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Prolyl endopeptidase or prolyl oligopeptidase (PEP or POP) is highly expressed in brain, and associated with autism spectrum disorders, dementia, aging and various psychological disorders, such as schizophrenia, mania, and neurodegeneration. To design highly potent and novel POP inhibitors, structure-based virtual screening was carried out using pharmacophore modeling and molecular docking studies. The docking based active compounds [incensole (1), incensole acetate (2), incensone (3), incensfuran (4), and epi-incensole acetate (5)] were selected and their dynamic behavior was studied through molecular dynamic simulation. Later, the top-ranked [predicted active, (1-5)] and lower-ranked [predicted in-active, (6-10)] compounds were tested by in-vitro assay. The in-vitro results showed that all top-ranked compounds (1-5) found significantly active against POP enzyme with IC

Proteases are enzymes that are not only essential for life but also industrially important. Understanding the substrate recognition mechanisms of proteases is important to enhance the use of proteases. The fungus Aspergillus produces a wide variety of proteases, including PEP, which is a prolyl endoprotease from A. niger. Although PEP exhibits amino acid sequence similarity to the serine peptidase family S28 proteins (PRCP and DPP7) that recognize Pro-X bonds in the terminal regions of peptides, PEP recognizes Pro-X bonds not only in peptides but also in proteins. To reveal the structural basis of the prolyl endoprotease activity of PEP, we determined the structure of PEP by X-ray crystallography at a resolution of 1.75 Å. The PEP structure shows that PEP has a wide-open catalytic pocket compared to its homologs. The characteristic catalytic pocket structure of PEP is predicted to be important for the recognition of protein substrates.

Oxidative stress (OS) is a common toxic feature in various neurodegenerative diseases. Therefore, reducing OS could provide a potential approach to achieve neuroprotection. Prolyl oligopeptidase (PREP) is a serine protease that is linked to neurodegeneration, as endogenous PREP inhibits autophagy and induces the accumulation of detrimental protein aggregates. As such, inhibition of PREP by a small-molecular inhibitor has provided neuroprotection in preclinical models of neurodegenerative diseases. In addition, PREP inhibition has been shown to reduce production of reactive oxygen species (ROS) and the absence of PREP blocks stress-induced ROS production. However, the mechanism behind PREP-related ROS regulation is not known. As we recently discovered PREP's physiological role as a protein phosphatase 2A (PP2A) regulator, we wanted to characterize PREP inhibition as an approach to reduce OS. We studied the impact of a PREP inhibitor, KYP-2047, on hydrogen peroxide and ferrous chloride induced ROS production and on cellular antioxidant response in HEK-293 and SHSY5Y cells. In addition, we used HEK-293 and SH-SY5Y PREP knock-out cells to validate the role of PREP on stress-induced ROS production. We were able to show that absence of PREP almost entirely blocks the stressinduced ROS production in both cell lines. Reduced ROS production and smaller antioxidant response was also seen in both cell lines after PREP inhibition by 10 mu M KYP-2047. Our results also revealed that the OS reducing mechanism of PREP inhibition is related to reduced activation of ROS producing NADPH oxidase through enhanced PP2A activation. In conclusion, our results suggest that PREP inhibition could also provide neuroprotection by reducing OS, thus broadening the scope of its beneficial effects on neurodegeneration.

Aspergillus niger prolyl endopeptidase (An-PEP) has become a research focus because of its advantages in specifically cleaving the C-terminal peptide bond of proline residues, especially it was an industrial food-grade acidic PEP. Aqueous two-phase system (ATPS) was first applied for separating An-PEP from fermentation broth. Via response surface method (RSM) experiment, an effectively separation of An-PEP was achieved by ATPS containing27% (w/w) ethanol and 14.5% (w/w) (NH4)2SO4 at pH 6.0 with the recovery of 90.29 ± 0.23% and purification coefficient of 15.35 ± 0.30. The purified An-PEP was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), fourier transform infrared (FTIR) and fluorescence spectrometry. The optimum temperature and pH of An-PEP were 40 °C and 4.5–5.0, respectively. An-PEP was activated and stabilized by Ca2+ but inhibited by Fe3+. The enzymatic application of purified An-PEP was evaluated by hydrolyzing egg white protein (EWP) to prepare bioactive peptides. The obtained hydrolysates had good scavenging ability of OH and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, angiotensin converting enzyme (ACE) inhibitory activity and anti-gout activity. This research realized a low-cost, high-efficiency and simple separation technology of An-PEP and provided a broader idea for the preparation of bioactive peptides and the application of An-PEP.

Prolyl oligopeptidase (POP) is a potential therapeutic target for treatment of several neurological disorders and α-synucleinopathies including Parkinson's disease. Most of the known POP inhibitors failed in the clinical trials due to poor pharmacokinetic properties and blood-brain impermeability. Therefore, a training set of 30 structurally diverse compounds with a wide range of inhibitory activity against POP was used to generate a quantitative pharmacophore model, Hypo 3, to identify potential POP inhibitors with desirable drug-like properties. Validations through test set, cost analysis, and Fisher's randomization methods proved that Hypo 3 accurately predicted the known inhibitors among inactive compounds. Hypo 3 was employed as 3D query for virtual screening on an in-house drug-like chemical database containing compounds with good brain permeability and ADMET parameters. Database screening with Hypo 3 resulted in 99 compounds that were narrowed down to 21 compounds through molecular docking. Among them, five compounds were identified in our earlier studies, while two compounds showed in vitro POP inhibition. The current study proposed new 16 virtually screened compounds as potential inhibitors against POP that possess Gold docking score in the range of 64.61–75.74 and Chemscore of −32.25 to −38.35. Furthermore, the top scoring four hit compounds were subjected to molecular dynamics simulations to reveal their appropriate binding modes and assessing binding free energies. The hit compounds interacted with POP effectively via hydrogen bonds with important active site residues along with hydrophobic interactions. Moreover, the hit compounds had key inter-molecular interactions and better binding free energies as compared to the reference inhibitor. A potential new hydrogen bond interaction was discovered between Hit 2 with the Arg252 residue of POP. To conclude, we propose four hit compounds with new structural scaffolds against POP for the lead development of POP-based therapeutics for neurological disorders.

Changes in prolyl oligopeptidase (PREP) expression levels, protein distribution, and activity correlate with aging and are reported in many neurodegenerative conditions. Together with decreased neuropeptide levels observed in aging and neurodegeneration, and PREP's ability to cleave only small peptides, PREP was identified as a druggable target. Known PREP non-enzymatic functions were disregarded or attributed to PREP enzymatic activity, and several potent small molecule PREP inhibitors were developed during early stages of PREP research. These showed a lot of potential but with variable results in experimental memory models, however, the initial excitement was short-lived and all of the clinical trials were discontinued in either Phase I or II clinical trials for unknown reasons. Recently, PREP's ability to form protein-protein interactions, alter cell proliferation and autophagy has gained more attention than earlier recognized catalytical activity. Of new findings, particularly the aggregation of alpha-synuclein (aSyn) that is seen in the presence of PREP is especially interesting because PREP inhibitors are capable of altering aSyn-PREP interaction in a manner that reduces the aSyn dimerization process. Therefore, it is possible that PREP inhibitors that are altering interactions could have different characteristics than those aimed for strong inhibition of catalytic activity. Moreover, PREP co-localization with aSyn, tau, and amyloid-beta hints to PREP's possible role not only in the synucleinopathies but in other neurodegenerative diseases as well. This commentary will focus on less well-acknowledged non-enzymatic functions of PREP that may provide a better approach for the development of PREP inhibitors for the treatment of neurodegenerative disorders.

Two new iridoids (1–2) and a new decomposition product of valepotriates (3), together with fifteen known compounds (4–18) were isolated from the roots and rhizomes of Valeriana polystachya Smith, a native species from the Pampa Biome. Their structures were elucidated by means of NMR spectroscopy, mass spectrometry and optical rotation. The structures of 3 and 18 were further confirmed by single crystal X-ray diffraction analysis. In the group of the isolated compounds, 6β-hydroxysitostenone, hydroxymaltol and isovillosol were isolated from the Valeriana genus for the first time. The extracts and isolated compounds were evaluated for their in vitro activities against acetylcholinesterase (AChE) and prolyloligopeptidase (POP). Compounds 7, 9 and 11 showed weak inhibitory activity against AChE, while 3 and 5 displayed exceptional POP inhibitory activity, with IC50 values of 5.3 ± 0.07 and 7.9 ± 0.4 μM, respectively.

Polyozellin is a p-terphenyl compound which was isolated from Polyozellus multiplex, and exhibits an inhibitory activity against prolyl oligopeptidase (POP). Its structure was assigned as 1 having a p-terphenyl skeleton including a p-substituted dibenzofuran moiety by spectroscopic analyses and chemical means. This paper describes the total syntheses of the proposed structure 1 for polyozellin and its o-isomer 2, revising the structure of polyozellin to the latter. These syntheses involved a double Suzuki-Miyaura coupling using chlorophenylboronic acid as a common key building block, and Cu mediated Ullmann cyclization as key steps. The inhibitory activities of synthetic compounds against POP and cancer cells were also evaluated.

Proteins are the most prominent bioactive component in deer antler velvet. The aim of the present study was to track the fate of protein of antler velvet by protein digestomics. The peptide profile identified by LC-MS/MS and the in vitro bioactivity of antler velvet aqueous extract (AAE) were investigated in simulated gastrointestinal digestion. A total of 23, 387 and 417 peptides in AAE, gastric and pancreatic digests were identified using LC-MS/MS, respectively. Collagens, the predominant proteins, released 34 peptides in gastric digests and 146 peptides in pancreatic digests. The gastric and pancreatic digests presented dipeptidyl peptidase IV (DPP-IV) and prolyl endopeptidase (PEP) inhibition activities. Four peptides from digests were proved to be DPP-IV and PEP inhibitory peptides. The results showed that the peptides released from antler velvet protein contributed to the bioactivity of antler velvet during digestion.

Introduction: Prolyl oligopeptidase (prolyl endopeptidase, Prep), a multifunctional protease hydrolyzing -Pro-X-peptide bonds, is highly expressed in the mouse placenta, but the function during development is not known. We explored the possibility of Prep's involvement in placental differentiation. Methods: We cultured trophoblast stem cells (TSCs) derived from the E6.5 mouse embryo and investigated the detailed expression pattern of Prep during their differentiation. Prep-specific inhibitors were added to the TSC culture, and the effect on the differentiation was assessed by microscopic observation and the expression of marker gene for each placental cell. Results: During TSC differentiation for 6 days, Prep was constantly detected at mRNA, protein, and activity levels, and the proteinwas found mainly in the cytoplasm. The addition of 30 mu M and 10 mu M SUAM-14746, a Prep-specific inhibitor, effectively inhibited the differentiation into spongiotrophoblasts (SpTs) and trophoblast giant cells (TGCs), while the TSC viability was not affected. 5 mu M SUAM-14746 impaired the differentiation into SpTs, and 1 mu M SUAM-14746 exhibited no effects. Another Prep-specific inhibitor, KYP-2047, did not affect the differentiation. We confirmed efficient inhibition of Prep enzymatic activity in TSCs by both inhibitors. Conclusion: The dose-dependent effect of SUAM-14746 on TSCs suggests that Prep plays an important role in the differentiation into SpTs and TGCs in the mouse placenta. (C) 2017 Elsevier Ltd. All rights reserved.

Cognitive deficits are considered a key feature of schizophrenia, and they usually precede the onset of the illness and continue after psychotic symptoms appear. Current antipsychotic drugs have little or no effect on the cognitive deficits of this disorder. Prolyl oligopeptidase (POP) is an 81-kDa monomeric serine protease that is expressed in brain and other tissues. POP inhibitors have shown neuroprotective, anti-amnesic and cognition-enhancing properties. Here we studied the potential of IPR19, a new POP inhibitor, for the treatment of the cognitive symptoms related to schizophrenia. The efficacy of the inhibitor was evaluated in mouse models based on subchronic phencyclidine and acute dizocilpine administration, and in adult offspring from mothers with immune reaction induced by polyinosinic:polycytidylic acid administration during pregnancy. Acute IPR19 administration (5 mg/kg, i.p.) reversed the cognitive performance deficits of the three mouse models in the novel object recognition test, T-maze, and eight-arm radial maze. The compound also ameliorates deficits of the prepulse inhibition response. The in vitro inhibitory efficacy and selectivity, brain penetration and exposure time after injection of IPR19 were also addressed. Our results indicate that the inhibition of POP using IPR19 may offer a promising strategy to develop drugs to ameliorate the cognitive deficits of schizophrenia.

Prolyl oligopeptidase (POP, EC 3.4.21.26) is a cytosolic serine protease that hydrolyses proline containing small peptides. The members of prolyl oligopeptidase family play important roles in many physiological processes such as neurodegenerative diseases, maturation and degradation of peptide hormones. Thus the enzyme has been purified and characterized from various sources to elucidate the potential use as therapeutics. In this study recombinant Myxococcus xanthus prolyl oligopeptidase expressed in E. coli was purified 60.3 fold, using metal-chelate affinity and gel permeation chromatography. The recombinant enzyme had a monomeric molecular weight of 70 kDa. Isoelectric point of the enzyme was found to be approximately 6.3 by two-dimensional polyacrylamide gel electrophoresis. The optimum pH and temperature was estimated as 7.5 and 37 °C, respectively. The purified enzyme was stable in a pH range of 6.0–8.5 and thermally stable up to 37 °C. The K m and V max values were 0.2 mM and 3.42 μmol/min/mg. The proteolytic activity was inhibited by active-site inhibitors of serine protease, Z-Pro-Prolinal, PMSF, and metal ions, Cd 2+ , and Hg 2+ . Furthermore, the hydrolysis efficiency of the recombinant prolyl oligopeptidase was investigated with wheat gluten.

Prolyl oligopeptidase (PREP) has been implicated in neurodegeneration and neuroinflammation and has been considered a drug target to enhance memory in dementia. However, the true physiological role of PREP is not yet understood. In this paper, we report the phenotyping of a mouse line where the PREP gene has been knocked out. This work indicates that the lack of PREP in mice causes reduced anxiety but also hyperactivity. The cortical volumes of PREP knockout mice were smaller than those of wild type littermates. Additionally, we found increased expression of diazepam binding inhibitor protein in the cortex and of the somatostatin receptor-2 in the hippocampus of PREP knockout mice. Furthermore, immunohistochemistry and tail suspension test revealed lack of response of PREP knockout mice to lipopolysaccharide insult. Further analysis revealed significantly increased levels of polysialylated-neural cell adhesion molecule in PREP deficient mice. These findings might be explained as possible alteration in brain plasticity caused by PREP deficiency, which in turn affect behaviour and brain development.

Incubation of sodium caseinate (NaCN) and purified α-casein (αs-CN) with an Aspergillus niger derived prolyl endoproteinase (An-PEP) for 1, 2, 3, 4, 8 and 24 h resulted in the generation of potent angiotensin converting enzyme (ACE) inhibitory hydrolysates. An ACE IC50 of 21.1±5.1 μg/ml was obtained on incubation of An-PEP with NaCN for 4 h. Fractionation of the NaCN hydrolysates using 3 kDa centrifugal filters resulted in highly active permeate fractions, the most potent being obtained from the 3 h hydrolysate (ACE IC50=2.9±0.3 μg/ml). The hydrolytic specificity of An-PEP for purified α-CN was assessed using UPLC ESI MS/MS. The analysis confirmed An-PEP's cleavage preference for the C-terminal side of Pro and also confirmed that An-PEP has the ability to cleave at the C-terminal of Ala, Leu, Arg and His residues.

Berberine, a naturally occurring compound, possesses an interesting multipotent pharmacological profile potentially applicable for Alzheimer’s disease (AD) treatment. In this study, a series of novel 22 berberine derivatives was developed and tested in vitro. Berberine core was substituted at position 9-O of its aromatic ring region. All the hybrids under the study revealed multi-targeted profile inhibiting prolyl oligopeptidase, acetylcholinesterase and butyrylcholinesterase highlighting 4a, 4g, 4j, 4l and 4s possessing balanced activities in the micromolar range. The top-ranked candidates in terms of the most pronounced potency against POP, AChE and BChE can be classified as 4d, 4u and 4v, bearing 4-methylbenzyl, (naphthalen-2-yl)methylene and 1-phenoxyethyl moieties, respectively. In vitro data were corroborated by detailed kinetic analysis of the selected lead molecules. 4d, 4u and 4v were also inspected for their potential to inhibit aggregation of two abberant proteins in AD, namely amyloid beta and tau, indicating their potential disease-modifying properties. To explain the results of our study, we carried out docking simulation to the active sites of the respective enzyme with the best berberine derivatives, along with QSAR study. We also investigated compounds’ potential permeability through blood-brain barrier by applying parallel artificial membrane permeation assay and addressed their cytotoxicity profile.

A novel prolyl endopeptidase from Stenotrophomonas maltophilia, SmPEP, was discovered and characterized. The specific activity of the recombinant SmPEP expressed by Escherichia coli BL21 (DE3), was 68.3 U/mg at pH 8.0 and 37 °C. In order to improve the substrate specificity for long-chain peptide, rational design was applied based on the structure constructed by homology modeling. Inter-domain sites within the β-propeller domain were chosen for the mutation to weaken the inter-domain interaction and form an open conformation for long-chain substrate entering into the active site. The substrate specificity on a designed long-chain substrate, PQPQLPYPQPQLP, of the mutants F263A and E184 G increased 8.77 and 5.75 times respectively versus wild-type. After the saturated mutation of the both sites, the reactive rate of mutant F263 V on 13-mer peptide was 10.2 times higher than that of the wild-type. Then the mutant F263 V was used in the hydrolysis of casein, and the ACE inhibitory activity of the hydrolysate was significantly improved compared with wild type enzyme, which verified the efficiency of the design strategy.

Fibroblast activation protein (FAP) is a non-classical serine protease expressed predominantly in conditions accompanied by tissue remodeling, particularly cancer. Due to its plasma membrane localization, FAP represents a promising molecular target for tumor imaging and treatment. The unique enzymatic activity of FAP facilitates development of diagnostic and therapeutic tools based on molecular recognition of FAP by substrates and small-molecule inhibitors, in addition to conventional antibody-based strategies. In this review, we provide background on the pathophysiological role of FAP and discuss its potential for diagnostic and therapeutic applications. Furthermore, we present a detailed analysis of the structural patterns crucial for substrate and inhibitor recognition by the FAP active site and determinants of selectivity over the related proteases dipeptidyl peptidase IV and prolyl endopeptidase. We also review published data on targeting of the tumor microenvironment with FAP antibodies, FAP-targeted prodrugs, activity-based probes and small-molecule inhibitors. We describe use of a recently developed, selective FAP inhibitor with low-nanomolar potency in inhibitor-based targeting strategies including synthetic antibody mimetics based on hydrophilic polymers and inhibitor conjugates for PET imaging. In conclusion, recent advances in understanding of the molecular structure and function of FAP have significantly contributed to the development of several tools with potential for translation into clinical practice.

The plasma activity of nine aminopeptidases was monitored over a year in first-episode psychotic patients. We observed significant differences in aminopeptidase B (APB), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPPIV), but not in puromycin-sensitive aminopeptidase (PSA), prolyl endopeptidase (PEP), cysteine aminopeptidase (Cys-AP), aspartate aminopeptidase (Asp-AP), glutamate aminopeptidase (Glu) or piroglutamate aminopeptidase (PGI) in these patients compared to controls, and also a progressive increase in plasma activity, correlated to changes in scores on clinical scales, Global Assessment of Functioning scale (GAF) and Hamilton Depression Rating Scale (HDRS), at 1 month of follow-up. At 1 month after diagnosis, the median score obtained by patients on the GAF was negatively associated with the plasma activity of APB and PEP measured at the beginning of the psychotic episode, indicating a role as a negative prognostic factor that can predict psychiatric symptomatology. In the case of HDRS, scores at 1 month after diagnosis were found to be positively associated with the initial plasma activity of DPPIV, APN and PSA, indicating that their initial elevation is a negative prognostic factor that can predict subsequent depressive symptomatology. Taken together, these results suggest a pathophysiological involvement of plasma peptidases and indicate that aminopeptidase activity can predict the course of first-episode psychosis patients, acting as a prognostic indicator.

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyses proline-containing peptides at the carboxy terminus of proline residues. It has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore may have important clinical implications. Thirty-one isoquinoline alkaloids of various structural types, previously isolated in our laboratory, were screened for their ability to inhibit prolyl oligopeptidase. Promising results have been showed by alkaloids californidine (IC50=55.6±3.5 μM), dihydrosanquinarine (IC50=99.1±7.6 μM), corypalmine (IC50=128.0±10.5 μM) and N-methyllaurotetanine (IC50=135.0±11.7 μM).

According to the World Health Organization (WHO), approximately 450 million people suffer from mental or neurological disorders and five of the ten leading causes of disability and premature death worldwide are psychiatric conditions. Social, biological and neurological sciences provided extensive understanding into the role of risk and protective factors in the development of mental disorders and poor mental health. Altered activity of a number of enzymes, such as prolyl endopeptidase (PEP, EC 3.4.21.26), has been linked to the prevention and treatment of a number of mental disorders, including anxiety, depression and Alzheimer's disease. The inhibition of PEP has potential for use in the prevention and in the treatment of mental disorders. The objective of this work was to identify PEP-inhibitory peptides from meat proteins using in silico methods. In this paper, five proteins commonly found in meat by-products were evaluated as a substrate for use in the generation of PEP inhibitory peptides. These include serum albumin, collagen and myosin. These proteins were cleaved in silico using BIOPEP and ExPASy PeptideCutter and the generated peptides were compared to known PEP-inhibiting peptides in the database of BIOPEP. A number of novel PEP inhibitory peptide sequences were identified in this study, including PPL, APPH, IPP and PPG with corresponding IC50 values of 2.86, 3.95, 4.02 and 2.70 mM, respectively. This work demonstrates the usefulness of in silico analysis for predicting the release of PEP-inhibiting peptides from meat proteins.

The characteristics and functional properties of gelatine from freshwater fish skin (Barbus callensis) were investigated. The gelatine extraction efficiency was improved by an acid-swelling process in the presence of barbel crude acid protease extract. Barbel skin gelatine (BSG) contained 92.15% protein, 0.31% lipid and 0.72% ash. The amino acid profile of BSG showed a high percentage of imino acids. The electrophoretic profile showed that BSG is mainly composed of α- and β-components. BSG showed an excellent solubility and possessed interfacial properties, which were governed by the protein concentration. Biological activities of the hydrolysates obtained after digestion of BSG with several commercial proteases were evaluated. The results suggested that these hydrolysates are a good source of natural inhibitors of dipeptidyl peptidase-IV and prolyl endopeptidase and could potentially be used as dietary ingredients in the management of type 2-diabetes and/or neuropathological disorders. © 2014 Elsevier Ltd. All rights reserved., This work was funded by Ministry of Higher Education and Scientific Research, Tunisia, and also by the Spanish Ministry of Foreign Affairs (MAEC-AECID).

Tumor microenvironments (TMEs) are composed of cancer cells, fibroblasts, extracellular matrix, microvessels, and endothelial cells. Two prolyl endopeptidases, fibroblast activation protein (FAP) and prolyl oligopeptidase (POP), are commonly overexpressed by epithelial-derived malignancies, with the specificity of FAP expression by cancer stromal fibroblasts suggesting FAP as a possible therapeutic target. Despite overexpression in most cancers and having a role in angiogenesis, inhibition of POP activity has received little attention as an approach to quench tumor growth. We developed two specific and highly effective pseudopeptide inhibitors, M83, which inhibits FAP and POP proteinase activities, and J94, which inhibits only POP. Both suppressed human colon cancer xenograft growth >90% in mice. By immunohistochemical stains, M83- and J94-treated tumors had fewer microvessels, and apoptotic areas were apparent in both. In response to M83, but not J94, disordered collagen accumulations were observed. Neither M83- nor J94-treated mice manifested changes in behavior, weight, or gastrointestinal function. Tumor growth suppression was more extensive than noted with recently reported efforts by others to inhibit FAP proteinase function or reduce FAP expression. Diminished angiogenesis and the accompanying profound reduction in tumor growth suggest that inhibition of either FAP or POP may offer new therapeutic approaches that directly target TMEs.

α-Synuclein (aSyn) aggregation, mitochondrial dysfunction and oxidative damage has been shown to be related to the death of dopaminergic neurons in Parkinson's disease (PD). Prolyl oligopeptidase (PREP) is proposed to increase aSyn aggregation, and PREP inhibition has been shown to inhibit the aggregation process in vitro and in vivo. In this study, we investigated the effects of a specific PREP inhibitor, KYP-2047, on rotenone induced aSyn aggregation and increased the production of reactive oxygen species (ROS) in cells overexpressing A53T mutation of aSyn. Rotenone, a mitochondrial toxin that induces oxidative damage and aSyn aggregation, associated with PD pathology, was selected as a model for this study. The results showed that rotenone induced the formation of high-molecular-weight aSyn oligomers, and this was countered by simultaneous incubation with KYP-2047. Inhibition of PREP also decreased the production of ROS in [A53T]aSyn overexpressing cells, leading to improved cell viability.

Several investigative prolyl oligopeptidase (PREP) inhibitors have been shown to improve learning and memory in various preclinical trials but the mechanism of action behind these effects remains unclear. Since hippocampal and cortical acetylcholine (ACh) is known to play an important role in cognitive processes, the effects of two potent PREP inhibitors, JTP-4819 and KYP-2047, on extracellular ACh levels in hippocampus and medial prefrontal cortex were assessed using in vivo microdialysis. Conscious rats were treated with a single dose (15 or 50 μmol/kg i.p.) of JTP-4819, KYP-2047 or vehicle, and extracellular ACh levels were monitored for 5 h after treatment. In hippocampus, KYP-2047 had no significant effect on the ACh levels, although a trend towards decreased levels was observed at the higher dose. JTP-4819 had no significant effect on the hippocampal ACh levels at the lower dose (15 μmol/kg), but the higher dose (50 μmol/kg) significantly decreased ACh levels in hippocampus by about 25%. In cortex, the smaller dose (15 μmol/kg) of KYP-2047 decreased ACh levels maximally by 25%, and a similar (ns) effect was also observed after the higher dose. JTP-4819 had no effect at the lower dose, but the higher dose decreased ACh levels maximally by about 30%. In conclusion, the present results suggest that the cognition-enhancing effects of investigative PREP inhibitors are not due to enhanced cholinergic transmission in hippocampus or cortex.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway conveys signals from receptor tyrosine kinases (RTKs) to regulate cell metabolism, proliferation, survival, and motility. Previously we found that prolylcarboxypeptidase (PRCP) regulate proliferation and survival in breast cancer cells. In this study, we found that PRCP and the related family member prolylendopeptidase (PREP) are essential for proliferation and survival of pancreatic cancer cells. Depletion/inhibition of PRCP and PREP-induced serine phosphorylation and degradation of IRS-1, leading to inactivation of the cellular PI3K and AKT. Notably, depletion/inhibition of PRCP/PREP destabilized IRS-1 in the cells treated with rapamycin, blocking the feedback activation PI3K/AKT. Consequently, inhibition of PRCP/PREP enhanced rapamycin-induced cytotoxicity. Thus, we have identified PRCP and PREP as a stabilizer of IRS-1 which is critical for PI3K/AKT/mTOR signaling in pancreatic cancer cells.

Kynapcin-12 is a prolyl oligopeptidase (POP) inhibitor isolated from Polyozellus multiplex, and its structure was assigned as 1 having a p-hydroquinone moiety by spectroscopic analyses and chemical means. This Letter describes the total syntheses of the proposed structure 1 for kynapcin-12 and 2′,3′-diacetoxy-1,5′,6′,4″-tetrahydroxy-p-terphenyl 2 isolated from Boletopsis grisea, revising the structure of kynapcin-12 to the latter. These syntheses involved double Suzuki–Miyaura coupling, CAN oxidation, and LTA oxidation as key steps. The inhibitory activities of synthetic compounds against POP and cancer cells were also evaluated.

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2–S′2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.

Prolyl oligopeptidase (PREP) cleaves short peptides at the C-side of proline. Although several proline containing neuropeptides have been shown to be efficiently cleaved by PREP in vitro, the actual physiological substrates of this peptidase are still a matter of controversy. The aim of this study was to evaluate the changes in the peptidome of rat tissues caused by a repeated 4-day administration of the potent and specific PREP inhibitor KYP-2047, using our recently developed iTRAQ-based technique. We found tissue-dependent changes in the levels of specific subsets of peptides mainly derived from cytosolic proteins. Particularly in the kidney, where the levels of cytochrome c oxidase were found decreased, many of the altered peptides originated from mitochondrial proteins being involved in energy metabolism. However, in the hypothalamus, we found significant changes in peptides derived front hormone precursors. We could not confirm a role of PREP as the metabolising enzyme for beta-endorphin, galanin, octadecaneuropeptide, neuropeptide-glutamic acid-isoleucine, substance P. somatostatin, enkephalin and neuropeptide Y. Furthermore, changes in the degradation patterns of some of these neuropeptides, and also most of those derived from other larger proteins, did not follow specificity to proline. After a 4-day treatment, we found a significant amount of peptides, all derived from secreted pro-proteins, being cleaved with pair of basic residue specificity. In vitro experiments indicated that PREP modifies the endogenous dibasic residue specific proteolysis, in a KYP-2047 sensitive v/ay. These findings suggest that PREP may act indirectly within the routes leading to the specific peptide changes that we observed. The data reported here suggest a wider tissue specific physiological role of l'REP rather than the mere metabolism of proline containing active peptides and hormones. (C) 2012 Elsevier Masson SAS. All rights reserved.

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189-209 (loop A), 577-608 (loop B), and 636-646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.

Prolyl oligopeptidase (PREP, EC 3.4.21.26) hydrolyzes neuropeptides, such as neurotensin and substance P in vitro, but its importance in the in vivo metabolism of these peptides has not been proved. This is the first report where intracerebral microdialysis combined with highly sensitive radioimmunoassay has been used to investigate the effect of PREP inhibition on the brain extracellular peptide levels in conscious rats. We show that PREP inhibition by KYP-2047 (50 μmol/kg = 17 mg/kg, intraperitoneally, that effectively inhibits PREP in the brain), has no effect on the neurotensin and substance P levels in the striatum extracellular space. This provides a further piece of evidence in support of the proposition that PREP is not significantly responsible for the in vivo cleavage of substance P or neurotensin, and that occasional positive cognitive effects associated with some PREP inhibitors are not mediated through elevated extracellular levels of these peptides. Direct regulation of peptide processing by PREP is not likely because the enzyme is located intracellularly and the peptide substrates are mostly extracellular.

Background and objective Resistance of thrombi to plasmin digestion depends primarily on the amount of α(2)-antiplasmin (α(2)AP) incorporated within fibrin. Circulating prolyl-specific serine proteinase, antiplasmin-cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met-α(2)AP between -Pro12-Asn13- to yield Asn-α(2)AP, which is crosslinked to fibrin approximately 13× more rapidly than Met-α(2)AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met-α(2)AP to Asn-α(2)AP and thereby enhance endogenous fibrinolysis. Methods and results We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl-Arg-(8-amino-3,6-dioxaoctanoic acid)-D-Ala-L-boroPro selectively inhibited APCE vs. DPPIV, with an apparent K(i) of 5.7 nm vs. 6.1 μm, indicating that an approximately 1000-fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent K(i) of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose-dependent decrease of APCE-mediated Met-α(2)AP cleavage, which ultimately shortened plasminogen activator-induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC(50) value in plasma remaining comparable to that in phosphate buffer. Conclusion These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.

Prolyl endopeptidase (PEP) is a serine protease that cleaves small peptides at the carboxyl side of L-proline. PEP has been reported to have important functions in the brain being implicated in learning and memory processes, psychological disorders and neurodegenerative diseases. Several PEP substrates have been shown to play a role during brain development and this observation led us to investigate the expression of PEP mRNA in the rat brain and spinal cord, from embryo to adult stages. In situ hybridization revealed that PEP mRNA is expressed early, from embryonic day 15, notably in germinative areas including the neocortical, hippocampal, pallidal, thalamic, anterior hypothalamic, tectal, cerebellar, pontine and medullary neuroepithelia. PEP mRNA was also found in the differentiating fields of the olfactory bulb, the orbital and cingulate cortex, the hippocampal formation, the cortical plate and the subventricular zone of the cortex. Quantitative RT-PCR analysis in various brain areas and the spinal cord showed that PEP mRNA levels are more abundant during the perinatal stages, coinciding with a period of neuronal migration and differentiation. From then on, PEP mRNA expression decreased, reaching its lowest levels at adulthood. Overall, the present data support the possibility that PEP exerts specific functions related to neurodevelopment besides those proposed to date.

Prolyl oligopeptidase (POP) is a serine protease highly expressed in the brain that hydrolyses peptide bonds at the carboxyl terminal of prolyl residues. There is evidence that this enzyme participates in several functions of the central nervous system. Scutellaria racemosa Pers demonstrated significant and selective POP inhibition. Fractionation of the hydroalcoholic extract resulted in the isolation of four main constituents identified for the first time from S. racemosa Pers, the triterpenoid lupeol (1) and the flavonoids oroxylin A (5,7-dihydroxy-6-methoxyflavone, 2), hispidulin (4',5,7-trihydroxy-6-methoxyflavone, 3), and oroxyloside (oroxylin A 7-O-glucuronide, 4). Inhibitory assays indicated that 3 and 4 at a concentration of 100 microM inhibit 43 and 34% of total POP activity, respectively.

Prolyl endopeptidase (EC 3.4.21.26) (PEP) is a serine peptidase that converts several biologically active peptides. This enzyme has been linked to several neurological, digestive, cardiovascular and infectous disorders. However, little is known about its involvement in neoplastic processes. This study analyzes fluorimetrically cytosolic and membrane-bound PEP activity in a large series (n=122) of normal and neoplastic tissues from the kidney, colon, oral cavity, larynx, thyroid gland and testis. Cytosolic PEP activity significantly increased in clear cell renal cell carcinoma, urothelial carcinoma of the renal pelvis and head and neck squamous cell carcinoma. Both cytosolic and membrane-bound PEP activity were also increased in colorectal adenomatous polyps. These data suggest the involvement of PEP in some mechanisms that underlie neoplastic processes.

The broad inhibitory spectrum of aldehydes and the possibility that amino acid residues modulate their specificity point to the potential of using peptidyl aldehydes as activity-based probes. Here, we establish the potential of peptidyl aldehydes in activity-based proteomics by synthesizing different probes and using them to specifically label a well-known serine protease in an activity-dependent manner. From our results, peptidyl aldehydes emerge as promising activity-based probes that enable multiple enzymatic-class detection by substrate recognition and can be used in diverse activity-based proteomics applications like protein identification and activity profiling.

The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, alpha-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstream of the toxin regions and as the last predicted amino acid in the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide and was inhibited by the specific POP inhibitor benzyloxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.

Seasonal quantitative patterns of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolyl-imino (PIP) aminopeptidases and prolyl oligopeptidase (POP) activities in soluble (SF) and solubilized membrane-bound (MF) fractions from ductus deferens, vagina and uterus were studied to evaluate their relationships with the reproductive cycle and the extensive long-term spermatozoa storage (LTSS) of the Neotropical rattlesnake Crotalus durissus terrificus. APB, PIP and POP were detected only in SF, while other peptidases were detected in SF and MF. APB, APN-PI and APN-PS were predominant in most tissues in all seasons. Peptidase activities had a common pattern of increment during the dry season (winter/autumn), which coincides with the mating period (autumn) and LTSS in the female (winter), as well as the reduction of spermatozoa motility and maintenance of fertilization capacity of spermatozoa. The high CAP activity in the soluble fraction of the vagina during winter, compared to summer (time of parturition) and spring, coincides with the relaxation of this tissue. In the soluble fraction, the low PAP-I activity of the ductus deferens coincided with its high activity in the vagina during the winter; and the inverse occurred in summer, which is consistent with the physiological process of preserving spermatozoon viability. In conclusion, the studied peptidase activities had seasonal and tissue-specific characteristics, which suggest a relevant role in the reproductive physiology of C. d. terrificus.

Prolyl oligopeptidase (POP) is an endopeptidase which cleaves short proline-containing neuropeptides, and it is involved in memory and learning. POP also has an intercellular function mediated through the inositol pathway, and has been involved in cell death. POP has been early considered as a housekeeping enzyme, but the recent research indicates that POP expression is regulated across tissues and intracellularly. In the brain, POP is exclusively expressed in neurons and most abundantly in pyramidal neurons of cerebral cortex, in the CA1 field neurons of hippocampus and in cerebellar Purkinje's cells. Intracellularly, POP is mainly present in the cytoplasm and some in intracellular membranes, like rough endoplasmic reticulum and Golgi apparatus. In this paper, we systematically studied the levels of expression of POP along the life of cerebellar granule cells (CGC) in culture and the distribution of POP within different intracellular compartments. We used the tight-binding inhibitor JTP-4819 covalently coupled with fluorescein (FJTP) as a tool to study the changes on expression and localization of POP protein. Our results indicate that POP activity levels are regulated during the life of the neurons. POP was found mainly in cytoplasm and neuronal projections, but at an early developmental phase significant amounts were found also in nuclei. Along the life of the neurons, POP activity fluctuated in 7-day cycles. In young neurons, the cytosolic POP activity was low but increased by maturation so that the activity peak coincided with full differentiation. Over aging, cytoplasmic POP was concentrated around nucleus, but the activity decreased with time. POP was also present in vesicles across the neuron. No major changes were seen in the nuclear or membrane bound POP over aging until activity disappeared upon neuronal death. This is the first time when POP was found in the nuclei of human neuronal cells. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized within the catalytic site of a human chimeric POP protein which provided a more detailed understanding of how these inhibitors interacted with the key residues within the catalytic pocket.

Prolyl oligopeptidase (POP, EC 3.4.21.26) is a member of a family of serine peptidases with post-proline cleaving activity towards peptides. It is located in the cytosol in active form but without hydrolytic activity on proteins or peptides higher than 30 amino acids. Its function is not well defined, but it is involved in central nervous system disorders. Here, we studied the substrate specificity of wild type POP (POPwt) and its C255T variant lacking the non-catalytic Cys(255). This residue is located in the seven-bladed beta-propeller domain that regulates the activity of POP. Fluorescence resonance energy transfer (FRET) peptides were used with sequences derived from bradykinin-containing region of human kininogen and flanked by Abz (ortho-aminobenzoic acid) and EDDnp [N-ethylenediamine-(2,4-dinitrophenyl)]. The peptide Abz-GFSPFRQ-EDDnp was taken as leader substrate for the synthesis of five series of peptides modified at the P(3), P(2), P'(1), P'(2) and P'(3) residues. The optimal amino acids in each position for POPwt resulted in the sequence RRPYIR that is very similar to the C-terminal sequence of neurotensin. The cyclic peptides c(G((n))FSPFR) (n=1-4) were hydrolyzed by POP; their cycloretro and cycloretro-inverso analogues were inhibitors in the micromolar range. The differences between POPwt and its C255T mutant in the hydrolysis of the series derived from Abz-GFSPFRQ-EDDnp were restricted to the non-prime site of the substrates. The kinetic data of hydrolysis and inhibition by the cyclic peptides are consistent with the structures of POP-substrate/inhibitor complexes and with the substrate specificity data obtained with linear FRET peptides. All together, these results give information about the POP-substrate/inhibitor interactions that further complete knowledge of this important oligopeptidase.

Real-time (TaqMan) PCR assays were developed to detect the strawberry angular leaf spot pathogen Xanthomonas fragariae ( Xf ) and the strawberry bacterial blight pathogen Xanthomonas arboricola pv. fragariae ( Xaf ). The Xf PCR ( Xf gyrB ) was designed within regions of the gyraseB gene, unique to Xf , after generating gyraseB DNA sequence data from Xf and other closely related strains. The Xaf PCR ( Xaf pep ) was designed within regions of the pep prolyl endopeptidase gene that were unique to Xaf , after generating pep DNA sequence data from Xf and Xaf strains. The Xf gyrB PCR detected only Xf strains amongst a panel of 20 Xanthomonas -related spp. and pathovars. The Xaf pep PCR assay detected all Xaf strains tested plus two other (of three tested) X. arboricola pathovars. An existing genomic DNA extraction protocol was modified to facilitate detection of both pathogens to 10 3 cells per strawberry leaf disc.

The prolyl oligopeptidase TNA1_POP was found to be encoded in the genome of the hyperthermophilic archaeon Thermococcus sp. NA1 and showed high similarities to its archaeal homologs (76-83%). The enzyme was found to be a single polypeptide composed of 616 amino acids with conserved signature domains. A recombinant TNA1_POP expressed in Escherichia coli was capable of hydrolyzing succinyl-Ala-Pro-p-nitroanilide (Suc-Ala-Pro-pNA) with temperature and pH optimums of 80 degrees C and 7, respectively. TNA1_POP activity appeared to be significantly activated by pre-incubation at 80 degrees C and 90 degrees C with the optimum temperature unchanged. The heat-activated enzyme exhibited a k(cat) approximately twofold higher than that of the unheated enzyme, however, both enzymes showed the same K(m). TNA1_POP was thermostable at 80 degrees C retaining 80% of its heat-activated activity even after 23 h, but it lost its enzymatic activity at 90 degrees C with a half-life of 3 h. The loss of the enzymatic activity at 90 degrees C seemed to be caused by the autodegradation of the enzyme, not by thermal denaturation, as supported by circular dichroism spectropolarimetry. Autodegradation fragments ranging from 2 to 18 kDa were mapped by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

Prolyl endopeptidase and pyroglutamyl peptidase I are enzymes which participate in the degradation of thyrotropin-releasing hormone (TRH), a hormone which is thought to play an important role in the development of organs and tissues. Here, we have characterized the ontogeny of TRH degrading enzyme activity in the brain cortex, lung, heart, kidney and liver. Overall, prolyl endopeptidase activity was found to be 2 to 5 fold higher in newborn vs. adult rat tissues, with the exception of the soluble form in the liver and the particulate form in the lung. In contrast, the developmental profile of pyroglutamyl peptidase I activity was found to be more variable and tissue dependent. These results corroborate the idea that both enzymes play important, tissue-specific roles during the development and maturation of rat organs.

The structure-activity relationship of various N-acyl-Gly-, N-acyl-Sar-, and N-blocked-boroPro derivatives against three prolyl peptidases was explored. Several N-acyl-Gly- and N-blocked-boroPro compounds showed low nanomolar inhibitory activity against fibroblast activation protein (FAP) and prolyl oligopeptidase (POP) and selectivity against dipeptidyl peptidase-4 (DPP4). N-Acyl-Sar-boroPro analogs retained selectivity against DPP4 and potent POP inhibitory activity but displayed decreased FAP inhibitory activity.

In order to replace the P2–P1 amide group, different 1-cycloalkenyls and 2-aryls were studied in the place of the P1 pyrrolidine group of a 4-phenylbutanoyl- l -Pro-pyrrolidine structure, which is a well-known prolyl oligopeptidase inhibitor SUAM-1221. The 1-cyclopentenyl and the 2-thienyl groups gave novel compounds, which were equipotent with the corresponding pyrrolidine-analog SUAM-1221. It was shown that the P2–P1 amide group of POP inhibitors can be replaced by an α,β-unsaturated carbonyl group or the aryl conjugated carbonyl group.

A series of ionizable prolyl oligopeptidase inhibitors were developed through the introduction of a pyridyl group to the P3 position of the prolyl oligopeptidase inhibitor structure. The study was performed on previously developed prolyl oligopeptidase inhibitors with proline mimetics at the P2 position. The 3-pyridyl group resulted in equipotent compounds as compared to the parent compounds. It was shown that the pyridyl group improves water solubility and, in combination with a 5(R)-tert-butyl-l-prolyl group at the P2 position, good lipophilicity can be achieved.

Prolyl oligopeptidase (POP) is a serine protease that specifically hydrolyses small peptides at the carboxyl end of the proline residue. POP has gained pharmaceutical interest, since its inhibitors have been shown to have antiamnesic properties in rat. We examined the effect of the 2( S )-substituents CN and COCH 2 OH at the P1 site of the parent inhibitors isophthalic acid 2( S )-(cyclopentanecarbonyl)pyrrolidine- l -prolyl-pyrrolidine amide and 4-phenylbutanoyl- l -prolyl-pyrrolidine and bulky 5- t -butyl group at the P2 site l -prolyl residue of the parent inhibitor 4-phenylbutanoyl- l -prolyl-pyrrolidine on the binding kinetics to the enzyme. In addition, we studied the duration of POP inhibition in the rat tissues in vivo after i.p. administration. CN and COCH 2 OH substituents at the P1 site pyrrolidine group were found to greatly increase the affinity of the inhibitor and the enzyme–inhibitor complex half-life. In addition, 5- t -butyl group at the P2 site l -prolyl residue increased the dissociation half-life of the enzyme–inhibitor complex, without much affecting the inhibitory potency. The duration of the inhibition in the rat tissues followed the inhibition kinetic properties in that the compounds with fast dissociation produced shorter inhibition in the rat tissues than the compounds with slow dissociation. The duration of POP inhibition of compounds was evidently not governed by their serum clearance. The fact that the in vivo pharmacodynamic behaviour of POP inhibitors can be predicted by their in vitro-properties may be of importance when designing therapeutically useful POP inhibitors.

In 11 patients with a recessive congenital disorder, which we refer to as "the hypotonia-cystinuria syndrome," microdeletion of part of the SLC3A1 and PREPL genes on chromosome 2p21 was found. Patients present with generalized hypotonia at birth, nephrolithiasis, growth hormone deficiency, minor facial dysmorphism, and failure to thrive, followed by hyperphagia and rapid weight gain in late childhood. Since loss-of-function mutations in SLC3A1 are known to cause isolated cystinuria type I, and since the expression of the flanking genes, C2orf34 and PPM1B, was normal, the extended phenotype can be attributed to the deletion of PREPL. PREPL is localized in the cytosol and shows homology with prolyl endopeptidase and oligopeptidase B. Substitution of the predicted catalytic residues (Ser470, Asp556, and His601) by alanines resulted in loss of reactivity with a serine hydrolase-specific probe. In sharp contrast to prolyl oligopeptidase and oligopeptidase B, which require both aminoterminal and carboxyterminal sequences for activity, PREPL activity appears to depend only on the carboxyterminal domain. Taken together, these results suggest that PREPL is a novel oligopeptidase, with unique structural and functional characteristics, involved in hypotonia-cystinuria syndrome.

The function of prolyl oligopeptidase (PO) has been associated with several disorders of the central nervous system. The purpose of this study was to identify endogenous substrates for recombinant porcine PO in porcine brain. The smaller polypeptides were extracted from total brain homogenates and fractionated by two-dimensional chromatography prior to incubation with PO. Shifts in the mass spectrum between the control and the incubated sample, marked potential substrates. Using MSMS peptide sequencing techniques, we identified several fragments of intracellular proteins as potential substrates, which opens new perspectives for finding the function of PO in the intracellular space.