Production of the prolyl endoprotease (PEP) from Aspergillus sp. FSDE 16 by solid-state fermentation (SSF) and use for producing a gluten-free beer.

Beer is a beverage that contains gluten and cannot be consumed by people with celiac disease. In this context, the enzyme prolyl endoprotease (PEP) can be used to reduce the gluten content in beer. The present study aimed to produce the PEP from Aspergillus sp. FSDE 16 using solid-state fermentation with 5 conditions and comparing with a similar commercial enzyme produced from Aspergillus niger in the production of a gluten-free beer. The results of the performed cultures showed that during the culture, the most increased protease activity (54.46 U/mL) occurred on the 4th day. In contrast, for PEP, the highest activity (0.0356 U/mL) was obtained on the 3rd day of culture in condition. Regarding beer production, cell growth, pH, and total soluble solids showed similar behavior over the 7 days for beers produced without enzyme addition or with the addition of commercial enzyme and with the addition of the enzyme extract produced. The addition of the enzyme and the enzyme extract did not promote changes, and all the beers produced showed similar and satisfactory results, with acid pH between 4 and 5, total soluble solids ranging from 4.80 to 5.05, alcohol content ranging from 2.83% to 3.08%, and all beers having a dark character with deep amber and light copper color. Gluten removal was effectively using the commercial enzyme and the enzyme produced according to condition (v) reaching gluten concentrations equal to 17 ± 5.31 and 21.19 ± 11.28 ppm, respectively. In this way, the production of the enzyme by SSF and its application in the removal of gluten in beer was efficient.

Isolation and characterization of natural inhibitors of post-proline specific peptidases from the leaves of Cotinus coggygria Scop.

ETHNOPHARMACOLOGICAL RELEVANCE: Cotinus coggygria has a number of applications in traditional medicine most of which are connected with its anti-inflammatory and anti-oxidant properties. Since inflammation and oxidative stress are recognized as triggering factors for cancer, anti-cancer activity has also been documented and the possible mechanisms of this activity are under investigation. Important components of C. coggygria extracts are shown to be hydrolysable gallotannins of which pentagalloyl-O-glucose has been studied in details. This compound inhibits various enzymes including prolyl oligopeptidase which is involved in tumorigenesis and tumour growth. According to our pilot studies, oligo-O-galloylglucoses with more than five galloyl residues are also presented in the herb of Bulgarian origin, but their activities have not been examined. AIM OF THE STUDY: To establish an extraction method by which it is possible to concentrate high molecular hydrolysable gallotannins from dried leaves of Cotinus coggygria and to determine their inhibitory properties towards prolyl oligopeptidase and fibroblast activation protein α. MATERIALS AND METHODS: Dried leaves of C. coggygria were extracted using different solvents in single-phase or biphasic systems under various extraction conditions. Main compounds of the extracts were identified by using high performance liquid chromatography and liquid chromatography - high resolution mass spectrometry. The extracts' inhibitory properties towards prolyl oligopeptidase and fibroblast activation protein α were studied on recombinant human enzymes by enzyme kinetic analyses using a fluorogenic substrate. RESULTS: Ethyl acetate/water (pH 3.0) extraction of dried plant leaves proved to be the most efficient method for isolation of high molecular hydrolysable gallotannins which can be further concentrated by precipitation of dicyclohexylammonium salts in ethyl acetate. The main components of those extracts were oligo-O-galloyl glucoses with more than five gallic acid residues. They were shown to inhibit both enzymes studied but were about 30 times more effective inhibitors of prolyl oligopeptidase. CONCLUSIONS: C. coggygria from Bulgarian origin is shown to possess a substantial quantity of oligo-O-galloyl glucoses with more than five gallic acid residues which has not been described thus far in the same herb from other sources. An extraction method useable for concentrating those compounds is established. They are found to inhibit prolyl oligopeptidase with a very good selectivity to fibroblast activation protein α. The previously described antitumor activity of this plant may be at least in part due to the inhibition of the above enzymes which has been shown to participate in the genesis and development of various types of tumors.

Evaluation of prolyl oligopeptidase and acetylcholinesterase inhibition by Phyllanthus tenellus Roxb. from Brazil.

Phyllanthus tenellus Roxb. (Phyllanthaceae) is a plant used in Brazilian folk medicine for the treatment of intestinal infections and diabetes. Despite its use in traditional medicine, it was reported that P. tenellus extract may cause several effects in the central nervous system (CNS) of animals, such as agitation and signs of depression. The aim of this study was to determine the main constituents of P. tenellus methanol extract and to investigate whether the extract is able to inhibit the enzymes prolyl oligopeptidase (POP), acetylcholinesterase (AChE) and dipeptidyl peptidase-IV (DPP-IV). Corilagin (1) was isolated as the main constituent of the P. tenellus extract, along with rutin (2) and vitexin-2″-O-rhamnoside (3). The extract presented the ability to inhibit mainly POP. Dichloromethane and ethyl acetate fractions showed the highest inhibitory potency against POP (IC50 values of 1.7 ± 0.4 and 11.7 ± 2 µg/mL, respectively). All fractions were inactive against AChE. Corilagin displayed selective POP inhibition in a dose-dependent manner, with IC50= 19.7 ± 2.6 µg/mL. Corilagin exhibited moderate capacity to pass through the phospholipid membrane by passive diffusion, presenting effective permeability (Pe) of 1.26 × 10-7 cm/s.

Post-Weaning Social Isolation Disturbs Gene Expression in Rat Brain Structures.

Post-weaning social isolation of male Wistar rats for 10 weeks led to an increase of their aggressiveness, sensorimotor reactivity, and cognitive deficiency, manifesting in training disorders evaluated by the acoustic startle response (amplitude of the response decreasing). Expression of gene encoding serine protease prolyl endopeptidase (EC 3.4.21.26) in the frontal cortex was higher than in control rats kept in groups, while the level of mRNA of the gene encoding dipeptidyl peptidase IV (EC 3.4.14.5) did not differ from the control in any of the brain structures. The levels of serotonin transporter gene mRNA in the striatum and hypothalamus were higher than in the control. No appreciable changes in the expression of genes encoding tryptophan hydroxylase-2 and monoaminoxidase A and B in the frontal cortex, striatum, amygdala, hypothalamus, and hippocampus were detected. The data indicated the involvement of genes associated with the serotoninergic system in the mechanisms of mental disorders induced by post-weaning social isolation and suggest the gene encoding prolyl endopeptidase as a candidate gene involved in the pathogenesis of these disorders.

Chemical constituents from Valeriana polystachya Smith and evaluation of their effects on the acetylcholinesterase and prolyl oligopeptidase activities.

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, 6ß-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 ±â€¯0.07 and 7.9 ±â€¯0.4 µM, respectively.

A randomised, double-blind, cross-over trial to evaluate bread, in which gluten has been pre-digested by prolyl endoprotease treatment, in subjects self-reporting benefits of adopting a gluten-free or low-gluten diet.

The aim of the present study was to determine if the enzyme Aspergillus niger prolyl endoprotease (ANPEP), which degrades the immunogenic proline-rich residues in gluten peptides, can be used in the development of new wheat products, suitable for gluten-sensitive (GS) individuals. We have carried out a double-blind, randomised, cross-over trial with two groups of adults; subjects, self-reporting benefits of adopting a gluten-free or low-gluten diet (GS, n 16) and a control non-GS group (n 12). For the trial, volunteers consumed four wheat breads: normal bread, bread treated with 0·8 or 1 % ANPEP and low-protein bread made from biscuit flour. Compared with controls, GS subjects had a favourable cardiovascular lipid profile - lower LDL (4·0 (sem 0·3) v. 2·8 (sem 0·2) mmol/l; P=0·008) and LDL:HDL ratio (3·2 (sem 0·4) v. 1·8 (sem 0·2); P=0·005) and modified haematological profile. The majority of the GS subjects followed a low-gluten lifestyle, which helps to reduce the gastrointestinal (GI) symptoms severity. The low-gluten lifestyle does not have any effect on the quality of life, fatigue or mental state of this population. Consumption of normal wheat bread increased GI symptoms in GS subjects compared with their habitual diet. ANPEP lowered the immunogenic gluten in the treated bread by approximately 40 %. However, when compared with the control bread for inducing GI symptoms, no treatment effects were apparent. ANPEP can be applied in the production of bread with taste, texture and appearance comparable with standard bread.

Cholinesterase and Prolyl Oligopeptidase Inhibitory Activities of Alkaloids from Argemone platyceras (Papaveraceae).

Alzheimer's disease is an age-related, neurodegenerative disorder, characterized by cognitive impairment and restrictions in activities of daily living. This disease is the most common form of dementia with complex multifactorial pathological mechanisms. Many therapeutic approaches have been proposed. Among them, inhibition of acetylcholinesterase, butyrylcholinesterase, and prolyl oligopeptidase can be beneficial targets in the treatment of Alzheimer's disease. Roots, along with aerial parts of Argemone platyceras, were extracted with ethanol and fractionated on an alumina column using light petrol, chloroform and ethanol. Subsequently, repeated preparative thin-layer chromatography led to the isolation of (+)-laudanosine, protopine, (-)-argemonine, allocryptopine, (-)-platycerine, (-)-munitagine, and (-)-norargemonine belonging to pavine, protopine and benzyltetrahydroisoquinoline structural types. Chemical structures of the isolated alkaloids were elucidated by optical rotation, spectroscopic and spectrometric analysis (NMR, MS), and comparison with literature data. (+)-Laudanosine was isolated from A. platyceras for the first time. Isolated compounds were tested for human blood acetylcholinesterase, human plasma butyrylcholinesterase and recombinant prolyl oligopeptidase inhibitory activity. The alkaloids inhibited the enzymes in a dose-dependent manner. The most active compound (-)-munitagine, a pavine alkaloid, inhibited both acetylcholinesterase and prolyl oligopeptidase with IC50 values of 62.3 ± 5.8 µM and 277.0 ± 31.3 µM, respectively.

Chemical composition and evaluation of prolyl oligopeptidase and acetylcholinesterase inhibitory activities of Leonurus Sibiricus L. from Brazil.

Chemical investigation of the aerial parts of Leonurus sibiricus L. used in Brazilian folk medicine led to the identification of the following constituents: the labdane-type diterpenoid leojaponin, the phytosterols ß-sitosterol and ß-sitosterol glucoside and the alkaloid leonurine. The crude extracts obtained from methanol and methanol/1% HCl and pure compounds isolated from L. sibirius were investigated as acetylcholinesterase (AChE) and prolyl oligopeptidase (POP) inhibitors. Extracts obtained by maceration were active against POP (53-58%), but showed weak activity against AChE. The isolated leojaponin and leonurine were evaluated as POP inhibitors.

Isolation of prolyl endopeptidase inhibitory peptides from a sodium caseinate hydrolysate.

Prolyl endopeptidase (PEP) has been associated with neurodegenerative disorders, and the PEP inhibitors can restore the memory loss caused by amnesic compounds. In this study, we investigated the PEP inhibitory activity of the enzymatic hydrolysates from various food protein sources, and isolated and identified the PEP inhibitory peptides. The hydrolysate obtained from sodium caseinate using bromelain (SC/BML) displayed the highest inhibitory activity of 86.8% at 5 mg mL(-1) in the present study, and its IC50 value against PEP was 0.77 mg mL(-1). The F-5 fraction by RP-HPLC (reversed-phase high performance liquid chromatography) from SC/BML showed the highest PEP inhibition rate of 88.4%, and 9 peptide sequences were identified. The synthetic peptides (1245.63-1787.94 Da) showed dose-dependent inhibition effects on PEP as competitive inhibitors with IC50 values between 29.8 and 650.5 µM. The results suggest that the peptides derived from sodium caseinate have the potential to be PEP inhibitors.

Chemical Composition and Inhibitory Effects of Hypericum brasiliense and H. connatum on Prolyl Oligopeptidase and Acetylcholinesterase Activities.

BACKGROUND: The genus Hypericum (family Clusiaceae) comprises various species that are used in traditional medicine, such as wound healing, antidepressant, and anticancer agents. OBJECTIVE: The aim of this study was to evaluate the inhibitory capacity of extracts and fractions from two Hypericum species used in the Brazilian folk medicine (H. brasiliense and H. connatum) against the enzymes prolyl oligopeptidase (POP), dipeptidyl peptidase-IV (DPP-IV), and acetylcholinesterase (AChE), as well as to identify their main active constituents. METHODS: Dried aerial parts of H. connatum and H. brasiliense were subjected to extraction with 8:2 methanol-H2O. Each hydroalcoholic extract was fractioned resulting in ethyl acetate and aqueous fractions. The activity of POP, DPP-IV and AChE was determined in vitro in 96-well microplates. RESULTS: The main components identified in the plant extracts were chlorogenic acid (1), quercitrin (2), rutin (3), quercetin (4), and isoquercitrin (5). Hydroalcoholic extracts, ethyl acetate and aqueous fractions showed high POP inhibitory activity with IC50 values of 2.6 to 3.7 µg/mL. AChE and DPP-IV inhibitory effects were very low for all extracts and substances. CONCLUSION: Chlorogenic acid (1) and quercetin (4) were the main constituent responsible for the activity observed against POP. Parallel artificial membrane permeability assay of ethyl acetate fractions of both species showed that the metabolite that can effectively pass through the lipid membrane is 4, the aglycone form of 2, 3 and 5.

Alkaloids from Peumus boldus and their acetylcholinesterase, butyrylcholinesterase and prolyl oligopeptidase inhibition activity.

Eleven isoquinoline alkaloids (1-11) were isolated from dried leaves of Peumus boldus Mol. by standard chromatographic methods. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analysis, and by comparison with literature data. Compounds isolated in sufficient amount were evaluated for their acetylcholinesterase, and butyrylcholinesterase inhibition activity using Ellman's method. In the prolyl oligopeptidase assay, Z-Gly-Pro-p-nitroanilide was used as substrate. Promising butyrylcholinesterase inhibition activities were demonstrated by two benzylisoquinoline alkaloids, reticuline (8) and N-methylcoclaurine (9), with IC50 values of 33.6 ± 3.0 µM and 15.0 ± 1.4 µM, respectively. Important prolyl oligopeptidase inhibition activities were shown by N-methyllaurotetanine (6) and sinoacutine (4) with IC50 values of 135.4 ± 23.2 µM and 143.1 ± 25.4 µM, respectively. Other tested compounds were considered inactive.

Isoquinoline alkaloids as prolyl oligopeptidase inhibitors.

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).

Chemical composition of bioactive alkaloid extracts from some Narcissus species and varieties and their biological activity.

Alkaloid extracts of eight Narcissus (Amaryllidaceae) species and varieties were studied with respect to their acetylcholinesterase (HuAChE) and butyrylcholinesterase (HuBuChE) inhibitory activity and alkaloid patterns. Thirty alkaloids were determined by GC/MS, and twenty-five of them identified from their mass spectra, retention times and retention indexes. Promising HuAChE inhibition activity was demonstrated by six Narcissus taxa and HuBuChE inhibition by N. jonquila cv. Double Campernelle and N. nanus cv. Elka with IC50 values of 24.1 +/- 1.9 microg/mL and 25.1 +/- 1.8 microg/mL, respectively. Two alkaloids were isolated in pure form using preparative TLC and identified as the galanthamine type alkaloid narwedine and the lycorine type alkaloid incartine. Both compounds were tested for their biological activity. They were considered inactive in HuAChE/HuBuChE assays, but showed promising prolyl oligopeptidase inhibition activities with IC50 values of 0.95 +/- 0.12 mM and 0.91 g 0.09 mM, respectively.

Hypothalamic prolyl endopeptidase (PREP) regulates pancreatic insulin and glucagon secretion in mice.

Prolyl endopeptidase (PREP) has been implicated in neuronal functions. Here we report that hypothalamic PREP is predominantly expressed in the ventromedial nucleus (VMH), where it regulates glucose-induced neuronal activation. PREP knockdown mice (Prep(gt/gt)) exhibited glucose intolerance, decreased fasting insulin, increased fasting glucagon levels, and reduced glucose-induced insulin secretion compared with wild-type controls. Consistent with this, central infusion of a specific PREP inhibitor, S17092, impaired glucose tolerance and decreased insulin levels in wild-type mice. Arguing further for a central mode of action of PREP, isolated pancreatic islets showed no difference in glucose-induced insulin release between Prep(gt/gt) and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prep(gt/gt) and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prep(gt/gt) mice have elevated sympathetic outflow and norepinephrine levels in the pancreas, and propranolol treatment reversed glucose intolerance in these mice. Finally, re-expression of PREP by bilateral VMH injection of adeno-associated virus-PREP reversed the glucose-intolerant phenotype of the Prep(gt/gt) mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function.

Effect of prolyl endopeptidase inhibitor benzyloxycarbonyl-methionyl-2(S)-cyanopyrrolidine on activity of proline-specific peptidases in brain structures of rats with experimental MPTP-induced depressive syndrome.

A noncompetitive synthetic inhibitor of prolyl endopeptidase benzyloxycarbonyl-methionyl-2(S)-cyanopyrrolidine (1.0 mg/kg intraperitoneally for 2 weeks) prevented the increase in activity of prolyl endopeptidase in the frontal cortex, striatum, and hypothalamus and activation of dipeptidyl peptidase IV in the frontal cortex of rats with experimental dopamine deficiency-dependent depressive syndrome caused by administration of proneurotoxin MPTP (2 weeks). Our results suggest that the antidepressive effect of prolyl endopeptidase inhibitor is at least partly related to prevention of enzyme activation in the frontal cortex. The antistress effect of this substance can be associated with prevention of enzyme activation in the hypothalamus.

In vitro evaluation of caffeoyl and cinnamoyl derivatives as potential prolyl oligopeptidase inhibitors.

A screening of the natural product chlorogenic acid, isolated from the Brazilian medicinal plant Hypericum brasiliense, caffeic acid, cinnamic acid, and p-methoxycinnamic acid, and derivatives of caffeoylquinic, caffeoyl, and cinnamoyl against the enzymes prolyl oligopeptidase and dipeptidyl peptidase IV was carried out. Caffeoylquinic, caffeoyl, and cinnamoyl derivatives were prepared using simple derivatization procedures and through coupling reactions with the amino acid proline. The dipeptidyl peptidase IV assay showed inhibitory activity of the tested compounds at a high concentration (500 µM) in the range of 81.5-7.2 %. In contrast, the derivatives methyl ester and 1,7-acetonide obtained from chlorogenic acid, and caffeic acid and its methyl ester derivative showed selectivity and activity as prolyl oligopeptidase inhibitors, with IC50 values of 3 to 14 mM.

Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study.

AIM: To assesses the safety and efficacy of Aspergillus niger prolyl endoprotease (AN-PEP) to mitigate the immunogenic effects of gluten in celiac patients. METHODS: Patients with initial diagnosis of celiac disease as confirmed by positive serology with subtotal or total villous atrophy on duodenal biopsies who adhere to a strict gluten-free diet (GFD) resulting in normalised antibodies and mucosal healing classified as Marsh 0 or I were included. In a randomised double-blind placebo-controlled pilot study, patients consumed toast (approximately 7 g/d gluten) with AN-PEP for 2 wk (safety phase). After a 2-wk washout period with adherence of the usual GFD, 14 patients were randomised to gluten intake with either AN-PEP or placebo for 2 wk (efficacy phase). Measurements at baseline included complaints, quality-of-life, serum antibodies, immunophenotyping of T-cells and duodenal mucosa immunohistology. Furthermore, serum and quality of life questionnaires were collected during and after the safety, washout and efficacy phase. Duodenal biopsies were collected after the safety phase and after the efficacy phase. A change in histological evaluation according to the modified Marsh classification was the primary endpoint. RESULTS: In total, 16 adults were enrolled in the study. No serious adverse events occurred during the trial and no patients withdrew during the trial. The mean score for the gastrointestinal subcategory of the celiac disease quality (CDQ) was relatively high throughout the study, indicating that AN-PEP was well tolerated. In the efficacy phase, the CDQ scores of patients consuming gluten with placebo or gluten with AN-PEP did not significantly deteriorate and moreover no differences between the groups were observed. During the efficacy phase, neither the placebo nor the AN-PEP group developed significant antibody titers. The IgA-EM concentrations remained negative in both groups. Two patients were excluded from entering the efficacy phase as their mucosa showed an increase of two Marsh steps after the safety phase, yet with undetectable serum antibodies, while 14 patients were considered histologically stable on gluten with AN-PEP. Also after the efficacy phase, no significant deterioration was observed regarding immunohistological and flow cytometric evaluation in the group consuming placebo compared to the group receiving AN-PEP. Furthermore, IgA-tTG deposit staining increased after 2 wk of gluten compared to baseline in four out of seven patients on placebo. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits. CONCLUSION: AN-PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN-PEP.

Alkaloids from Chlidanthus fragrans and their acetylcholinesterase, butyrylcholinesterase and prolyl oligopeptidase activities.

Eleven Amaryllidaceae alkaloids (1-11) were isolated from fresh bulbs of Chlidanthus fragrans Herb. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic experiments. Complete NMR assignments were achieved for deoxypretazzetine (1). All compounds were evaluated for their erythrocytic acetylcholinesterase and serum butyrylcholinesterase inhibition activity using Ellman's method. In the prolyl oligopeptidase assay, Z-Gly-Pro-p-nitroanilide was used as substrate. In biological assays, only the crinine type Amaryllidaceae alkaloid undulatine showed promising acetylcholinesterase and prolyl oligopeptidase inhibition activity with IC50 values of 23.0 +/- 1.0 microM and 1.96 +/- 0.12 mM, respectively. Other isolated compounds were considered inactive.

Anti-inflammatory and neuroactive properties of selected fruit extracts.

Epidemiological evidence supports inverse associations between fruit and vegetable intake and incidence of cardiovascular disease and neurodegeneration. Dietary botanicals with salient health benefits include berries and leafy vegetables. Molecular pharmacology research has ascribed these benefits primarily to phenolic constituents and antioxidant activity. The current investigation sought to eluicidate pharmacologic activity of two novel preparations of berry and spinach extracts in vitro. Blueberry and cranberry exhibited the greatest antioxidant activity. In a dose-dependent manner, a proprietary mixture of cranberry and blueberry extracts inhibited inhibitor of κB kinase ß, a central node in inflammatory signal transduction. A proprietary mixture of blueberry, strawberry, and spinach extracts inhibited prolyl endopeptidase, a regulator of central neuropeptide stability and an emerging therapeutic target in neurology and psychiatry. These results indicate specific molecular targets of blended dietary plants with potential relevance to inflammation and neurological health.

Virtual screening and computational optimization for the discovery of covalent prolyl oligopeptidase inhibitors with activity in human cells.

Our docking program, Fitted, implemented in our computational platform, Forecaster, has been modified to carry out automated virtual screening of covalent inhibitors. With this modified version of the program, virtual screening and further docking-based optimization of a selected hit led to the identification of potential covalent reversible inhibitors of prolyl oligopeptidase activity. After visual inspection, a virtual hit molecule together with four analogues were selected for synthesis and made in one-five chemical steps. Biological evaluations on recombinant POP and FAPα enzymes, cell extracts, and living cells demonstrated high potency and selectivity for POP over FAPα and DPPIV. Three compounds even exhibited high nanomolar inhibitory activities in intact living human cells and acceptable metabolic stability. This small set of molecules also demonstrated that covalent binding and/or geometrical constraints to the ligand/protein complex may lead to an increase in bioactivity.