Circulating Dipeptidyl Peptidase 3 Modulates Systemic and Renal Hemodynamics Through Cleavage of Angiotensin Peptides.

BACKGROUND: High circulating DPP3 (dipeptidyl peptidase 3) has been associated with poor prognosis in critically ill patients with circulatory failure. In such situation, DPP3 could play a pathological role, putatively via an excessive angiotensin peptides cleavage. Our objective was to investigate the hemodynamics changes induced by DPP3 in mice and the relation between the observed effects and renin-angiotensin system modulation. METHODS: Ten-week-old male C57Bl/6J mice were subjected to intravenous injection of purified human DPP3 or an anti-DPP3 antibody (procizumab). Invasive blood pressure and renal blood flow were monitored throughout the experiments. Circulating angiotensin peptides and catecholamines were measured and receptor blocking experiment performed to investigate the underlying mechanisms. RESULTS: DPP3 administration significantly increased renal blood flow, while blood pressure was minimally affected. Conversely, procizumab led to significantly decreased renal blood flow. Angiotensin peptides measurement and an AT1R (angiotensin II receptor type 1) blockade experiment using valsartan demonstrated that the renovascular effect induced by DPP3 is due to reduced AT1R activation via decreased concentrations of circulating angiotensin II, III, and IV. Measurements of circulating catecholamines and an adrenergic receptor blockade by labetalol demonstrated a concomitant catecholamines release that explains blood pressure maintenance upon DPP3 administration. CONCLUSIONS: High circulating DPP3 increases renal blood flow due to reduced AT1R activation via decreased concentrations of circulating angiotensin peptides while blood pressure is maintained by concomitant endogenous catecholamines release.

Effects of Dipeptidyl Peptidase-4 Inhibitor and Angiotensin-Converting Enzyme Inhibitor on Experimental Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease in the United States and worldwide. Proteinuria is a major marker of the severity of injury. Dipeptidyl peptidase-4 inhibitor (DPP-4I) increases incretin-related insulin production and is, therefore, used to treat diabetes. We investigated whether DPP4I could have direct effect on kidney independent of its hypoglycemic activity. We, therefore, tested the effects of DPP4I with or without angiotensin-converting enzyme inhibitor (ACEI) on the progression of diabetic nephropathy and albuminuria in a murine model of DKD. eNOS-/-db/db mice were randomized to the following groups at age 10 weeks and treated until sacrifice: baseline (sacrificed at week 10), untreated control, ACEI, DPP4I, and combination of DPP4I and ACEI (Combo, sacrificed at week 18). Systemic parameters and urine albumin-creatinine ratio were assessed at baseline, weeks 14, and 18. Kidney morphology, glomerular filtration rate (GFR), WT-1, a marker for differentiated podocytes, podoplanin, a marker of foot process integrity, glomerular collagen IV, and alpha-smooth muscle actin were assessed at the end of the study. All mice had hyperglycemia and proteinuria at study entry at week 10. Untreated control mice had increased albuminuria, progression of glomerular injury, and reduced GFR at week 18 compared with baseline. DPP4I alone reduced blood glucose and kidney DPP-4 activity but failed to protect against kidney injury compared with untreated control. ACEI alone and combination groups showed significantly reduced albuminuria and glomerular injury, and maintained GFR and WT-1+ cells. Only the combination group had significantly less glomerular collagen IV deposition and more podoplanin preservation than the untreated control. DPP-4I alone does not decrease the progression of kidney injury in the eNOS-/-db/db mouse model, suggesting that targeting only hyperglycemia is not an optimal treatment strategy for DKD. Combined DPP-4I with ACEI added more benefit to reducing the glomerular matrix.

Update on Mast Cell Proteases as Drug Targets.

Mast cell granules are packed with proteases, which are released with other mediators by degranulating stimuli. Several of these proteases are targets of potentially therapeutic inhibitors based on hypothesized contributions to diseases, notably asthma and ulcerative colitis for ß-tryptases, heart and kidney scarring for chymases, and airway infection for dipeptidyl peptidase-I. Small-molecule and antibody-based ß-tryptase inhibitors showing preclinical promise were tested in early-phase human trials with some evidence of benefit. Chymase inhibitors were given safely in Phase II trials without demonstrating benefits, whereas dipeptidyl peptidase-I inhibitor improved bronchiectasis, in effects likely related to inactivation of the enzyme in neutrophils.

Dipeptidyl peptidase-4 inhibitor-related renal disease.

BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used to treat type 2 diabetes (T2D). Lowering blood glucose is expected also to reduce the progression of diabetic nephropathy. We experienced a patient with T2D who achieved good glycemic control with a DPP-4 inhibitor but experienced rapid deterioration of renal function. Therefore, we performed a retrospective study of similar patients treated at our hospital. METHODS: Out of 56 patients with biopsy-proven diabetic nephropathy who underwent native kidney biopsy at Toranomon Hospital from January 2018 through December 2022, we selected 22 patients who had been receiving DPP-4 inhibitors for at least 9 months at the time of kidney biopsy. Of these patients, we evaluated 16 diagnosed with class IIa diabetic nephropathy according to Tervaert's pathologic classification. The yearly estimated glomerular filtration rate (eGFR) slope in the 16 patients was arranged from the highest to the lowest slope. Ten patients with a large eGFR slope had thrombotic microangiopathy (TMA)-like lesions characterized by glomerular endothelial cell proliferation and GBM duplication on kidney biopsy (group A), whereas the remaining 6 patients did not have TMA-like lesions (group B). RESULTS: Group A had a median (interquartile range [IQR]) eGFR of 18.2 (16.2, 26.2) and a yearly median (IQR) eGFR slope of -11.2 (-17.6, -9.2) mL/min/1.73 m2 after of DPP-4 administration, whereas group B had a median (IQR) eGFR of 31.5 (21.9, 34.8) mL/min/1.73 m2 and a yearly median (IQR) eGFR slope of -1.6 (-3.1, -0.3). Renal function declined significantly more rapidly in group A than in group B, and proteinuria was higher in group A than in group B (median [IQR], 3.4 [2.6, 4.4] g/day vs 0.8 [0.4, 1.3] g/day, respectively). Five patients in group A progressed to dialysis during follow-up, but none of the patients in group B did. Median (IQR) hemoglobin A1c was 6.2 % (6.0 %, 6.6 %) in group A and 5.8 % (5.7 %, 6.6 %) in group B. CONCLUSION: DPP-4 inhibitors promote vascular endothelial regeneration, but when this effect occurs in the glomerulus, glomerular endothelial cell proliferation leads to TMA-like lesions, which may cause an increase in proteinuria and rapid decline in renal function.

Dipeptidyl peptidase-1 inhibition with brensocatib reduces the activity of all major neutrophil serine proteases in patients with bronchiectasis: results from the WILLOW trial.

BACKGROUND: Brensocatib is an oral, selective, reversible inhibitor of dipeptidyl peptidase-1 (DPP-1), responsible for activating neutrophil serine proteases (NSPs) including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG). In chronic inflammatory lung diseases such as non-cystic fibrosis bronchiectasis (NCFBE), neutrophils accumulate in the airways resulting in excess active NSPs that cause damaging inflammation and lung destruction. METHODS: The 24-week WILLOW trial (NCT03218917) was a randomized, double-blind, placebo-controlled, parallel-group trial in patients with NCFBE conducted at 116 sites across 14 countries. In this trial, treatment with brensocatib was associated with improvements in clinical outcomes including time to first exacerbation, reduction in exacerbation frequency and a reduction in NE activity in sputum. An exploratory analysis of NE activity in white blood cell (WBC) extracts and NE, PR3 and CatG activity in sputum was conducted to further characterize brensocatib's effect and identify potential correlated effects. RESULTS: NE, PR3 and CatG activities were reduced in sputum and NE activity was reduced in WBC extracts in a dose-dependent manner after four weeks of brensocatib treatment, with a return to baseline four weeks after the end of treatment. Brensocatib produced the greatest reduction in the sputum activity of CatG, followed by NE and then PR3. Positive correlations among the sputum NSPs were observed both at baseline and in response to treatment, with the strongest correlation among the sputum NSPs for NE and CatG. CONCLUSIONS: These results suggest a broad anti-inflammatory effect of brensocatib underlying its clinical efficacy observed in NCFBE patients. TRIAL REGISTRATION: The study was approved by the corresponding ethical review boards of all participating centers. The trial was approved by the Food and Drug Administration and registered at clinicaltrials.gov (NCT03218917) on July 17, 2017 and approved by the European Medicines Agency and registered at the European Union Clinical trials Register (EudraCT No. 2017-002533-32). An independent, external data and safety monitoring committee (comprising physicians with pulmonary expertise, a statistician experienced in the evaluation of clinical safety, and experts in periodontal disease and dermatology) reviewed all adverse events.

The antiatherosclerotic action of 1G244 - An inhibitor of dipeptidyl peptidases 8/9 - is mediated by the induction of macrophage death.

BACKGROUND: Targeting cell death to induce favorable functional and morphological changes within atherosclerotic plaques has long been postulated as a promising anti-atherosclerotic strategy. In this regard, inhibition of dipeptidyl peptidases 8/9 has received special attention in the context of chronic inflammatory diseases due to its regulatory role in macrophage death in vivo. METHODS: The present study investigates the influence of prolonged treatment with 1G244 - an inhibitor of dipeptidyl peptidases 8/9 - on the development of the advanced atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods. RESULTS: 1G244 administration has led to a reduction in atherosclerotic plaque size in an apoE-knockout mice model. Moreover, it reduced the content of in-plaque macrophages, attributed by immunohistochemical phenotyping to the pro-inflammatory M1-like activation state of these cells. Inhibition of dipeptidyl peptidases 8/9 augmented the lytic form of death response of activated macrophages in-vitro. CONCLUSIONS: In summary, inhibition of DPP 8/9 elicited an anti-atherosclerotic effect in apoE-/- mice, which can be attributed to the lytic form of death induction in activated macrophages, as assessed by the in vitro BMDM model. This, in turn, results in a reduction of the plaque area without its transformation towards a rupture-prone morphology.

Importance of Fibrosis in the Pathogenesis of Uterine Leiomyoma and the Promising Anti-fibrotic Effects of Dipeptidyl Peptidase-4 and Fibroblast Activation Protein Inhibitors in the Treatment of Uterine Leiomyoma.

Uterine fibroid or leiomyoma is the most common benign uterus tumor. The tumor is primarily composed of smooth muscle (fibroid) cells, myofibroblast, and a significant amount of extracellular matrix components. It mainly affects women of reproductive age. They are uncommon before menarche and usually disappear after menopause. The fibroids have excessive extracellular matrix components secreted by activated fibroblast cells (myofibroblast). Myofibroblast has the characteristics of fibroblast and smooth muscle cells. These cells possess contractile capability due to the expression of contractile proteins which are normally found only in muscle tissues. The rigid nature of the tumor is responsible for many side effects associated with uterine fibroids. The current drug treatment strategies are primarily hormone-driven and not anti-fibrotic. This paper emphasizes the fibrotic background of uterine fibroids and the mechanisms behind the deposition of excessive extracellular matrix components. The transforming growth factor-ß, hippo, and focal adhesion kinase-mediated signaling pathways activate the fibroblast cells and deposit excessive extracellular matrix materials. We also exemplify how dipeptidyl peptidase-4 and fibroblast activation protein inhibitors could be beneficial in reducing the fibrotic process in leiomyoma. Dipeptidyl peptidase-4 and fibroblast activation protein inhibitors prevent the fibrotic process in organs such as the kidneys, lungs, liver, and heart. These inhibitors are proven to inhibit the signaling pathways mentioned above at various stages of their activation. Based on literature evidence, we constructed a narrative review on the mechanisms that support the beneficial effects of dipeptidyl peptidase-4 and fibroblast activation protein inhibitors for treating uterine fibroids.

Expanded substrate specificity supported by P1' and P2' residues enables bacterial dipeptidyl-peptidase 7 to degrade bioactive peptides.

Dipeptide production from extracellular proteins is crucial for Porphyromonas gingivalis, a pathogen related to chronic periodontitis, because its energy production is entirely dependent on the metabolism of amino acids predominantly incorporated as dipeptides. These dipeptides are produced by periplasmic dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11. Although the substrate specificities of these four DPPs cover most amino acids at the penultimate position from the N terminus (P1), no DPP is known to cleave penultimate Gly, Ser, Thr, or His. Here, we report an expanded substrate preference of bacterial DPP7 that covers those residues. MALDI-TOF mass spectrometry analysis demonstrated that DPP7 efficiently degraded incretins and other gastrointestinal peptides, which were successively cleaved at every second residue, including Ala, Gly, Ser, and Gln, as well as authentic hydrophobic residues. Intravenous injection of DPP7 into mice orally administered glucose caused declines in plasma glucagon-like peptide-1 and insulin, accompanied by increased blood glucose levels. A newly developed coupled enzyme reaction system that uses synthetic fluorogenic peptides revealed that the P1' and P2' residues of substrates significantly elevated kcat values, providing an expanded substrate preference. This activity enhancement was most effective toward the substrates with nonfavorable but nonrepulsive P1 residues in DPP7. Enhancement of kcat by prime-side residues was also observed in DPP11 but not DPP4 and DPP5. Based on this expanded substrate specificity, we demonstrate that a combination of DPPs enables proteolytic liberation of all types of N-terminal dipeptides and ensures P. gingivalis growth and pathogenicity.

Neurofilament light is a treatment-responsive biomarker in CLN2 disease.

OBJECTIVE: Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is a rare, progressive, fatal neurodegenerative pediatric disorder resulting from deficiencies of the lysosomal enzyme tripeptidyl peptidase 1 that are caused by mutations in TPP1. Identifying biomarkers of CLN2 disease progression will be important in assessing the efficacy of therapeutic interventions for this disorder. Neurofilament light is an intrinsic component of healthy neurons; elevated circulating extracellular neurofilament light is a biomarker of neuropathology in several adult-onset neurological diseases. Our objective was to assess whether circulating neurofilament light is a biomarker that is responsive to enzyme replacement therapy (ERT) in CLN2 disease. METHODS: Using an ultrasensitive immunoassay, we assessed plasma neurofilament light changes during disease progression in a canine model of CLN2 disease and in ERT clinical trial CLN2 disease patients. RESULTS: In tripeptidyl peptidase 1 (TPP1)-null dogs (N = 11), but not in control dogs [N = 6 (TPP1+/- ) and N = 27 (WT)], neurofilament light levels increased more than tenfold above initial low baseline levels during disease progression. Before treatment in 21 human subjects with CLN2 disease (age range: 1.72-6.85 years), neurofilament light levels were 48-fold higher (P < 0.001) than in 7 pediatric controls (age range: 8-11 years). Pretreatment neurofilament light did not significantly correlate with disease severity or age. In CLN2 disease subjects receiving ERT, neurofilament light levels decreased by 50% each year over more than 3 years of treatment. INTERPRETATION: Our data indicate that circulating neurofilament light is a treatment-responsive biomarker in CLN2 disease and could contribute to understanding of the pathophysiology of this devastating pediatric disorder.

TPP1 Delivery to Lysosomes with Extracellular Vesicles and their Enhanced Brain Distribution in the Animal Model of Batten Disease.

Extracellular vesicles (EVs) are promising natural nanocarriers for delivery of various types of therapeutics. Earlier engineered EV-based formulations for neurodegenerative diseases and cancer are reported. Herein, the use of macrophage-derived EVs for brain delivery of a soluble lysosomal enzyme tripeptidyl peptidase-1, TPP1, to treat a lysosomal storage disorder, Neuronal Ceroid Lipofuscinoses 2 (CLN2) or Batten disease, is investigated. TPP1 is loaded into EVs using two methods: i) transfection of parental EV-producing macrophages with TPP1-encoding plasmid DNA (pDNA) or ii) incorporation therapeutic protein TPP1 into naive empty EVs. For the former approach, EVs released by pretransfected macrophages contain the active enzyme and TPP1-encoding pDNA. To achieve high loading efficiency by the latter approach, sonication or permeabilization of EV membranes with saponin is utilized. Both methods provide proficient incorporation of functional TPP1 into EVs (EV-TPP1). EVs significantly increase stability of TPP1 against protease degradation and provide efficient TPP1 delivery to target cells in in vitro model of CLN2. The majority of EV-TPP1 (≈70%) is delivered to target organelles, lysosomes. Finally, a robust brain accumulation of EV carriers and increased lifespan is recorded in late-infantile neuronal ceroid lipofuscinosis (LINCL) mouse model following intraperitoneal administration of EV-TPP1.

Pathophysiological Implications of Dipeptidyl Peptidases.

Dipeptidyl peptidases (DPPs) belong to one of the protease families classified under EC 3.4.14 in the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. DPPs family consists of eight members in the mammalian species. They play a role in oligopeptide N-terminal processing and degradation of bioactive peptides. Over the past 20 years, most of the studies have been focused on DPP 4 that has important roles in metabolism and immunity. A large number of pharmacological inhibitors against DPP 4 have been tested rigorously and some of them are now used in the treatment of type 2 diabetes and obesity. In addition, current researches cast a spotlight on other physiological and pathological functions of DPP family members such as DPP 3 for the purpose of investigating their application as novel therapeutic compounds. In this review, we provide an update about the pathophysiological functions of DPPs, and discuss the future potential of the DPP family as pharmacological and therapeutic agents and targets.

Novel Therapeutic Role for Dipeptidyl Peptidase III in the Treatment of Hypertension.

Dipeptidyl peptidase III (DPP III) cleaves dipeptide residues from the N terminus of polypeptides ranging from 3 to 10 amino acids in length and is implicated in pathophysiological processes through the breakdown of certain oligopeptides or their fragments. In this study, we newly identified the biochemical properties of DPP III for angiotensin II (Ang II), which consists of 8 amino acids. DPP III quickly and effectively digested Ang II with Km = 3.7×10(-6) mol/L. In the in vivo experiments, DPP III remarkably reduced blood pressure in Ang II-infused hypertensive mice without alteration of heart rate. DPP III did not affect hemodynamics in noradrenalin-induced hypertensive mice or normotensive mice, suggesting specificity for Ang II. When DPP III was intravenously injected every other day for 4 weeks after Ang II osmotic minipump implantation in mice, Ang II-induced cardiac fibrosis and hypertrophy were significantly attenuated. This DPP III effect was at least similar to that caused by an angiotensin receptor blocker candesartan. Furthermore, administration of DPP III dramatically reduced the increase in urine albumin excretion and kidney injury and inflammation markers caused by Ang II infusion. Both DPP III and candesartan administration showed slight additive inhibition in the albumin excretion. These results reveal a novel potential use of DPP III in the treatment of hypertension and its protective effects on hypertension-sensitive organs, such as the heart and kidneys.

Intrathecal tripeptidyl-peptidase 1 reduces lysosomal storage in a canine model of late infantile neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by mutations in the gene encoding tripeptidyl-peptidase 1 (TPP1). LINCL patients accumulate lysosomal storage materials in the CNS accompanied by neurodegeneration, blindness, and functional decline. Dachshunds homozygous for a null mutation in the TPP1 gene recapitulate many symptoms of the human disease. The objectives of this study were to determine whether intrathecal (IT) TPP1 treatment attenuates storage accumulation and functional decline in TPP1-/- Dachshunds and to characterize the CNS distribution of TPP1 activity. TPP1 was administered to one TPP1-/- and one homozygous wild-type (WT) dog. An additional TPP1-/- and WT dog received vehicle. Four IT administrations of 32 mg TPP1 formulated in 2.3 mL of artificial cerebrospinal fluid (aCSF) or vehicle were administered monthly via the cerebellomedullary cistern from four to seven months of age. Functional decline was assessed by physical and neurological examinations, electrophysiology, and T-maze performance. Neural tissues were collected 48 h after the fourth administration and analyzed for TPP1 activity and autofluorescent storage material. TPP1 was distributed at greater than WT levels in many areas of the CNS of the TPP1-/- dog administered TPP1. The amount of autofluorescent storage was decreased in this dog relative to the vehicle-treated affected control. No improvement in overall function was observed in this dog compared to the vehicle-treated TPP1-/- littermate control. These results demonstrate for the first time in a large animal model of LINCL widespread delivery of biochemically active TPP1 to the brain after IT administration along with a decrease in lysosomal storage material. Further studies with this model will be necessary to optimize the dosing route and regimen to attenuate functional decline.

N-terminally modified glucagon-like peptide-1(7-36) amide exhibits resistance to enzymatic degradation while maintaining its antihyperglycaemic activity in vivo.

Glucagon-like peptide-1(7-36)amide (tGLP-1) is inactivated by dipeptidyl peptidase (DPP) IV by removal of the NH(2)-terminal dipeptide His(7)-Ala(8). We examined the degradation of NH(2)-terminally modified His(7)99% of His(7)-glucitol tGLP-1 remained intact at 12 h. His(7)-glucitol tGLP-1 was similarly resistant to plasma degradation in vitro. His(7)-glucitol tGLP-1 showed greater resistance to degradation in vivo (92% intact) compared to tGLP-1 (27% intact) 10 min after i.p. administration to Wistar rats. Glucose homeostasis was examined following i.p. injection of both peptides (12 nmol/kg) together with glucose (18 mmol/kg). Plasma glucose concentrations were significantly reduced and insulin concentrations elevated following peptides administration compared with glucose alone. The area under the curve (AUC) for glucose for controls (AUC 691+/-35 mM/min) was significantly lower after administration of tGLP-1 and His(7)-glucitol tGLP-1 (36 and 49% less; AUC 440+/-40 and 353+/-31 mM/min, respectively; P<0.01). This was associated with a significantly higher AUC for insulin (98-99% greater; AUC 834+/-46 and 838+/-39 ng/ml/min, respectively; P<0.01) after tGLP-1 and His(7)-glucitol tGLP-1 administration compared to controls (421+/-30 ng/ml/min). In conclusion, His(7)-glucitol tGLP-1 resists plasma DPP IV degradation while retaining potent antihyperglycaemic and insulin-releasing activities in vivo.

Activation of thrombin-inactivated single-chain urokinase-type plasminogen activator by dipeptidyl peptidase I (cathepsin C).

Single-chain urokinase-type plasminogen activator (scu-PA) is inactivated by thrombin, which cleaves the peptide bond between Arg156 and Phe157. In a search for potential activators of thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), we found that the lysosomal aminopeptidase dipeptidyl-peptidase I or cathepsin C efficiently activates tcu-PA/T. Cathepsin C was as active towards tcu-PA/T as the bacterial proteinase thermolysin and about 300-times more active than plasmin. The activation by cathepsin C proceeded in a concentration-dependent and time-dependent manner with a pH optimum between 5 and 7. Furthermore, the effect of cathepsin C was inhibited by cystatin and stimulated by cysteine, typical for the action of a thiol proteinase. As no degradation of the tcu-PA/T molecule by cathepsin C was visible on SDS/PAGE, we suggest that activation of tcu-PA/T occurs by cleavage between Lys158-Ile159 and removal of the two N-terminal amino acid residues (Phe157-Lys158) of the B chain of tcu-PA/T. We conclude that both thrombin and dipeptidyl-peptidases like cathepsin C might play a regulatory role in the plasminogen-plasmin system by inactivating scu-PA and activating tcu-PA/T, respectively.

Dipeptidyl peptidase IV: development, design, synthesis and biological evaluation of inhibitors.

Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) is a highly specific serine protease which cleaves off N-terminal dipeptides from peptides with a penultimate proline or alanine. The enzyme is widely distributed in mammalian cells and tissues, but specific activities differ greatly. In the hematopoietic system it is found almost exclusively on T cells, where it is identified as CD26, a T cell activation molecule. DPP IV may be involved in the metabolism of peptides, intestinal assimilation and cell adhesion and it plays an integral role in T cell activation. DPP IV inhibitors may provide help during the further elucidation of the biological function(s) of this enzyme. Moreover, because of the integral role the enzyme plays in T cell activation, specific inhibition of DPP IV may constitute a new way of immune modulation. N-Peptidyl-O-acylhydroxylamines and boronic acid analogues of proline and alanine are two known DPP IV inhibitors. The major drawbacks for their therapeutic use are for the hydroxylamines, the toxicity and for the boronic acid derivatives, the chemical liability. A low toxicity, acceptable stability and a high specificity are essential criteria for the design of inhibitors that are suitable, not only for experimental, but also for therapeutic use. Therefore we proposed 5 types of potential DPP IV inhibitors: azapeptides, azetidines, Michael substrates, reduced peptides and phosphonic acids. All the synthesized compounds possess a substrate-like structure, which is a pre-requisite for recognition by the enzyme. We choose for a modified proline or alanine at the penultimate position, substituted with glycine, alanine, valine, isoleucine or phenylalanine at the N-terminus. The prepared compounds were screened biologically at a 5 mM concentration with a fluorometric method using Gly-Pro-4-Me-2NA as substrate.

Anti-CD26 monoclonal antibodies can reversibly arrest human T lymphocytes in the late G1 phase of the cell cycle.

Three different anti-CD26 monoclonal antibodies (mAbs) are described, which specifically inhibited proliferation of human T lymphocytes after stimulation with PHA, tetanus toxoid or soluble anti-CD3 mAb. Anti-CD26 mAbs induced in T cells a dose-dependent shift of the maximum of DNA synthesis, which was due to a transitory arrest of cells in the cell cycle. This cell cycle arrest was found to occur in the late phase of G1 since the expression of the T cell activation markers CD25-, CD71-, and HLA-DR-positive cells was the same in anti-CD26 mAb-containing and control cultures. Propidium iodide staining further confirmed the assumption that the arrest occurs in the first round of the cell cycle before S phase cells were detectable. Because the cells were arrested before consuming IL-2 in the S phase, we observed in accumulation of IL-2 in anti-CD26 mAb-containing cultures, whereas IFN-gamma production by PHA-activated T lymphocytes was reduced. These data indicate that CD26 is involved in the processes of T cell activation and proliferation.

In vitro degradation of apo-, zinc-, and cadmium-metallothionein by cathepsins B, C, and D.

Metallothionein (MT) has been extensively studied over the past several years because of its probable role in endogenous metal homeostasis and cellular protection. A large body of knowledge now exists describing the physicochemical properties of MT as well as the mechanisms involved in MT induction. It has been well established that MT protects tissues from metal toxicity by chelating metals that would otherwise be available to interact with and disrupt vital cell functions. Information on the degradation of metal-saturated MT and the fate of the metals associated with it would be extremely important in predicting metal toxicity. Lysosomes have been targeted as a possible subcellular site for the turnover of MT; however, the susceptibility of MT to degradation by specific acidic proteases (i.e., cathepsins) has not been described. Therefore, the purpose of the present study was to examine the relative abilities of cathepsins B, C, and D to degrade Zn7-MT, Cd7-MT, and apo-MT in vitro. In so doing, the effects of metal species, degree of metal saturation, and pH on the degradation processes were evaluated. Time course experiments revealed that apo-MT was rapidly degraded by all three cathepsins. Cathepsin B degraded apo-MT approximately 36-fold more rapidly than cathepsin C and 45-fold more rapidly than cathepsin D. Therefore, under the in vitro conditions used in this study, the relative potency of the cathepsins tested was cathepsin B much much greater than cathepsin C greater than cathepsin D. In comparison, metal-saturated MT was more than 1000-fold more resistant to degradation by the cathepsins tested. In order to determine how much metal was needed to protect MT against degradation, apo-MT was reconstituted with increasing molar equivalents of Zn2+. The results suggest that as metal to apo-MT ratios increase, less apo-MT substrate is available to the protease and degradation decreases.

Activities of dipeptidyl peptidases in BXSB mice and MRL/lpr mice with lupus erythematosus-like syndrome.

Activities of peptidases were examined in tissues of male BXSB and male MRL/Mp-lpr/lpr (MRL/lpr) mice which are animal models of human systemic lupus erythematosus. Female BXSB and male MRL/+ + mice without histopathological changes were used as controls. Activity of DPP II in the spleen, kidney, and liver showed an increase at 13 and 20 weeks of age, while that of DPP IV was decreased at 20 weeks of age, as compared to control mice. The ratio of DPP II/DPP IV activities in the tissues was significantly increased and these findings agree with our previous results in the tissues of NZB mice and in the serum of patients with lupus erythematosus, underscoring the importance of hydrolytic enzymes in the pathogenesis of autoimmune diseases.

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.