4-Substituted boro-proline dipeptides: Synthesis, characterization, and dipeptidyl peptidase IV, 8, and 9 activities

The boroProline-based dipeptidyl boronic acids were among the first DPP-IV inhibitors identified, and remain the most potent known. We introduced various substitutions at the 4-position of the boroProline ring regioselectively and stereoselectively, and incorporated these aminoboronic acids into a series of 4-substituted boroPro-based dipeptides. Among these dipeptidyl boronic acids, Arg-(4S)-boroHyp (4q) was the most potent inhibitor of DPP-IV, DPP8 and DPP9, while (4S)-Hyp-(4R)-boroHyp (4o) exhibited the most selectivity for DPP-IV over DPP8 and DPP9.     

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.     

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.     

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.     

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.     

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.     

Type 2 diabetes--therapy with dipeptidyl peptidase IV inhibitors

The sole application of an inhibitor of the dipeptidyl peptidase DP IV (also DP 4, CD26, DPP-IV or DPP-4) to a mammal subsequently leading to improved glucose tolerance marks a major breakthrough in metabolic research bearing the potential of a new revolutionary diabetes therapy. This was demonstrated in rat applying the specific DP IV inhibitor isoleucyl thiazolidine. It was published in 1996 for the first time that a specific DP IV inhibitor in a given dose was able to completely block glucagon-like peptide-1 (GLP-1) degradation in vivo resulting in improved insulin response accompanied, by accelerated peripheral glucose disposal. Later on, these results were confirmed by several research teams applying DP IV inhibitors intravenously or orally. Today, the DP IV inhibition for the treatment of metabolic disorders is a validated principle. Now, more than 10 years after the initial animal experiments, first DP IV inhibitors as investigational drugs are tested in phase 3 clinical trials.     

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.     

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 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.     

Extended investigation of the substrate specificity of dipeptidyl peptidase IV from pig kidney.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney was investigated, using a series of substrates, in which the amino-acid residue in position P1, a structural derivative of proline, was altered with respect to ring size and substituents. It was demonstrated that dipeptidyl peptidase IV hydrolyses substrates of the type Ala-X-pNA, where X is proline (Pro), (R)-thiazolidine-4-carboxylic acid (Thz), (S)-pipecolic acid (Pip), (S)-oxazolidine-4-carboxylic acid (Oxa), or (S)-azetidine-2-carboxylic acid (Aze). The ring size and ring structure of the residue in the P1 position influence the rate of enzyme-catalysed hydrolysis of the substrate. The highest kcat value (814 s-1) was found for Ala-Aze-pNA. In contrast, the kcat value for Ala-Pro-pNA is nearly 55 s-1. With all substrates of this series, the rate-limiting step of the hydrolysis by dipeptidyl peptidase IV is the deacylation reaction. Compounds of substrate-like structure, in which the P2 residue has an R-configuration, are not hydrolysed by dipeptidyl peptidase IV.     

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.     

Possible ligand release pathway of dipeptidyl peptidase IV investigated by molecular dynamics simulations

Dipeptidyl peptidase IV (DPP4) is an important target for the treatment of Type II diabetes mellitus. The crystal structure of DPP4 demonstrates that there are two possible pathways to the active site, a side opening and a β propeller opening. However, it still lacks quantitative evidence to illustrate which pathway is more favorable for inhibitor to enter into or release from the active site. In this study, conventional and steered molecular dynamics simulations were performed to explore the details of inhibitor Q448 release from the active site of DPP4 via the two potential pathways. The comparisons of force and work together with potentials of mean force results suggested that the side opening might be more favorable for the inhibitor to pass through. Moreover, Glu205-Glu206 and Phe357 were recognized as two "key residues" in the active site for inhibitor binding. Accordingly, suggestions for further inhibitor design were provided.     

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.     

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.     

Potent and selective proline derived dipeptidyl peptidase IV inhibitors

In-house screening of the Merck sample collection identified proline derived homophenylalanine 3 as a DPP-IV inhibitor with modest potency (DPP-IV IC50=1.9 microM). Optimization of 3 led to compound 37, which is among the most potent and selective DPP-IV inhibitors discovered to date.     

Nonpeptide small-molecular inhibitors of dipeptidyl peptidase IV: N-phenylphthalimide analogs

A novel series of nonpeptide small-molecular dipeptidyl peptidase IV (DPP-IV) inhibitors with an N-phenylphthalimide skeleton has been developed. Some of the compounds, including 4-amino-(2,6-dimethylphenyl)phthalimides (7), 4- and 5-hydroxy-(2,6-diethylphenyl)phthalimide (11 and 14), 4-hydroxy-(2,6-diisopropylphenyl)phthalimide (12), and thiocarbonyl analogs of (2,6-diisopropylphenyl)phthalimide and their 4,5,6,7-tetrafluorinated derivative (18, 19 and 20), were more potent than the well-known DPP-IV-specific inhibitor, Pro-boroPro (PBP). Among them, 18 was revealed to be a DPP-IV-specific inhibitor, while the others also showed inhibitory activity toward another peptidase, aminopeptidase N (APN).     

Snake venom dipeptidyl peptidase IV: taxonomic distribution and quantitative variation

The present study examined the taxonomic distribution of dipeptidyl peptidase IV (DPP IV) activity in venoms of 59 ophidian taxa, representing seven subfamilies of the Families Elapidae and Viperidae. DPP IV activity is extremely variable at all taxonomic levels. It ranged from essentially none in laticaudine, hydrophiine, and some bungarine and elapine venoms, to 10.72 μmol 4-methoxy-β-naphthylamine liberated per min per 200 μg venom, for Ophiophagus hannah. Intra- and interpopulational variation were examined among eight populations of prairie rattlesnakes (Crotalus viridis viridis), Great Basin rattlesnakes (Crotalus viridis lutosus) and southern Pacific rattlesnakes (Crotalus viridis helleri). Among these populations, the mean weighted range of variation was 4.9-fold, and even among litter mates of C. v. lutosus, DPP IV activity varied as much as 5.6-fold. The two most salient findings, the near ubiquity of DPP IV in snake venoms and its great quantitative variability, even among full siblings, are paradoxical. The widespread distribution of the enzyme suggests an important role in envenomation, while the variable activity levels suggest that DPP IV and by extension, other individual enzymatic constituents, may not be under much individual selective pressure.