRH at its target sites.

Inactivation of pyroglutamyl aminopeptidase by N alpha-carbobenzoxy-L-pyroglutamyl chloromethyl ketone.

Pyroglutamyl aminopeptidase [pyrrolidone-carboxylate peptidase: EC 3.4.11.8] from Bacillus amyloliquefaciens was inactivated rapidly and irreversibly by N alpha-carbobenzoxy-L-pyroglutamyl chloromethyl ketone (Z-PGCK). The second-order rate constant of the inactivation was 1.1 x 10(5) M-1.s-1, a value which is comparable to that of the clostripain-TLCK reaction. The D-isomer of this chloromethyl ketone derivative was almost inert toward the enzyme under the same conditions. The inactivation reaction was prevented by the presence of a poor substrate, pyroglutamyl-valine. The PCMB-inactivated enzyme, that was reversibly reactivated by 2-mercaptoethanol, failed to react with Z-PGCK. These results suggest that this chloromethyl ketone derivative reacts as an affinity label, presumably with the active site cysteinyl residue of the enzyme, as was reported for L-pyroglutamyl chloromethyl ketone.

Pyroglutamyl peptidase II inhibition specifically increases recovery of TRH released from rat brain slices.

Pyroglutamyl peptidase II (EC 3.4.19-) is a highly specific membrane-bound thyrotropin releasing hormone (TRH) degrading enzyme. To study the functional significance of pyroglutamyl peptidase II in TRH degradation, we synthesized the reversible inhibitor N-1-carboxy-2-phenylethyl (Nimbenzyl)-histidyl-beta-naphthylamide (CPHNA). CPHNA inhibited the enzyme with a Ki of 8 microM, but had no effect no TRH receptors or no prolyl endopeptidase (EC 3.4.21.26). It weakly inhibited cytosolic pyroglutamyl peptidase I (EC 3.4.19.3). CPHNA at a concentration of 10(-4) M increased both the basal and potassium stimulated recovery of TRH released from hypothalamic slices by approximately two-fold. An even higher recovery was observed in slices from brain regions with relatively high levels of pyroglutamyl peptidase II. CPHNA had no effect on the basal recovery of gamma-aminobutyric acid or Met-enkephalin released from brain slices but decreased the potassium stimulated recovery of both Metenkephalin and gamma-aminobutyric acid. These data further support the involvement of pyroglutamyl peptidase II in the extracellular inactivation of brain TRH.

Evaluation of some substrates and potential inhibitors of tissue pyroglutamyl aminopeptidase I.

A fluorometric enzyme assay was developed to evaluate the ability of a variety of compounds to bind to and/or inhibit pyroglutamyl aminopeptidase I. Among these compounds were a series of chloromethyl ketone analogues of thyrotropin releasing hormone (TRH) which had previously been shown to possess TRH-like activity in the central nervous system and have now been found to be good inhibitors of pyroglutamyl aminopeptidase. Thus, it is suggested that the observed TRH-like CNS activity could derive indirectly from inhibition of endogenous TRH degradation by pyroglutamyl aminopeptidase I.

Specific inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase do not change the in vitro release of TRH or its content in rodent brain.

Pyroglutamyl diazomethyl ketone and N-benzyloxycarbonyl prolyl prolinal, specific inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase respectively, were used to study the possible role of these enzymes in the regulation of thyrotropin releasing hormone turnover. In vitro thyrotropin releasing hormone release by male rat hypothalamic slices was studied. Combined in vitro treatment with 10(-5)M of both inhibitors totally inhibited both enzymatic activities. The treatment did not affect basal or 56 mM K+ induced thyrotropin releasing hormone release or thyrotropin releasing hormone levels in slices. Repeated combined intraperitoneal injections of the two inhibitors for up to 12 hours produced a 70%-95% reduction in mouse brain pyroglutamyl peptidase I specific activity and a 65%-85% reduction in prolyl endopeptidase specific activity. Thyrotropin releasing hormone levels were unaffected by this treatment in all regions tested. The data suggest that these two enzymes are not involved in the intra- or extracellular control of thyrotropin releasing hormone levels in brain or hypophysis.

Possible role of neuropeptide degrading enzymes on thyroliberin secretion in fetal hypothalamic cultures grown in serum free medium.

In the present work, we have looked for the presence of two tissular neuropeptide degrading activities, the pyroglutamate aminopeptidase (PAP) and the post-proline cleaving enzyme (PPCE), in dissociated brain cell cultures. These two activities are present in extracts of cells grown in serum-free medium and are detected at a very low level in incubation media. Depolarization of hypothalamic neurons by 60 mM K+ does not specifically increase the level of PAP and PPCE in the medium. We have also used an inhibitor of PPCE: Z-Gly-ProCHN2. This compound can be left in contact with living cells without any toxicity, and in certain conditions of incubation blocks totally and irreversibly both PAP and PPCE. This blockade results in increased levels of TRH, intracellular as well as released into the medium, spontaneously and upon K+ depolarization. These results evidence the role of degradation processes in the mechanisms regulating peptide turn-over.

Evaluation of the role of prolyl endopeptidase and pyroglutamyl peptidase I in the metabolism of LHRH and TRH in brain.

Intraneuronal peptide regulatory mechanisms are still poorly understood. The cytosolic enzymes prolyl endopeptidase (EC 3.4.21.26) and pyroglutamyl peptidase I (E.C.3.4.19.3) degrade both TRH and LHRH. Previous studies from this laboratory have not supported a role for these enzymes in the control of TRH levels. These studies have now been extended to cell and organ cultures and examine the effects of enzyme inhibition on LHRH. Exposure of dispersed hypothalamic cells or median eminences in culture to Z-Pro-Prolinal and pyroglutamyl diazomethyl ketone, specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I respectively, did not change TRH content or recovery of released TRH. In vivo and in vitro treatment with these inhibitors did not modify the content of LHRH or recovery of this peptide upon release from several brain regions except in the olfactory bulb where an unexpected decrease in levels was observed. Olfactory bulb levels of TRH also decreased but only after prolonged in vivo inhibitor treatment. The decrease in olfactory bulb LHRH and TRH could not be accounted for by enzyme induction and is likely due to a non-specific or indirect effect of the inhibitors on the processing of these peptides. These studies demonstrate that levels of LHRH and TRH in brain are not controlled by cytosolic peptidases.

Metabolism of thyrotropin-releasing hormone in human cerebrospinal fluid. Isolation and characterization of pyroglutamate aminopeptidase activity.

Pyroglutamate aminopeptidase, which catalyzes metabolism of thyrotropin-releasing hormone (TRH) to cyclo(His-Pro), is the major enzyme of TRH metabolism in human CSF. The partially purified CSF pyroglutamate aminopeptidase has a pH optimum between 6.0 and 7.4, and a Km of 15.9 +/- 3.1 microM. A number of potential competitive inhibitors of the enzymatic activity were examined, of which luteinizing hormone-releasing hormone and bombesin were the most effective. An examination of the structure of various peptides that inhibit pyroglutamate aminopeptidase activity indicated that the enzyme generally prefers a substrate having amino-terminal pyroglutamic acid (pGlu) and a COOH-terminal that is either blocked or distant from amino-terminal pGlu. Heavy metals, EDTA and reducing agents inactivated the enyzme, whereas benzamidine, phenylmethylsulfonylfluoride, trypsin inhibitor and alkylating agents had little or no effect on the enzymatic activity. Thiol-oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid), however, considerally inhibited the enzymatic activity. We hypothesize that CSF pyroglutamate aminopeptidase may play a role in the biologic actions of TRH.

Calcium-dependent modulation by ethanol of mouse synaptosomal pyroglutamyl aminopeptidase activity under basal and K(+)-stimulated conditions.

We studied the in vitro effects of ethanol (25, 50 and 100 mM) on pyroglutamyl aminopeptidase activity (pGluAP), which has been reported as thyrotrophin-releasing-hormone-degrading activity. pGluAP was measured in presence or absence of calcium, under basal and K(+)-stimulated conditions, in synaptosomes and their incubation supernatant, using pyroglutamyl-beta-naphthylamide as substrate. In basal conditions, in synaptosomes, pGluAP was inhibited by ethanol in a calcium-independent way. In the supernatant, the response differed depending on the concentration of ethanol. Depolarization with K(+) modified pGluAP in synaptosomes and supernatant depending on the presence or not of calcium. In synaptosomes, in absence of calcium, the activity was inhibited at the highest concentrations of ethanol. In contrast, in the supernatant, under depolarizing conditions, ethanol increases pGluAP in absence of calcium. These changes may be in part responsible of the behavioural changes associated to alcohol intake.

Assessment of the role of TRH in the release of [3H]-dopamine from rat nucleus accumbens-lateral septum slices.

We have studied [3H]-dopamine ([3H]-DA) release from rat nucleus accumbens lateral septum slices in response to various paradigms aimed at increasing endogenous or exogenous thyrotropin releasing hormone (TRH) concentrations in the extracellular space. High KCl concentrations significantly enhanced [3H]-DA release by fourfold. TRH (10(-4) or 5 x 10(-4) M) did not affect [3H]-DA release. The release of [3H]-DA was not stimulated by TRH either in the presence of N-1-carboxy-2-phenylethyl (N(im)benzyl)-histidyl-beta naphthylamide, a specific pyroglutamyl peptidase II inhibitor, or that of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I. None of the peptidase inhibitors modified the [3H]-DA release by themselves. These results suggest that the TRH stimulation of [3H]-DA release in vitro observed in previous studies is not due to peptide inactivation but may be due to a nonspecific effect. TRH enhancement of DA release in nucleus accumbens in vivo may not be the result of a direct effect of TRH on DA terminals.

Purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle.

The purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle are described. The characteristics of leucyl aminopeptidase were as follows: optimum activity was at pH 9.5 in the presence of 5 mmol/l Mg2+ or 0.5 mmol Mn2+. No activation of enzyme activity was obtained following addition of other divalent cations or sulphhydryl reagents. Only the leucyl-AMC and methionyl-AMC derivatives were appreciably hydrolysed. The mol mass was estimated as 280 kDa. Approx. 50% inhibition of activity was obtained following addition of p-hydroxymercuriphenyl sulphonate (10 mumol/l), N-ethyl maleimide (2 mmol/l), o-phenanthroline (5 mmol/l), bacitracin (1 mmol/l), amastatin (1 microgram/ml) and bestatin (0.1 mumol/l); no inhibition of activity was obtained in the presence of phenylmethanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). The following oligopeptides were hydrolysed by the enzyme: luliberin 7-10, proctolin and [Leu5]enkephalin; oligopeptides not appreciably hydrolysed included neurotensin, angiotensin-I, substance-P and bradykinin. Pyroglutamyl aminopeptidase had the following characteristics: optimum activity was at pH 8.5 in the presence of 1 mmol/l dithiothreitol (an absolute requirement for maintenance of enzyme activity). Maximum activity was obtained in the absence of divalent cations. Only the pyroglutamyl-AMC derivative was appreciably hydrolysed. The mol mass of this enzyme was estimated as 22 kDa. Approximately 50% inhibition of activity was obtained on addition of phenanthroline (4 mmol/l) and antipain (7 microgram/ml); no inhibition of activity was obtained following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). Only oligopeptides with a pyroglutamyl N-terminal residue (thyroliberin, neurotensin, and luliberin) were hydrolysed by the enzyme.

Prodrugs of peptides. IV: Bioreversible derivatization of the pyroglutamyl group by N-acylation and N-aminomethylation to effect protection against pyroglutamyl aminopeptidase.

Various N-acyl derivatives and N-Mannich bases of the model compound L-pyroglutamyl benzylamide were synthesized to assess their suitability as prodrug forms for the N-terminal pyroglutamyl residue occurring in several peptides, with the aim of improving peptide delivery characteristics. Whereas pyroglutamyl benzylamide was rapidly hydrolyzed by pyroglutamyl aminopeptidase, the N-acyl derivatives and N-Mannich bases (N-aminomethyl derivatives) were totally resistant to cleavage by the enzyme. On the other hand, these derivatives are readily bioreversible, the conversion to the parent pyroglutamyl amide taking place either by spontaneous hydrolysis at physiological pH, as demonstrated for the N-Mannich bases, or by plasma enzymes, as shown for the N-acyl derivatives. The results suggest that by appropriate N-acylation or N-aminomethylation it may be feasible to protect pyroglutamyl-containing peptides against cleavage by pyroglutamyl aminopeptidase and to obtain a release of the parent peptide in the organism, hence improving the delivery characteristics of such peptides.

Inhibition of a model protease--pyroglutamate aminopeptidase by a natural oligosaccharide gum from Hakea gibbosa.

The purpose of this study was to investigate the effect of the oligosaccharide gum from Hakea gibbosa on the activity of a model protease enzyme pyroglutamate aminopeptidase (5-oxoprolyl peptidase; EC 3.4.19.3) and to elucidate the mechanism responsible for the decreased activity. Enzyme kinetic studies were conducted at 37 degrees C in 100 mM potassium phosphate buffer with 10 mM EDTA, 5% (v/v) glycerol, and 5 mM DTT (pH 8) for 15 min and were performed both in the presence and absence of the gum. Enzymatic activity was determined by a colorimetric assay using the specific substrate L-pyroglutamic acid beta-napthylamide. The enzyme kinetics was studied at various substrate and gum concentrations. The velocity of the reaction was determined by the amount of the product (beta-napthylamine) liberated at each substrate and gum concentration. The Ks and Vmax of the enzyme in the absence of the gum were 24.40+/-2.14 microM and 502.95+/-28.90 nmoles x min(-1) x mg protein(-1), respectively. As the concentration of the gum was gradually increased from 0.1 to 2%, the value of the Vmax decreased from 318.94+/-21.46 to 158.83+/-24.51 nmoles x min(-1) x mg protein(-1) while Ks increased from 17.42+/-4.6 to 63.03+/-1.89 microM. The mechanism for the inhibition of the enzyme by Hakea appeared to be a mixed-linear type (a type of non-competitive inhibition) as suggested from Hanes-Woolf, Dixon and Cornish-Bowden plots. The turnover number, kcat, calculated for the enzyme also decreased from 14.09+/-0.81 to 4.45+/-0.69 min(-1) as the concentration of the inhibitor was incrementally increased from 0 to 2% (w/v). The K(i) and alphaK(i) calculated from Dixon and Cornish-Bowden plots were found to be 0.31+/-0.11% (w/v) and 1.33+/-0.42% (w/v), respectively. The natural gum from Hakea gibbosa was effective in non-competitively inhibiting the enzyme pyroglutamate aminopeptidase. Thus, the natural gum may be a promising additive not only for its sustained-release and mucoadhesive properties as shown previously, but also for its ability to slow the enzymatic degradation of therapeutic polypeptides incorporated in dosage forms.

Benarthin: a new inhibitor of pyroglutamyl peptidase. III. Synthesis and structure-activity relationships.

Benarthin, a new inhibitor of pyroglutamyl peptidase (PG-peptidase), has been isolated from the culture filtrate of Streptomyces xanthophaeus MJ244-SF1. The structure of benarthin has been determined to be L-(2,3-dihydroxybenzoyl)arginyl-L-threonine. This structure was confirmed by the total synthesis of benarthin. Moreover, we synthesized benarthin derivatives to obtain information on the relationship between structure and inhibitory activity. The results indicated that the catechol group of benarthin is the essential moiety for the inhibition of PG-peptidase.