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1.
Can J Physiol Pharmacol ; 95(10): 1117-1124, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28384411

ABSTRACT

The renin-angiotensin system (RAS) generates, maintains, and makes worse hypertension and cardiovascular diseases (CVDs) through its biologically active component angiotensin II (Ang II), that causes vasoconstriction, sodium retention, and structural alterations of the heart and the arteries. A few endogenous vasodilators, kinins, natriuretic peptides, and possibly angiotensin (1-7), exert opposite actions and may provide useful therapeutic agents. As endothelial autacoids, the kinins are potent vasodilators, active natriuretics, and protectors of the endothelium. Indeed, the kallikrein-kinin system (KKS) is considered the dominant mechanism for counteracting the detrimental effects of the hyperactive RAS. The 2 systems, RAS and KKS, are controlled by the angiotensin-converting enzyme (ACE) that generates Ang II and inactivates the kinins. Inhibitors of ACE can reduce the impact of Ang II and potentiate the kinins, thus contributing to restore the cardiovascular homeostasis. In the last 20 years, ACE-inhibitors (ACE-Is) have become the drugs of first choice for the treatments of the major CVDs. ACE-Is not only reduce blood pressure, as sartans also do, but by protecting and potentiating the kinins, they can reduce morbidity and mortality and improve the quality of life for patients with CVDs. This paper provides a brief review of the literature on this topic.


Subject(s)
Cardiovascular Diseases/metabolism , Cardiovascular System/metabolism , Kallikrein-Kinin System , Renin-Angiotensin System , Angiotensin I/metabolism , Angiotensin II Type 1 Receptor Blockers/therapeutic use , Angiotensin-Converting Enzyme 2 , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/physiopathology , Cardiovascular System/drug effects , Cardiovascular System/physiopathology , Humans , Kallikrein-Kinin System/drug effects , Peptide Fragments/metabolism , Peptidyl-Dipeptidase A/metabolism , Proto-Oncogene Mas , Proto-Oncogene Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Renin-Angiotensin System/drug effects , Signal Transduction
2.
Biol Chem ; 397(4): 365-72, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26565554

ABSTRACT

Several studies have shown the potential therapeutic utility of kinin B1 receptor (B1R) peptide agonists in neurological and ischemic cardiovascular diseases and brain cancer. Preclinical safety studies are a prerequisite for further drug development. The objectives of this study were to determine the acute toxicity and pharmacokinetics of the peptide B1R agonist, SarLys[dPhe8]desArg9-bradykinin (NG29), as trifluoroacetate (TFacetate) or acetate salt form, following intravenous injection in rats. A maximum tolerated dose (MTD) of NG29-TFacetate was established at 75 mg/kg from the results of a dose range-finding study (up to 200 mg/kg). The short-term (4-day) repeat-dose toxicity study of NG29, using its MTD value, showed that NG29-acetate exhibited minimal non-adverse clinical pathology changes in hematology, coagulation, clinical chemistry and urine parameters and severe kidney histopathological changes characterized by renal tubular degeneration. No such effects were observed with NG29-TFacetate. At the injection site, NG29-TFacetate was considered to be more locally irritating when compared to the acetate form. The extent of exposure and half-life values of NG29-TFacetate were comparable to the acetate form (AUC0-α of 10.2 mg/l*h vs. 9.9 mg/l*h; T1/2 of 2.3 h vs. 2.4 h). This study shows that in rats NG29-TFacetate exhibits a superior tolerability profile compared with the peptide acetate form.


Subject(s)
Peptides/adverse effects , Peptides/pharmacokinetics , Receptor, Bradykinin B1/agonists , Animals , Dose-Response Relationship, Drug , HEK293 Cells , Half-Life , Humans , Injections, Intravenous , Male , Maximum Tolerated Dose , Peptides/administration & dosage , Peptides/chemistry , Rats , Rats, Wistar
3.
Biol Chem ; 397(4): 297-304, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26408609

ABSTRACT

This paper is divided into two sections: the first contains the essential elements of the opening lecture presented by Pr. Regoli to the 2015 International Kinin Symposium in S. Paulo, Brazil on June 28th and the second is the celebration of Dr. Regoli's 60 years of research on vasoactive peptides. The cardiovascular homeostasis derives from a balance of two systems, the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). The biologically active effector entity of RAS is angiotensin receptor-1 (AT-1R), and that of KKS is bradykinin B2 receptor (B2R). The first mediates vasoconstriction, the second is the most potent and efficient vasodilator. Thanks to its complex and multi-functional mechanism of action, involving nitric oxide (NO), prostacyclin and endothelial hyperpolarizing factor (EDHF). B2R is instrumental for the supply of blood, oxygen and nutrition to tissues. KKS is present on the vascular endothelium and functions as an autacoid playing major roles in cardiovascular diseases (CVDs) and diabetes. KKS exerts a paramount role in the prevention of thrombosis and atherosclerosis. Such knowledge emphasizes the already prominent value of the ACE-inhibitors (ACEIs) for the treatment of CVDs and diabetes. Indeed, the ACEIs, thanks to their double action (block of the RAS and potentiation of the KKS) are the ideal agents for a rational treatment of these diseases.


Subject(s)
Kinins/metabolism , Receptors, Peptide/metabolism , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/metabolism , Diabetes Mellitus/drug therapy , Diabetes Mellitus/metabolism , Humans
4.
Vascul Pharmacol ; 64: 1-10, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25579779

ABSTRACT

Hypertension is characterized by an imbalance between the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II AT-1 receptor antagonists (also known as sartans or ARBs) are potent modulators of these systems and are highly effective as first-line treatments for hypertension, diabetic nephropathies, and diseases of the brain and coronary arteries. However, these agents are mechanistically distinct and should not be considered interchangeable. In this mini-review, we provide novel insights into the often neglected roles of the KKS in the beneficial, protective, and reparative actions of ACEIs. Indeed, ACEIs are the only antihypertensive drugs that properly reduce the imbalance between the RAS and the KKS, thereby restoring optimal cardiovascular homeostasis and significantly reducing morbidity and the risk of all-cause mortality among individuals affected by hypertension and other cardiovascular diseases.


Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Antihypertensive Agents/pharmacology , Hypertension/drug therapy , Kallikrein-Kinin System/physiology , Renin-Angiotensin System/physiology , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Antihypertensive Agents/therapeutic use , Humans , Hypertension/metabolism , Hypertension/pathology , Kallikrein-Kinin System/drug effects , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Renin-Angiotensin System/drug effects
6.
Peptides ; 52: 82-9, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24361511

ABSTRACT

We previously showed that R-954 (AcOrn[Oic(2),(αMe)Phe(5),dßNal(7),Ile(8)]desArg(9)-bradykinin) is a potent, selective and stable peptide antagonist of the inducible GPCR kinin B1 receptor. This compound shows potential applications for the treatment of several diseases, including cancer and neurological disturbances of diabetes. To enable clinical translation, more information regarding its pharmacological, pharmacokinetics (PK) and toxicological properties at preclinical stage is warranted. This was the principal objective of the present study. Herein, specificity of R-954 was characterized in binding studies on 133 human molecular targets to reveal minor cross-reactivities against the angiotensin AT2 and the bombesin receptors (110- and 330-fold lower affinity than for B1R, respectively). The pharmacokinetic of R-954 was studied in both normal and streptozotocin-diabetic anaesthetized rats providing half-lives of 1.9-2.7h. R-954 does not appear to be metabolized in the rat circulation and in several rat tissue homogenates, as the kidney, lung and liver. It appears to be excreted as parent drug in the bile (21%) and in urine. A preliminary toxicological profile of R-954 was obtained in rats under various administration routes. R-954 appears to be well tolerated. Overall, these results indicate that R-954 exhibits favorable preclinical pharmacological/PK characteristics and encouraging safety profiles, suitable for early studies in humans.


Subject(s)
Bradykinin B1 Receptor Antagonists , Bradykinin/analogs & derivatives , Animals , Bradykinin/adverse effects , Bradykinin/pharmacokinetics , Bradykinin/pharmacology , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Humans , Neoplasms/drug therapy , Neoplasms/metabolism , Nervous System Diseases/drug therapy , Nervous System Diseases/metabolism , Rats , Rats, Sprague-Dawley , Rats, Wistar
7.
Peptides ; 37(1): 86-97, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22732666

ABSTRACT

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.


Subject(s)
Neurokinin-1 Receptor Antagonists , Pyridines/pharmacology , Analysis of Variance , Animals , Atropine/pharmacology , CHO Cells , Calcium Signaling , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Cricetinae , Dose-Response Relationship, Drug , Female , Gerbillinae , Guinea Pigs , HEK293 Cells , Humans , Ileum/drug effects , Ileum/metabolism , Ileum/physiology , In Vitro Techniques , Injections, Intraperitoneal , Injections, Spinal , Male , Mice , Muscarinic Antagonists/pharmacology , Muscle Contraction/drug effects , Nociception/drug effects , Protein Binding , Pyridines/administration & dosage , Rats , Receptors, Neurokinin-1/metabolism , Substance P/administration & dosage , Substance P/antagonists & inhibitors , Substance P/physiology
8.
Pharmacol Ther ; 135(1): 94-111, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22537664

ABSTRACT

In recent years, ACE Inhibitors (ACEIs) and Angiotensin II receptor antagonists (also known as AT1 receptor antagonists (AT1-RAs), angiotensin receptor blockers (ARBs), or Sartans), have become the drugs of choice for the treatment of hypertension, heart and renal failure, coronary artery diseases, myocardial infarction and diabetes. By suppressing angiotensin and potentiating bradykinin effects, ACEIs and ARBs activate hemodynamic, metabolic and cellular mechanisms that not only reduce high blood pressure, but also protect the endothelium, the heart, the kidney and the brain, namely the target organs which are at risk in cardiovascular diseases. Major therapeutic benefits of these drugs are the reduction of cardiovascular events and the amelioration of the quality of life and of the patient survival. Results from large clinical trials have established that ACEIs and ARBs are efficient and safe drugs, suitable for the chronic treatments of cardiovascular diseases. Side effects are rare and easily manageable in most cases. The following is a brief review of the basic actions and mechanisms by which two opposing systems, the renin-angiotensin (RAS) and the kallikrein-kinin (KKS), interact in the regulation of cardiovascular and fluid homeostasis to keep the balance in healthy life and correct the imbalance in pathological conditions. Here we discuss how and why imbalances created by overactive RAS are best corrected by treatments with ACEI or AT1-RAs.


Subject(s)
Cardiovascular Agents/therapeutic use , Cardiovascular Diseases/drug therapy , Kallikrein-Kinin System/drug effects , Kinins/metabolism , Angiotensin II Type 1 Receptor Blockers/therapeutic use , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/physiopathology , Homeostasis , Humans , Kallikreins/metabolism , Kinins/therapeutic use , Practice Guidelines as Topic , Receptors, Bradykinin/drug effects , Receptors, Bradykinin/metabolism , Renin-Angiotensin System/drug effects , Signal Transduction/drug effects
9.
CNS Neurosci Ther ; 17(3): 178-98, 2011 Jun.
Article in English | MEDLINE | ID: mdl-20497197

ABSTRACT

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions via selective activation of the N/OFQ peptide receptor (NOP). [(pF)Phe(4) Aib(7) Arg(14) Lys(15) ]N/OFQ-NH(2) (UFP-112) is an NOP receptor ligand designed using a combination of several chemical modifications in the same peptide sequence that increase NOP receptor affinity/potency and/or reduce susceptibility to enzymatic degradation. In the present review article, we summarize data from the literature and present original findings on the in vitro and in vivo pharmacological features of UFP-112. Moreover, important biological actions and possible therapeutic indications of NOP receptor agonists are discussed based on the results obtained with UFP-112 and compared with other peptide and nonpeptide NOP receptor ligands.


Subject(s)
Opioid Peptides/pharmacology , Receptors, Opioid/agonists , Animals , Drug Design , Humans , Ligands , Opioid Peptides/chemistry , Pain/drug therapy , Pain/physiopathology , Peptides/pharmacology , Synaptic Transmission/drug effects , Nociceptin Receptor
10.
Med Res Rev ; 30(5): 751-77, 2010 Sep.
Article in English | MEDLINE | ID: mdl-19824051

ABSTRACT

Neuropeptide S (NPS) is the last neuropeptide identified via reverse pharmacology techniques. NPS selectively binds and activates a previous orphan GPCR, now named NPSR, producing intracellular calcium mobilization and increases in cAMP levels. Biological functions modulated by the NPS/NPSR system include anxiety, arousal, locomotion, food intake, memory, and drug addiction. The primary sequence of NPS (in humans SFRNGVGTGMKKTSFQRAKS) is highly conserved among vertebrates especially at the N-terminus. Ala- and D-scan studies demonstrated that this part of the molecule is crucial for biological activity. Focused structure-activity studies performed on Phe(2), Arg(3), and Asn(4) confirmed this indication and revealed the chemical requirements of these positions for NPSR binding and activation. The sequence Gly(5)-Val(6)-Gly(7) seems to be important for shaping the bioactive conformation of the peptide. Structure-activity studies on Gly(5) enabled identification of the first generation of peptidergic NPSR pure antagonists including [D-Cys(tBu)(5)]NPS and [D-Val(5)]NPS whose antagonist properties were confirmed in vivo. Finally, the pharmacological features of substituted bicyclic piperazine molecules (e.g. SHA 68 (3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide) were recently published making available the first generation of nonpeptide NPSR antagonists. The use in future studies of NPSR antagonists will be of paramount importance for understanding which biological functions are controlled by the NPS/NPSR system and for defining the therapeutic potential of selective NPSR ligands.


Subject(s)
Chemistry, Pharmaceutical , Neurobiology , Neuropeptides/metabolism , Neuropeptides/pharmacology , Receptors, Neuropeptide/chemistry , Receptors, Neuropeptide/metabolism , Amino Acid Sequence , Animals , Humans , Ligands , Molecular Sequence Data , Neuropeptides/chemistry
11.
J Allergy Clin Immunol ; 124(6): 1303-10.e4, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19796797

ABSTRACT

BACKGROUND: Activation of bradykinin-mediated B2 receptor has been shown to play an important role in the onset of angioedema associated with C1 inhibitor deficiency. This finding has led to the development of novel therapeutic drugs such as the B2 receptor antagonist icatibant. However, it is unclear whether other receptors expressed on endothelial cells contribute to the release of kinins and vascular leakage in these patients. The recognition of their role may have obvious therapeutic implications. OBJECTIVE: Our aim was to investigate the involvement of B1 and gC1q receptors in in vitro and in vivo models of vascular leakage induced by plasma samples obtained from patients with C1 inhibitor deficiency. METHODS: The vascular leakage was evaluated in vitro on endothelial cells by a transwell model system and in vivo on rat mesentery microvessels by intravital microscopy. RESULTS: We observed that the attack phase plasma from C1 inhibitor-deficient patients caused a delayed fluorescein-labeled albumin leakage as opposed to the rapid effect of bradykinin, whereas remission plasma elicited a modest effect compared with control plasma. The plasma permeabilizing effect was prevented by blocking the gC1q receptor-high-molecular-weight kininogen interaction, was partially inhibited by B2 receptor or B1 receptor antagonists, and was totally prevented by the mixture of the 2 antagonists. Involvement of B1 receptor was supported by the finding that albumin leakage caused by attack phase plasma was enhanced by IL-1beta and was markedly reduced by brefeldin A. CONCLUSION: Our data suggest that both B1 receptor and gC1q receptor are involved in the vascular leakage induced by hereditary and acquired angioedema plasma.


Subject(s)
Angioedema/physiopathology , Capillary Permeability/drug effects , Complement C1 Inhibitor Protein/metabolism , Membrane Glycoproteins/metabolism , Receptor, Bradykinin B1/metabolism , Receptors, Complement/metabolism , Adrenergic beta-Antagonists/pharmacology , Adrenergic beta-Antagonists/therapeutic use , Angioedema/drug therapy , Animals , Antibodies, Monoclonal/pharmacology , Blood Vessels/drug effects , Blood Vessels/immunology , Blood Vessels/metabolism , Bradykinin/analogs & derivatives , Bradykinin/pharmacology , Bradykinin/therapeutic use , Bradykinin B1 Receptor Antagonists , Brefeldin A/pharmacology , Capillary Permeability/physiology , Cell Line, Tumor , Complement C1 Inhibitor Protein/genetics , Hereditary Angioedema Types I and II/drug therapy , Hereditary Angioedema Types I and II/physiopathology , Humans , Immunologic Factors/pharmacology , Interleukin-1beta/pharmacology , Male , Protein Synthesis Inhibitors/pharmacology , Rats , Rats, Inbred WKY
12.
Bioorg Med Chem ; 17(14): 5080-95, 2009 Jul 15.
Article in English | MEDLINE | ID: mdl-19527931

ABSTRACT

Twelve derivatives of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide (Comp 24) were synthesized and tested in binding experiments performed on CHO(hNOP) cell membranes. Among them, a novel interesting NOP receptor antagonist (compound 35) was identified by blending chemical moieties taken from different NOP receptor ligands. In vitro in various assays, Compound 35 consistently behaved as a pure, highly potent (pA(2) in the range 8.0-9.9), competitive and NOP selective antagonist. However compound 35 was found inactive when challenged against N/OFQ in vivo in the mouse tail withdrawal assay. Thus, the usefulness of the novel NOP ligand compound 35 is limited to in vitro investigations.


Subject(s)
Narcotic Antagonists , Piperidines/chemistry , Piperidines/pharmacology , Receptors, Opioid/metabolism , Spiro Compounds/chemistry , Spiro Compounds/pharmacology , Animals , Behavior, Animal/drug effects , CHO Cells , Cricetinae , Cricetulus , Electric Stimulation , Guinea Pigs , Humans , Ileum/drug effects , Male , Mice , Piperidines/administration & dosage , Piperidines/chemical synthesis , Protein Binding , Rats , Rats, Sprague-Dawley , Spiro Compounds/administration & dosage , Spiro Compounds/chemical synthesis , Structure-Activity Relationship , Vas Deferens/drug effects , Nociceptin Receptor
13.
J Med Chem ; 52(13): 4068-71, 2009 Jul 09.
Article in English | MEDLINE | ID: mdl-19473027

ABSTRACT

Neuropeptide S (NPS) regulates various biological functions by activating the NPS receptor (NPSR). Previous studies demonstrated that the substitution of Gly(5) with d-amino acids generates NPSR antagonists. Eleven [d-Xaa(5)]NPS derivatives were synthesized and pharmacologically tested measuring [Ca(2+)](i) in HEK293(mNPSR) cells. The results confirmed that the [d-Xaa(5)] substitution promotes antagonist activity with potency inversely related to the side chain size and allowed identification of the novel potent NPSR peptide antagonist [(t)Bu-d-Gly(5)]NPS.


Subject(s)
Neuropeptides/chemistry , Receptors, G-Protein-Coupled/antagonists & inhibitors , Calcium/analysis , Cell Line , Drug Discovery , Humans , Structure-Activity Relationship
14.
Eur J Pharmacol ; 614(1-3): 50-7, 2009 Jul 01.
Article in English | MEDLINE | ID: mdl-19445927

ABSTRACT

Compound 24, 1-benzyl-N-[3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl] pyrrolidine-2-carboxamide was recently identified as a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In this study, the in vitro and in vivo pharmacological profiles of Compound 24 were investigated. In vitro studies were performed measuring receptor and [(35)S]GTPgammaS binding and calcium mobilization in cells expressing the recombinant NOP receptor as well as using N/OFQ sensitive tissues. In vivo studies were conducted using the tail withdrawal assay in mice. Compound 24 produced a concentration-dependent displacement of [(3)H]N/OFQ binding to CHO(hNOP) cell membranes showing high affinity (pK(i) 9.62) and selectivity (1000 fold) over classical opioid receptors. Compound 24 antagonized with high potency the following in vitro effects of N/OFQ: stimulation of [(35)S]GTPgammaS binding in CHO(hNOP) cell membranes (pA(2) 9.98), calcium mobilization in CHO(hNOP) cells expressing the Galpha(qi5) chimeric protein (pK(B) 8.73), inhibition of electrically evoked twitches in the mouse (pA(2) 8.44) and rat (pK(B) 8.28) vas deferens, and in the guinea pig ileum (pK(B) 9.12). In electrically stimulated tissues, Compound 24 up to 1 microM did not modify the effects of classical opioid receptor agonists. Finally in vivo, in the mouse tail withdrawal assay, Compound 24 at 10 mg/kg antagonized the pronociceptive and antinociceptive effects of 1 nmol N/OFQ given supraspinally and spinally, respectively. Under the same experimental conditions Compound 24 did not affect the antinociceptive action of 3 nmol endomorphin-1 injected intrathecally. The present study demonstrated that Compound 24 is a pure, competitive, and highly potent non-peptide NOP receptor selective antagonist.


Subject(s)
Lysine/analogs & derivatives , Narcotic Antagonists , Animals , Behavior, Animal/drug effects , CHO Cells , Calcium/metabolism , Cricetinae , Cricetulus , Electric Stimulation , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Gene Expression Regulation/drug effects , Guanosine 5'-O-(3-Thiotriphosphate)/chemistry , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Ligands , Lysine/chemical synthesis , Lysine/metabolism , Lysine/pharmacology , Male , Mice , Peptides/chemistry , Receptors, Opioid/metabolism , Sulfur Isotopes/chemistry , Nociceptin Receptor
15.
Peptides ; 30(6): 1130-6, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19463746

ABSTRACT

Urotensin-II (U-II) activates the U-II receptor (UT) to modulate a range of biological responses at both central and peripheral sites. Previous studies have demonstrated that the sequence Trp(7)-Lys(8)-Tyr(9) of the cyclic portion of the peptide is crucial for biological activity. Here, we describe a focused structure-activity study of Tyr(9) which has been replaced with a series of non-coded amino acids in the U-II(4-11) template. Thirteen analogs were synthesized and pharmacologically tested for intracellular calcium mobilization in HEK293 cells stably expressing the rat UT receptor. The results of this study demonstrated the following Tyr(9) structure-activity features: (i) the position of the OH group of the side chain is not important for biological activity, (ii) the distance of the phenol moiety from the peptide backbone and its conformational freedom are crucial for UT receptor recognition, (iii) this position is important not only for receptor occupation but also for its activation since the 3,5-diiodoTyr(9) chemical modification generated a potent partial agonist. This pharmacological activity of [3,5-diiodoTyr(9)]U-II(4-11) was confirmed in bioassay experiments performed using the rat thoracic aorta as a U-II sensitive preparation.


Subject(s)
Receptors, G-Protein-Coupled/agonists , Tyrosine/metabolism , Urotensins/chemistry , Urotensins/pharmacology , Animals , Aorta, Thoracic/metabolism , Cell Line , Humans , Molecular Conformation , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/agonists , Recombinant Proteins/metabolism , Structure-Activity Relationship
16.
Naunyn Schmiedebergs Arch Pharmacol ; 379(6): 599-607, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19183962

ABSTRACT

In this study, the Galpha(qi5) protein was used to force the human nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor to signal through the Ca(2+) pathway in CHO cells. [Ca(2+)](i) levels were monitored using the fluorometer FlexStation II and the Ca(2+) dye Fluo 4 AM. Concentration response curves were generated with a panel of full and partial agonists, while NOP antagonists were assessed in inhibition-response curves. The following rank order of potency of antagonists was measured: SB - 612111 > J - 113397 = Trap - 101 > or = UFP - 101 > [Nphe1]N/OF Q(1 - 13)NH2 >> naloxone, which is superimposable to literature findings. The rank order of potency of full and partial agonists is also similar to that obtained in previous studies with the exception of a panel of ligands (UFP-112, Ro 64-6198, ZP120, UFP-113) whose potency was relatively low in the Galpha(qi5)-NOP receptor calcium assay. Interestingly, these NOP ligands are characterized by slow kinetic of interaction with the NOP receptor, as demonstrated by bioassay experiments. These results demonstrated that the FlexStation II-Galpha(qi5)-NOP receptor calcium assay represents an adequate and useful screening for NOP receptor ligands, particularly for antagonists.


Subject(s)
Calcium Signaling/physiology , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Receptors, Opioid/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Benzimidazoles/metabolism , Benzimidazoles/pharmacology , CHO Cells , Calcium Signaling/drug effects , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Humans , Piperidines/metabolism , Piperidines/pharmacology , Receptors, Opioid/agonists , Nociceptin Receptor
17.
J Pharmacol Exp Ther ; 328(2): 549-55, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18971372

ABSTRACT

Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as the NPS receptor (NPSR). In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [D-Cys(tBu)(5)]NPS was identified. [D-Cys(tBu)(5)]NPS up to 100 microM did not stimulate calcium mobilization in human embryonic kidney (HEK) 293 cells stably expressing the mouse NPSR; however, in a concentration-dependent manner, the peptide inhibited the stimulatory effects elicited by 10 and 100 nM NPS (pK(B), 6.62). In Schild analysis experiments [D-Cys(tBu)(5)]NPS (0.1-100 microM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA(2) value of 6.44. Ten micromolar [D-Cys(tBu)(5)]NPS did not affect signaling at seven NPSR unrelated G-protein-coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmol i.c.v. reduced the percentage of animals losing the RR in response to 15 mg/kg diazepam and their sleeping time. [d-Cys(tBu)(5)]NPS (1-10 nmol) was inactive per se but dose-dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal-promoting action of 1 nmol NPS could be detected in wild-type but not in mutant mice. In conclusion, [D-Cys(tBu)(5)]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist but with moderate potency. Moreover, using this tool together with receptor knockout mice studies, we demonstrated that the arousal-promoting action of NPS is because of the selective activation of the NPSR protein.


Subject(s)
Neuropeptides/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Animals , Cell Line , Humans , Male , Mice , Peptides/pharmacology
18.
Regul Pept ; 152(1-3): 67-72, 2009 Jan 08.
Article in English | MEDLINE | ID: mdl-18977249

ABSTRACT

Peptide and non-peptide kinin receptor antagonists were evaluated in cutaneous inflammation models in mice. Topical and i.p. application of kinin B(1) and B(2) receptor antagonists caused a significant inhibition of the capsaicin-induced cutaneous neurogenic inflammatory response. The calculated mean ID(50) for Hoe140 and SSR240612 were 23.83 (9.14-62.14) nmol/kg and 0.23 (0.15-0.36) mg/ear, respectively. The I(max) observed for Hoe140, SSR240612, R-715, FR173657, and FR plus SSR were 61+/-5%, 56+/-3%, 65+/-10%, 48+/-8%, and 52+/-4%, respectively. Supporting these results, double B(1) and B(2) kinin receptors knockout mice showed a significant inhibition of capsaicin-induced ear oedema (42+/-7%). However, mice with a single deletion of either B(1) or B(2) receptors exhibited no change in their capsaicin responses. In contrast, all of the examined kinin receptor antagonists were unable to inhibit the oedema induced by TPA and the results from knockout mice confirmed the lack of kinin receptor signaling in this model. These findings show that kinin receptors are present in the skin and that both kinin receptors seem to be important in the neurogenic inflammatory response. Moreover, non-peptide antagonists were very effective in reducing skin inflammation when topically applied, thereby suggesting that they could be useful tools in the treatment of some skin inflammatory diseases.


Subject(s)
Bradykinin B1 Receptor Antagonists , Bradykinin B2 Receptor Antagonists , Dermatitis/drug therapy , Dioxoles/therapeutic use , Quinolines/therapeutic use , Sulfonamides/therapeutic use , Animals , Capsaicin/administration & dosage , Dioxoles/administration & dosage , Female , Male , Mice , Mice, Knockout , Quinolines/administration & dosage , Receptor, Bradykinin B1/genetics , Receptor, Bradykinin B1/metabolism , Receptor, Bradykinin B2/genetics , Receptor, Bradykinin B2/metabolism , Sulfonamides/administration & dosage
19.
J Med Chem ; 52(2): 524-9, 2009 Jan 22.
Article in English | MEDLINE | ID: mdl-19113861

ABSTRACT

Neuropeptide S (NPS), the endogenous ligand of a previously orphan receptor now named NPSR, regulates various biological functions in the brain, including arousal, locomotion, anxiety, and food intake. Here we report on a focused structure-activity study of Gly5, which has been replaced with L and D amino acids. Fifteen NPS related peptides were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated that peptide potency is inversely related to the side chain size, while peptide efficacy strongly depends on the relative L and D configuration, with the L amino acids favoring agonist while D amino acids display antagonist pharmacological activity. [D-Val5]NPS behaved as NPSR pure antagonist in HEK293(mNPSR) cells showing the highest potency (pK(B) 7.56) among this series of peptides. The antagonist action of [D-Val5]NPS was confirmed in vivo in mice, where the peptide at a dose of 10 nmol completely blocked the stimulatory effect of 0.1 nmol NPS on locomotor activity.


Subject(s)
Neuropeptides/chemistry , Neuropeptides/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Amino Acid Sequence , Animals , Cell Line , Dose-Response Relationship, Drug , Humans , Locomotion/drug effects , Male , Mice , Molecular Sequence Data , Neuropeptides/chemical synthesis , Structure-Activity Relationship
20.
Bioorg Med Chem ; 16(19): 8841-5, 2008 Oct 01.
Article in English | MEDLINE | ID: mdl-18793857

ABSTRACT

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe(2)-Arg(3)-Asn(4) of the peptide is crucial for biological activity. Here, we report on a focused structure-activity study of Arg(3) and Asn(4) that have been replaced with a series of coded and non-coded amino acids. Thirty-eight human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 3 structure-activity features: (i) the guanidine moiety and its basic character are not crucial requirements, (ii) an aliphatic amino acid with a linear three carbon atom long side chain is sufficient to bind and fully activate NPSR, (iii) the receptor pocket allocating the position 3 side chain can accommodate slightly larger side chains at least to a certain degree [hArg, Arg(NO2) or Arg(Me)2 but not Arg(Tos)]. Position 4 seems to be more sensitive to amino acids replacement compared to position 3; in fact, all the amino acid replacements investigated produced either an important decrease of biological activity or generated inactive derivatives suggesting a pivotal role of the Asn(4) side chain for NPS bioactivity.


Subject(s)
Amino Acid Substitution , Neuropeptides/pharmacology , Amino Acid Sequence , Arginine/chemistry , Arginine/metabolism , Asparagine/chemistry , Asparagine/metabolism , Binding Sites , Calcium/metabolism , Cell Line , Humans , Kidney/embryology , Kidney/metabolism , Kidney/pathology , Ligands , Molecular Sequence Data , Neuropeptides/chemical synthesis , Phenylalanine/chemistry , Phenylalanine/metabolism , Structure-Activity Relationship
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