Lead discovery of quinoxalinediones as an inhibitor of dipeptidyl peptidase-IV (DPP-IV) by high-throughput screening

N-Ureido-quinoxalinedione derivatives have been discovered as leads for a novel series of dipeptidyl peptidase-IV (DPP-IV) inhibitors through high-throughput screening of our chemical library. A brief structure-activity relationship of the compounds was investigated. Among them, entry 5 showed the most potent inhibitory activity. The nitro group in quinoxaline moiety and the aromatic sulfonyl substituted ureido functional group seem to be important to increase the potency dramatically.     

Aminopiperidine-fused imidazoles as dipeptidyl peptidase-IV inhibitors

A new series of DPP-4 inhibitors derived from piperidine-fused benzimidazoles and imidazopyridines is described. Optimization of this class of DPP-4 inhibitors led to the discovery of imidazopyridine 34. The potency, selectivity, cross-species DMPK profiles, and in vivo efficacy of 34 is reported.     

Reduced serum dipeptidyl peptidase-IV after metformin and pioglitazone treatments

Dipeptidyl peptidase-IV (DPP-IV) regulates metabolism by degrading incretins involved in nutritional regulation. Metformin and pioglitazone improve insulin sensitivity whereas glyburide promotes insulin secretion. Zucker diabetic rats were treated with these antidiabetic agents for 2 weeks and DPP-IV activity and expression were determined. Serum DPP-IV activity increased whereas tissue activity decreased as the rats aged. Treatment of rats with metformin, pioglitazone, and glyburide did not alter DPP-IV mRNA expression in liver or kidney. Metformin and pioglitazone significantly (P<0.05) reduced serum DPP-IV activity and glycosylated hemoglobin. Glyburide did not lower DPP-IV activity or glycosylated hemoglobin. Regression analysis showed serum DPP-IV activity correlated with glycosylated hemoglobin (r=0.92) and glucagon-like peptide-1 levels (r=-0.49). Metformin, pioglitazone, and glyburide had no effect on serum DPP-IV activity in vitro, indicating these are not competitive DPP-IV inhibitors. We propose the in vivo inhibitory effects observed with metformin and pioglitazone on serum DPP-IV activity results from reduced DPP-IV secretion.     

Modulation of substance P signaling by dipeptidyl peptidase-IV enzymatic activity in human glioma cell lines

Dipeptidyl peptidase-IV (DPP-IV, CD26) is a serine protease almost ubiquitously expressed on cell surface and present in body fluids. DPP-IV has been suggested to proteolytically modify a number of biologically active peptides including substance P (SP) and the chemokine stromal cell derived factor-1alpha (SDF-1alpha, CXCL12). SP and SDF-1alpha have been implicated in the regulation of multiple biological processes and also induce responses that may be relevant for glioma progression. Both SP and SDF-1alpha are signaling through cell surface receptors and use intracellular calcium as a second messenger. The effect of DPP-IV on intracellular calcium mobilization mediated by SP and SDF-1alpha was monitored in suspension of wild type U373 and DPP-IV transfected U373DPPIV glioma cells using indicator FURA-2. Nanomolar concentrations of SP triggered a transient dose dependent increase in intracellular calcium rendering the cells refractory to repeated stimulation, while SDF-1 had no measurable effect. SP signaling in DPP-IV overexpressing U373DPPIV cells was not substantially different from that in wild type cells. However, preincubation of SP with the DPP-IV overexpressing cells lead to the loss of its signaling potential, which could be prevented with DPP-IV inhibitors. Taken together, DPP-IV may proteolytically inactivate local mediators involved in gliomagenesis.     

Discovery of conformationally rigid 3-azabicyclo[3.1.0]hexane-derived dipeptidyl peptidase-IV inhibitors

The induction of conformationally restricted N-(aryl or heteroaryl)-3-azabicyclo[3.1.0]hexane derivatives at P₂ region of compounds of 2-cyanopyrrolidine class was explored to develop novel DPP-IV inhibitors. The synthesis, structure–activity relationship, and selectivity against related proteases are delineated.     

Probing prime substrate binding sites of human dipeptidyl peptidase-IV using competitive substrate approach

Dipeptidyl peptidase-IV is a cell surface protease which plays an important role in glucose homeostasis through proteolytic inactivation of incretin hormones, primarily glucagon like peptide-1 (GLP-1). Substrate N-terminal amino acid (S2-S1) specificity is rather clearly defined, while no substantial information is available on the significance of amino acid interactions towards the C-terminus after the scissile bond (so called prime S1'-S4' or distant S5'-S28' sites). In the present study the increasing length of the peptide towards prime sites (S1'-S4') resulted in approximately 7-fold decrease in Km. Moreover, the Km for GLP-1 cleavage was comparable to that of an S2-S4' peptide, suggesting that few, if any, important enzyme-substrate interactions occur beyond the active site. Effect of substrate length on kcat was less obvious, but kcat/Km showed an increasing trend when His-Ala-pNA (representing the natural two N-terminal residues) was compared to GLP-1. To probe the impact of increasing substrate length on the free energy of activation (as has been suggested for elastase and chymotrypsin) we performed temperature studies. To adequately interpret thermodynamic data we sought to understand what steps limit the kcat expression. Steady-state parameters of the reactions catalyzed by serine proteases are composed of microscopic constants describing binding, acylation, and deacylation steps. Viscosity and pre-steady-state studies suggested that His-Ala-pNA cleavage is limited in the deacylation half-reaction, most likely the product release step. Thus, the free energy of activation, as calculated from the Eyring equation, is underestimated (at least for His-Ala-pNA) and the effect of substrate length on the acylation step (and transition-state stabilization) could not be unambiguously assessed.     

Synthesis, biological evaluation and structural determination of beta-aminoacyl-containing cyclic hydrazine derivatives as dipeptidyl peptidase IV (DPP-IV) inhibitors

Inhibitors of dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes. A series of beta-aminoacyl-containing cyclic hydrazine derivatives were synthesized and evaluated as DPP-IV inhibitors. One member of this series, (R)-3-amino-1-(2-benzoyl-1,2-diazepan-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one (10f), showed potent in vitro activity, good selectivity and in vivo efficacy in mouse models. Also, the binding mode of compound 10f was determined by X-ray crystallography.     

Structural basis of proline-specific exopeptidase activity as observed in human dipeptidyl peptidase-IV

Inhibition of dipeptidyl peptidase IV (DPP-IV), the main glucagon-like peptide 1 (GLP1)-degrading enzyme, has been proposed for the treatment of type II diabetes. We expressed and purified the ectodomain of human DPP-IV in Pichia pastoris and determined the X-ray structure at 2.1 A resolution. The enzyme consists of two domains, the catalytic domain, with an alpha/beta hydrolase fold, and a beta propeller domain with an 8-fold repeat of a four-strand beta sheet motif. The beta propeller domain contributes two important functions to the molecule that have not been reported for such structures, an extra beta sheet motif that forms part of the dimerization interface and an additional short helix with a double Glu sequence motif. The Glu motif provides recognition and a binding site for the N terminus of the substrates, as revealed by the complex structure with diprotin A, a substrate with low turnover that is trapped in the tetrahedral intermediate of the reaction in the crystal.     

Glucagon-like peptide 1 (GLP-1) secretion and plasma dipeptidyl peptidase IV (DPP-IV) activity in morbidly obese patients undergoing biliopancreatic diversion.

BACKGROUND: The physiological inhibitory control of glucagon-like Peptide 1 (GLP-1) on gastric emptying and the contribution of this peptide in the regulation of food intake as a satiety factor suggest that impaired secretion and/or activity of GLP-1 may be involved in the pathogenesis of obesity. We investigated food-mediated GLP-1 secretion as well as plasma activity of dipeptidyl-peptidase IV (DPP-IV), the enzyme responsible for rapid inactivation of the circulating peptide, in morbidly obese patients, before and after weight loss resulting from biliopancreatic diversion. METHODS: Twenty-two morbidly obese non-diabetic patients (BMI = 47.5 +/- 1.8) and 9 age-matched healthy volunteers were studied. A mixed meal (700 kcal) was administered to all subjects and blood samples were collected at 0, 15, 30, 60, 120 min for the determination of circulating glucose, insulin, GLP-1 (7 - 36 amide) concentrations and plasma DPP-IV activity. The patients repeated the test meal after 50 % overweight reduction resulting from surgical treatment (BMI = 33.8 +/- 1.1). RESULTS: While nutrient ingestion significantly increased plasma GLP-1 levels in the control group (30', 60': p < 0.01), the test-meal failed to modify basal peptide values in the obese patients, and an overall reduction in circulating GLP-1 occurred during the observation period (p < 0.001). Plasma DPP-IV activity in the same patients resulted as being significantly higher than controls, both at fasting and in response to the meal (p < 0.05). With respect to preoperative values, an overall increase in circulating GLP-1 levels occurred in all patients following biliopancreatic diversion (p < 0.001). Plasma DPP-IV activity, on the other hand, continued to be abnormally increased, even after considerable weight loss (p < 0.05 vs. controls). CONCLUSIONS: First: In morbid obesity, the accelerated inactivation of circulating GLP-1 could at least partially account for plasma peptide levels lower than normal, the defective availability of such a satiety factor possibly contributing to eating behaviour abnormalities; Second: plasma DPP-IV hyperactivity in the obese did not seem to be affected by the overweight degree, the increase in postoperative GLP-1 levels mainly resulting from hyperstimulation of GLP-1 secretory cells due to surgical manipulation of gastrointestinal tract. If the abnormally accelerated degradation of GLP-1 in obesity is confirmed, selective DPP-IV inhibitors could actually represent an ideal approach to obesity management.     

Inhibition of recombinant dipeptidyl peptidase III by synthetic hemorphin-like peptides

In order to find the most effective antagonist for dipeptidyl peptidase III degrading enkephalin, we synthesized hemorphin-like pentapeptides with aliphatic or aromatic amino acids at the N-termini, such as VVYPW, LVYPW, IVYPW, YVYPW, FVYPW and WVYPW. Among those pentapeptides, IVYPW and WVYPW showed the strongest inhibitory activity toward rDPP III. The K(i) values of IVYPW and WVYPW were 0.100+/-0.011 and 0.126+/-0.015 microM (mean+/-S.E.), respectively. The order of K(i) values was Ile> or =Trp>Phe> or =Tyr>Leu>Ala>Val>Ser>Gly. rDPP III activity is inhibited in a non-competitive manner by these peptides. The peptide VYPW did not inhibit rDPP III activity, but the sequence is essential for the expression of inhibitory activity.     

Inhibition of dipeptidyl peptidase IV (DPP IV) by 2-(2-amino-1-fluoro-propylidene)-cyclopentanecarbonitrile,a fluoroolefin containing peptidomimetic

Novel, potent inhibitors of dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5, CD26), containing the fluoroolefin peptide isostere psi [CFz.dbnd6;C], have been prepared via the intermediacy of the Peterson fluoroolefination reaction. The nitrile containing inhibitors were found to inhibit dipeptidyl peptidase IV competitively with K(i) values for the l-3 and u-3 inhibitors of 7.69 and 6.03 microM, respectively. In contrast to earlier reported fluoroolefin containing inhibitors, the nitriles underwent no detectable degradation at pH 7.6 under buffered conditions.     

Metabolism of glucagon by dipeptidyl peptidase IV (CD26)

Glucagon is a 29-amino acid polypeptide released from pancreatic islet alpha-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymatic metabolism of glucagon was assessed using sensitive mass spectrometric techniques to identify the molecular products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential production of glucagon(3-29) and glucagon(5-29). In human serum, degradation to glucagon(3-29) was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degradation, monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degradation and inactivation of glucagon. These findings have important implications for the determination of glucagon levels in human plasma.     

Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin.

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3 X 10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2 X 10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0 X 10(7) M-1 X s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2'-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.     

Effect of a dipeptidyl peptidase-IV inhibitor, des-fluoro-sitagliptin, on neointimal formation after balloon injury in rats

Background

Recently, it has been suggested that enhancement of incretin effect improves cardiac function. We investigated the effect of a DPP-IV inhibitor, des-fluoro-sitagliptin, in reducing occurrence of restenosis in carotid artery in response to balloon injury and the related mechanisms.

Methods and Findings

Otsuka Long-Evans Tokushima Fatty rats were grouped into four: control (normal saline) and sitagliptin 100, 250 and 500 mg/kg per day (n = 10 per group). Sitagliptin or normal saline were given orally from 1 week before to 2 weeks after carotid injury. After 3 weeks of treatment, sitagliptin treatment caused a significant and dose-dependent reduction in intima-media ratio (IMR) in obese diabetic rats. This effect was accompanied by improved glucose homeostasis, decreased circulating levels of high-sensitivity C-reactive protein (hsCRP) and increased adiponectin level. Moreover, decreased IMR was correlated significantly with reduced hsCRP, tumor necrosis factor-α and monocyte chemoattractant protein-1 levels and plasminogen activator inhibitor-1 activity. In vitro evidence with vascular smooth muscle cells (VSMCs) demonstrated that proliferation and migration were decreased significantly after sitagliptin treatment. In addition, sitagliptin increased caspase-3 activity and decreased monocyte adhesion and NFκB activation in VSMCs.

Conclusions

Sitagliptin has protective properties against restenosis after carotid injury and therapeutic implications for treating macrovascular complications of diabetes.     

Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors

Preclinical and clinical studies suggest that whey proteins can reduce postprandial glucose levels and stimulate insulin release in healthy subjects and in subjects with type 2 diabetes by reducing dipeptidyl peptidase-4 (DPP-4) activity in the proximal bowel and hence increasing intact incretin levels. Our aim was to identify DPP-4 inhibitors among short peptides occurring in hydrolysates of β-lactoglobulin, the major whey protein found in the milk of ruminants. We proved that the bioactive peptide Ile-Pro-Ala can be regarded as a moderate DPP-4 inhibitor.     

Studies on dipeptidyl(amino)peptidase IV (glycyl-proline naphthylamidase)

Summary The activity of dipeptidyl(amino)peptidase IV (DAP IV, glycylproline naphthylamidase) was discovered in the endothelial cells of the venous part of capillary bed and of small venules of many organs of the rat, mini-pig, rabbit, cock as well as man. In aortae, large arteries and veins only a portion of vase vasorum displays a positive reaction. Glycyl-proline-4-methoxy-2-naphthylamide (Gly-Pro-MNA) is the substrate of choice both from the viewpoint of enzyme kinetics as well as localization. Phenylalanyl-proline-4-methoxy-2-naphthylamide (Phe-Pro-MNA) is cleaved less easily, however, it enables a good localization. 1- and 2-naphthylamine derivatives of glycylproline display better kinetic properties than Phe-Pro-MNA, however they enable a satisfactory localization under special conditions only.The recommended diazonium salt for the routine is Fast Blue B. The enzyme is quite firmly associated with the structure and chloroform-acetone preextraction of cryostat sections does not influence its activity significantly while improving the localization. Block fixation in aldehydes inhibits the enzyme activity (glutaraldehyde more than formaldehyde). The osmificated azo-dye originated of 4-methoxy-2-naphthylamine and Fast Blue B or hexazonium-p-rosaniline is still partially soluble in solvents used for the usual embedding in epoxyresins for electron microscopical examination. This is a drawback for a reliable demonstration of DAP IV in endothelial cells on the electronmicroscopical level using the epoxy-resin technique. DAP IV of the endothelium is inhibited totally by DFP (10^–3M), partially by E 600 (10^–3); and slightly by phenanthroline (10^–3M). It is unaffected by EDTA (10^–3M) and N-ethyl maleimide (10^–3M).The combined demonstration of alkaline phosphatase and DAP IV in the same section renders a reliable demonstration of the capillary bed in many organs.The contribution of DAP IV activity of the capillary endothelium to the total DAP IV activity in a particular organ is decisive in the myocardium, striated muscle, aorta and lung; it represents about one half of the total activity in spleen and pancreas and is less expressed in the liver, intestine and particularly in the kidney.In the jejunum of patients sufferring coeliac sprue the activity of capillary endothelium in the propria is decreased or not demonstrable in the acute stage. After a gluten-free diet it is restituted. The activity of DAP IV does not change significantly in aortae of the rabbit and man with atherosclerosis. In plaques of human aortae the capillary endothelium reacts at the most. Vasa vasorum in the adventitia overlying large plaques, which penetrate into the media, display a high DAP IV activity and their number can be increased. In plaques of arteries of cocks there is a positive DAP IV reaction in foam cells. DAP IV does not belong to the enzymes indicating early changes in atherogenesis.The function of DAP IV in the endothelium is not known. It may be a part of the machinery influeneing the protein part of the endothelial coat or may participate in the degradation of some vasoactive peptides.     

Localization of angiotensin converting enzyme (dipeptidyl carboxypeptidase) in swine sperm by immufluorescence

Testis and epididymis are known to have high amounts of angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). We investigated the localization of the enzyme in these tissues by an immunofluorescent technique and found that the enzyme was localized in the spermatids and residual bodies in the Sertoli cells of the testis. Furthermore, the enzyme was shown to be present in the cytoplasmic droplet of epididymal sperm and also in detached cytoplasmic droplets in semen. The enzyme was not detected in the interstitium of testis and epididymis except for the endothelial cells of the vessel.     

Inhibition of phosphofructokinases by copper(II)

The biochemical inhibition by Cu2+ on eight phylogenetically and biochemically different phosphofructokinases (PFKs) was investigated. The enzymes screened included representatives from thermophilic and mesophilic bacteria, a hyperthermophilic archaeon and a eukaryote, covering all three phosphoryl donor subtypes (ATP, ADP and pyrophosphate). The sensitivities of the enzymes to Cu2+ varied greatly, with the archaeal ADP-PFK being the least and the eukaryote ATP-PFK being the most sensitive. The bacterial ATP- and pyrophosphate-dependent PFKs showed intermediate sensitivity with the exception of the Spirochaeta thermophila enzyme (pyrophosphate-dependent) which was relatively resistant.     

Inhibition of transketolase by hexacyanoferrate(III)

The effect of hexacyanoferrate(III) on the catalytic activity of transketolase has been studied. This oxidant inactivates only one of two active sites of the enzyme, the one with a higher affinity to the coenzyme (thiamine diphosphate). The second active site does not lose its catalytic activity. These observations indicate that the active sites of holotransketolase, being indiscernible by data of X-ray analysis, exhibit functional nonequivalence.