Prolyl Endopeptidase Inhibitor Research Articles

Prolyl Endopeptidase Is Involved in Filaggrinolysis and Cornification

Filaggrin (FLG) is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it co-localized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis (RHEs) using benzyloxycarbonyl-Pro-Prolinal (ZPP) induced the accumulation of FLG monomers. Down-regulation of PREP expression in RHEs using RNA interference confirmed the impact of PREP on FLG metabolism, and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, Western blotting and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification like loricrin. Consequently, at the functional level, the trans-epidermal electric resistance was drastically reduced.

Discovery of a Tunable Heterocyclic Electrophile 4-Chloro-pyrazolopyridine That Defines a Unique Subset of Ligandable Cysteines.

Electrophilic small molecules with novel reactivity are powerful tools that enable activity-based protein profiling and covalent inhibitor discovery. Here, we report a reactive heterocyclic scaffold, 4-chloro-pyrazolopyridine (CPzP) for selective modification of proteins via a nucleophilic aromatic substitution (SNAr) mechanism. Chemoproteomic profiling reveals that CPzPs engage cysteines within functionally diverse protein sites including ribosomal protein S5 (RPS5), inosine monophosphate dehydrogenase 2 (IMPDH2), and heat shock protein 60 (HSP60). Through the optimization of appended recognition elements, we demonstrate the utility of CPzP for covalent inhibition of prolyl endopeptidase (PREP) by targeting a noncatalytic active-site cysteine. This study suggests that the proteome reactivity of CPzPs can be modulated by both electronic and steric features of the ring system, providing a new tunable electrophile for applications in chemoproteomics and covalent inhibitor design.

Exploring bi-carbazole-linked triazoles as inhibitors of prolyl endo peptidase via integrated in vitro and in silico study

A serine protease called prolyl endopeptidase (PEP) hydrolyses the peptide bonds on the carboxy side of the proline ring. The excessive PEP expression in brain results in neurodegenerative illnesses like dementia, Alzheimer’s disease, and Parkinson's disease. Results of the prior studies on antioxidant activity, and the non-cytotoxic effect of bi-carbazole-linked triazoles, encouraged us to extend our studies towards its anti-diabetic potential. Hence, for this purpose all compounds 1–9 were evaluated to reveal their anti-prolyl endo peptidase activity. Fortunately, seven compounds resulted into significant inhibitory capability ranging from 26 to 63 µM. Among them six compounds 4–9 exhibited more potent inhibitory activity with IC50 values 46.10 ± 1.16, 42.30 ± 1.18, 37.14 ± 1.21, 26.29 ± 0.76, 28.31 ± 0.64 and 31.11 ± 0.84 µM respectively, while compound 3 was the least active compound in the series with IC50 value 63.10 ± 1.58 µM comparing with standard PEP inhibitor bacitracin (IC50 = 125 ± 1.50 µM). Moreover, mechanistic study was performed for the most active compounds 7 and 8 with Ki values 24.10 ± 0.0076 and 23.67 ± 0.0084 µM respectively. Further, the in silico studies suggested that the compounds exhibited potential interactions and significant molecular conformations, thereby elucidating the structural basis for their inhibitory effects.

Germinated rice: an overview of gaba, phenolic components and antioxidant activity

This article provides an overview of various studies reporting the correlation and mechanism of increased GABA, phenolic components, and antioxidant reactions in sprouted rice. Sprouted rice is viewed as a functional food because it contains nutrients such as GABA, antioxidants, and other metabolites that are excellent for health. The germination process significantly increases the availability of nutrients such as GABA by 15.4-fold and antioxidant activity by 0.43%. Germination not only adds to the nutrients already present but also introduces new components such as inositol, g-oryzanol, ferulic acid, phytic acid, zinc, tocotrienols, potassium, and prolyl endopeptidase inhibitors. Thus, sprouted rice products can be used as a means of dietary improvement and also as health-promoting foods.

Therapeutic Effect of Prolyl Endopeptidase Inhibitor in High-fat Diet-induced Metabolic Dysfunction-associated Fatty Liver Disease.

Prolyl endopeptidase (PREP) is a serine endopeptidase that participates in many pathological processes including inflammation, oxidative stress, and autophagy. Our previous studies found that PREP knockout exhibited multiple benefits in high-fat diet (HFD) or methionine choline-deficient diet-induced metabolic dysfunction-associated fatty liver disease (MAFLD). However, cumulative studies have suggested that PREP performs complex functions during disease development. Therefore, further understanding the role of PREP in MAFLD development is the foundation of PREP intervention. In this study, an HFD-induced MAFLD model at different time points (4, 8, 12, and 16 weeks) was used to explore dynamic changes in the PREP proline-glycine-proline (PGP)/N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) system. To explore its potential value in MAFLD treatment, saline, or the PREP inhibitor, KYP-2047, was administered to HFD-induced MAFLD mice from the 10th to 16th weeks. PREP activity and expression were increased in HFD-mice compared with control mice from the 12th week onwards, and increased PREP mainly resulted in the activation of the matrix metalloproteinase 8/9 (MMP8/9)-PREP-PGP axis rather than the thymosin β4-meprin α/PREP-AcSDKP axis. In addition, KYP-2047 reduced HFD-induced liver injury and oxidative stress, improved lipid metabolism through the suppression of lipogenic genes and the induction of β-oxidation-related genes, and attenuated hepatic inflammation by decreasing MMP8/9 and PGP. Moreover, KYP2047 restored HFD-induced impaired autophagy and this was verified in HepG2 cells. These findings suggest that increased PREP activity/expression during MAFLD development might be a key factor in the transition from simple steatosis to steatohepatitis, and KYP-2047 might possess therapeutic potential for MAFLD treatment.

Structure-activity relationship and biochemical evaluation of novel fibroblast activation protein and prolyl endopeptidase inhibitors with α-ketoamide warheads.

Peptidomimetic inhibitors of fibroblast activation protein (FAP) are regarded as promising tools for tumor targeting invivo. Even though several peptidomimetic compounds with nanomolar potency have been described, broad chemical space for further modification remained unexplored. Therefore, we set to analyze the structure-activity relationship (SAR) of pseudopeptide compound series with α-ketoamide warheads in order to explore the contributions of the P1' and P2' moieties to the inhibitory potency. A series of novel inhibitors bearing varied P1' and/or P2' moieties was synthesized by combining a Passerini reaction-Amine Deprotection-Acyl Migration (PADAM) approach with peptide coupling and subsequent oxidation. The resulting compounds inhibited FAP and the related prolyl endopeptidase (PREP) with potencies in the nanomolar to sub-nanomolar range. The most potent FAP inhibitor IOCB22-AP446 (6d, IC50=89 pM) had about 36-fold higher inhibition potency than the most potent inhibitor published to date. The compounds were selective over FAP's closest homolog DPP-IV, were stable in human and mouse plasma and in mouse microsomes, and displayed minimal cytotoxicity in tissue cultures.

Inhibition of Prolyl Oligopeptidase Prevents Consequences of Reperfusion following Intestinal Ischemia.

Background: Intestinal ischemia/reperfusion injury (IRI) remains a clinical event that contributes to high morbidity and mortality rates. Intestinal epithelium is exposed to histological and vascular changes following tissue ischemia. Prolyl endopeptidase (PREP), involved in inflammatory responses, could be targeted for recovery from the permanent consequences following intestinal ischemia. Our aim was to investigate the role of PREP inhibitor KYP-2047 in tissue damage, angiogenesis, and endothelial barrier permeability after intestinal IRI in mice. Methods: KYP-2047 treatments were performed 5 min prior to intestinal damage. Intestinal IRI was induced in mice by clamping the superior mesenteric artery and the celiac trunk for 30 min, followed by 1 h of reperfusion. Results: PREP inhibition by KYP-2047 treatment reduced intestinal IR-induced histological damage and neutrophil accumulation, limiting inflammation through decrease of NF-ĸB nuclear translocation and fibrotic processes. KYP-2047 treatment restored barrier permeability and structural alteration following intestinal IRI, attenuating neovascular processes compromised by ischemia/reperfusion. Additionally, loss of epithelial cells during intestinal ischemia occurring by apoptosis was limited by KYP-2047 treatment, which showed strong effects counteracting apoptosis and DNA damage. Conclusions: These findings provide the first evidence that PREP inhibition through KYP-2047 inhibitor use could be a validate strategy for resolving alterations of intestinal epithelium the pathophysiology of intestinal disease.

Bioactive Natural Products from Endophytic Fungus Aspergillus nidulans Associated with Nyctanthes arbor-tristis Linn.

Endophytic fungi are a substantial source of bioactive secondary metabolites. Present studies on Aspergillus nidulans―an endophytic fungus from Nyctanthes arbor-tristis Linn. afforded one new 1, 5-dihydroxy-3-methylxanthone-6-carboxylic acid methyl ester (1) and one known 5, 10-dihydro-phenazine-1-carboxylic acid (2) compound. The structures were elucidated by spectral techniques (Mass, 1D and 2D NMR), and their urease, carbonic anhydrase and prolyl endopeptidase inhibitory activities were evaluated. Compound 1 showed notable prolyl endopeptidase inhibition (IC50 = 3.13 ± 0.09 µM) and compound 2 showed significant carbonic anhydrase (IC50 =14.3 ± 1.05 µM) and urease (IC50 = 24.3 ± 1.15 µM) inhibition.

4-Methyltetrahydropyran as a Convenient Alternative Solvent for Olefin Metathesis Reaction: Model Studies and Medicinal Chemistry Applications.

A number of metathesis reactions were successfully conducted in 4-methyltetrahydropyran, including both standard model dienes, as well as more complex substrates, such as analogues of biologically active compounds and active pharmaceutical ingredients. To place this solvent in a context of pharmaceutical R + D, larger-scale syntheses of SUAM 1221, a prolyl endopeptidase inhibitor with potential application in Alzheimer disease treatment, and a derivative of sildenafil, an analogue of the popular Viagra drug, were executed. In the latter case, despite all the setup being made in air, the metathesis reaction at a 33 g scale proceeded very well with relatively low catalyst loading and without need of aqueous workup or column chromatography.

Prolyl endopeptidase inhibitor Y-29794 blocks the IRS1-AKT-mTORC1 pathway and inhibits survival and in vivo tumor growth of triple-negative breast cancer

ABSTRACT Prolyl endopeptidase (PREP), also known as prolyl oligopeptidase (POP), is an enzyme that cleaves short peptides (<30 amino acids in length) on the C-terminal side of proline. PREP is highly expressed in multiple carcinomas and is a potential target for cancer therapy. A potent inhibitor of PREP, Y-29794, causes long-lasting inhibition of PREP in mouse tissues. However, there are no reports on Y-29794 effects on cancer cell and tumor proliferation. Using cell line models of aggressive triple-negative breast cancer (TNBC), we show here that Y-29794 inhibited proliferation and induced death in multiple TNBC cell lines. Cell death induced by Y-29794 coincided with inhibition of the IRS1-AKT-mTORC1 survival signaling pathway, although stable depletion of PREP alone was not sufficient to reduce IRS1-AKT-mTORC1 signaling or induce death. These results suggest that Y-29794 elicits its cancer cell killing effect by targeting other mechanisms in addition to PREP. Importantly, Y-29794 inhibited tumor growth when tested in xenograft models of TNBC in mice. Induction of cell death in culture and inhibition of xenograft tumor growth support the potential utility of Y-29794 or its derivatives as a treatment option for TNBC tumors.

Molecular Docking Studies and Anti-Alzheimer’s Potential of Isolated Compounds from Tinospora cordifolia

Inhibition of acetylcholinesterase (AChE) is a promising strategy for the management of Alzheimer’s disease (AD). Prolyl endopeptidase (PEP) is a post-proline cleaving enzyme that plays an important role in learning and memory. This study explore the AChE and PEP inhibitory activities of compounds from T. cordifolia. Their affinity for different binding sites in silico was also evaluated. Ellman’s and β-naphtyl acetate-fast blue salt (NA-FB) assay methods were used to determine the cholinesterase inhibitory activity. PEP inhibition was determined by colorimetric assay according to Yoshimoto. Isolation of bioactive compounds was done by different chromatographic methods and structures established using spectroscopy. Molecular docking was carried out using MOE software suite modeled structures based on the coordinates of PDB ID: 3IVM and 10CE. The ethyl acetate fraction had the best AChE inhibition with an IC50 value of 1.4574 ± 0.47 mg/mL at 5 mg/mL. Nine compounds identified as β-sitosterol, tinosporide, 8-hydroxycolumbin, oxoglaucine, corydine, liriodenine, N-formylanonaine, palmatine, 1-octacosanol were isolated from different fractions with oxoglaucine being reported for the first time in the genus Tinospora. Oxoglaucine, liriodenine and N-formylanonaine demonstrated good AChE inhibitory activity with IC50 values of 0.8033 ± 0.09 mg/mL, 0.8076 ± 0.07 mg/mL and 0.8190 ± 0.06 mg/mL at 1 mg/mL respectively. Oxoglaucine and corydine at 1 mM inhibited prolyl endopeptidase with IC50 values of 0.7802 ± 3.1 mM and 0.7884 ± 3.17 mM respectively. Molecular docking revealed hydrophobic, hydrogen bonding, and Pi-stacking interactions between the ligands and the active site of enzymes. Compounds from Tinospora cordifolia are a possible lead for the development of drugs that may be useful in the management of AD.

Prolyl endopeptidase gene disruption attenuates high fat diet-induced nonalcoholic fatty liver disease in mice by improving hepatic steatosis and inflammation.

BackgroundProlyl endopeptidase (PREP) is a serine endopeptidase that regulates inflammatory responses. PREP inhibitors can reduce hepatocyte lipid accumulation and may participate in the progression of nonalcoholic fatty liver disease (NAFLD). We investigated whether disruption of PREP regulates hepatic steatosis and inflammation in mice with NAFLD.MethodsWild-type and PREP gene disrupted mice were randomly divided into low-fat diet wild-type (LFD-WT), high-fat diet wild-type (HFD-WT), low-fat diet PREP disruption (LFD-PREPgt), and high-fat diet PREP disruption (HFD-PREPgt) groups. Animals were euthanized at the endpoint of 32 weeks. The NAFLD activity score and number of inflammatory cells were determined by hematoxylin-eosin staining and immunohistochemical staining of liver tissue. The expression levels of inflammation- and lipid metabolism-associated genes in the liver and serum were detected by quantitative reverse transcription PCR, mass spectrometry, or enzyme-linked immunosorbent assay.ResultsThe body weight and epididymal fat tissue index of the HFD-PREPgt mice were significantly decreased compared with that of the HFD-WT mice. Moreover, the NAFLD activity score and liver function were attenuated in the HFD-PREPgt mice. Fat accumulation and the level of expression of mRNAs associated with lipid metabolism and proinflammatory responses were improved in the HFD-PREPgt mice. The number of CD68-positive cells in liver tissue and the serum levels of inflammation-associated factors were significantly decreased in the HFD-PREPgt mice compared with those in the HFD-WT mice. Further mechanistic investigations indicated that the protective effect of PREP disruption on liver inflammation was associated with the suppressed production of matrix metalloproteinases (MMPs) and proline-glycine-proline (PGP) and the inhibition of neutrophil infiltration.ConclusionsLoss of PREP lowers the severity of hepatic steatosis and inflammatory responses in a high-fat diet-induced nonalcoholic steatohepatitis model. PREP inhibition may protect against NAFLD.

Effect of cyanopyrrolidine derivatives on the activity of prolylendopeptidase, acute exudative inflammation and visceral pain in mice

Cyanopyrrolidine derivatives benzyloxycarbonyl-methionyl-cyanopyrrolidine (ZMetPrdN), benzyloxycarbonylphenylalanyl- cyanopyrrolidine (ZPhePrdN), tert-butyl-hydroxycarbonyl-glycyl-cyanopyrrolidine (BocGlyPrdN), tert-butyl-hydroxycarbonyl-methionyl-cyanopyrrolidine (BocMetPrdN) are inhibitors of prolylendopeptidase (PREP; EC 3.4.21.26) with an IC50 of 2 nM to 12 nM. ZMetPrdN, ZPhePrdN and BocMetPrdN additionally inhibited dipeptidyl peptidase IV (DPP-4; EC 3.4.14.5) with an IC50 of 1100 nM to 3200 nM. All the compounds have antinociceptive properties in the acetic acid writhing test in mice. But only cyanopyrrolidine derivatives with aromatic substituents decrease exudative inflammation. The cyanopyrrolidine derivatives also increase PREP activity and compensatorily reduce DPP-4 activity in the serum of mice three hours after the induction of inflammation. Thus, cyanopyrrolidine derivatives exhibit antinociceptive and antiexudative properties in part via their effect on PREP.

Chemical constituents from Parrotia persica- Structural derivatization and their potential prolyl endopeptidase inhibition activity.

The current study was aimed to evaluate the prolyl endopeptidase (PEP) inhibitory activity of glutinol (1), azadiradione (2), quercetin 3-O-β-d-glactopyranoside (3), catechin (4), quercetin (5), naringenin (6) isolated from Parrotia persica C. A. Mey. Naringenin (6) was further derivatized into 7-O-propargylnaringenin (7), 4',6',4″-O-propargylchalcone (8), and 4',4″-O-propargylchalcone (9). All compounds 1-9 were evaluated for their PEP inhibition activity. PEP is associated with several diseases, including dementia, and Alzheimer's disease (AD). Azadiradione (2) was less active with IC50=356.80±2.9µM, whereas quercetin (5) showed a potent activity with IC50=37.12±2.2µM, as compared to IC50=125.00±1.5µM of standard drug bacitracin. Naringenin (6) was found to be inactive, whereas its new analogues 7-9 were identified as potent inhibitors of PEP with IC50=35.20, 41.20, and 29.60µM, respectively. Kinetic studies of active compounds indicated their modes of inhibition. Compounds 7-9 were found to be mixed-type inhibitors, while compound 5 was found to be non-competitive inhibitor.

Abstract P2014: Prolyl Endopeptidase (PEP) is the Main Angiotensin (1-7) Forming Enzyme From Angiotensin II (1-8) in the Circulation: Implications for Hypertension

The AngII(1-8)-Ang(1-7) axis of the Renin Angiotensin System (RAS) encompasses three enzymes that form Ang(1-7) directly from AngII: Angiotensin Converting Enzyme 2 (ACE2), Prolyl Carboxypeptidase (PRCP) and Prolyl Endopeptidase (PEP). We studied their relative contribution to Ang(1-7) formation in-vivo and also ex-vivo in serum, lungs and kidneys using models of genetic ablation coupled with pharmacological inhibitors. In WT mice infusion of AngII resulted in a rapid increase of plasma Ang(1-7). In a model of ACE2 and PRCP double genetic ablation AngII infusion resulted in a similar increase in Ang(1-7) as in WT (505 ± 46 vs 482 ± 63 pg/ml, respectively), showing that the bulk of Ang(1-7) formation in circulation is essentially independent of ACE2 and PRCP. By contrast, a PEP inhibitor, ZPP, obliterated the rise in plasma Ang(1-7) after infusing AngII(1-8) to control WT mice. In a genetic mouse model of PEP deficiency (PEP -/- ) the increase in Ang(1-7) was also blunted as compared to WT mice (277±41 and 423±25 pg/ml, respectively p=0.01). The rate of recovery from acute AngII(1-8)-induced hypertension, in PEP -/- mice moreover, was delayed as compared to WT mice (Figure). In ex vivo studies PEP inhibition with ZZP reduced Ang(1-7) formation from Ang(1-8) in serum and in lung lysates. By contrast, in kidney lysates the absence of ACE2, but not PEP, obliterated Ang1-7 formation from added AngII. We conclude that PEP is the main enzyme responsible for AngII conversion to Ang(1-7) in the circulation and in the lungs. PEP, not ACE2, is responsible for protecting against AngII-induced hypertension.

Benzyloxycarbonyl-proline-prolinal (ZPP): Dual complementary roles for neutrophil inhibition

Neutrophil influx and activation contributes to organ damage in several major lung diseases. This inflammatory influx is initiated and propagated by both classical chemokines such as interleukin-8 and by downstream mediators such as the collagen fragment cum neutrophil chemokine Pro-Gly-Pro (PGP), which share use of the ELR + CXC receptor family. Benzyloxycarbonyl-proline-prolinal (ZPP) is known to suppress the PGP pathway via inhibition of prolyl endopeptidase (PE), the terminal enzyme in the generation of PGP from collagen. However, the structural homology of ZPP and PGP suggests that ZPP might also directly affect classical glutamate-leucine-arginine positive (ELR+) CXC chemokine signaling. In this investigation, we confirm that ZPP inhibits PE in vitro, demonstrate that ZPP inhibits both ELR + CXC and PGP-mediated chemotaxis in human and murine neutrophils, abrogates neutrophil influx induced by murine intratracheal challenge with LPS, and attenuates human neutrophil chemotaxis to sputum samples of human subjects with cystic fibrosis. Cumulatively, these data demonstrate that ZPP has dual, complementary inhibitory effects upon neutrophil chemokine/matrikine signaling which make it an attractive compound for clinical study of neutrophil inhibition in conditions (such as cystic fibrosis and chronic obstructive pulmonary disease) which evidence concurrent harmful increases of both chemokine and matrikine signaling.

Antiparkinsonian Action of the Prolyl Endopeptidase Inhibitor in Mice

Prolyl endopeptidase is involved in neurodegeneration, proliferation, neuroinflammation, and neuron differentiation. It has been observed that severity of the neurodegenerative process correlates with an increasing level of prolyl endopeptidase in the brain. According to recent research, prolyl endopeptidase might interact with prion-like proteins, which are believed to play the key role in the development of neurodegenerative diseases. Consequently, prolyl endopeptidase inhibitors might be a promising group of chemicals to study in neurodegenerative diseases, particularly Parkinson’s disease. The aim of this research was to determine the effects of benzyloxycarbonyl-methionyl-2(S)-cyanopyrrolidine, a new inhibitor of prolyl endopeptidase, using haloperidol catalepsy test in SHK outbred mice and Parkinson-like syndrome induced by a single systemic injection of neurotoxic 1-methyl-4-phenyl-tetrahydropiridine at a dose of 30 mg/kg in C57Bl/6 inbred mice. As a result, the compound reduces the severity of the main extrapyramidal symptoms: rigidity and motor deficits. However, anticataleptic activity was not found. According to our results, the prolyl endopeptidase inhibitor does not affect the dopaminergic neurons directly but provides possible neuroprotection to the neurons.

Investigation of umami and kokumi taste‐active components in bovine bone marrow extract produced during enzymatic hydrolysis and Maillard reaction

AbstractUmami‐ and kokumi‐taste‐active peptides in Maillard reaction products of enzymatic hydrolysate of bovine bone marrow extract (BBME) were purified and identified by ultrafiltration, gel filtration chromatography, reverse‐phase high‐performance liquid chromatography, liquid chromatography/quadrupole‐time of flight mass spectrometry (LC‐Q‐TOF‐MS/MS) and sensory evaluation. Eight umami and kokumi taste oligopeptides were found in the enzymatic hydrolysate of BBME. After Maillard reaction, the numbers of oligopeptides increased to twelve, such as Ala‐His, Ala‐Val‐His, Gly‐Pro, His‐Gly, Pro‐Ala‐His, Pro‐Val‐Asn‐His, Phe‐Glu‐Ala, Ala‐Ala‐Cys‐Arg, Leu‐Met, Leu‐Thr‐His, Pro‐Gly‐His, Ser‐Thr‐Gly‐Arg, which are linked to a variety of functional properties, such as ACE inhibitor, prolyl endopeptidase inhibitor and dipeptidyl peptidase IV inhibitor, etc. Eight peptides, including Cys‐Pro‐Arg, Ser‐Gly‐Val‐Glu, Pro‐Cys, Cys‐Glu, Cys‐Met‐Thr, Phe‐Glu‐Ala, Ala‐Gln and Leu‐Met, were synthesised and then subjected to sensory evaluation. The evaluation showed that among the eight peptides, Cys‐Pro‐Arg, Pro‐Cys and Leu‐Met led to the distinctly enhanced kokumi taste in blank beef soup.

Tryptophan-Containing Dual Neuroprotective Peptides: Prolyl Endopeptidase Inhibition and Caenorhabditis elegans Protection from β-Amyloid Peptide Toxicity.

Neuroprotective peptides represent an attractive pharmacological strategy for the prevention or treatment of age-related diseases, for which there are currently few effective therapies. Lactoferrin (LF)-derived peptides (PKHs) and a set of six rationally-designed tryptophan (W)-containing heptapeptides (PACEIs) were characterized as prolyl endopeptidase (PEP) inhibitors, and their effect on β-amyloid peptide (Aβ) toxicity in a Caenorhabditis elegans model of Alzheimer’s disease (AD) was evaluated. Two LF-derived sequences, PKH8 and PKH11, sharing a W at the C-terminal end, and the six PACEI heptapeptides (PACEI48L to PACEI53L) exhibited significant in vitro PEP inhibition. The inhibitory peptides PKH11 and PACEI50L also alleviated Aβ-induced paralysis in the in vivo C. elegans model of AD. Partial or total loss of the inhibitory effect on PEP was achieved by the substitution of W residues in PKH11 and PACEI50L and correlated with the loss of protection against Aβ toxicity, pointing out the relevance of W on the neuroprotective activity. Further experiments suggest that C. elegans protection might not be mediated by an antioxidant mechanism but rather by inhibition of Aβ oligomerization and thus, amyloid deposition. In conclusion, novel natural and rationally-designed W-containing peptides are suitable starting leads to design effective neuroprotective agents.

Abstract P415: ACE N-domain Regulates High-glucose Mediated Interleukin-1 beta Production by Renal Epithelial Cells Independently From Angiotensin II Generation

Research studies demonstrated that interleukin (IL)-1β contributes to the development of diabetic nephropathy and hypertension. However, the origin and regulation of IL-1β synthesis during diabetic kidney injury are still unknown. Here, we hypothesize that renal epithelial cells produce IL-1β in response to a high glucose stress and that angiotensin converting enzyme (ACE) plays a key role in this process. To study this, we isolated proximal tubular (PT) epithelial cells from wild-type (WT) and mice lacking either the ACE N-domain (NKO) or the C-domain (CKO) catalytic activity. These cells were exposed to normal (5 mM) or high (30 mM) glucose for 24 hours. IL-1β produced by PT cells were assessed by ELISA and RT-PCR. High glucose induced WT PT cells to release significant amounts of IL-1β (from 5±1 to 70±6 pg/ml, p&lt;0.001; n=6). When WT PT cells were exposed to a high glucose media in the presence of an ACE inhibitor (lisinopril, 10 mM), IL-1β levels were significantly reduced (from 70±6 to 38±6 pg/ml, p&lt;0.01). In contrast, AT1 receptor blockade by losartan did not change the amount of IL-1β produced by WT PT cells. To determine which ACE domain is associated with IL-1β production, NKO and CKO PT cells were exposed to high glucose. Strikingly, NKO PT cells released lower amounts of IL-1β when exposed to high glucose compared to WT (NKO: 15±7 vs. WT: 79±9 pg/ml, p&lt;0.01, n=4). No differences were observed between WT and CKO PT cells. Since the ACE N-domain degrades the anti-inflammatory tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), we tested whether the lower IL-1β production in NKO PT cells was due to an accumulation of AcSDKP. For this, we pre-treated NKO PT cells with a prolyl endopeptidase inhibitor (S17092, 50μM) to prevent the production of AcSDKP. Notably, this treatment increased the IL-1β response to high glucose in NKO PT cells (2.1±0.3-fold increase, p&lt;0.01, n=4). Our data indicate that: 1) PT cells can sense and respond to high glucose by secreting IL-1β and 2) the absence of the ACE N-domain blunts the production of IL-1β through a mechanism that involves AcSDKP accumulation. In conclusion, ACE might contribute to the inflammatory response that underlays diabetic nephropathy independently from angiotensin II generation.