Discovery of sulfonamides and 9-oxo-2,8-diazaspiro[5,5]undecane-2-carboxamides as human kynurenine aminotransferase 2 (KAT2) inhibitors.

Human kynurenine aminotransferase 2 (KAT2) inhibitors could be potentially used to treat the cognitive deficits associated with bipolar disease and schizophrenia. Although, there has been active drug research activity by several industrial and academic groups in developing KAT2 inhibitors over the years, no such compound has proceeded to the clinics. Here, we report two different chemical series of reversible KAT2 inhibitors with sub-micromolar activities. The first series was identified by a high-throughput screening of a diverse random library and the second one by structure-based virtual screening. Two novel crystal structures of KAT2 complexed with different reversible inhibitors were also deposited to the Protein databank which could be useful for future drug discovery efforts.

Simple, direct, and informative method for the assessment of CYP2C19 enzyme inactivation kinetics.

Many clinically relevant drug interactions involving cytochrome P450 inhibition are mediated by mechanism-based inactivation (MBI). Time-dependent inhibition is one of the major features distinguishing between reversible inhibition and MBI. It thus provides a useful screening approach for early drug interaction risk assessment. Accordingly, we developed an easy and informative fluorometric method for the assessment of CYP2C19 enzyme inactivation kinetics. Dibenzylfluorescein (DBF) is widely used as a profluorescent probe substrate for P450 activity and inhibition assays, but its use has been considered to be limited to traditional endpoint assays. We monitored CYP2C19-catalyzed metabolism of DBF using synthesized fluorescein benzyl ester and fluorescein benzyl ether along with commercially available fluorescein as intermediate standards. Furthermore, we demonstrated the use of DBF in a kinetic assay as a progress curve analysis for straightforward determination of whether a compound is a time-dependent inactivator of CYP2C19. The recombinant human CYP2C19 inactivation kinetics of isoniazid, ticlopidine, and tranylcypromine were evaluated, and their key kinetic parameters were measured from the same experiment. The known mechanism-based inactivators, isoniazid and ticlopidine, exhibited clear time-dependent inactivation with K(I) and k(inact) values of 250.5 ± 34 µM and 0.137 ± 0.006 min(-1) and 1.96 ± 0.5 µM and 0.135 ± 0.009 min(-1), respectively. Tranylcypromine did not display any time-dependent inhibition, which is consistent with its reported mechanism of competitive inhibition. In summary, DBF is suitable for use in the progress curve analysis approach and can be used as an initial screen to identify compounds that require more detailed investigations in drug interaction optimization.

Prolyl endopeptidase is involved in cellular signalling in human neuroblastoma SH-SY5Y cells.

Prolyl endopeptidase (PREP), probably acting through the inositol cycle, has been implicated in memory and learning. However, the physiological role of PREP is unknown. It has been shown that PREP expression, regulated in cerebellar granule cells, has probably a role in cell proliferation and differentiation. Here, we report the levels and subcellular distribution of PREP in human neuroblastoma SH-SY5Y cells in proliferating conditions and under differentiation induced by retinoic acid (RA). We analysed the levels of cell signalling intermediates, growth behavior and gene expression, and differentiation morphology changes, upon PREP inhibition. After induction of differentiation, PREP activity was found decreased in the nucleus but increased to high levels in the cytoplasm, due in part to increased PREP transcription. The levels of inositol (1,4,5)-trisphosphate revealed no correlation with PREP activity, but phosphorylated extracellular signal-regulated kinases 1 and 2 were decreased by PREP inhibition during early stages of differentiation. Morphological evaluation indicated that PREP inhibition retarded the onset of differentiation. PREP activity regulated gene expression of protein synthesis machinery, intracellular transport and kinase complexes. We conclude that PREP is a regulatory target and a regulatory element in cell signalling. This is the first report of a direct influence of a cell signalling molecule, RA, on PREP expression.

Characterization of membrane-bound prolyl endopeptidase from brain.

Prolyl oligopeptidase (POP) is a serine protease that cleaves small peptides at the carboxyl side of an internal proline residue. Substance P, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. POP has been implicated in a variety of brain processes, including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension, and psychiatric disorders. Although POP has been considered to be a soluble cytoplasmic peptidase, significant levels of activity have been detected in membranes and in extracellular fluids such as serum, cerebrospinal fluid, seminal fluid, and urine, suggesting the existence of noncytoplasmic forms. Furthermore, a closely associated membrane prolyl endopeptidase (PE) activity has been previously detected in synaptosomes and shown to be different from the cytoplasmic POP activity. Here we isolated, purified and characterized this membrane-bound PE, herein referred to as mPOP. Although, when attached to membranes, mPOP presents certain features that distinguish it from the classical POP, our results indicate that this protein has the same amino acid sequence as POP except for the possible addition of a hydrophobic membrane anchor. The kinetic properties of detergent-soluble mPOP are fully comparable to those of POP; however, when attached to the membranes in its natural conformation, mPOP is significantly less active and, moreover, it migrates anomalously in SDS/PAGE. Our results are the first to show that membrane-bound and cytoplasmic POP are encoded by variants of the same gene.

Prolyl oligopeptidase: a potential target for the treatment of cognitive disorders.

Prolyl oligopeptidase (POP) is a ubiquitous post-proline cleaving enzyme that is highly expressed in brain. Current knowledge about the biochemical features of POP and the pharmacological action of its specific inhibitors has indicated that POP participates in several aspects of the central nervous system (CNS), including learning, memory and mood. Furthermore, a role has been suggested for POP in pathological processes such as eating and mood disorders, hypertension and cell-cycle disturbances, in addition to its proposed connection with the neurodegenerative processes which occur in Alzheimer's, Huntington's and Parkinson's diseases. The milestones responsible for the accelerated development of POP inhibitors include the discovery that these compounds reverse memory loss in animal models of drug- or lesion-induced amnesia and the observation that the expression of POP correlates with age. Today, several POP inhibitors have already been evaluated in preclinical trials as potential drugs for the treatment of natural memory deficits that occur with aging or the pathological memory loss characteristic of Alzheimer's disease. Thus, the results that are emerging from basic research on POP function will facilitate the fine-tuning of more efficient drugs to target this protease.

Prolamin hydrolysis in wheat sourdoughs with differing proteolytic activities.

The prolamins of wheat, rye, and barley contain structures that are harmful to gluten-sensitive people, and an extensive degradation of these prolamins during food processing might eliminate this problem. Sourdough fermentation is a cereal food process during which some protein degradation occurs. In this study, the prolamin hydrolysis that occurred in a high-proteolytic-activity germinated-wheat sourdough (GWSD) was compared with that of wheat sourdough systems which contained moderate or no proteolytic activities. Virtually all of the wheat prolamins (gliadins and glutenins) were degraded during the GWSD fermentation. Quantification of its prolamin levels confirmed that extensive prolamin hydrolysis had occurred in the GWSD. This hydrolysis was attributed to the cysteine proteinase activities of the germinated wheat. The use of high-proteolytic sourdoughs in baking could make it possible to prepare new low-prolamin cereal-based products for use by gluten-sensitive people, who could then diversify their diets by including these whole-grain containing products into their every-day diets.

Beneficial effect of prolyl oligopeptidase inhibition on spatial memory in young but not in old scopolamine-treated rats.

The effects of a novel prolyl oligopeptidase (POP) inhibitor KYP-2047 on spatial memory of young (3-month-old) and old (8- to 9-month-old) scopolamine-treated rats (0.4 mg/kg intraperitoneally) was investigated in the Morris water maze. In addition, the concentrations of promnesic neuropeptide substrates of POP, substance P and neurotensin in various brain areas after acute and chronic POP inhibition were measured in young rats. In addition, inositol-1,4,5-trisphosphate (IP(3)) levels were assayed in rat cortex and hippocampus after effective 2.5-day POP inhibition. KYP-2047 (1 or 5 mg/kg 30 min. before daily testing) dose-dependently improved the escape performance (i.e. latency to find the hidden platform and swimming path length) of the young but not the old rats in the water maze. POP inhibition had no consistent effect on substance P levels in cortex, hippocampus or hypothalamus, and only a modest increase in neurotensin concentration was observed in the hypothalamus after a single dose of KYP-2047. Moreover, IP(3) concentrations remained unaffected in cortex and hippocampus after POP inhibition. In conclusion, the behavioural data support the earlier findings of the promnesic action of POP inhibitors, but the mechanism of the memory-enhancing action remains unclear.

Binding kinetics and duration of in vivo action of novel prolyl oligopeptidase inhibitors.

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.

Synthesis and characterization of the novel fluorescent prolyl oligopeptidase inhibitor 4-fluoresceinthiocarbamoyl-6-aminocaproyl-L-prolyl-2(S)-(hydroxyacetyl)pyrrolidine.

The synthesis and characterization of the first fluorescent prolyl oligopeptidase inhibitor 4-fluoresceinthiocarbamoyl-6-aminocaproyl-L-prolyl-2(S)-(hydroxyacetyl)pyrrolidine is described. This compound has an IC50 value of 0.83 nM and a dissociation half-life of 160 min, and its fluorescence signal is detectable using standard filters for fluorescein. These properties make this compound a suitable probe for visualizing prolyl oligopeptidase in various applications.

Dicarboxylic acid azacycle l-prolyl-pyrrolidine amides as prolyl oligopeptidase inhibitors and three-dimensional quantitative structure-activity relationship of the enzyme-inhibitor interactions.

A series of dicarboxylic acid azacycle l-prolyl-pyrrolidine amides was synthesized, and their inhibitory activity against prolyl oligopeptidase (POP) from porcine brain was tested. Three different azacycles were tested at the position beyond P3 and six different dicarboxylic acids at the P3 position. l-Prolyl-pyrrolidine and l-prolyl-2(S)-cyanopyrrolidine were used at the P2-P1 positions. The IC(50) values ranged from 0.39 to 19000 nM. The most potent inhibitor was the 3,3-dimethylglutaric acid azepane l-prolyl-2(S)-cyanopyrrolidine amide. Molecular docking (GOLD) was used to analyze binding interactions between different POP inhibitors of this type and the POP enzyme. The data set consisted of the novel inhibitors, inhibitors published previously by our group, and well-known reference compounds. The alignments were further analyzed using comparative molecular similarity indices analysis. The binding of the inhibitors was consistent at the P1-P3 positions. Beyond the P3 position, two different binding modes were found, one that favors lipophilic structures and one that favors nonhydrophobic structures.

Slow-binding inhibitors of prolyl oligopeptidase with different functional groups at the P1 site.

POP (prolyl oligopeptidase) specifically hydrolyses a number of small proline-containing peptides at the carboxy end of the proline residue and POP inhibitors have been shown to have cognition-enhancing properties. It has been noted that certain functional groups at the P1 site of the inhibitor, which correspond to the substrate residue on the N-terminal side of the bond to be cleaved, increase the inhibitory potency. However, detailed mechanistic and kinetic analysis of the inhibition has not been studied. In the present study, we examined the effect of different functional groups at the P1 site of the parent inhibitor isophthalic acid bis-(L-prolylpyrrolidine) amide on the binding kinetics to POP. Addition of CHO, CN or COCH(2)OH groups to the P1 site increased the inhibitory potency by two orders of magnitude (K(i)=11.8-0.1 nM) and caused a clear slow-binding inhibition. The inhibitor containing a CHO group had the lowest association rate constant, k(on)=(2.43+/-0.12) x 10(5) M(-1) x s(-1), whereas the inhibitor with a CN group exhibited the fastest binding, k(on)=(12.0+/-0.08)x10(5) M(-1) x s(-1). In addition, the dissociation rate was found to be crucially dependent on the type of the functional group. Compounds with COCH(2)OH and CHO groups had much longer half-lives of dissociation (over 5 h) compared with the compound with the CN group (25 min), although the K(i) values of the compounds were relatively similar. A possibility to optimize the duration of inhibition by changing the functional group at the P1 site is important when planning therapeutically useful POP inhibitors.

New prolyl oligopeptidase inhibitors developed from dicarboxylic acid bis(l-prolyl-pyrrolidine) amides.

Isophthalic acid bis(l-prolyl-pyrrolidine) amide is a very potent prolyl oligopeptidase inhibitor, but it has a log P value of -0.2, which is very low for a compound targeted to the brain. Therefore, these types of compounds were further modified to improve the structure-activity relationships, with the focus on increasing the log P value. The inhibitory activity against prolyl oligopeptidase from pig brain was tested in vitro. The most promising compounds resulted from replacing the pyrrolidinyl group at the P5 site by cycloalkyl groups, such as cyclopentyl and cyclohexyl groups, and by a phenyl group. These compounds are slightly more potent, and they have a significantly higher log P value. The potency of these compounds was further increased by replacing the pyrrolidinyl group at the P1 site by 2(S)-cyanopyrrolidinyl and 2(S)-(hydroxyacetyl)pyrrolidinyl groups.

Dicarboxylic acid bis(L-prolyl-pyrrolidine) amides as prolyl oligopeptidase inhibitors.

New dicarboxylic acid bis(L-prolyl-pyrrolidine) amides were synthesized, and their inhibitory activity against prolyl oligopeptidase from pig brain was tested in vitro. As compared with earlier described prolyl oligopeptidase inhibitors, these new compounds have in common an L-prolyl-pyrrolidine moiety, but the typical lipophilic acyl end group is replaced by another L-prolyl-pyrrolidine moiety connected symmetrically with a short dicarboxylic acid linker. These compounds are a new type of peptidomimetic prolyl oligopeptidase inhibitor.

A cyclopent-2-enecarbonyl group mimics proline at the P2 position of prolyl oligopeptidase inhibitors.

With the aim to replace the natural amino acid proline by a proline mimetic structure, a cyclopent-2-enecarbonyl moiety was studied at the P2 position of prolyl oligopeptidase (POP) inhibitors. The cyclopent-2-enecarbonyl moiety proved to be an excellent proline mimetic at the P2 position of POP inhibitors. The replacement is particularly useful when increased lipophilicity is needed.

Substrate-dependent, non-hyperbolic kinetics of pig brain prolyl oligopeptidase and its tight binding inhibition by JTP-4819.

Prolyl oligopeptidase (POP) is a cytosolic serine protease that hydrolyses small peptides at the carboxyl end of the proline residue. It has raised pharmaceutical interest, since its inhibitors have been shown to have antiamnesic properties. We studied prolyl oligopeptidase kinetics with two 7-amino-4-methylcoumarin derivatives: Z-Gly-Pro-AMC and Suc-Gly-Pro-AMC. Z-Gly-Pro-AMC was found to obey standard Henri-Michaelis-Menten kinetics with a K(m) of 30+/-3 microM, whereas Suc-Gly-Pro-AMC exhibited substrate inhibition kinetics with K(m) and K(is) of 510+/-150 and 270+/-90 microM, respectively. Autodock simulations revealed that either the succinyl or the AMC-end of Suc-Gly-Pro-AMC may bind to the S'1 subsite of the active site. We believe that non-specifically bound Suc-Gly-Pro-AMC allows the simultaneous binding of second substrate molecule to the active site and this leads in substrate inhibition. In addition, we demonstrated that the inhibition type of a well characterized prolyl oligopeptidase inhibitor, JTP-4819, is competitive tight binding with a K(ic) of 0.045+/-0.008 nM. We suggest that due to the high concentration of prolyl oligopeptidase in the brain (0.12 nmol/g pig brain), the tight binding nature of the inhibition should be considered when using brain homogenate as the enzyme source in prolyl oligopeptidase inhibition measurements. This is of importance in studying structure-activity relationships of potent prolyl oligopeptidase inhibitors.

Gene Expression Profiling of Gliadin Effects on Intestinal Epithelial Cells Suggests Novel Non-Enzymatic Functions of Pepsin and Trypsin.

Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p<0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The results rather suggest that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study suggests novel non-enzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation.

Enzymatic detoxification of gluten by germinating wheat proteases: implications for new treatment of celiac disease.

INTRODUCTION: Currently the only treatment for celiac disease is a lifelong gluten-free diet. The diet is, however, often burdensome, and thus new treatment options are warranted. We isolated proteases from germinating wheat grain naturally meant for total digestion of wheat storage proteins and investigated whether these enzymes can diminish toxic effects of gluten in vitro and ex vivo. METHODS: Pepsin and trypsin digested (PT) gliadin was pretreated with proteases from germinating wheat, whereafter the degradation was analyzed by HPLC-MS (high-performance liquid chromatography and mass spectroscopy) and sodium dodecyl sulphate polyacrylamide gel electrophoresis. The toxicity of cleaved PT-gliadin products was assessed in Caco-2 epithelial cells, celiac patient-derived T cells, and in human small intestinal mucosal organ culture biopsies. RESULTS: Proteases from germinating wheat degraded gliadin into small peptide fragments, which, unlike unprocessed PT-gliadin, did not increase epithelial permeability, induce cytoskeletal rearrangement or changes in ZO-1 expression in Caco-2 cells. Pretreated gliadin did not stimulate T cell proliferation in vitro or enhance the production of autoantibodies to culture supernatants and the activation of CD25+ lymphocytes in the organ culture to the same extent as unprocessed PT-gliadin. DISCUSSION: Germinating wheat enzymes reduce the toxicity of wheat gliadin in vitro and ex vivo. Further studies are justified to develop an alternative therapy for celiac disease.

Cellular and subcellular distribution of rat brain prolyl oligopeptidase and its association with specific neuronal neurotransmitters.

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes proline-containing peptides shorter than 30-mer. It has been suggested that POP is associated with cognitive functions and inositol 1,4,5-triphosphate (IP(3)) signaling. However, little is known about the distribution and physiological role of POP in the brain. We used immunohistochemistry to determine the cellular and subcellular distribution of POP in the rat brain. POP was specifically expressed in the glutamatergic pyramidal neurons of the cerebral cortex, particularly in the primary motor and somatosensory cortices, and also in the CA1 field of hippocampus. Purkinje cells of the cerebellum were also intensively immunostained for POP. Double immunofluorescence indicated that POP was present in the gamma-aminobutyric acid (GABA)ergic and cholinergic interneurons of the thalamus and cortex but not in the nigrostriatal dopaminergic neurons. POP did not colocalize with astrocytic markers in any part of the rat brain. We used postembedding immunoelectron microscopy to determine the distribution of POP at the subcellular level. POP was mainly present in neuronal cytosol and membranes, hardly at all in neuronal plasma membrane, but more extensively in intracellular membranes such as the rough endoplasmic reticulum and Golgi apparatus. Our findings point to a role for POP--evidently modifying neuropeptide levels--in excitatory and inhibitory neurotransmission in the central nervous system via glutamatergic, GABAergic, and cholinergic neurotransmission systems. Furthermore, according to our results, POP may be involved in thalamocortical neurotransmission, memory and learning functions of the hippocampal formation, and GABAergic regulation of voluntary movements. Subcellular distribution of POP points to a role in protein processing and secretion.

Distribution of prolyl oligopeptidase in the mouse whole-body sections and peripheral tissues.

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyses proline-containing peptides shorter than 30-mer, including many bioactive peptides. The distribution of POP in the brain has been studied but little is known about the distribution of peripheral POP. We used immunohistochemistry to localize POP in mouse whole-body sections and at the cellular level in peripheral tissues. Furthermore, we used a POP activity assay to reveal the associations between POP protein and its enzymatic activity. The highest POP protein densities were found in brain, kidney, testis and thymus, but in the liver the amounts of POP protein were small. There were remarkable differences between the distribution of POP protein and activity. The highest POP activities were found in the liver and testis while kidney had the lowest activity. In peripheral tissues, POP was present in various cell types both in the cytoplasm and nucleus of the cells, in contrast to the brain where no nuclear localization was detected. These findings support the proposed role of POP in cell proliferation in peripheral tissues. The dissociation of the distribution of POP protein and its enzymatic activity points to nonhydrolytic functions of POP and to strict endogenous regulation of POP activity.

Distribution of immunoreactive prolyl oligopeptidase in human and rat brain.

Prolyl oligopeptidase (POP) is a serine endoprotease that hydrolyses peptides shorter than 30-mer. POP may have a role in inositol 1,4,5-triphosphate (IP(3)) signaling and in the actions of antidepressants, and POP inhibitors have exhibited antiamnesic and neuroprotective properties. However, little is known about the distribution of POP protein in the brain. We used immunohistochemistry to localize POP enzyme in the human whole hemisphere and in the rat whole brain. In humans, the highest POP densities were observed in caudate nucleus and putamen, hippocampus and cortex. In the rat, the highest POP densities were found in substantia nigra, hippocampus, cerebellum and caudate putamen. In general, the distribution of POP in human and rat brains was very similar and resembled that of IP(3) receptors. Our findings are support for a role of POP in movement regulation, cognition and possibly in IP(3) signaling. The expression of POP in processing nuclei further supports its function beyond neuropeptide metabolism.