Demonstration of two molecular forms of dipeptidyl peptidase IV in normal human serum

The heterogeneity of dipeptidyl peptidase IV (EC 3.4.14.5) was investigated in normal human serum. Thin-layer analytical isoelectric focusing revealed the presence of multiple molecular forms of the enzyme, their isoelectric points being in the pH range of 3.30-4.25. The maximum of enzyme activity appeared around pH 3.50. After treatment with neuraminidase the pI shifted to 4.70-5.40 with two maxima at pH 5.00 and 5.15. The Triton X-100 solubilized as well as the papain-treated-Triton X-100 solubilized enzyme from the whole human adult jejunal biopsy were also found to be heterogeneous. They focused--both before and after neuraminidase treatment--at pH values different from those of the enzyme of normal human serum. There was almost no pI shift after neuraminidase treatment of the intestinal enzyme from adult enterobiopsy. Electrophoresis in continuous polyacrylamide gradient gels as well as gel chromatography on Bio-Gel A-1.5m revealed two molecular forms of dipeptidyl peptidase IV in normal human serum. The estimated relative molecular mass of the major enzyme form was 250 000 in both the separation techniques used. On the other hand, the apparent relative molecular mass of the minor enzyme form was 450 000 as assessed by gradient gel electrophoresis, and 550 000, when estimated by gel chromatography. The Km values for glycyl-L-proline-4-nitroanilide as substrate with the major and minor forms of the serum enzyme were 1.60 +/- 0.39 X 10(-4) mol/l and 1.60 +/- 0.13 X 10(-4) mol/l, respectively. Our results indicate that the dipeptidyl peptidase IV in normal human serum is a heterogeneous enzyme as far as its charge and molecular size are concerned.     

A kinetic fluorometric assay of dipeptidyl peptidase IV in viable human blood mononuclear cells

A continuous-rate fluorometric assay of dipeptidyl peptidase IV (DP-IV) in viable human blood mononuclear cells using 7-(L-glycyl-L-prolylamido)-4-methylcoumarin as the substrate is described. The assay method is accurate, rapid, and highly sensitive for measuring the level of cell-surface bound DP-IV activity in suspension of blood mononuclear cells, as well as of other viable cells bearing this enzyme. We believe that the kinetic assay is suitable for studying the regulation of expression and the role of plasma membrane-bound DP-IV on the cellular level.     

Purification and properties of dipeptidyl peptidase IV from human urine

Dipeptidyl peptidase IV (DPP-IV) (EC 3.4.14.5) has been purified from normal human urine using immunoaffinity chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of urinary DPP-IV was estimated to be 280 kDa by gel filtration. The Km value for the hydrolysis of (Gly-L-Pro)-4-methylcoumaryl-7-amide tosylate was calculated to be 1.25 +/- 0.25 X 10(-4) M; the pH optimum and pI were 8.7 and 6.5, respectively. These properties were the same as those of renal DPP-IV. Both renal and this urinary DPP-IV were inhibited by diisopropylfluorophosphate and activated by phospholipid. From these results we suppose that urinary DPP-IV may be derived from the kidney rather than from the blood     

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.     

Fluoroolefins as amide bond mimics in dipeptidyl peptidase IV inhibitors

The synthesis, selectivity, rat pharmacokinetic profile, and drug metabolism profiles of a series of potent fluoroolefin-derived DPP-4 inhibitors (4) are reported. A radiolabeled fluoroolefin 33 was shown to possess a high propensity to form reactive metabolites, thus revealing a potential liability for this class of DPP-4 inhibitors.     

Dipeptidyl peptidase IV activity in commercial solutions of human serum albumin

Due to the heterogeneous nature of commercial human serum albumin (cHSA), other components, such as the protease dipeptidyl peptidase IV (DPP-IV), possibly contribute to the therapeutic effect of cHSA. Here, we provide evidence for the first time that DPP-IV activity contributes to the formation of aspartate–alanine diketopiperazine (DA-DKP), a known immunomodulatory molecule from the N terminus of human albumin. cHSA was assayed for DPP-IV activity using a specific DPP-IV substrate and inhibitor. DPP-IV activity was assayed at 37 and 60 °C because cHSA solutions are pasteurized at 60 °C. DPP-IV activity in cHSA was compared with other sources of albumin such as a recombinant albumin (rHSA). In addition, the production of DA-DKP was measured by negative electrospray ionization/liquid chromatography mass spectrometry (ESI⁻/LCMS). Significant levels of DPP-IV activity were present in cHSA. This activity was abolished using a specific DPP-IV inhibitor. Fully 70 to 80% DPP-IV activity remained at 60 °C compared with the 37 °C incubate. No DPP-IV activity was present in rHSA, suggesting that DPP-IV activity is present only in HSA produced using the Cohn fractionation process. The formation of DA-DKP at 60 °C was observed with the DPP-IV inhibitor significantly decreasing this formation. DPP-IV activity in cHSA results in the production of DA-DKP, which could account for some of the clinical effects of cHSA.     

Study of dipeptidyl peptidase IV as a surface marker of human natural killer cells

The enriched populations of natural killer (NK) cells have been obtained using two different approaches. The first one was based on the separation in a density gradient of Percoll and further formation of rosettes with sheep erythrocytes. This method allowed to isolate the population containing approximately 70% of CD16(+)-cells. The second approach consisted in separating lymphocytes on a flow cytofluorometer FACS II. Using this method the population with 80% CD16(+)-cells was isolated from PBMC. Studies on morphological, phenotypical and functional characteristics of the first population revealed that NK cells constituted 70% of total number of cells; T-lymphocytes, 8-lymphocytes and monocytes constituted the minor population (10%, 8% and 1% respectively). Activity of DPIV was determined on both cell populations obtained. As it was shown, approximately 27% of the cells isolated using percoll density gradient and 22% of the cells after the separation on a flow cytofluorometer carried the enzyme molecules on the cell surface. The results of the present study apparently indicate that part of NK cells (about 10%) is characterised by the presence of DPIV on the cell surface.     

Ketopyrrolidines and ketoazetidines as potent dipeptidyl peptidase IV (DPP IV) inhibitors

In this paper, the synthesis and structure-activity relationships (SAR) of two classes of electrophile-based dipeptidyl peptidase IV (DPP IV) inhibitors, the ketopyrrolidines and ketoazetidines, is discussed. The SAR of these series demonstrate that the 2-thiazole, 2-benzothiazole, and 2-pyridylketones are optimal S1' binding groups for potency against DPP IV. In addition, both cyclohexyl glycine (CHG) and octahydroindole carboxylate (OIC) serve as the most potent S2 binding groups within each series. Stereochemistry at the alpha-position of the central ring is relevant to potency within the ketopyrrolidines series, but not in the ketoazetidine series. Finally, the ketoazetidines display enhanced stability over the corresponding ketopyrrolidines, while maintaining their potency. In fact, certain stabilized ketoazetidines can maintain their in vitro potency and inhibit DPP IV in the plasma for up to 6h.     

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.     

New fluorinated pyrrolidine and azetidine amides as dipeptidyl peptidase IV inhibitors

Cyclohexylglycine amides of various fluorinated pyrrolidines and azetidines were prepared and tested for activity against dipeptidyl peptidase IV and in vivo in the KK mouse model of type 2 diabetes. The tetrafluoropyrrolidide, cis-3,4-difluoropyrrolidide and the fluorinated azetidides displayed unexpectedly strong activity.     

Dipeptidyl peptidase IV in the immune system. Effects of specific enzyme inhibitors on activity of dipeptidyl peptidase IV and proliferation of human lymphocytes.

Dipeptidyl peptidase IV (DP IV) is a membrane peptidase playing a significant role in the process of activation and proliferation of human thymus-derived lymphocytes. This conclusion is drawn from (1) the induction of this enzyme on mitogen-activated T lymphocytes (cf. Sch?n, E. & Ansorge, S. (1990) Biol. Chem. Hoppe-Seyler 371, 699-705) and (2) the impairment of different functions of activated T cells in the presence of specific inhibitors and antibodies against DP IV (Sch?n, E. & al. (1987) Eur. J. Immunol 17, 1821-1826). This paper is aimed at testing new active site-specific peptide inhibitors for their efficiency as inhibitors of lymphocyte DP IV and DNA synthesis of mitogen-stimulated lymphocytes. These inhibitors comprise (i) diacylhydroxylamine derivatives of Xaa-Pro or Xaa-Ala peptides, (ii) different oligopeptides with N-terminal Xaa-Pro-sequences, and (iii) amino-acid amides of the pyrrolidide and the thiazolidide type. The thiazolidides of epsilon-(4-nitrobenzyloxycarbonyl)-L-lysine and of L-isoleucine as well as Ala-Pro-nitrobenzoylhydroxylamine are the most effective inhibitors in both test systems, yielding half-maximal inhibitory concentrations in the micromolar range. Cell viability was not impaired in this effective concentration range. Other inhibitors of DP IV are one to two orders of magnitude less efficient in the suppression of lymphocyte proliferation.     

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.     

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-l-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-l-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.     

Structure-based design and synthesis of benzimidazole derivatives as dipeptidyl peptidase IV inhibitors

A novel series of non-covalent, benzimidazole-based inhibitors of DPP-4 has been developed from a small fragment hit using structure-based drug design. A highly versatile synthetic route was created for the development of SAR, which led to the discovery of potent and selective inhibitors with excellent pharmaceutical properties.     

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).     

Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity

Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.     

Synthesis and biological evaluation of homopiperazine derivatives with beta-aminoacyl group as dipeptidyl peptidase IV inhibitors

Compounds with homopiperazine skeleton are designed to find a potent DPP-IV inhibitor without inhibiting CYP. Thus a series of beta-aminoacyl-containing homopiperazine derivatives was synthesized and evaluated. Compounds with acid moiety were found to be potent inhibitors of DPP-IV without inhibiting CYP 3A4. More specifically, compound 7m showed nanomolar activity with no inhibition towards five subtypes of CYPs, was considered as a prototype for further derivatization. Based on its X-ray co-crystal structure with human DPP-IV, we identified compounds 7s and 7t which showed good in vitro activity, no CYP inhibition, and good selectivity.     

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.