Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar K(i) values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.     

Molecular characterization of dipeptidyl peptidase activity in serum: soluble CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides.

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a serine type protease with an important modulatory activity on a number of chemokines, neuropeptides and peptide hormones. It is also known as CD26 or adenosine deaminase (ADA; EC 3.5.4.4) binding protein. DPPIV has been demonstrated on the plasmamembranes of T cells and activated natural killer or B cells as well as on a number of endothelial and differentiated epithelial cells. A soluble form of CD26/DPPIV has been described in serum. Over the past few years, several related enzymes with similar dipeptidyl peptidase activity have been discovered, raising questions on the molecular origin(s) of serum dipeptidyl peptidase activity. Among them attractin, the human orthologue of the mouse mahogany protein, was postulated to be responsible for the majority of the DPPIV-like activity in serum. Using ADA-affinity chromatography, it is shown here that 95% of the serum dipeptidyl peptidase activity is associated with a protein with ADA-binding properties. The natural protein was purified in milligram quantities, allowing molecular characterization (N-terminal sequence, glycosylation type, CD-spectrum, pH and thermal stability) and comparison with CD26/DPPIV from other sources. The purified serum enzyme was confirmed as CD26.     

Purification and characterization of barley dipeptidyl peptidase IV

Barley (Hordeum vulgare L.) storage proteins, which have a high content of proline (Pro) and glutamine, are cleaved by cysteine endoproteases to yield peptides with a Pro next to the N-terminal and/or C-terminal amino acid residues. A peptidase cleaving after Xaa-Pro- at the N terminus of peptides was purified from green barley malt. It was identified as a serine-type dipeptidyl peptidase (DPP), based on inhibitor studies, and the nature of the cleavage product. It is a monomeric glycoprotein with an apparent molecular mass of 105 kD (85 kD after deglycosylation), with a pI of 3.55 and a pH optimum at 7.2. Substrate specificity was determined with a series of fluorogenic peptide substrates with the general formula Xaa-Pro-AMC, where Xaa is an unspecified amino acid and AMC is 7-amino-4-methylcoumarin. The best substrates were Xaa = lysine and arginine, while the poorest were Xaa = aspartic acid, phenylalanine, and glutamic acid. The K(m) values ranged from 0.071 to 8.9 microM, compared with values of 9 to 130 microM reported for mammalian DPP IVs. We discuss the possible role of DPP IV in the degradation of small Pro-containing peptides transported from the endosperm to the embryo of the germinating barley grain.     

Identification and characterization of human DPP9, a novel homologue of dipeptidyl peptidase IV

We used an in silico approach to identify new cDNAs with homology to dipeptidyl peptidase IV (DPP IV). DPP IV (EC 3.4.14.5) is a serine protease with a rare enzyme activity having an important role in the regulation of various processes, such as blood glucose control and immune responses. Here, we report the identification and characterization of a novel DPP IV-like molecule, termed dipeptidyl peptidase-like protein 9 (DPP9). The deduced amino acid sequence of DPP9 has a serine protease motif, GWSYG, identical to that found in DPP IV. The presence of this motif, together with a conserved order and spacing of the Ser, Asp, and His residues that form the catalytic triad in DPP IV, places DPP9 in the "DPP IV gene family". Northern blots showed that DPP9 is ubiquitously expressed, with the highest expression levels in skeletal muscle, heart, and liver, and the lowest in brain. In vitro translation of the cloned full-length DPP9 sequence resulted in a DPP9 protein product that migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position similar to the predicted protein size of 98 kDa. Consistent with the lack of predicted transmembrane domains and a signal sequence, DPP9 was found in a soluble, putative cytosolic form. A DPP9 orthologue in mice was identified by expressed sequence tag database searches and verified by cDNA cloning.     

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors’ affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.     

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 1: identification of dipeptide derived leads

Dipeptide derivatives bearing various P2 residues and pyrrolidine derivatives as P1 mimics were evaluated in order to identify lead structures for the development of DPP8 and DPP9 inhibitors. Structure–activity-relationship data obtained in this way led to the preparation of a series of α-aminoacyl ((2S, 4S)-4-azido-2-cyanopyrrolidines). These compounds were shown to be nanomolar DPP8/9 inhibitors with modest overall selectivity toward DPP IV and DPP II.     

Synthesis and structure-activity relationships of potent 4-fluoro-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors

Dipeptidyl peptidase IV (DPP-IV) inhibitors are promising antidiabetic drugs, and several drugs are in the developmental stage. We previously reported that the introduction of fluorine to the 4-position of 2-cyanopyrrolidine enhanced the DPP-IV inhibitory effect. In the present report, we examined the structure-activity relationship (SAR) of 2-cyano-4-fluoropyrrolidine with N-substituted glycine at the 1-position. We report the identification of a potent and stable DPP-IV inhibitor (TS-021) with a long-term persistent plasma drug concentration and a potent antihyperglycemic activity.     

Glutamic acid analogues as potent dipeptidyl peptidase IV and 8 inhibitors

To find potent and selective inhibitors of dipeptidyl peptidase IV (DPP-IV), we synthesized a series of 2-cyanopyrrolidine with P2-site 4-substituted glutamic acid derivatives and tested their activities against DPP-IV, DPP8, and DPP-II. Analogues that incorporated a bulky substituent at the first carbon position of benzylamine or isoquinoline showed over 30-fold selectivity for DPP-IV over both DPP8 and DPP-II. From structure-activity relationship studies, we speculate that the S2 site of DPP8 might be similar to that of DPP-IV, while DPP-IV inhibitor with N-substituted glycine in the P2 site and/or with a moiety involving in hydrophobic interaction with the side chain of Phe357 might provide a better selectivity for DPP-IV over DPP8.     

Expression,purification, and characterization of human dipeptidyl peptidase IV/CD26 in Sf9 insect cells

The human dipeptidyl peptidase IV/CD26 (DPPIV/CD26) is a multifunctional type-II membrane bound glycoprotein. As a receptor of collagen I and fibronectin it mediates cell-cell and cell-matrix adhesion, and by interacting with extracellular adenosine deaminase and CD45 it is involved in regulatory and costimulatory events in the immune system. DPPIV/CD26 has a very distinct substrate specificity, and is potentially capable of truncating many cytokines, chemokines, and peptide hormones. In this study, we describe the overexpression, purification, and characterization of human DPPIV/CD26 in Spodoptera frugiperda (Sf9) cells, using the baculovirus system. Overexpression of DPPIV/CD26 was confirmed by measurement of its peptidase specificity, SDS-PAGE, and Western blot analyses. Expression rates were between 6.4 and 17.6 mg protein per liter suspension culture (1.5 x 10(9)cells). The N-linked oligosaccharide composition was examined and compared with that of mammalian cell-expressed DPPIV/CD26. Two-step purification by immunoaffinity chromatography and size-exclusion fast protein liquid chromatography (SE-FPLC) led to highly stable protein with significant peptidase activity. A subsequent gel filtration step on a Superdex 200 column yielded 2mg homogeneous dimeric DPPIV/CD26 (per liter insect cell culture) for crystallographic studies. Protein homogeneity was confirmed by silver staining of non-denaturating PAGE gels and by MALDI-TOF analysis of tryptic peptides.     

Selective fluorescence probes for dipeptidyl peptidase activity-fibroblast activation protein and dipeptidyl peptidase IV

Development of suitable tools to assess enzyme activity directly from their complex cellular environment has a dramatic impact on understanding the functional roles of proteins as well as on the discovery of new drugs. In this study, a novel fluorescence-based chemosensor strategy for the direct readout of dipeptidase activities within intact living cells is described. Selective activity-based probes were designed to sense two important type II transmembrane serine proteases, fibroblast activation protein (FAP) and dipeptidyl peptidase IV (DPP-IV). These serine proteases have been implicated in diverse cellular activities, including blood coagulation, digestion, immune responses, wound healing, tumor growth, tumor invasion, and metastasis. Here, we validated that Ac-GPGP-2SBPO and GPGP-2SBPO probes are excellent reporters of both proteolytic activities. Furthermore, the novel probes can differentiate between FAP and DPP-IV proteolytic activities in cellular assay. Potentially, this assay platform is immediately useful for novel drug discovery.     

Synthesis and biological activity of sulphostin analogues, novel dipeptidyl peptidase IV inhibitors

The structure of sulphostin (1), a novel dipeptidyl peptidase IV (DPP-IV) inhibitor, is consisted of three key functional groups, including a characteristic amino(sulfoamino)phosphinyl group, on a piperidine ring. To examine the relationship between its structure and the inhibitory activity against DPP-IV, various analogues were synthesized and their activities were investigated. These results indicated that all of the functional groups on the piperidine ring were crucial to the DPP-IV inhibitory activity of sulphostin, and that the sulfonic acid group, which constructed the amino(sulfoamino)phosphinyl group, contributed to the stability of the compound. Moreover, those functional groups should be adjoined on the piperidine ring for the inhibitory activity. The size of the nitrogen-containing heterocyclic ring including piperidine appeared to scarcely affect the activity. In the present study, the sulfonic acid-deficient five-membered ring analogue 27a showed the strongest inhibitory activity (IC50=11 nM).     

4-Amino cyclohexylglycine analogues as potent dipeptidyl peptidase IV inhibitors

Substituted 4-amino cyclohexylglycine analogues were evaluated for DP-IV inhibitory properties. Bis-sulfonamide 15e was an extremely potent 2.6 nM inhibitor of the enzyme with excellent selectivity over all counterscreens. 2,4-difluorobenzenesulfonamide 15b and 1-naphthyl amide 16b, however, combined an acceptable in vitro profile with good pharmacokinetic properties in the rat, and 15b was orally efficacious at 3 mpk in an OGTT in lean mice.     

Purification and characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.     

Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes.

Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.     

Synthesis and structure-activity relationships of potent 3- or 4-substituted-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors

Dipeptidyl peptidase IV (DPP-IV) inhibitors have attracted attention as potential drugs for use in the treatment of type 2 diabetes because they prevent degradation of glucagon-like peptide-1 (GLP-1) and extend its duration of action. A series of 2-cyanopyrrolidines are among the most potent of DPP-IV inhibitors. We focused our attention on substitutions at the 3- or 4-position of 2-cyanopyrrolidines and synthesized and evaluated various derivatives. Among them, the 4-fluoro derivative was found to exhibit better DPP-IV inhibitory activity and higher plasma drug concentrations after oral administration to rats than the 4-unsubstituted derivative. We report here on the synthesis and biological data of the aforementioned derivatives.     

4-arylcyclohexylalanine analogs as potent, selective, and orally active inhibitors of dipeptidyl peptidase IV

A novel series of 4-arylcyclohexylalanine DPP-4 inhibitors was synthesized and tested for inhibitory activity as well as selectivity over the related proline-specific enzymes DPP-8 and DPP-9. Optimization of this series led to 28 (DPP-4 IC(50)=4.8 nM), which showed an excellent pharmacokinetic profile across several preclinical species. Evaluation of 28 in an oral glucose tolerance test demonstrated that this compound effectively reduced glucose excursion in lean mice.     

Presence of dipeptidyl peptidase II, dipeptidyl peptidase IV, and prolyl endopeptidase in effusion from patients with serous otitis media

We have measured for the first time, using specific substrates and specific fluorometric analyses, activities of three pathophysiologically important peptidases, i.e., dipeptidyl peptidase II, dipeptidyl peptidase IV, and prolyl endopeptidase in effusions from 45 patients with chronic otitis media with effusion. In 20 patients, DPP II and DPP IV were assayed simultaneously in effusions and sera. Activity of PEP was also estimated in effusions and sera from 25 patients. The mean values (+/- SD) of DPP II and DPP IV (n = 45) and PEP (n = 25) in effusion from patients with OME were 0.020 +/- 0.007, 0.66 +/- 0.04, and 0.040 +/- 0.006 nmole/min/mg protein, and 0.21 +/- 0.01, 16.2 +/- 1.87, and 1.90 +/- 0.23 nmole/min/ml of effusion, respectively. The mean values (+/- SD) for DPP II, DPP IV, and PEP in sera were 2.82 +/- 0.18, 54.8 +/- 1.23, and 3.73 +/- 0.33 nmole/min/ml of serum, respectively, which were similar to our previously reported values. Activities of DPP II, DPP IV, and PEP of serous effusions were comparable to those in serum. However, there was no significant correlation between their activities in serum and effusion. This may suggest that the major source of these enzymes in effusions may not be serum but the cells in the middle ear.     

Activities of dipeptidyl peptidase II, dipeptidyl peptidase IV, prolyl endopeptidase, and collagenase-like peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis.

We examined the activities of peptidases in the synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Dipeptidyl peptidase II (DPP II), prolyl endopeptidase (PEP), and collagenase-like peptidase (CLP) activities were higher in knee joint synovial membrane from patients with RA than in that from patients with OA. DPP II and PEP activities in knee joint synovial membrane of patients with RA increased in parallel with the increase in joint fluid volume, whereas DPP IV activity decreased in parallel with the increase in joint fluid volume. These results suggest that these peptidases in the synovial membrane may play some role in immunological disturbances in the joints of patients with RA. Measurement of these peptidases in synovial membrane may be useful in the diagnosis of the severity of local joint inflammation.     

Novel N-substituted 4-hydrazino piperidine derivative as a dipeptidyl peptidase IV inhibitor

A novel class of N-substituted 4-hydrazino piperidine derivatives were designed, synthesized and evaluated for DPP IV inhibition. The SAR studies on the N-substituted piperidine led to the discovery of compound 22e as a potent DPP IV inhibitor (IC₅₀ 88 nM), which is highly selective over other peptidases. In vivo efficacy indicates that compound 22e stimulates insulin release in response to glucose load and improves glucose tolerance in n5-STZ and Zucker Diabetic Fatty (ZDF) rats.