Dipeptidyl peptidase IV inhibitors: a promising new therapeutic approach for the management of type 2 diabetes

Glucagon-like peptide-1 is an insulinotropic hormone with antidiabetic potential due to its spectrum of effects, which include glucose-dependent stimulation of insulin and inhibition of glucagon secretion, tropic effects on the pancreatic beta-cells, inhibition of gastric emptying and the reduction of appetite. Glucagon-like peptide-1 is, however, extremely rapidly inactivated by the serine peptidase, dipeptidyl peptidase IV, so that the native peptide is not useful clinically. A new approach to utilise the beneficial effects of glucagon-like peptide-1 in the treatment of type 2 diabetes has been the development of orally active dipeptidyl peptidase IV inhibitors. Preclinical studies have demonstrated that this approach is effective in enhancing endogenous levels of glucagon-like peptide-1, resulting in improved glucose tolerance in glucose-intolerant and diabetic animal models. In recent studies of 3-12 months duration in patients with type 2 diabetes, dipeptidyl peptidase IV inhibitors have proved efficacious, both as monotherapy and when given in combination with metformin. Fasting and postprandial glucose concentrations were reduced, leading to reductions in glycosylated haemoglobin levels, while beta-cell function was preserved. Current information suggests dipeptidyl peptidase IV inhibitors are body weight neutral and are well tolerated. A number of dipeptidyl peptidase IV inhibitors are now in the late stages of clinical development. These have different properties, in terms of their duration of action and anticipated dosing frequency, but data from protracted dosing studies is presently not available to allow comparison of their clinical efficacy.     

Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis

The dipeptidyl peptidase IV gene family contains the four peptidases dipeptidyl peptidase IV, fibroblast activation protein, dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Dipeptidyl peptidase IV and fibroblast activation protein are involved in cell-extracellular matrix interactions and tissue remodeling. Fibroblast activation protein is upregulated and dipeptidyl peptidase IV is dysregulated in chronic liver disease. The effects of dipeptidyl peptidase 8 and dipeptidyl peptidase 9 on cell adhesion, cell migration, wound healing and apoptosis were measured by using green fluorescent protein fusion proteins to identify transfected cells. Dipeptidyl peptidase 9-overexpressing cells exhibited impaired cell adhesion, migration in transwells and monolayer wound healing on collagen I, fibronectin and Matrigel. Dipeptidyl peptidase 8-overexpressing cells exhibited impaired cell migration on collagen I and impaired wound healing on collagen I and fibronectin in comparison to the green fluorescent protein-transfected controls. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 enhanced induced apoptosis, and dipeptidyl peptidase 9 overexpression increased spontaneous apoptosis. Mechanistic investigations showed that neither the catalytic serine of dipeptidyl peptidase 8 or dipeptidyl peptidase 9 nor the Arg-Gly-Asp integrin-binding motif in dipeptidyl peptidase 9 were required for the impairment of cell survival, cell adhesion or wound healing. We have previously shown that the in vitro roles of dipeptidyl peptidase IV and fibroblast activation protein in cell-extracellular matrix interactions and apoptosis are similarly independent of catalytic activity. Dipeptidyl peptidase 9 overexpression reduced beta-catenin, tissue inhibitor of matrix metalloproteinases 2 and discoidin domain receptor 1 expression. This is the first demonstration that dipeptidyl peptidase 8 and dipeptidyl peptidase 9 influence cell-extracellular matrix interactions, and thus may regulate tissue remodeling.     

An Essential Signal Peptide Peptidase Identified in an RNAi Screen of Serine Peptidases of Trypanosoma brucei

The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival.     

Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase). Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline. In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R. The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid. Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase). Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.     

Metformin effects on dipeptidylpeptidase IV degradation of glucagon-like peptide-1

There is current interest in the use of inhibitors of dipeptidyl peptidase IV (DP IV) as therapeutic agents to normalize glycemic excursions in type 2 diabetic patients. Data indicating that metformin increases the circulating amount of active glucagon-like peptide-1 (GLP-1) in obese nondiabetic subjects have recently been presented, and it was proposed that metformin might act as a DP IV inhibitor. This possibility has been investigated directly using a number of in vitro methods. Studies were performed on DP IV enzyme from three sources: 20% human serum, purified porcine kidney DP IV, and recombinant human DP IV. Inhibition of DP IV hydrolysis of the substrate Gly-Pro-pNA by metformin was examined spectrophotometrically. Effects of metformin on GLP-1([7-36NH2]) degradation were assessed by mass spectrometry. In addition, surface plasmon resonance was used to establish whether or not metformin had any effect on GLP-1([7-36NH2]) or GLP-1([9-36NH2]) interaction with immobilized porcine or human DP IV. Metformin failed to alter the kinetics of Gly-Pro-pNA hydrolysis or GLP-1 degradation tested according to established methods. Surface plasmon resonance recordings indicated that both GLP-1([7-36NH2]) and GLP-1([9-36NH2]) show micromolar affinity (K(D)) for DP IV, but neither interaction was influenced by metformin. The results conclusively indicate that metformin does not act directly on DP IV, therefore alternative explanations for the purported effect of metformin on circulating active GLP-1 concentrations must be considered.     

Peptidases of the rumen bacterium, Prevotella ruminicola

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested.Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P. ruminicola. Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.     

Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes.

Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma-glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney.     

Circulation and degradation of GIP and GLP-1.

The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted from the intestinal K- and L-cells, respectively, but are immediately subject to rapid degradation. GLP-1 is found in two active forms, amidated GLP-1 (7-36) amide and glycine-extended GLP-1 (7-37), while GIP exists as a single 42 amino acid peptide. The aminopeptidase, dipeptidyl peptidase IV (DPP IV), which is found in the endothelium of the local capillary bed within the intestinal wall, is important for the initial inactivation of both peptides, with GLP-1 being particularly readily degraded. DPP IV cleavage generates N-terminally truncated metabolites (GLP-1 (9-36) amide / (9-37) and GIP (3-42)), which are the major circulating forms. Subsequently, the peptides may be degraded by other enzymes and extracted in an organ-specific manner. However, other endogenous metabolites have not yet been identified, possibly because existing assays are unable either to recognize them or to differentiate them from the primary metabolites. Neutral endopeptidase 24.11 has been demonstrated to be able to degrade GLP-1 in vivo, but its relevance in GIP metabolism has not yet been established. Intact GLP-1 and GIP are inactivated during passage across the hepatic bed by DPP IV associated with the hepatocytes, and further degraded by the peripheral tissues, while the kidney is important for the final elimination of the metabolites.     

Purification of Two Dipeptidyl Aminopeptidases II from Rat Brain and Their Action on Proline-Containing Neuropeptides

From the soluble and membrane fractions of rat brain homogenate, two enzymes that liberate dipeptides of the type Xaa-Pro from chromogenic substrates were purified to homogeneity. The two isolated dipeptidyl peptidases had similar molecular and catalytic properties: For the native proteins, molecular weights of 110,000 were estimated; for the denatured proteins, the estimate was 52,500. Whereas the soluble peptidase yielded one band of pI 4.2 after analytical isoelectric focusing, two additional enzymatic active bands were detected between pI 4.2 and 4.3 for the membrane-associated form. As judged from identical patterns after neuraminidase treatment, both peptidases contained no sialic acid. A pH optimum of 5.5 was estimated for the hydrolysis of Gly-Pro- and Arg-Pro-nitroanilide. Substrates with alanine instead of proline in the penultimate position were hydrolyzed at comparable rates. Acidic amino acids in the ultimate N-terminal position of the substrates reduced the activities of the peptidases 100-fold as compared with corresponding substrates with unblocked neutral or, especially, basic termini. The action of the dipeptidyl peptidase on several peptides with N-terminal Xaa-Pro sequences was investigated. Tripeptides were rapidly hydrolyzed, but the activities considerably decreased with increasing chain length of the peptides. Although the tetrapeptide substance P 1-4 was still a good substrate, the activities detected for the sequential liberation of Xaa-Pro dipeptides from substance P itself or casomorphin were considerably lower. Longer peptides were not cleaved. The peptidases hydrolyzed Pro-Pro bonds, e.g., in bradykinin 1-3 or 1-5 fragments, but bradykinin itself was resistant. The enzymes were inhibited by serine protease inhibitors, like diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, and by high salt concentrations but not by the aminopeptidase inhibitors bacitracin and bestatin. Based on the molecular and catalytic properties, both enzymes can be classified as species of dipeptidyl peptidase II (EC 3.4.14.2) rather than IV (EC 3.4.14.5). However, some catalytic properties differentiate the brain enzyme from forms of dipeptidyl peptidase II of other sources.     

The importance of the nine-amino acid C-terminal sequence of exendin-4 for binding to the GLP-1 receptor and for biological activity

Exendin-4, a 39-amino acid (AA) peptide, is a long-acting agonist at the glucagon-like peptide-1 (GLP-1) receptor. Consequently, it may be preferable to GLP-1 as a long-term treatment for type 2 diabetes mellitus. Exendin-4 (Ex-4), unlike GLP-1, is not degraded by dipeptidyl peptidase IV (DPP IV), is less susceptible to degradation by neutral endopeptidase, and possesses a nine-AA C-terminal sequence absent from GLP-1. Here we examine the importance of these nine AAs for biological activity of Ex-4, a sequence of truncated Ex-4 analogs, and native GLP-1 and GLP-1 analogs to which all or parts of the C-terminal sequence have been added. We found that removing these AAs from Ex-4 to produce Ex (1-30) reduced the affinity for the GLP-1 receptor (GLP-1R) relative to Ex-4 (IC50: Ex-4, 3.22+/-0.9 nM; Ex (1-30), 32+/-5.8 nM) but made it comparable to that of GLP-1 (IC50: 44.9+/-3.2 nM). The addition of this nine-AA sequence to GLP-1 improved the affinity of both GLP-1 and the DPP IV resistant analog GLP-1 8-glycine for the GLP-1 receptor (IC50: GLP-1 Gly8 [GG], 220+/-23 nM; GLP-1 Gly8 Ex (31-39), 74+/-11 nM). Observations of the cAMP response in an insulinoma cell line show a similar trend for biological activity.     

The SUMO1-E67 Interacting Loop Peptide Is an Allosteric Inhibitor of the Dipeptidyl Peptidases 8 and 9

The intracellular peptidases dipeptidyl peptidase (DPP) 8 and DPP9 are involved in multiple cellular pathways including antigen maturation, cellular homeostasis, energy metabolism, and cell viability. Previously we showed that the small ubiquitin-like protein modifier SUMO1 interacts with an armlike structure in DPP9, leading to allosteric activation of the peptidase. Here we demonstrate that the E67-interacting loop (EIL) peptide, which corresponds to the interaction surface of SUMO1 with DPP9, acts as a noncompetitive inhibitor of DPP9. Moreover, by analyzing the sensitivity of DPP9 arm mutants to the EIL peptide, we mapped specific residues in the arm that are important for inhibition by the EIL, suggesting that the peptide acts as an allosteric inhibitor of DPP9. By modifying the EIL peptide, we constructed peptide variants with more than a 1,000-fold selectivity toward DPP8 (147 nm) and DPP9 (170 nm) over DPPIV (200 μm). Furthermore, application of these peptides to cells leads to a clear inhibition of cellular prolyl peptidase activity. Importantly, in line with previous publications, inhibition of DPP9 with these novel allosteric peptide inhibitors leads to an increase in EGF-mediated phosphorylation of Akt. This work highlights the potential use of peptides that mimic interaction surfaces for modulating enzyme activity.     

Novel aspects of cellular action of dipeptidyl peptidase IV/CD26

The cellular dipeptidyl peptidase IV (DPIV, E.C.3.4.14.5, CD26) is a type II membrane peptidase with various physio-logical functions. Our main knowledge on DPIV comes from studies of soluble DPIV which plays a role in regulation of glucose homeostasis by inactivation of the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic poly-peptide. It has been reported that membrane-bound DPIV plays a crucial role in the immune system and in other tissues and cells, but the knowledge on the action of cellular DPIV and its regulation is limited. In this study, we show particularly for immune cells that DPIV and not DP8 or DP9 is the most potent member of the DPIV family in regulating cellular immune functions. Moreover, we provide evidence that soluble and cellular DPIV differ in functions and hand-ling of substrates and inhibitors owing to the different accessibility of peptide substrates to the two access paths of DPIV. The different functions are based on the favored access path of the central pore of cellular DPIV and a special central pore binding site which assists substrate access to the active site of the enzyme. The newly discovered central pore binding site mediates an autosterical regulation of cellular DPIV and is its most crucial target site to regulate cellular functions such as growth and cytokine production. Neuropeptide Y (NPY) processing by cellular DPIV was found to be inhibited by ligands which interact with the central pore binding site. This finding suggests a crucial role of the immunosuppressive cytokine NPY in the function of DPIV in growth regulation.     

Applications of dipeptidyl peptidase IV inhibitors in diabetes mellitus

A number of alternative therapies for type 2 diabetes are currently under development that take advantage of the actions of the incretin hormones glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide on the pancreatic beta-cell. One such approach is based on the inhibition of dipeptidyl peptidase IV (DP IV), the major enzyme responsible for degrading the incretins in vivo. DP IV exhibits characteristics that have allowed the development of specific inhibitors with proven efficacy in improving glucose tolerance in animal models of diabetes and type 2 human diabetics. While enhancement of insulin secretion, resulting from blockade of incretin degradation, has been proposed to be the major mode of inhibitor action, there is also evidence that inhibition of gastric emptying, reduction in glucagon secretion and important effects on beta-cell differentiation, mitogenesis and survival, by the incretins and other DP IV-sensitive peptides, can potentially preserve beta-cell mass, and improve insulin secretory function and glucose handling in diabetics.     

Role of hepatocyte nuclear factor 1alpha and 1beta in the transcriptional regulation of human dipeptidyl peptidase IV during differentiation of Caco-2 cells

Caco-2 cells undergo differentiation to an enterocytic-like cell when maintained in a post-confluent state for 1-2 weeks. During this period Caco-2 cells begin to express high levels brush border membrane associated enzymes such as dipeptidyl peptidase IV. Using the dipeptidyl peptidase IV gene promoter in electrophoretic mobility shift assays, we have shown for the first time that levels of hepatocyte nuclear factor 1alpha increase three- to fourfold during Caco-2 cell differentiation. Transient cotransfection experiments with 3T3 cells using dipeptidyl peptidase IV promoter constructs and expression vectors containing hepatocyte nuclear factor 1alpha and beta show that the ratio of alpha and beta modulates reporter gene expression. These results suggest that the increase in levels of hepatocyte nuclear factor 1alpha that occur during intestinal cell differentiation, are important for expression of dipeptidyl peptidase IV and other intestinal proteins.     

Conformation of N-terminal HIV-1 Tat (fragment 1-9) peptide by NMR and MD simulations.

The N-terminal portion of HIV-1 Tat covering residues 1-9 is a competitive inhibitor of dipeptidyl peptidase IV (DP IV). We have used 1H NMR techniques, coupled with molecular dynamics methods, to determine the conformation of this peptide in the three diverse media: DMSO-d6, water (pH 2.7) and 40% HFA solution. The results indicate that in both DMSO-d6 and HFA the peptide has a tendency to acquire a type I beta-turn around the segment Asp5-Pro6-Asn7-IIe8. The N-terminal end is seen to be as a random coil. In water, the structure is best described as a left-handed polyproline type II (PPII) helix for the mid segment region Asp2 to Pro6. The structures obtained in this study have been compared with an earlier report on Tat (1-9).     

Simultaneous determination of incretin hormones and their truncated forms from human plasma by immunoprecipitation and liquid chromatography-mass spectrometry

The incretins, glucose-dependent insulinotropic peptide (GIP(1-42)) and glucagon-like peptide 1 (GLP-1(7-36)), are involved in regulation of gastric emptying, glucose homeostasis, body fat regulation and the glucose-induced insulin secretion from the endocrine pancreas. After release in the circulation both peptides are rapidly degraded by the exopeptidase dipeptidyl peptidase IV (DP IV) to the inactive polypeptides GIP(3-42) and GLP-1(9-36). In vivo stabilization of the active incretins by orally available DP IV-inhibitors is now widely accepted as a new therapeutic approach in antidiabetic treatment. In order to demonstrate the pharmacodynamic effect of DP IV-inhibitors, it is necessary to measure the plasma levels of active and inactive forms of GIP and GLP-1. We previously described an immunoprecipitation method as sample preparation and concentration in combination with a LC-MS analysis for determination of active and inactive GIP. We could improve the efficiency and suitability of this method by reduction of the necessary sample volume to 1.0 ml and simultaneous measurement of GIP(1-42), GIP(3-42) and GLP-1(7-36), GLP-1(9-36), without loss of sensitivity. An LOQ of approximately 5 and 11 pmol/l was maintained for GIP and GLP-1, respectively.     

The degradation of bioactive peptides and proteins by dipeptidyl peptidase IV from human placenta

The degradation of several bioactive peptides and proteins by purified human dipeptidyl peptidase IV is reported. It was hitherto unknown that human gastrin-releasing peptide, human chorionic gonadotropin, human pancreatic polypeptide, sheep prolactin, aprotinin, corticotropin-like intermediate lobe peptide and (Tyr-)melanostatin are substrates of this peptidase. Kinetic constants were determined for the degradation of a number of other natural peptides, including substance P, the degradation of which has been described earlier in a qualitative manner. Generally, small peptides are degraded much more rapidly than proteins. However, the Km-values seem to be independent of the peptide chain length. The influence of the action of dipeptidyl peptidase IV on the biological function of peptides and proteins is discussed.     

Biological activity of GLP-1-analogues with N-terminal modifications

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and improves glycemic control in type 2 diabetes. In serum the peptide is degraded by dipeptidyl peptidase IV (DPP IV). The resulting short biological half-time limits the therapeutic use of GLP-1. Therefore, various GLP-1 analogues with alterations in cleavage positions were synthesized. GLP-1-receptor binding was investigated in RINm5F cells. Biological activity of the GLP-1 analogues was investigated in vitro by measuring cAMP production in RINm5F cells. GLP-1 analogues with modifications in position 2 were not cleaved by DPP IV and showed receptor affinity and in vitro biological activity comparable to native GLP-1. Analogues with alterations in positions 2 and 8, 2 and 9 or 8 and 9 showed a significant decrease in receptor affinity and biological activity. In vivo biological activity was tested in pigs. GLP-1 analogues were administered subcutaneously followed by an intravenous bolus injection of glucose. Plasma glucose and insulin were monitored over 4 h. Compared to native GLP-1, analogues with an altered position 2 showed similar or increased potency and biological half-time. Other GLP-1 analogues were less active. Despite the lack of degradation of these GLP-1 analogues by DPP IV in vitro, their biological action is as short as that of GLP-1, except for desamino-GLP-1, indicating that other degradation enzymes are important in vivo. Alterations of GLP-1 in positions 8 or 9 result in a loss of biological activity without extending biological half-time.     

Thioxo amino acid pyrrolidides and thiazolidides: new inhibitors of proline specific peptidases

Aminopeptidase P (APP), dipeptidyl peptidase II (DP II), dipeptidyl peptidase IV (DP IV) and prolyl oligopeptidase (POP) are proline specific peptidases. Hence, they are able to cleave peptide bonds containing the imino acid proline. Amino acid pyrrolidides (Pyrr) and thiazolidides (Thia) are well-known product analogue inhibitors of DP IV and POP. For the first time we describe the influence of a thioxo amide bond, incorporated into these compounds, on the inhibition of the proline specific peptidases. Taking into account the substrate specificity of these peptidases, we have synthesized Xaa-psi[CS-N]-Pyrr and Xaa-psi[CS-N]-Thia of the amino acids Ala, Phe, Val and Ile. The inhibition constants were determined for the above mentioned proline specific peptidases isolated from different sources. As a result, the serine proteases DP II, DP IV and POP were inhibited competitively, whereas metal-dependent APP displayed a linear mixed-type inhibition with inhibition constants up to 10(-4) M. Thioxylation of Xaa-Pyrr and Xaa-Thia led to a slight decrease of inhibition of DP IV and POP compared to Xaa-Pyrr and Xaa-Thia, though the inhibition constants were still in the range up to 10(-7) M. As Xaa-Thia exist as two isomers, we investigated isomer specific inhibition with regard to DP IV. Thus, our studies have revealed that DP IV was only inhibited by the Z isomer of the Xaa-psi[CS-N]-Thia. For the first time, Xaa-Pyrr and Xaa-Thia were characterized as inhibitors of DP II with inhibition constants in the micromolar range. In contrast to DP IV inhibition, the Xaa-psi[CS-N]-Pyrr and Xaa-psi[CS-N]-Thia have proven to be more potent inhibitors of DP II than the corresponding Xaa-Pyrr and Xaa-Thia. Thus, these Xaa-psi[CS-N]-Thia are new potent inhibitors especially suitable for DP II with K(i) values ranging in the upper nanomolar concentration.     

Biosynthesis and degradation of altered immature forms of intestinal dipeptidyl peptidase IV in a rat strain lacking the enzyme.

We have used a strain of rat (Fischer 344) lacking brush border membrane dipeptidyl peptidase IV activity to examine its effect on the intestinal assimilation of prolyl peptides. In addition, we have examined the biochemical basis for the enzyme deficiency. An analysis of several brush border membrane hydrolases in different regions of the small intestine demonstrates that these rats lack only dipeptidyl peptidase IV. They also have a greatly reduced ability to hydrolyze and absorb in vivo peptides of the NH2-X-Pro-Y type which are known substrates for the enzyme. Immunoblot analysis with polyclonal and monoclonal antibody indicates that the animals lack an identifiable dipeptidyl peptidase IV protein in intestinal epithelial cells. Levels and types of dipeptidyl peptidase IV mRNA were analyzed in several tissues and found to be similar to that of control animals. Biosynthetic labeling of intestinal explants revealed that two distinct forms (102 and 108 kDa) of dipeptidyl peptidase IV are initially synthesized by deficient rats, in contrast to the single protein (106 kDa) observed in normal animals. Pulse-chase labeling experiments (+/- endoglycosidase H) show that these two altered forms of dipeptidyl peptidase IV, although initially glycosylated with N-linked high mannose carbohydrate, fail to be processed to the mature complex glycosylated form and undergo intracellular degradation.