中文网站正在持续更新中,请密切关注我们康肽生物的最新动态,或点击访问右上角的英文官方网站 www.phoenixpeptide.com
PHOENIX PHARMACEUTICALS, INC. TOP HOME PAGE
Top » Catalog English Version | My Account | 联系我们 | China



 多肽



 标记多肽 



 多肽激素文库



 抗体 



 免疫试剂盒 



 生物标志物阵列 



 多肽样品检测



 自定义肽链合成及GMP



 产品目录索取



 样品准备



 提问和解答


DX 600 & WFML

A novel ACE2-specific peptide inhibitor & Rat Renin Inhibitor

Novel peptide inhibitors of angiotensin-converting enzyme 2

Angiotensin-converting enzyme 2 (ACE2), a recently identified human homolog of ACE, is a novel metallocarboxypeptidase with specificity, tissue distribution, and function distinct from those of ACE. ACE2 may play a unique role in the renin-angiotensin system and mediate cardiovascular and renal function. Here we report the discovery of ACE2 peptide inhibitors through selection of constrained peptide libraries displayed on phage. Six constrained peptide libraries were constructed and selected against FLAG-tagged ACE2 target. ACE2 peptide binders were identified and classified into five groups, based on their effects on ACE2 activity. Peptides from the first three classes exhibited none, weak, or moderate inhibition on ACE2. Peptides from the fourth class exhibited strong inhibition, with equilibrium inhibition constants (K(i) values) from 0.38 to 1.7 microm. Peptides from the fifth class exhibited very strong inhibition, with K(i) values < 0.14 microm. The most potent inhibitor, DX600, had a K(i) of 2.8 nm. Steady-state enzyme kinetic analysis showed that these potent ACE2 inhibitors exhibited a mixed competitive and non-competitive type of inhibition. They were not hydrolyzed by ACE2. Furthermore, they did not inhibit ACE activity, and thus were specific to ACE2. Finally, they also inhibited ACE2 activity toward its natural substrate angiotensin I, suggesting that they would be functional in vivo. As novel ACE2-specific peptide inhibitors, they should be useful in elucidation of ACE2 in vivo function, thus contributing to our better understanding of the biology of cardiovascular regulation. Our results also demonstrate that library selection by phage display technology can be a rapid and efficient way to discover potent and specific protease inhibitors.

Huang L, et al. J Biol Chem. 2003 May 2;278(18):15532-40.

 

Angiotensin IV in the central nervous system

The mammalian brain harbors a renin-angiotensin system (RAS), which is independent from the peripheral RAS. Angiotensin II is a well-studied member of the RAS and exerts most of the known angiotensin-mediated effects on fluid and electrolyte homeostasis, autonomic activity, neuroendocrine regulation, and behavior. This review summarizes a mass of compelling new evidence for the biological role of an active (3-8) fragment of angiotensin II, named angiotensin IV. Angiotensin IV binds to a widely distributed binding site in the brain, but which is different from the known angiotensin II receptors AT1 and AT2. Angiotensin IV has been implicated in a number of physiological actions, including the regulation of blood flow, the modulation of exploratory behavior, and processes attributed to learning and memory. Furthermore, angiotensin IV may also be involved in neuronal development. Collectively, the available evidence suggests that angiotensin IV is a potent neuropeptide, involved in a broad range of brain functions. 

Von Bohlen Und Halbach O. Cell Tissue Res 2003 Jan;311(1):1-9

 

Angiotensin-converting enzyme 2 is an essential regulator of heart function

Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Here we show that angiotensin-converting enzyme 2 (ace2) maps to a defined quantitative trait locus (QTL) on the X chromosome in three different rat models of hypertension. In all hypertensive rat strains, ACE2 messenger RNA and protein expression were markedly reduced, suggesting that ace2 is a candidate gene for this QTL. Targeted disruption of ACE2 in mice results in a severe cardiac contractility defect, increased angiotensin II levels, and up regulation of hypoxia-induced genes in the heart. Genetic ablation of ACE on an ACE2 mutant background completely rescues the cardiac phenotype. But disruption of ACER, a Drosophila ACE2 homologue, results in a severe defect of heart morphogenesis. These genetic data for ACE2 show that it is an essential regulator of heart function in vivo.

Crackower MA, et al. Nature. 2002 Jun 20;417(6891):822-8.

 

AT(4) receptor is insulin-regulated membrane amino peptidase: potential mechanisms of memory enhancement

Although angiotensin IV (Ang IV) was thought initially to be an inactive product of Ang II degradation, it was subsequently shown that the hexapeptide markedly enhances learning and memory in normal rodents and reverses the memory deficits seen in animal models of amnesia. These central nervous system effects of Ang IV are mediated by binding to a specific site, known as the AT(4) receptor, which is found in appreciable levels throughout the brain and is concentrated particularly in regions involved in cognition. This field of research was redefined by the identification of the AT(4) receptor as the trans membrane enzyme, insulin-regulated membrane amino peptidase (IRAP). Here, we explore the potential mechanisms by which Ang IV binding to IRAP leads to the facilitation of learning and memory.

Albiston AL, et al. Trends Endocrinol Metab 2003 Mar;14(2):72-7

 

Cellular targets for angiotensin II fragments: pharmacological and molecular evidence

Although angiotensin II has long been considered to represent the end product of the renin-angiotensin system (RAS), there is accumulating evidence that it encompasses additional effector peptides with diverse functions. In this respect, angiotensin IV (Ang IV) formed by deletion of the two N terminal amino acids, has sparked great interest because of its wide range of physiological effects. Among those, its facilitatory role in memory acquisition and retrieval is of special therapeutic relevance. High affinity binding sites for this peptide have been denoted as AT(4)- receptors and, very recently, they have been proposed to correspond to the membrane-associated OTase/ IRAP amino peptidase. This offers new opportunities for examining physiological roles of Ang IV in the fields of cognition, cardiovascular and renal metabolism and pathophysiological conditions like diabetes and hypertension. Still new recognition sites may be unveiled for this and other angiotensin fragments. Recognition sites for Ang-(1-7) (deletion of the C terminal amino acid) are still elusive and some of the actions of angiotensin III (deletion of the N terminal amino acid) in the CNS are hard to explain on the basis of their interaction with AT(1)-receptors only. A more thorough cross-talk between in vitro investigations on native and transfected cell lines and in vivo investigations on healthy, diseased and transgenic animals may prove to be essential to further unravel the molecular basis of the physiological actions of these small endogenous angiotensin fragments.

Vauquelin G, et al. J Renin Angiotensin Aldosterone Syst 2002 Dec;3(4):195-204

 

Comparative effects of angiotensin IV and two hemorphins on angiotensin-converting enzyme activity

The role of angiotensin IV (Ang IV) in the regulation of angiotensin-converting enzyme (ACE) was studied in vitro. This study demonstrates that this active fragment appeared as a novel endogenous ACE inhibitor. Inhibitory kinetic studies revealed that Ang IV acts as a purely competitive inhibitor with a K(i) value of 35 microM. Ang IV was found to be quite resistant to ACE hydrolysis opposite to hemorphins which are both ACE inhibitors and substrates. In order to confirm a putative role of Ang IV and hemorphins in the Renin-Angiotensin system (RAS) regulation, we studied their influence on Ang I conversion. We noticed that 16.7 microM of both peptides decreased more than 50% of Ang I conversion to Ang II in vitro. The capacity of hemorphins, particularly LVVH-7, and Ang IV to inhibit ACE activity here suggests a synergistic relation between these two peptides and the regulation of RAS.

Fruitier-Arnaudin I, et al. Peptides 2002 Aug;23(8):1465-70

 

Neuropeptide conversion to bio active fragments - an important pathway in neuromodulation

Biosynthetic pathways for the formation of neuroactive peptides and the processes for their inactivation include several enzymatic steps. In addition to enzymatic processing and degradation, several neuropeptide's have been shown to undergo enzymatic conversion to fragments with retained or modified biological activity. This has most clearly been demonstrated for e.g. opioid peptides, tachykinins, calcitonin gene-related peptide (CGRP) as well as for peptides belonging to the renin-angiotensin system. Sometimes the released fragment shares the activity of the parent compound. However, in many cases the conversion reaction is linked to a change in the receptor activation profile, i.e. the generated fragment acts on and stimulates a receptor not recognized by the parent peptide. This review will describe the characteristics of certain neuropeptide fragments having the ability to modify the biological action of the peptide from which they are derived. Focus will be directed to the tachykinins, the opioid peptides, angiotensin's as well as to CGRP, bradykinin and nociceptin. The kappa opioid receptor selective opioid peptide, dynorphin, recognized for its ability to produce dysphoria, is converted to the delta opioid receptor agonist Leu-enkephalin, with euphoric properties. The tachykinins, typified by substance P (SP), is converted to the bioactive fragment SP(1-7), a heptapeptide mimicking some but opposing other effects of the parent peptide. The bioactive angiotensin II, known to bind to and stimulate the AT-1 and AT-2 receptors, is converted to angiotensin IV (i.e. angiotensin 3-8) with preference for the AT-4 sites or to angiotensin (1-7), not recognized by any of these receptors. Both angiotensin IV and angiotensin (1-7) are biologically active. For example angiotensin (1-7) retains some of the actions ascribed for angiotensin II but is shown to counteract others. Thus, it is obvious that the activity of many neuroactive peptides is modulated by bioactive fragments, which are formed by the action of a variety of peptidases. This phenomenon appears to represent an important regulatory mechanism that modulates many neuropeptide systems but is generally not acknowledged.

Hallberg M, Nyberg F. Curr Protein Pept Sci 2003 Feb.;4(1):31-44

 

Effects of angiotensin's II and IV on blood pressure, renal function, and PAI-1 expression in the heart and kidney of the rat

The role of angiotensin II (AII) and angiotensin IV (AIV) as inducers of PAI-1 expression during hypertension was studied in vivo. A 2-week infusion of AII (300 ng/kg/min) via an osmotic pump increased systolic blood pressure (171 2 vs. 138 6 mm Hg), urinary protein excretion (32 6 vs. 14 2 mg/day), and renal (2.2 0.5 vs. 1.0 0.1) and cardiac (1.8 0.3 vs. 1.0 0.1) gene expression of plasminogen activator inhibitor 1 (PAI-1). AIV infusion did not affect any of the above with the exception of PAI-1 gene expression which was increased in the left ventricles (1.7 0.3 vs. 1.0 0.1). AII-infused rats displayed a decreased creatinine clearance (538 75 vs. 898 96 ml/min) and hypertrophic left ventricles (0.275 0.006 vs. 0.220 0.011 g/100 g). Our results demonstrate that AII but not AIV infusion is associated with increased renal PAI-1 gene expression.

Abrahamsen CT, et al. Pharmacology 2002 Sep;66(1):26-30

 

Angiotensin IV is a potent agonist for constitutive active human AT1 receptors. Distinct roles of the N-and C-terminal residues of angiotensin II during AT1 receptor activation

The octapeptide hormone, angiotensin II (Ang II), exerts its major physiological effects by activating AT(1) receptors. In vivo Ang II is degraded to bioactive peptides, including Ang III (angiotensin-(2-8)) and Ang IV (angiotensin-(3-8)). These peptides stimulate inositol phosphate generation in human AT(1) receptor expressing CHO-K1 cells, but the potency of Ang IV is very low. Substitution of Asn(111) with glycine, which is known to cause constitutive receptor activation by disrupting its interaction with the seventh trans membrane helix (TM VII), selectively increased the potency of Ang IV (900-fold) and angiotensin-(4-8), and leads to partial agonism of angiotensin-(5-8). Consistent with the need for the interaction between Arg(2) of Ang II and Ang III with Asp(281), substitution of this residue with alanine (D281A) decreased the peptide's potency without affecting that of Ang IV. All effects of the D281A mutation were superseded by the N111G mutation. The increased affinity of Ang IV to the N111G mutant was also demonstrated by binding studies. A model is proposed in which the Arg(2)-Asp(281) interaction causes a conformational change in TM VII of the receptor, which, similar to the N111G mutation, eliminates the constraining intra molecular interaction between Asn(111) and TM VII. The receptor adopts a more relaxed conformation, allowing the binding of the C-terminal five residues of Ang II that switches this "pre activated" receptor into the fully active conformation.

Le MT, et al. J Biol Chem 2002 Jun 28;277(26):23107-10

 

Stimulation of collagen gel contraction by angiotensin II and III in cardiac fibroblasts

OBJECTIVE: The aim of the present study was to investigate whether angiotensin II (Ang II), angiotensin III (Ang III) or Ang II (2-8), angiotensin IV (Ang IV) or Ang II (3-8) and Ang II (1-7), Ang II (4-8), Ang II (5-8) and Ang II (1-4) can stimulate collagen gel contraction in cardiac fibroblasts in serum-free conditions. METHODS: Cardiac fibroblasts (from male adult Wistar rats) from passage 2 were cultured to confluency and added to a hydrated collagen gel in a Dulbecco's Modified Eagle's Medium, with or without foetal bovine serum, for one, two or three days. The area of the collagen gels embedded with cardiac fibroblasts was determined by a densitometric analysis. Collagen gel contraction was characterized by a decrease in the gel area. RESULTS: Ang II dose-dependently stimulated the contraction of collagen mediated by cardiac fibroblasts after one, two or three days of incubation in a serum-free medium. Telmisartan completely blocked the Ang II-induced collagen contraction by cardiac fibroblasts. PD 123319 and des-Asp(1)-Ile(8)-Ang II had no effect on the Ang II-induced collagen contraction by cardiac fibroblasts. Ang III also stimulated the contraction of collagen mediated by cardiac fibroblasts after one, two or three days of incubation in a serum-free medium. des-Asp(1)-Ile(8)-Ang II and telmisartan completely blocked the Ang III-induced collagen gel contraction by cardiac fibroblasts. des-Asp(1)-Ile(8)-Ang II, however, had no effect on the Ang II-induced collagen gel contraction by cardiac fibroblasts. Ang IV and Ang II (4-8), (5-8), (1-7) and (1-4), however, had no effect on collagen gel contraction by cardiac fibroblasts. Addition of telmisartan, PD 123319 or des-Asp(1)-Ile(8)-Ang II alone did not affect collagen gel contraction by cardiac fibroblasts. CONCLUSION: Our data demonstrate that the effects of Ang II on the collagen gel contraction by adult rat cardiac fibroblasts in serum-free conditions are Ang II type 1(AT(1))-receptor- mediated, because they are abolished by the specific AT(1)-receptor antagonist, telmisartan, and not by the AT(2)-receptor antagonist PD 123319 or by the Ang III antagonist des-Asp(1)-Ile(8)-angiotensin. The Ang III- stimulated contraction of collagen by cardiac fibroblasts is completely blocked by the Ang III receptor antagonist, des-Asp(1)-Ile(8)-angiotensin II, and by telmisartan.

Lijnen P, et al. J Renin Angiotensin Aldosterone Syst 2002 Sep;3(3):160-6

 

Angiotensin peptide assay

Trunk blood was obtained from 8-wk-old salt-replete SHR (n = 8) or salt-depleted SHR (n = 8) for determination of plasma concentrations of angiotensin peptides by RIA . Blood was collected into chilled Vacutainer tubes containing a mixture of peptidase inhibitors: 25 mM EDTA, 0.44 mM 1,20-orthophenanthrolene monohydrate (Sigma, St. Louis, MO), 1 mM sodium parachloromercuribenzoate, and 3 µM WFML (rat renin inhibitor: acetyl-His-Pro-Phe-Val-Statine-Leu-Phe). After 20 min on ice, blood samples were centrifuged at 3,000 rpm for 20 min, and aliquots of plasma were stored at -80°C until assayed for angiotensin peptides. Plasma was extracted on a Sep-Pak C18 column according to our previously published protocol . The sample was eluted, reconstituted, and split for the RIA of ANG I, ANG II.

ACE2

Ki determination of DX600 peptide using ACE2 assays with the synthetic substrate. DX600, at concentrations ranging from 5 to 73 nM, was pre incubated with 7 nM ACE2. The substrate M-2195 was added at concentrations ranging from 12 to 50 µM.

A, Dixon plot. Filled squares, 12.6 µM substrate (M-2195); open squares, 15.2 µM; filled triangles, 17.7 µM; ×, 20.2 µM; open triangles, 22.8 µM; filled circles, 27.8 µM; and open circles, 45.6 µM.

B, Dixon secondary plot. The slope at each substrate concentration in A was plotted against the reciprocal substrate concentration. Data were fitted to a linear regression (y = mx + b, where m = Km/(Ki × Vmax) = 1.7077, b = 0.0089). Km (20.6 µM) and Vmax (4.3 farads/s) were obtained by a fit of the data in the absence of inhibitor to the Michaelis-Menten equation by nonlinear regression analysis. Ki was calculated to be 2.8 nM from the equation Ki = Km/(Vmax × m).

 

ACE2

DX600 was a specific inhibitor to ACE2. A, effects of DX600 on ACE2 and ACE activity. Increasing concentrations of DX600 peptides were incubated with ACE2 (20 nM) or ACE (7.5 nM) prior to the addition of the substrate M-2195 (50 µM). B, effects of ACE peptide inhibitor teprotide on ACE2 and ACE activity. Increasing concentrations of teprotide were incubated with ACE2 (20 nM) or ACE (7.5 nM) prior to the addition of the substrate M-2195. The relative enzymatic activity was plotted against the peptide concentration. ACE2, filled circle; ACE, open circle.

 

DX600

Kd determination of DX600 and DX512 peptides. The binding affinities of the very strong inhibitors were analyzed by BIAcore as described under "Experimental Procedures." Shown here are representative sensor grams of DX600 (A) and DX512 (B). The data (response units, RU) were background corrected and plotted against time (seconds). The wavy lines depict actual data, and the solid lines depict fitted data. DX600 was assayed at 100, 50, 25, and 12.5 nM, corresponding to respective curves from top to bottom. DX512 was assayed at 500, 250, 125, and 62.5 nM, corresponding to respective curves from top to bottom.

 

DX600

DX600 and DX512 peptides were stable ACE2 inhibitors. DX600 (A) and DX512 (B), at both low and high concentrations, were each incubated with ACE2 (20 nM) for 10 min or 20 h at room temperature prior to the addition of M-2195. The relative ACE2 activity was plotted against the peptide concentration. Open square, 10 min; filled square, 20 h.

Huang L. L., et al. J. Biol. Chem., Vol. 278, Issue 18, 15532-15540, May 2, 2003


ANG1-7

Maximal change in blood pressure produced by 5% dextrose in water (Veh), PD-123319, [D-Ala7]ANG-(1-7), or the combination of PD-123319 and [D-Ala7]ANG-(1-7) in AT1-blocked, salt-depleted SHR. PD-123319 was infused at 0.12 µmol/min, and [D-Ala7]ANG-(1-7) was infused at 10 pmol/min. To assess the effect of fluid infused, Veh was infused at 0.2 ml/min. Individual drugs were infused at a rate of 0.1 ml/min; combined drug infusion such as PD-123319 and [D-Ala7]ANG-(1-7) amounted to 0.2 ml/min. a P < 0.05 for Veh vs. PD-123319; b P < 0.05 for Veh vs. [D-Ala7]ANG-(1-7); c P < 0.05 for Veh vs. PD-123319 + [D-Ala7]ANG-(1-7); d P < 0.05 for PD-123319 vs. [D-Ala7]ANG-(1-7); e P < 0.05 for PD-123319 vs. PD-123319 + [D-Ala7]ANG-(1-7).

 

Effect of [D-Ala7]ANG-(1-7) infusion in salt-depleted SHR in the absence and presence of AT1 receptor blockade. [D-Ala7]ANG-(1-7) infusion in conscious salt-depleted SHR rapidly increased mean arterial pressure (MAP) irrespective of whether AT1 receptors had been previously blocked by losartan (32.5 µmol/kg iv).

Nakamura S., et al. Am J Physiology Regul Integr Comp Physiology 284: R164-R173, 2003

angiotensin;BKNew

%002-26%;%002-18%


分类搜索
关键字搜索
按字母搜索
A B C D E F G H I J K L M N
O P Q R S T U V W X Y Z

Copyright © 2024 PHOENIX BIOTECH