Non-peptide Antagonist Research Articles

Bv8/PK2 and prokineticin receptors: a druggable pronociceptive system

Mammalian Bv8 (also called prokineticin 2) is a secreted protein that regulates diverse biological processes including pain perception. It belongs to a new family of chemokines, which activate two G-protein linked receptors (prokineticin receptor 1 and 2, PKR1 and PKR2) expressed in regions of the nervous system associated with pain and in cells participating to immuno-inflammatory responses. Primary sensitive neurons co-express PKRs and the transient potential receptor vanilloid 1, cooperating in nociceptor sensitization. Bv8, strongly upregulated in neutrophils and other inflammatory cells, is a main pronociceptive mediator in inflamed tissues, where it sensitizes peripheral nociceptors, stimulates neutrophil chemotaxis and modulates the release of inflammatory and pronociceptive cytokines. Availability of a nonpeptide PKR antagonist, leading to blockade of the Bv8/PKR system, ameliorates pain arising from tissue injury and reduces the time required for recovery from injury.

Efficacy and Safety of Tolvaptan in Heart Failure Patients with Sustained Volume Overload despite the Use of Conventional Diuretics: A Phase III Open-Label Study

Volume overload is a common complication associated with heart failure (HF) and is recommended to be treated with loop or thiazide diuretics. However, use of diuretics can cause serum electrolyte imbalances and diuretic resistance. Tolvaptan, a selective, oral, non-peptide vasopressin V2-receptor antagonist, offers a new option for treating volume overload in HF patients. The aim of this study was to investigate the efficacy and safety of tolvaptan in Japanese HF patients with volume overload. Fifty-one HF patients with volume overload, despite using conventional diuretics, were treated with 15 mg/day tolvaptan for 7 days. If the response was insufficient at Day 7, tolvaptan was continued for a further 7 days at either 15 mg/day or 30 mg/day. Outcomes included changes in body weight, symptoms and safety parameters. Thirty-six patients discontinued treatment within 7 days, therefore 15 patients entered the second phase of treatment. In two patients, tolvaptan was increased to 30 mg/day after 7 days. Body weight was reduced on Day 7 (-1.95 ± 1.98 kg; n = 41) and Day 14 (-2.35 ± 1.44 kg; n = 11, 15 mg/day). Symptoms of volume overload, including lower limb edema, pulmonary congestion, jugular venous distention and hepatomegaly, were improved by tolvaptan treatment for 7 or 14 days. Neither tolvaptan increased the incidence of severe or serious adverse events when administered for 7-14 days. This study confirms the efficacy and safety of 15 mg/day tolvaptan for 7-14 days in Japanese HF patients with volume overload despite conventional diuretics.

Structural requirements for the binding affinity of some small, non–peptide C5a receptor antagonists

Complement anaphylatoxin 5a (C5a) has been recognized as a potent therapeutic target for anti-inflammatory therapy, thus, blocking the action of C5a on its binding receptors may provide an effective treatment of a variety of inflammatory diseases. However, there have been few clinically available non-peptide C5a receptor antagonists disclosed at present. In pursuit of better anti-inflammatory drugs, quantitative structure–activity relationship studies were carried out in a series of non-peptide C5a receptor antagonists with binding activity using different physicochemical descriptors. The conventional best 2D-QSAR models were developed using a training set of 35 molecules and an external test set of 8 molecules by genetic function approximation (GFA) and stepwise multiple linear regression (Stepwise-MLR) with acceptable r^2^ of 0.773 and 0.863, r^2^~CV~ of 0.752 and 0.775, and r^2^~pred~ of 0.801 and 0.888, respectively, indicating binding activity strongly depends on thermodynamic properties as expressed by the hydrophobicity of molecules.

Modeling of Human Prokineticin Receptors: Interactions with Novel Small-Molecule Binders and Potential Off-Target Drugs

Background and MotivationThe Prokineticin receptor (PKR) 1 and 2 subtypes are novel members of family A GPCRs, which exhibit an unusually high degree of sequence similarity. Prokineticins (PKs), their cognate ligands, are small secreted proteins of ∼80 amino acids; however, non-peptidic low-molecular weight antagonists have also been identified. PKs and their receptors play important roles under various physiological conditions such as maintaining circadian rhythm and pain perception, as well as regulating angiogenesis and modulating immunity. Identifying binding sites for known antagonists and for additional potential binders will facilitate studying and regulating these novel receptors. Blocking PKRs may serve as a therapeutic tool for various diseases, including acute pain, inflammation and cancer.Methods and ResultsLigand-based pharmacophore models were derived from known antagonists, and virtual screening performed on the DrugBank dataset identified potential human PKR (hPKR) ligands with novel scaffolds. Interestingly, these included several HIV protease inhibitors for which endothelial cell dysfunction is a documented side effect. Our results suggest that the side effects might be due to inhibition of the PKR signaling pathway. Docking of known binders to a 3D homology model of hPKR1 is in agreement with the well-established canonical TM-bundle binding site of family A GPCRs. Furthermore, the docking results highlight residues that may form specific contacts with the ligands. These contacts provide structural explanation for the importance of several chemical features that were obtained from the structure-activity analysis of known binders. With the exception of a single loop residue that might be perused in the future for obtaining subtype-specific regulation, the results suggest an identical TM-bundle binding site for hPKR1 and hPKR2. In addition, analysis of the intracellular regions highlights variable regions that may provide subtype specificity.

Abstract C76: Inhibition of galectin-1 expression by OTX008, a novel calyx[4]arene, is associated with antiproliferative and antiangiogenic activity in human tumor xenograft models.

Abstract Background: Galectin-1 binds neuropilin-1, enhances VEGFR2 signalling, and contributes to membrane anchorage of H-RAS in cancer cells. Those effects may modulate cancer cell proliferation, apoptosis, cell cycle, and tumor angiogenesis. OTX008 is a non-peptide chemical antagonist designed to bind the amphipathic -sheet conformation of galectin-1. We previously showed that OTX008 displays direct antiproliferative effects in cancer cell lines (AACR 2011, Abstract n°685). In this study we focused on the antiproliferative and antiangiogenic effects of OTX008 in xenograft models. Material and Methods: A2780–1A9 (1A9) ovarian cells and SQ20B head and neck cells, selected for intermediate/high in vitro sensitivity to OTX008, were injected s.c. into female nu/nu athymic mice. When tumors became palpable, treatment was initiated with vehicle (PBS) or 5–10 mg/kg OTX008 i.p. q.d for 3 weeks. At the end of treatment, mice were sacrificed and tumors collected. mRNA expression was evaluated with qRT-PCR. Immunohistochemistry (IHC) was performed on OCT-embedded tumor stained with H&E, MIB1 (Ki67), VEGFR2, CD31, or galectin-1. Image quantification was done with Histolab software (Microvision, France) subtracting the background. Results: OTX008 inhibited tumor growth in both 1A9 and SQ20B xenografts. OTX008 also decreased the number of secondary tumors (subcutaneous metastases distant from the injection site) in SQ20B xenografts. 1A9 and SQ20B tumors treated with OTX008 expressed significantly lower galectin-1 protein as compared to control tumors (1A9: 11.103 +/− 2.103 μm2 vs 26.103 +/− 6.103 μm2, p<0.05; SQ20B: 3.103 μm2 +/− 0,6.103 vs 28.103 +/− 7.103 μm2, p<0.01). Consistent with antiproliferative effects of OTX008, treated tumors expressed lower MIB1. Tumors treated with OTX008 also showed lower VEGFR2 mRNA and protein levels. Consistent with antiangiogenic effects of OTX008, CD31 immunostaining showed that treated tumors had significantly lower vessels number (p<0.01 for SQ20B) and diameter (1A9: 0,5.103 +/−0,1.103 μm2 vs 6.103 +/− 2.103 μm2, p<0.01; SQ20B: 10.103 +/−1.103 μm2 vs 28.103 +/− 4.103 μm2, p<0.005), suggesting microvessel normalization. In order to confirm that these antiproliferative and antiangiogenic effects are mediated by galectin-1 in vivo, SQ20B cells were transfected with sh-galectin-1 and injected into nude mice. Inhibiting galectin-1 by shRNA reduced tumor growth comparable to the effects seen with OTX008. IHC analysis showed that inhibiting galectin-1 by shRNA recapitulated the effects of OTX008 in SQ20B xenografts, including a decrease of MIB1 (p<0.005) and VEGFR2 (p<0.05) expression in cancer cells, as well as in the number (p<0.05) and the diameter of tumor vessels (p<0.005). Conclusion: Antiproliferative activity of OTX008 in xenograft models seems to be associated with galectin-1 expression inhibition. In addition to this direct effect, qualitative and quantitative normalization of microvessels may also play a role in the antitumor activity of OTX008. Galectin-1 and VEGFR2 expression as well as quantitative assessment of microvessels are candidate biomarkers for further clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C76.

3D-Quantitative structure–activity relationship and docking studies of the tachykinin NK3 receptor

The tachykinin NK(3) receptor (NK(3)R) is a novel drug target for schizophrenia and drug abuse. Since few non-peptide antagonists of this G protein-coupled receptor are available, we have initiated this study to gain a better understanding of the structure-activity relationships of NK(3) antagonist compounds. We developed a 3D comparative molecular similarity index analysis (CoMSIA) model that gave cross-validated PLS values with q(2) >0.5 which were validated using a test set. We also describe the development of a homology model of the NK(3)R. The model was then used to develop a pharmacophore for docked ligands. This pharmacophore showed two aromatic, two hydrogen donor and one acceptor/aromatic points. These data will be useful for future structure-based drug discovery of ligands for the NK(3)R.

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

Bv8-Prokineticins and their Receptors: Modulators of Pain

Bv8 is a small protein secreted by frog skin. Mammalian homologues of Bv8, the prokineticins PK1 and PK2, and their G-protein coupled receptors PKR1 and PKR2 have been identified and linked to several biological effects as gut motility, circadian rhythms, neurogenesis, angiogenesis and cancer progression, haematopoiesis and nociception. In rodents, administration of amphibian Bv8 lowers nociceptor thresholds to a broad spectrum of physical and chemical stimuli. The prokineticin receptors are present in regions of the nervous system associated with pain; primary sensitive neurons expressing PKRs also express the vanilloid receptor TRPV1, providing an anatomical basis for PKR1/TRPV1 cooperative interaction in nociceptor sensitization. Bv8/PK2, strongly up-regulated in neutrophils and other inflammatory cells, is a main pronociceptive mediator in inflamed tissues. Indeed Bv8/PK2 produced by inflammatory cells is released at the site of inflammation where it sensitizes peripheral nociceptors, stimulates chemotaxis and modulates the release of inflammatory and pronociceptive cytokines. Availability of a non-peptide PKR antagonist, leading to blockade the PK/PKR system, ameliorates pain arising from tissue injury and, additionally, reduces the time required for recovery from injury.

Optimal Use of Platelet Glycoprotein IIb/IIIa Receptor Antagonists in Patients Undergoing Percutaneous Coronary Interventions

Discovery of the central role of platelets in the pathogenesis of acute coronary syndromes (ACS) and ischaemic complications of percutaneous coronary interventions (PCI) has led to the widespread use of oral and parenteral platelet inhibitors to treat these conditions. Glycoprotein (GP) IIb/IIIa (also known as α(IIb)β(3)) receptors on the surface of platelets play an essential role in platelet aggregation and serve as a key mediator in the formation of arterial thrombus. When activated, GP IIb/IIIa receptors bind to fibrinogen, which serves as the 'final common pathway' in platelet aggregation. Of the numerous agents developed for modulating platelet activity, intravenous platelet GP IIb/IIIa receptor antagonists are the most potent. There are four agents in clinical use, including abciximab, eptifibatide, tirofiban and lamifiban, although lamifiban is not approved for use in the US. While all agents block fibrinogen binding to GP IIb/IIIa, they do so by different mechanisms. Abciximab is a humanized form of a murine monoclonal antibody directed against GP IIb/IIIa, eptifibatide is a synthetic, cyclic heptapeptide that contains a lysine-glycine-aspartic acid (KGD) sequence that mimics the arginine-glycine-aspartic acid (RGD) sequence found on GP IIb/IIIa, tirofiban is a non-peptide antagonist derived by optimization of the tyrosine analogue that structurally mimicks the RGD-containing loop of the disintegrin echistatin, and lamifiban is a synthetic, non-cyclic, non-peptide, low-molecular-weight compound. In clinical trials, use of these agents reduces ischaemic adverse cardiovascular events in patients with ACS undergoing PCI, but at a cost of increased bleeding.

Identification of a novel RAMP-independent CGRP receptor antagonist

Identification of an HIV integrase inhibitor with micromolar affinity for the CGRP receptor led to the discovery of a series of structurally novel CGRP receptor antagonists. Optimization of this series produced compound 16, a low-molecular weight CGRP receptor antagonist with good pharmacokinetic properties in both rat and dog. In contrast to other nonpeptide antagonists, the activity of 16 was affected by the presence of divalent cations and showed evidence of an alternative, RAMP-independent CGRP receptor binding site.

The novel small molecule α9α10 nicotinic acetylcholine receptor antagonist ZZ-204G is analgesic

Chronic pain is inadequately managed with currently available classes of analgesic drugs. Recently, peptide antagonists of the α9α10 nicotinic acetylcholine receptor were shown to be analgesic. The present study was conducted to characterize a novel small molecule, non-peptide antagonist at nicotinic receptors. The tetrakis-quaternary ammonium compound ZZ-204G was evaluated for functional activity on cloned nicotinic receptors expressed in Xenopus oocytes. In-vivo efficacy was assessed in rat models of tonic inflammatory pain (formalin test), neuropathic pain (chronic constriction nerve injury), and thermal nociception (tail flick test). ZZ-204G was an antagonist at nicotinic receptors inhibiting the α9α10 subtype with an IC₅₀ of 0.51 (0.35-0.72) nM. Antagonist activity at other nicotinic subtypes (α1β1δε, α2β2, α2β4, α3β2, α3β4, α4β2, α4β4, α6/α3β2β3, α6/α3β4 and α7) was 10-1000-fold lower than at the α9α10 subtype. In competition binding assays, the k(i) of ZZ-204G at γ-aminobutyric acid(A), serotonin(3), γ-aminobutyric acid(B), κ- and μ-opioid receptors was 1000- to >10,000-fold lower than at α9α10 nicotinic receptors. Parenteral administration of ZZ-204G dose-dependently decreased nociceptive behaviors (paw flinches) in the formalin test and mechanical hyperalgesia in the chronic constriction nerve injury model of neuropathic pain. ZZ-204G was not antinociceptive in the tail flick assay. Results from the rotarod assay indicated that lower doses of ZZ-204G that were analgesic did not alter motor function. In summary, ZZ-204G represents a prototype small molecule antagonist for α9α10 nicotinic receptors and provides a novel molecular scaffold for analgesic agents with the potential to treat chronic inflammatory or neuropathic pain.

Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na+, K+-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na+, K+-ATPase, peptide effect on high affinity [3H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1×10−4M concentration. On the other hand, the single addition of 1×10−6M, 1×10−5M and 1×10−4M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [3H]-ouabain binding (in %) to 87±16; 74±16 and 34±17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [3H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased Kd value whereas failed to modify Bmax value, indicating a competitive type interaction of the peptide at Na+, K+-ATPase ouabain site. At variance, SR 48692 decreased Bmax value whereas it did not modify Kd value. [3H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30min earlier with 100μg and 250μg/kg SR 48692. It was observed that the 250μg/kg SR 48692 dose led to 19% decrease in basal [3H]-ouabain binding. After SR 48692 treatments, addition of 1×10−6M led to additive or synergic effect. Results suggested that [3H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.

ChemInform Abstract: Neuropeptide S Receptor: Recent Updates on Nonpeptide Antagonist Discovery

AbstractReview: 61 refs.

Effect of grapefruit juice on the pharmacokinetics of tolvaptan, a non-peptide arginine vasopressin antagonist, in healthy subjects

Tolvaptan is a selective vasopressin V2 receptor antagonist that can be given orally once daily for treatment of clinically significant hypervolemic and euvolemic hyponatremia (US and Europe) or extracellular volume expansion despite taking other diuretics (Japan). In vitro studies indicated that tolvaptan was a CYP3A4 substrate. A single-center, randomized, crossover trial of 60-mg tolvaptan with 240 mL of water or with 240 mL of reconstituted grapefruit juice (washout period of 72 h between doses) was conducted in 20 healthy subjects. Blood samples for tolvaptan plasma concentrations were obtained for 48 h postdose. All subjects completed the trial. Following co-administration with grapefruit juice, tolvaptan concentrations were elevated compared with tolvaptan alone for only 16 h postdose; consequently, the mean elimination half-life of tolvaptan was unchanged, 5.7 vs 5.1 h respectively. The mean maximal plasma concentration (C(max)) and the area under the curve (AUC(∞)) of tolvaptan were increased 1.86- and 1.56-fold respectively when co-administered with grapefruit juice. It appears that grapefruit juice increases the bioavailability of tolvaptan, but does not affect its systemic elimination. The adverse event profile was consistent with the aquaretic effect of tolvaptan as urinary frequency, thirst, and dry mouth were the most frequently reported events.

Peptide and Non-peptide Antagonists Targeting Endothelin Receptors in Physiology and Pathology

As for other peptides such as bradykinin, neurokinins and angiotensins, peptide antagonists for endothelin-1 (ET-1) have been early on developed towards the pharmacological characterization of both ET(A) and ET(B) receptors. Interestingly, unlike the previously mentioned three peptides, receptors for ET-1 were cloned and purified prior to the report of ET(A)and ET(B)receptor antagonists such as BQ-123 and BQ-788. The availability of such pharmacological tools and the use of molecular approaches have certainly fast-tracked the development of non-peptide ET receptor antagonists for clinical applications. Albeit rapid degradation by gastric enzymes and short half-life in plasma of peptide receptor antagonists limit their use in clinical settings, those molecules have been of importance in the identification of mediators and modulators of ET-1 induced properties in vitro and in vivo, as described further in this review. Peptide antagonists acting selectively or, with equivalent affinities against ET(A)and ET(B)receptors were reported prior to the advent of clinically relevant non-peptide blockers such as Bosentan. Confounding mechanisms involving, for example, the endogenous modulators nitric oxide and prostacyclin as well as allosteric interactions between ET receptor types, have also been clarified with the use of peptide antagonists for endothelins. Finally, peptide antagonists were also used to identify the precise pharmacology of ET-1 precursors such as big-endothelin-1 and ET-1 (1-31). The present review will thus attempt to summarize the knowledge to date and future perspectives related to use of peptide antagonists targeting endothelin receptors in physiological and pathological settings.

B1 and B2 kinin receptor participation in hyperproliferative and inflammatory skin processes in mice

Kinins are released during dermal injury and inflammation and seem to contribute to the pathogenesis of cutaneous diseases. Participation of kinins in skin inflammatory process was evaluated using knockout mice and non-peptide kinin receptor antagonists. Chronic skin inflammation was induced by multiple applications of TPA in mice ear. The B(2) knockout mice (B(2)(-/-)) showed a significant increase of ear weight (23 ± 10%) and epidermal cellular hyperproliferation and acanthosis formation upon histological analysis when compared with wildtype mice. Also, evaluation of PCNA levels by Western blot and immunohistochemistry confirmed the increase in the epidermis hyperproliferation in the ear skin of B(2)(-/-) mice. In contrast, no modification in these parameters was detected in B(1) knockout mice (B(1)(-/-)). However, mice lacking both kinin receptors (B(1)B(2)(-/-)) presented a considerable reduction of epidermis thickness and in PCNA levels. Following the establishment of skin inflammation (5th day of TPA application) treatment with the non-peptide antagonists SSR 240612 (B(1) receptor antagonist), FR 173657 (B(2) receptor antagonist), or SSR 240612 plus FR 173657 topically applied, caused a significant inhibition of ear weight (20 ± 5%, 34 ± 4% and 32 ± 6%, respectively). In the histological analysis, the antagonists produced a reduction in epidermal hyperplasia and acanthosis formation; but the treatment with a combination of the two antagonists did not increase efficacy. Kinin receptors seem to be involved in the control of the keratinocyte hyperproliferative process, and non-peptide kinin receptor antagonists may be useful tools in the treatment of hyperproliferative skin disorders.

Functional reconstitution of human neuropeptide Y (NPY) Y2 and Y4 receptors in Sf9 insect cells

The four functionally expressed human neuropeptide Y receptor subtypes (hY1R, hY2R, hY4R, hY5R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive Gi/o-proteins. The number of small molecules described as ligands for hY1R and hY5R exceeds by far those for hY2R. Potent non-peptidergic ligands for the hY4R are not available so far. Here, we report on the functional reconstitution of the hY2R and the hY4R in Sf9 insect cells using the baculovirus system. Sf9 cells were genetically engineered by infection with up to four different baculoviruses, combining the receptors with G-proteins of the Gi/o family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y2) and hPP (Y4), the GPCRs coupled to various Gi/Go-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY2R + Gαi2 and hY4R + Gαi2/Gαo + RGS4, combined with Gβ1γ2, yielded best signal-to-noise ratios. hY2R function was validated using both agonistic peptides (NPY, PYY, NPY13–36) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY4R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY2R and abolished hY4R function. Investigations with monovalent salts showed sensitivity of hY4R toward Na+, revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y4R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y2 and Y4 receptor ligands.

NPY receptors as potential targets for anti‐obesity drug development

The neuropeptide Y system has proven to be one of the most important regulators of feeding behaviour and energy homeostasis, thus presenting great potential as a therapeutic target for the treatment of disorders such as obesity and at the other extreme, anorexia. Due to the initial lack of pharmacological tools that are active in vivo, functions of the different Y receptors have been mainly studied in knockout and transgenic mouse models. However, over recent years various Y receptor selective peptidic and non-peptidic agonists and antagonists have been developed and tested. Their therapeutic potential in relation to treating obesity and other disorders of energy homeostasis is discussed in this review.

Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes

The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T(3.40) was selected based on sequence alignments both between subtypes and between species and G(2.68), L(4.60) and Q(6.55) also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3-36), NPY(13-36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3-36) increased for the mutants T(3.40)I and Q(6.55)A. Increased affinity was also observed for PYY to Q(6.55)A. PYY(3-36) displayed decreased affinity for G(2.68)N and L(4.60)A whereas binding of NPY(13-36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T(3.40)I, L(4.60)A and Q(6.55)A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.

The role of CXCR2 chemokine receptors in the oral squamous cell carcinoma

This study evaluated the relevance of CXCR2 chemokine receptors in oral squamous cell carcinoma, by means of in vitro and in vivo approaches. The in vitro incubation of the selective and non-peptide CXCR2 receptor antagonist N-(2-hydroxy-4-nitrophenyl)-N9-(2-bromophenyl) Urea (SB225002; 25 to 800 nM) produced a time- and concentration-dependent inhibition of SCC158 (rat) and HN30 (human) cell lines viability. Conversely, this antagonist did not significantly affect the viability of the immortalized keratinocyte lineage, HaCaT. Additionally, the incubation of human IL-8 and rat CINC-1 CXCR2 agonists produced a concentration-related increase on HN30 and SCC158 proliferation. The submucosal injection of SCC158 cells (5 × 10(6) cells) into the tongue of Fischer 344 rats induced tumor development, which displayed typical clinical features. Immunohistochemical analysis of rat tongue biopsies revealed a marked increase of CXCR2 receptor immunoreactivity, which was accompanied by augumented expression of VEGF and caspase-3. Our data suggests an important role for CXCR2 receptors in oral squamous cell carcinoma.