APN Activity Research Articles

Bufadienolides preferentially inhibit aminopeptidase N among mammalian metallo-aminopeptidases; relationship with effects on human melanoma MeWo cells

Bufadienolides are steroids that inhibit Na+/K+-ATPase; recent evidence shows that bufalin inhibits the activity of porcine aminopeptidase N (pAPN). We evaluated the selectivity of some bufadienolides on metallo-aminopeptidases. Among the enzymes of the M1 and M17 families, pAPN and porcine aminopeptidase A (pAPA) were the only targets of some bufadienolides. ѱ-bufarenogin, telocinobufagin, marinobufagin, bufalin, cinobufagin, and bufogenin inhibited the activity of pAPN in a dose-dependent manner in the range of 10−7–10−6 M. The inhibition mechanism was classical reversible noncompetitive for telocinobufagin, bufalin and cinobufagin. Bufogenin had the lowest Ki value and a non-competitive behavior. pAPA activity was inhibited by ѱ-bufarenogin, cinobufagin, and bufogenin, with a classical competitive type of inhibition. The models of enzyme-inhibitor complexes agreed with the non-competitive type of inhibition of pAPN by telocinobufagin, bufalin, cinobufagin, and bufogenin. Since APN is a target in cancer therapy, we tested the effect of bufadienolides on the MeWo APN+ human melanoma cell line; they induced cell death, but we obtained scant evidence that inhibition of APN contributed to their effect. Thus, APN is a selective target of some bufadienolides, and we suggest that inhibition of APN activity by bufadienolides is not a major contributor to their antiproliferative properties in MeWo cells.

A facile turn-on chemiluminescence probe for sensitive imaging on aminopeptidase N activity.

Aminopeptidase N, as a target for drug discovery, shows marked relationships with many diseases, especially liver injury and cancer. Here, we explored a chemiluminescence (CL) probe for sensing APN by tethering the APN-specific substrate group to the ortho-acrylated phenoxy-dioxetane scaffold. In this way, two CL probes (APN-CL and BAPN-CL) were designed with noncapped leucine and butoxy-carbonyl capped leucine as the protecting group to preserve the chemiexcitation energy. The uncovered leucine was demonstrated to be essential for detection of APN activity by comparing the CL intensity of two CL probes. Probe APN-CL was turned on upon APN cleavage, resulting in a high chemiluminescent emission, whereas the chemiexcitation energy of probe BAPN-CL was still restrained even with the high-level APN. The result was further elucidated by molecular docking simulations. Probe APN-CL exhibited a fast response and high sensitivity with a detection limit of 0.068 U/L, and an excellent specificity for the discrimination of APN from biological ions, small molecules, and other proteases commonly found in living system. By virtue of good stability and cell viability, probe APN-CL imaged abnormal levels of APN in tumour cells and tumour-bearing mice. Moreover, this probe APN-CL could be easily used to evaluate APN inhibitors and APN levels in plasma samples from 20 patients. Overall, as a facile and cost-effective probe, APN-CL will be a promising alternative in the early diagnosis of pathologies and for cost-effective screening of inhibitors.

De novo design of an ultrasensitive fluorogenic probe for aminopeptidase N sensing in living system

Aminopeptidase N is a widely expressed zinc-biding metalloprotease which plays important roles in maintaining normal physiological functions and the abnormal activity of APN is highly related chronic inflammatory disease and cancer progression. Thus, the detection and specific tracing of APN activity is of great importance in clinical diagnosis, drug discovery and disease related biological study. To date, enzyme activated fluorescent probe has attracted intense attention due to its high sensitivity and spatial-temporal resolution and biocompatibility in living system. Many researchers have attempted in constructing APN activity based probes and applicated in medical study such as tumor imaging, photodynamic therapy, and surgical navigation. However, these probes were all acquired by incorporating the fluorophore with its natural substrate. Thus, to discovery a new design strategy for APN targeting could provide efficient alternative method for APN probe construction and APN related chemical biology research. By adapting the unnatural amino-acid (2-Aminobutyric acid, Abu) which showed high affinity and specificity toward APN, we herein proposed a new probe design strategy for APN sensing and a near-infrared fluorescent probe TMN-Abu was constructed through this strategy. The in vitro assays showed that TMN-Abu possess high sensitivity toward APN (LOD = 0.57 ng/mL) and high binding affinity toward APN (Km = 19.1 ± 1.84 μM). Most importantly, TMN-Abu was successfully applicated into monitoring endogenous APN activity in living cells, tumor-bearing nude mice and for the first time applicated into diabetic model mice and APN activity dynamic change in biological events such as starvation and EGF stimulation.

Development of a novel latent electrochemical molecular substrate for the real-time monitoring of the tumor marker aminopeptidase N in live cells, whole blood and urine

Aminopeptidase N (APN/CD13) plays an important role in the growth and metastasis, of tumor, and is a potential biomarker for the post-treatment surveillance of cancer reoccurrence and progression of various malignancies. Thus, we have designed and prepared a convenient and ultrasensitive APN-targeting activity-based ratiometric electrochemical molecular substrate (Ala-AFC) for direct real-time monitoring of APN activity in biosamples. The APN in our experiment was used to hydrolyze the alanine moiety of the Ala-AFC probe and, as a result of this hydrolysis, realize concomitantly a cascade reaction to unmask the electrochemical reporter N-alkylated amino ferrocene (AAF). The Ala-AFC probe exhibited high sensitivity with a wide detection range of 0.05–110 ng mL-1 and a low limit of detection of 23.18 pg mL-1. The electrochemical signals were found to be distinctly specific for APN and free of interference from other electroactive biological species. Furthermore, the Ala-AFC probe was employed to monitor and quantify, in real-time, the activity of APN in tumor cells, whole blood, and urine. In addition, the results of our direct electrochemical quantifications of the amount of APN in whole blood and urine were found to be consistent with the results of the use of commercially available fluorometric assay kits to sense APN in serum and urine. Thus our approach shows promise as a point-of-care tool for cancer diagnostics and post-treatment surveillance of cancer reoccurrence.

Hyperlipidemic plasma molecules bind and inhibit adiponectin activity

IntroductionAdiponectin is a potent vascular protective molecule. Recent findings have suggested adiponectin resistance during early diabetes. However, the molecular mechanisms responsible remain unidentified. Here, we took an unbiased approach to identify whether hyperlipidemic plasma molecules exist that bind and inhibit adiponectin function, contributing to adiponectin resistance and diabetic vascular injury.MethodsAdult rats were randomly assigned to receive either a normal or a high‐fat diet for 8 weeks. Plasma was co‐immunoprecipitated with anti‐APN antibody and analyzed by mass spectrometry. The APN binding molecules and their effect upon APN biological activity were determined.ResultsAs expected, the high‐fat‐diet increased plasma triglyceride, total cholesterol, and low‐density lipoprotein. Importantly, the circulating APN level was significantly increased at this time point. Mass spectrometry identified 18 proteins with increased APN binding in hyperlipidemic plasma, among which four proteins critical in lipid metabolism, including apolipoprotein A1 (APOA1), APOA4, APOC1, and paraoxonase 1, were further investigated. Incubating recombinant APN with APOA1 markedly (P < 0.01), and incubating with APOC1 significantly (P < 0.05), inhibited APN activity as evidenced by the reduced AMPK activation in HUVECs. APOA4 and paraoxonase 1 incubation had no effect upon APN activity. Finally, plasma APOA1 was significantly increased (P < 0.05) in hyperlipidemic plasma compared with the control plasma.ConclusionsIt was demonstrated for the first time that increased APOA1 and APOC1 in hyperlipidemic plasma binds and inhibits APN activity. This result not only identifies a novel molecular mechanism responsible for adiponectin resistance during early stage diabetes, but also provides additional new insight into the diverse/controversial (protective and harmful) functions of high‐density lipoprotein.

Effect of non-competitive inhibitors of aminopeptidase N on viability of human and murine tumor cells

Abstract Cancer is the second leading cause of death worldwide. Peptidases participate in tumor development and growth. Mammalian neutral aminopeptidase (APN, EC 3.4.11.2, M1 family) catalyzes the cleavage of neutral and basic amino acids from the N-terminus of substrates. APN expression is dysregulated in several types of cancer, being a target for the development of new anticancer agents. Recently, we identified three new non-competitive inhibitors of soluble porcine APN (pAPN) by virtual screening (BTB11079, JFD00064, BTB07018, from Maybridge). In the present contribution we assayed their effect on the activity of APN in a microsomal preparation of porcine kidney cortex, a model of the physicochemical environment of the enzyme. These classical inhibitors had an IC50 value of 3–5 µM. Additionally, using a kinetic approach and a specific substrate, we quantified APN activity on the cell surface of human and murine lung, colon, prostate, and skin tumor cells. APN inhibitors reduced tumor cells viability, more efficiently in the higher APN activity tumor cell lines, but not in non-tumoral cells. BTB11079, JFD00064, BTB07018 effects on cell viability were stronger than that of bestatin, a positive control. Thus, these non-competitive APN inhibitors may be useful tools for cancer treatment.

Novel Human Aminopeptidase N Inhibitors: Discovery and Optimization of Subsite Binding Interactions.

Aminopeptidase N (APN/CD13) is a zinc-dependent M1 aminopeptidase that contributes to cancer progression by promoting angiogenesis, metastasis, and tumor invasion. We have previously identified hydroxamic acid-containing analogues that are potent inhibitors of the APN homologue from the malarial parasite Plasmodium falciparum M1 aminopeptidase (PfA-M1). Herein, we describe the rationale that underpins the repurposing of PfA-M1 inhibitors as novel APN inhibitors. A series of novel hydroxamic acid analogues were developed using a structure-based design approach and evaluated their inhibition activities against APN. N-(2-(Hydroxyamino)-2-oxo-1-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)ethyl)-4-(methylsulfonamido)benzamide (6ad) proved to be an extremely potent inhibitor of APN activity in vitro, selective against other zinc-dependent enzymes such as matrix metalloproteases, and possessed limited cytotoxicity against Ad293 cells and favorable physicochemical and metabolic stability properties. The combined results indicate that compound 6ad may be a useful lead for the development of anticancer agents.

Discovery of novel non-competitive inhibitors of mammalian neutral M1 aminopeptidase (APN)

Neutral metallo-aminopeptidase (APN) catalyzes the cleavage of neutral and basic amino acids from the N-terminus of protein or peptide substrates. APN expression is dysregulated in inflammatory diseases as well as in several types of cancer. Therefore, inhibitors of APN may be effective against cancer and inflammation. By virtual screening and enzymatic assays, we identified three non-competitive inhibitors (α > 1) of the porcine and human APN with Ki values in the μM range. These non-peptidic compounds lack the classical zinc-binding groups (ZBG) present in most of the APN inhibitors. Molecular docking simulations suggested the novel inhibitors suppress APN activity by an alternative mechanism to Zn coordination: they interacted with residues comprising the S1 and S5′ subsites of APN. Of note, these compounds also inhibited the porcine aminopeptidase A (pAPA) using a competitive inhibition mode. This indicated differences in the binding mode of these compounds with APN and APA. Based on sequence and structural analyses, we predicted the significance of targeting human APN residues: Ala-351, Arg-442, Ala-474, Phe-896 and Asn-900 for improving the selectivity of the identified compounds. Remarkably, the intraperitoneal injection of compounds BTB07018 and JFD00064 inhibited APN activity in rat brain, liver and kidney indicating good bio-distribution of these inhibitors in vivo. These data reinforce the idea of designing novel APN inhibitors based on lead compounds without ZBG.

Abstract 12836: Usefulness of Serum Levels of Adipocytokines and Estimated Δ-5 Desaturase Activity as Biomarkers Predicting Hepatic Steatosis in Patients With Coronary Artery Disease on Statin Therapy

Introduction: Nonalcoholic fatty liver disease (NAFLD) is closely linked to obesity or type 2 diabetes mellitus, which are risk factors for coronary artery disease (CAD), and also includes non-alcoholic steatohepatitis that often develops life-threatening liver cirrhosis. Imaging tests for NAFLD are more sensitive than liver biochemistries, but expensive and cumbersome as screening tests. An establishment of biomarkers for NAFLD is important for prevention of fatal liver diseases in CAD patients. Hypothesis: Serum levels of adipocytokines/adipokines and estimated Δ-5 desaturase (D5D) activity may be useful biomarkers predicting NAFLD in patients with CAD. Methods: A cross-sectional study was conducted on 127 patients with CAD on statin therapy. Dihomo-γ-linolenic acid (DGLA) and arachidonic acid (AA) in serum total lipids were measured by gas chromatography, and D5D activity was estimated by AA to DGLA ratio. Serum levels of adiponectin (Apn), leptin, resistin, visfatin and plasminogen activator inhibitor (PAI)-1, were determined by the respective ELISA. Abdominal computed tomography (CT) measured CT attenuation for liver (L-CTA) to estimate lipid content, and subjects with L-CTA &lt;50 HU were defined as NAFLD. Results: Simple linear regressions revealed that L-CTA was significantly correlated with serum levels of Apn, leptin and D5D activity (p &lt; 0.01, p = 0.03 and p &lt; 0.001, respectively), but not with those of resistin, visfatin or PAI-1. In multivariate logistic analysis among three factors consisting of a low Apn level: &lt; 6.8 μg/mL, a high leptin level: ≥ 3.4 ng/mL and a low D5D activity: ≤ 5.2 (the cutoff points were defined via ROC analysis against NAFLD), NAFLD was significantly associated with a high leptin level and a low D5D activity (adjusted odds ratio [OR]: 4.9, 95% confidence interval [CI]: 1.5-20) and OR: 15.6, 95%CI: 2.9-290, respectively), but weakly with a low Apn level (OR: 1.9, 95%CI: 0.6-6.5). A combination of these factors showed a strong association with NAFLD (area under the ROC curve: 0.84, p &lt; 0.0001). Conclusions: Serum levels of Apn, leptin and estimated D5D activity were significantly correlated with hepatic lipid content, and incrementally predict the presence of NAFLD in patients with CAD on statin therapy.

In vitro activity of some flavonoid derivatives on human leukemic myeloid cells: evidence for aminopeptidase-N (CD13) inhibition, antiproliferative and cell death properties

Leukemia cells from patients with acute myeloid leukemia (AML) display high proliferative capacity and are resistant to death. Membrane-anchored aminopeptidase-N/CD13 is a potential drug target in AML . Clinical research efforts are currently focusing on targeted therapies that induce death in AML cells. We previously developed a non-cytotoxic APN/CD13 inhibitor based on flavone-8-acetic acid scaffold, the 2',3-dinitroflavone-8-acetic acid (1) . In this context, among the variously substituted 113 compounds further synthesized and tested for evaluation of their effects on APN/CD13 activity, proliferation and survival in human AML U937 cells, eight flavonoid derivatives emerged: 2',3-dinitro-6-methoxy-flavone-8-acetic acid ( 2 ), four compounds ( 3 – 6 ) with the 3-chloro-2,3-dihydro-3-nitro-2-phenyl-4 H -1-benzopyran-4-one structure, and three ( 7 – 9 ) with the 3-chloro-3,4-dihydro-4-hydroxy-3-nitro-2-phenyl-2 H -1-benzopyran framework. Different structure-activity relationships were observed between APN/CD13 activity and growth/survival processes. We showed that compound 2 , but not benzopyran derivatives 3 – 9 , inhibited APN activity (although to a less degree than 1) . Both 1 and 2 did not affect AML cell proliferation and survival, indicating that CD13’s APN activity is not required for these processes. In contrast, benzopyran compounds 3 – 9 inhibited in a concentration-dependent manner the growth of U937 cells by inducing death as evidenced by phosphatidylserine externalization. Cell death was associated with the presence of geminal nitro group and chlorine at the 3-position of the 2 H -1-benzopyran scaffold. The presence of other substituents such as CH 2 COOH or CH 2 CH=CH 2 groups at the 8-position, NO 2 or I substituents at the 2'- or 3'-position, OCH 3 or OCH 2 C 6 H 5 groups at the 4'-position did not affect cell death. Importantly, the inhibitory effects evidenced with compounds 7 – 9 were not due to their potential decomposition into the corresponding ( Z )-(2chloro-2-nitroethenyl)benzene and salicylaldehyde. Based on these preliminary data, the 3-chloro,3-nitro-2 H -1-benzopyran derivatives could be classified as a new group of compounds with promising antitumor properties; this study therefore provides the opportunity to explore their potential efficiency in AML patients’ cells ex vivo .

Determination of enzyme activity in rabbit seminal plasma and its relationship with quality semen parameters

&lt;p&gt;The objective of this study was to determine rabbit seminal plasma enzyme activity. Furthermore, correlations between semen parameters and enzyme activity and male age were examined. The study was performed using 17 New Zealand White males from 5 to 9 mo old. Overall, 252 semen samples were collected from bucks from May to September. Semen characteristics were analysed and the seminal plasma was obtained by centrifugation. The activities of alanyl aminopeptidase (APN), aspartate transaminase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALKP) in the seminal plasma fluid were measured. Significant differences between males were found in APN, GGT, LDH, ALKP and ALT activities (P&amp;lt;0.05). No significant differences between enzyme activity and male age were found. We also observed significant positive correlations between male age and sperm concentration (r=0.26), progressive motility (r=0.17) and amplitude of lateral head displacement (r=0.21), and negative ones between male age and average path velocity (r=–0.56), velocity of the sperm head along its actual curvilinear path (r=–0.61), straight line velocity (r=–0.50), linearity index (r=–0.13), and cytoplasmic droplet (r=–0.33). Furthermore, a significant negative correlation between APN activity and the status of the acrosome (r=–0.20) and significant positive correlations between APN activity and the sperm abnormalities (r=0.21), GGT activity and sperm concentration (r=0.34) and the status of the acrosome (r=0.31), and ALKP activity and sperm concentration were observed (r=0.41). In our study, APN and GGT seem to be the most predictive enzymes for rabbit semen quality.&lt;/p&gt;

Design of a Hyperpolarized Molecular Probe for Detection of Aminopeptidase N Activity

AbstractAminopeptidase N (APN) is an important enzyme that is involved in tumor angiogenesis. Detection of APN activity can thus lead to early diagnosis and elucidation of tumor development. Although some molecular probes for APN have been developed, the detection of APN activity in opaque biological samples remains a challenge. To this end, we designed a hyperpolarized NMR probe [1‐13C]Ala‐NH2 which satisfies the prerequisites for APN detection, namely, sufficient retention of the hyperpolarized state, a high reactivity to APN, and an APN‐induced chemical shift change. The [1‐13C]Ala‐NH2 probe allowed sensitive detection of APN activity using 13C NMR spectroscopy.

Aminopeptidase N inhibitor 4cc synergizes antitumor effects of 5-fluorouracil on human liver cancer cells through ROS-dependent CD13 inhibition

Aminopeptidase N (APN, also known as CD13) is involved in cellular processes of various types of tumors and a potential anti-cancer therapeutic target. Here, we report the effect of an APN inhibitor 4cc in enhancing sensitivity of hepatocellular carcinoma (HCC) cell lines and xenograft model in response to 5-fluorouracil (5-FU) in vivo and in vitro. The treatment of the combination of 4cc with 5-FU, compared to the combination of bestain with 5-FU, markedly suppressed cell growth and induced apoptosis of HCC cells, accompanying the increase in the level of reactive oxygen species (ROS) and followed by a decrease in the mitochondrial membrane potential (ΔΨM). Furthermore, the combination of 4cc and 5-FU showed a significant inhibitory effect on the growth of HCC xenograft tumors. In addition, following the treatment of 4cc, APN activity and clonogenic formation and the number of CD13-positive cells in PLC/PRF/5 cells were significantly decreased, suggesting that 4cc may also inhibit liver cancer stem cells by CD13 inhibition. These results showed that the APN inhibitor 4cc synergizes antitumor effects of 5-FU on human liver cancer cells via ROS-mediated drug resistance inhibition and concurrent activation of the mitochondrial pathways of apoptosis.

Aminopeptidase N Activity Predicts 5-Year Survival in Colorectal Cancer Patients

BackgroundAminopeptidase N (APN; EC 3.4.11.2) is a membrane dimeric metallopeptidase involved in differentiation, development, and proliferative processes of several tissues. Recent studies have demonstrated the increased expression and activity of...

The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage.

ABSTRACTOsteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε–CD13 interaction could be a new therapeutic target in osteoarthritis.

Physiological responses to short-term fasting among herbivorous, omnivorous, and carnivorous fishes

We explored the integrated role of dietary specialization and feeding periodicity on the response of the gastrointestinal tract of teleosts fishes to short-term (7-10days) fasting and refeeding. Fasted and fed herbivorous grass carp (Ctenopharyngodon idella), omnivorous channel catfish (Ictalurus punctatus), and carnivorous largemouth bass (Micropterus salmoides) were compared for digestive organ masses, intestinal morphology, gastrointestinal pH, and the specific activities and total intestinal capacities of the intestinal hydrolases aminopeptidase (APN) and maltase and intestinal nutrient transporters. All three species experience intestinal hypertrophy with feeding as noted by significant increases in enterocyte dimensions. Of the three, only I. punctatus experienced a postprandial increase in intestinal length, and only C. idella experienced significant modulation of intestinal microvillus length. Feeding resulted in acidification of the stomachs of I. punctatus and M. salmoides. Predicted to exhibit a relatively modest set of postprandial responses because of their more frequent feeding habits, C. idella only experienced increases in APN and maltase activity with feeding and no significant regulation of nutrient uptake. Significant regulation of hydrolase activities and nutrient uptake were exhibited by I. punctatus and M. salmoides, with I. punctatus experiencing the most comprehensive set of responses. As predicted by food habits, there was an interspecific gradient in intestinal length and glucose uptake extending from longer intestines and greater glucose uptake for the herbivorous C. idella, intermediate lengths and glucose uptake for the omnivorous I. punctatus, and shorter intestines and reduced glucose uptake for the carnivorous M. salmoides. Among teleosts fishes, short episodes of fasting lead to significant alterations in intestinal form and function that are rapidly restored with feeding.

Structure- Activity Relationship Study and Function-Based Petidomimetic Design of Human Opiorphin with Improved Bioavailability Property and Unaltered Analgesic Activity

Human opiorphin inhibits enkephalin-inactivating ectopeptidases to produce analgesic and antidepressant-like effects in standard murine models via activation of μ and/or δ opioid pathways. It is an endogenous peptide regulator of enkephalin bioavailability. Opiorphin molecule, a QRFSR-peptide, is thus a promising prototype for the design of an improved class of analgesics. The major limitation on the clinical use of peptide drugs is their rapid degradation by circulating peptidases. Our goal was, therefore, to search for functional derivatives of opiorphin with improved metabolic stability. In order to identify the functional amino acid groups required for opiorphin inhibitory potency toward both AP-N and NEP human ectopeptidases, we used the Structure-Activity Relationship (SAR) method. From this data, a series of opiorphin derivatives was designed and selected. The best performing compound then underwent a complete metabolic profile using in vitro kinetic models. Finally, its safety profile relative to the native peptide as well as its efficacy in an in vivo rat model was evaluated. We demonstrated a tight structural selectivity in the functional interaction of opiorphin with both human NEP and AP-N targets by SAR studies. Nevertheless, we found that the addition of an N-terminal Zn-chelating group, a Cys-thiol group and the replacement of the first labile peptide bond by a polyethylene surrogate, a [CH2]6 linker,and, finally, the substitution of Ser4 by Ser-O-[CH2]8, results in a high performing C-[(CH2)6]-QRF[S-O-[CH2]8]-R peptidomimetic product. This designed opiorphin analog shows reinforced inhibitory potency toward human AP-N activity (more than 10-fold increase) and NEP activities (more than 40-fold increase) relative to the QRFSR native peptide. It also has increased metabolic stability in human plasma and yet retains full analgesic activity in the behavioral formalin-induced rat pain model. C-[(CH2)6]-QRF[S-O-[CH2]8]-R thus represents a very attractive and promising analgesic drug-candidate.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Neutral aminopeptidase and dipeptidyl peptidase IV in the development of collagen II-induced arthritis

This study evaluated the hypothesis that neutral (APN) and dipeptidyl-IV (DPPIV) aminopeptidase activity levels would be critical for the susceptibility to arthritis in collagen-induced model (CIA). The macroscopic signs of arthritis in CIA rats were checked and peripheral blood, synovial fluid and synovial tissue from knee joint were withdrawn. Soluble (SF) and solubilized membrane-bound (MF) fractions from the synovial tissue and peripheral blood mononuclear cells (PBMCs) were obtained. APN and DPPIV activities were fluorometrically quantified. Severe swelling in both the entire hind paws was the minimum criterion to select CIA rats with arthritis. These arthritic rats had high APN in plasma, synovial fluid and SF of the synovial tissue, together with low APN and DPPIV in MF of PBMCs and hallmark histological changes in tibio-tarsal joint. CIA rats with no macroscopic signs of arthritis were diagnosed as resistant and they had low APN in MF of the synovial tissue, low DPPIV in SF of PBMCs and high DPPIV in plasma together with histological aspects of tibio-tarsal joint similar to healthy control rats. Data suggested that APN and DPPIV activity levels are related to the development of arthritis, being protective or inducer of the susceptibility. Understanding what is controlling the compartment-specific changes of these peptidases and looking at ways in which to manipulate their activities may lead to a better knowledge of the arthritic processes and novel treatments.

Novel cyclic-imide peptidomimetics as aminopeptidase N inhibitors. Structure-based design, chemistry and activity evaluation. II

A novel class of potent aminopeptidase N inhibitors with 3-amino-cyclic-imide scaffold is described. The preliminary biological test revealed that all the compounds displayed high specific inhibitory activity against aminopeptidase N compared with previous work because of the existence of free amino group. Compounds containing hydroxamate group are more potent than carboxyl and ester derivatives. Compound 13f potentially inhibited APN activity with IC 50 value of 1.8 μM and displayed specific activity profiles in cells assay and in vivo anti-metastasis assay.