Human NK1 Receptor Research Articles

Structural motifs encoded by individual exons of the human neurokinin-1 receptor gene interact differentially with selective agonists and antagonists.

Three chimeric receptors were constructed by exchanging exons between human neurokinin NK1 and NK3 receptor genes. The N-terminal sequences of these chimeric receptors are encoded by exon 1, exon 1-2, or exon 1-3 of the NK1 receptor gene, whereas the remaining C-terminal sequences of these chimeric receptors are encoded by corresponding exons of the human NK3 receptor gene. Substance P bound with high affinities to all three chimeric receptors, suggesting that in addition to the common structures composed of conserved amino acid residues among neurokinin receptors, structural elements encoded by the first exon of the human NK1 receptor gene may also play an important role for substance P binding. On the contrary, potent NK1 antagonists L703,606 and SR140,333 did not show any detectable binding to these chimeric receptors. In accordance, sequences encoded by exon 4, and possibly exon 5, are likely to contain important structural motifs that may directly or indirectly influence the binding of these antagonists. Further comparison of the binding affinities of highly selective NK1 agonists, [Sar9, Met(O2)11] substance P, substance P methyl ester, and septide, revealed that each agonist may interact differently with the human NK1 receptor. These results show that the exon-exchanging technique can be a useful tool for studying structure-function relationships of receptors in which exon-intron junctions are fully conserved among receptor subtypes.

The Unpredicted High Affinities of a Large Number of Naturally Occurring Tachykinins for Chimeric NK1/NK3 Receptors Suggest a Role for an Inhibitory Domain in Determining Receptor Specificity

Three chimeric receptors were constructed by exchanging exon sequences between human NK1 and NK3 receptor genes. The resulting chimeric receptors not only retained high affinities for their natural ligands substance P and neurokinin B but also exhibited surprisingly high affinities for other naturally occurring tachykinins including neurokinin A, neuropeptide K, neuropeptide gamma, eledoisin, kassinin, physalaemin, and phyllomedusin. In contrast, these chimeric receptors displayed a wide range of variability in their affinities for non-naturally occurring ligands including selective agonists and antagonists of NK1, NK2, and NK3 receptors. Since the only common feature among these naturally occurring neurokinin peptides is the conserved C-terminal sequences, our data suggest that these conserved sequences must play the major role in conferring high affinity binding to the chimeric receptors. To explain the apparently "improved" affinities of these naturally occurring ligands for the chimeric receptors as compared with their affinities for the parent NK1 and NK3 receptors, we are proposing that certain inhibitory domains that are present in the NK1 and/or NK3 receptors are compromised in these chimeric receptors. Upon disruption of these inhibitory domains during the formation of chimeras, the naturally occurring ligands can interact more favorably with chimeric receptors through their conserved C-terminal sequences. Based on this hypothesis, the binding affinities of natural tachykinin ligands may be largely determined by their conserved C-terminal sequences, whereas receptor selectivities of these ligands are influenced more by the presence or absence of inhibitory domains rather than specific binding domains on their target receptors.

Use of a dipeptide chemical library in the development of non-peptide tachykinin NK3 receptor selective antagonists.

The use of a dipeptide library as the source of a micromolar chemical lead compound for the human tachykinin NK3 receptor is described. The screening of a dipeptide library through a cloned human NK3 receptor binding assay resulted in the identification of Boc(S)Phe(S)PheNH2 (1), which has subsequently been developed, following a 'peptoid' design strategy, into a series of high-affinity NK3 receptor selective antagonists. The structure-activity relationship of the C-terminal portion of this dipeptide lead was first explored and led to the identification of the urea derivative Boc(S)Phe(R)alphaMePheNH(CH2)7NHCONH2 (41, PD157672). This modified dipeptide has a Ke of 7 nM in blocking senktide-induced increases in intracellular calcium levels in human NK3 receptors stably expressed in CHO cells. Subsequent optimization of the N-terminal BocPhe group and the alphaMePhe residue side chain of 41 led to the identification of [S-(R*,S*)]-[2-(2,3-difluorophenyl)-1-methyl-1-[(7-ureidoheptyl)ca r bamoyl]ethyl]carbamic acid 2-methyl-1-phenylpropyl ester (60, PD161182), a non-peptide NK3 receptor selective antagonist. Compound 60 blocks the senktide-evoked increases in intracellular calcium levels in cloned human NK3 receptors stably expressed in CHO cells with Ke of 0.9 nM.

Pharmacological characterization of SR 142801: a new non-peptide antagonist of the neurokinin NK-3 receptor.

Pharmacological and biochemical assays were performed to characterize SR 142801, a new NK-3 receptor antagonist, and its [R]-enantiomer, SR 142806. The compounds were tested (1) in the guinea pig isolated ileum stimulated with [MePhe7]NKB (NK-3 system) in order to evaluate onset and duration of action and to estimate the apparent affinity of the antagonist in terms of pA2 at 140 min after application; (2) in 6 selected monoreceptor systems, the rabbit (rb) vena cava for the NK-1rb receptor, the rabbit pulmonary artery and the hamster (hs) urinary bladder for the NK-2rb and NK-2hs receptors, the rat (r) portal vein for the NK-3r receptor, and in two multireceptor systems adequately treated with NK-1 or NK-2 receptor antagonists to obtain monoreceptor-mediated biological responses (the rat urinary bladder treated with SR 48968 for evaluating the NK-1r and the guinea pig-gp-ileum treated with CP-99994 for measuring the antagonist affinity on the NK-3gp receptor), in order to evaluate the antagonist selectivity, and (3) in various plasma membrane preparations containing NK-3-binding sites from rats, guinea pigs, and man. The data presented indicate that SR 142801 is a potent, fairly selective non-peptide antagonist of the functional (pA2 9.4) and binding (Ki 0.11 nmol/l) site of the guinea pig and human (Ki 0.21 nmol/l) NK-3 receptors, while being much less active on the Nk-3 receptors of other species, particularly the rat (pA2 7.0; Ki 15 nmol/l). SR 142801 shows a slow onset of action and acts as a long-lasting irreversible antagonist, specific for neurokinin receptors, especially the NK-3 sites of guinea pigs and man.

The pharmacology of GR203040, a novel, potent and selective non‐peptide tachykinin NK1 receptor antagonist

1. The in vitro and in vivo pharmacology of GR203040 ((2S, 3S)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), a novel, highly potent and selective non-peptide tachykinin NK1 receptor antagonist, was investigated in the present study. 2. GR203040 potently inhibited [3H]-substance P binding to human NK1 receptors expressed in Chinese hamster ovary (CHO) and U373 MG astrocytoma cells, and NK1 receptors in ferret and gerbil cortex (pKi values of 10.3, 10.5, 10.1 and 10.1 respectively). GR203040 had lower affinity at rat NK1 receptors (pKi = 8.6) and little affinity for human NK2 receptors (pKi < 5.0) in CHO cells and NK3 receptors in guinea-pig cortex (pKi < 6.0). With the exception of the histamine H1 receptor (pIC50 = 7.5). GR203040 had little affinity (pIC50 < 6.0) at all non-NK1 receptors and ion channels examined. Furthermore, GR203040 produced only weak inhibition of Na+ currents in SH-SY5Y neuroblastoma and superior cervical ganglion cells (pIC50 values < 4.0). GR203040 produced only weak antagonism of Ca(2+)-evoked contractions of rat isolated portal vein (pKn = 4.1). The enantiomer of GR203040, GR205608 (2R, 3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), had 10,000 fold lower affinity at the human NK1 receptor expressed in CHO cells (pKi = 6.3). 3. In gerbil ex vivo binding experiments, GR203040 produced a dose-dependent inhibition of the binding of [3H]-substance P to cerebral cortical membranes (ED50 = 15 micrograms kg-1 s.c. and 0.42 mg kg-1 p.o.). At 10 micrograms kg-1 s.c., the inhibition of [3H]-substance P binding was maintained for > 6 h. In the rat, GR203040 was less potent (ED50 = 15.4 mg kg-1 s.c.) probably reflecting, at least in part, its lower affinity at the rat NK1 receptor. 4. In guinea-pig isolated ileum and dog isolated middle cerebral and basilar arteries, GR203040 produced a rightward displacement of the concentration-effect curves to substance P methyl ester (SPOMe) with suppression of the maximum agonist response (apparent pKB values of 11.9, 11.2 and 11.1 respectively). 5. In anaesthetized rabbits, GR203040 antagonized reductions in carotid arterial vascular resistance evoked by SPOMe, injected via the lingual artery (DR10 (i.e. the dose producing a dose-ratio of 10) = 1.1 micrograms kg-1, i.v.). At a dose 20 fold greater than its DR10 value (i.e. 22 micrograms kg-1, i.v.), significant antagonism was evident more than 2 h after GR203040 administration. 6. In anaesthetized rats, GR203040 (3 and 10 mg kg-1, i.v.) produced a dose-dependent inhibition of plasma protein extravasation in dura mater, conjunctiva, eyelid and lip in response to electrical stimulation of the trigeminal ganglion. 7. It is concluded that GR203040 is one of the most potent and selective NK1 receptor antagonists yet described, and as such, has considerable potential as a pharmacological tool to characterize the physiological and pathological roles of substance P and NK1 receptors. GR203040 may also have potential as a novel therapeutic agent for the treatment of conditions such as migraine, emesis and pain.

Co-expression of the neurokinin NK2 receptor and G-protein components in the fission yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe has proven useful for studying molecular interactions between a range of signal transduction components. We now report the first coexpression of a mammalian seven-transmembrane receptor and G-protein components in S. pombe. We selected the human neurokinin NK2 receptor together with its G-protein-signalling partner G q for this study. Yeast membrane fractions showed high levels of NK2 receptor-binding activity (1159 ± 534 ( n = 3) fmol/mg protein) although initial experiments with intact cells revealed an absence of receptors at the cell surface. Using a construct comprising the NK2 coding sequence fused with the signal sequence from an endogenous phosphatase ( phol), we detected ∼ 400 NK2 receptors/cell in unbroken yeast. Successful co-expression of the NK2 receptor with the G-protien subunits G αq, β1 or β2 and γ3 failed to modulate agonist binding, suggesting the absence of functional interaction between these components. As an alternative test of G αq function, we next expressed its downstream effector target phospholipase C-β1 (PLCβ1) in S. pombe. Al-though PLCβ1 undergoes powerful in vitro activation by G αq derived from baculovirus-infected Sf9 cells and mammalian cells, G αq expressed in S. pombe is totally ineffective. Similar results were also achieved with the G-protein subunit G α16. Together, these data suggest that seven-transmembrane receptors can be expressed in S. pombe at high levels and directed to the cell surface although their interaction with co-expressed G-proteins in undetectable. Production of inactive G α-chains in S. pombe may account for these observations.

4,4-Diphenyl-7-perhydrothiapyrano[3,4-c]pyrrolone, a new series of substance P receptors antagonists

The synthesis of 4,4-Diphenyl-7-perhydrothiapyrano[3,4-c]pyrrolones, a new series of substance P antagonists with high affinity for rat and human NK1 receptors is described.

C-terminal Truncation of the Neurokinin-2 Receptor Causes Enhanced and Sustained Agonist-induced Signaling: ROLE OF RECEPTOR PHOSPHORYLATION IN SIGNAL ATTENUATION

The G protein-linked receptor for neurokinin A (NKA) couples to stimulation of phospholipase C and, in some cells, adenylyl cyclase. We have examined the function of the C-terminal cytoplasmic domain in receptor signaling and desensitization. We constructed C-terminal deletion mutants of the human NK-2 receptor (epitope tagged) to remove potential Ser/Thr phosphorylation sites, and expressed them in both mammalian and insect cells. When activated, truncated receptors mediate stronger and more prolonged phosphoinositide hydrolysis than wild-type receptor; however, the amplitude and kinetics of the NKA-induced rise in cytosolic Ca2+ remain unaltered. Protein kinase C (PKC)-activating phorbol ester abolishes wild-type receptor signaling but not mutant receptor signaling. Mutant receptors also mediate enhanced and prolonged cAMP generation, at least in part via PKC activation. When expressed in COS cells or Sf9 insect cells, the wild-type receptor is phosphorylated; receptor phosphorylation increases after addition of either NKA or phorbol ester. In contrast, mutant receptors are not phosphorylated by either treatment. Our results suggest that C-terminal Ser/Thr phosphorylation sites in the NK-2 receptor have a critical role in both homologous and heterologous desensitization. Removal of these phosphorylation sites results in a receptor that mediates sustained activation of signaling pathways and is insensitive to inhibition by PKC.

Conversion of antagonist-binding site to metal-ion site in the tachykinin NK-1 receptor.

Mutational analysis of the tachykinin NK-1 (refs 1-7), NK-2 (ref. 8) and angiotensin AT-1 (refs 9, 10) receptors indicates that non-peptide antagonists act through residues located between the seven transmembrane segments, whereas natural peptide agonists bind mainly to residues scattered in the exterior part of the receptor. The presumed contact points for the prototype NK-1 antagonist CP96,345 cluster on opposing faces of the outer portions of transmembrane helices V and VI (refs 1-5). Here we show that systematic introduction of histidyl residues at this antagonist-binding site in the human NK-1 receptor gradually converts it into a high-affinity metal-ion-binding site without affecting agonist binding. In a double mutant with histidine residues substituted at the top of transmembrane segments V and VI, respectively, Zn2+ inhibits binding of radiolabelled agonist peptide and efficiently blocks phosphoinositol turnover induced by substance P. We propose that Zn2+ and CP96,345 act as 'allosteric competitive' antagonists by stabilizing inactive conformations of the mutant and the wild-type receptor respectively. Introduction of metal-ion-binding sites could be used as a general tool in the structural and functional characterization of helix-helix interactions in G-protein-coupled receptors, as well as in other membrane proteins.

Synthesis of perhydro-1,4-ethano-1,5-naphthyridine and perhydro-4,7-ethanopyrrolo[3,2-b]pyridine derivatives: potential NK1-receptor antagonists. X-Ray molecular structures of (4aR,8S,8aR)-6-oxo-8-phenylperhydro-1,4-ethano-1,5-naphthyridine and (4aR,7R,8R,8aR)-7,8-diphenylperhydro-1,4-ethano-1,5-naphthyridine

Derivatives of perhydro-1,4-ethano-1,5-naphthyridine and 4,7-ethanopyrrolo[3,2-b]pyridine were designed and synthesized as conformationally constrained analogues of the potent NK1-receptor antagonist CP-96,345. 2-Benzylidenequinuclidin-3-one 1 was used as the common starting material: (i) heterocyclizations of compound 1 with N-(carbamoylmethyl)pyridinium chloride gave unsaturated pyridone derivatives which, after catalytic hydrogenation, afforded 1,5-naphthyridines, and (ii) functionalization of compound 1 by nucleophilic 1,4-addition reactions, followed by reductive cyclizations, gave quinuclidine derivatives with fused five- or six-membered rings. The cyclization reactions proceeded stereoselectively and the relative stereochemistries were determined by a combination of molecular mechanics calculations. X-Ray crystallography, and NMR spectroscopy. The biological activities of the synthesized derivatives were evaluated by binding studies to human NK1-receptors in UC11MG cells. The compounds had low to moderate affinity for the NK1-receptor.

Pharmacological Characterization of Tachykinin-Induced Intracellular Calcium Rise in a Human NK2 Receptor Transfected Cell Line

We have examined the effect of various natural and synthetic tachykinins on the steady state Ca ++-rise ([Ca ++] i) in transfected chinese hamster ovary cells expressing recombinant human Neurokinin 2 (NK 2) receptors. The rank order of potency with natural tachykinins was NeurokininA > Neurokinin B> Eledoisin > Physaelamin > substance P. The selective NK 2 agonist, [β-Ala 8]NKA(4-10) was very potent,with an EC 50 value of 4.83 × 10 −9M whereas Senktide, MePhe 7NKB and Sar 9, (MetO 2) 11 substance P, selective NK 3 and NK 1 agonists, respectively, did not have any effect on [Ca ++] i in hrNK2CHO cells, suggesting a selective and preferential recognition and activation of NK 2 receptors in these cells. (±) SR 48968, a selective NK 2 antagonist, abolished the β-AlaNKA-induced [Ca ++] i with an IC 50 value of 0.7 nM. Two other peptidic NK 2 antagonists, MEN 10376 and L-658977, were less active with IC 50 values of 49 nM and 5.29 μM, respectively. In contrast, (±) CP-96,345 and (±)CP-99,994 and RP 67580, all selective NK 1 antagonists, did not have any effect on the β-AlaNKA-induced [Ca ++] i in hrNK2CHO cells (±) SR 140333, a potent and selective NK 1 antagonist, had a 35% inhibition under similar conditions. These data demonstrate a high selectivity and sensitivity to NK 2 receptor mediated [Ca ++] i in rhNK2R-CHO cells and may be of value as a rapid, selective test of drug action at the human NK 2 receptors in vitro.

Characterization of the interaction of N-acyl-L-tryptophan benzyl ester neurokinin antagonists with the human neurokinin-1 receptor.

We have recently shown that a series of N-acyl-L-tryptophan benzyl esters are potent substance P antagonists (Macleod, A. M., Merchant, K. J., Cascieri, M. A., Sadowski, S., Ber, E., Swain, C. J., and Baker, R. (1993) J. Med Chem. 14, 2044-2045). We now report the detailed characterization of the interaction of N-acetyl-L-tryptophan-3,5-bistrifluoromethyl benzyl ester (L-732,138) with the human neurokinin-1 (NK-1) receptor. L-732,138 inhibits the binding of 125I-substance P to the cloned human NK1 receptor expressed in Chinese hamster ovary cells with an IC50 of 2.3 +/- 0.7 nM. In contrast, it has 200-fold lower affinity for the cloned rat NK-1 receptor and has > 1000-fold lower affinity for the human NK-2 and NK-3 receptors. L-732,138 acts as a competitive antagonist of substance P, as shown by functional Schild analysis of the inhibition of substance P-induced inositol phosphate synthesis, by kinetic analysis of the dissociation rate, and by thermodynamic analysis of the equilibrium binding of 125I-substance P to the NK-1 receptor. L-732,138 also competitively inhibits the binding of the quinuclidine amine antagonist, [125I]L-703,606, to the receptor. The compound has 230- and 10-fold reduced affinity for mutant NK-1 receptors in which histidine 265 or histidine 197, respectively, are replaced with alanine. We have previously shown that these residues play key roles in the binding of quinuclidine antagonists to the NK-1 receptor. These results suggest that the tryptophan and quinuclidine series of NK-1 antagonists bind to similar binding sites on the human NK-1 receptor.

Benzomalvins, new substance P inhibitors from a Penicillium sp.

In the course of screening microbial broths for neurokinin receptor antagonists, a series of new benzodiazepines, benzomalvins A (1), B (2) and C (3), has been isolated from the culture broth of a fungus identified as a Penicillium sp. Benzomalvin A (1) showed inhibitory activity against substance P with Ki values of 12, 42 and 43 microM at the guinea pig, rat and human neurokinin NK1 receptors, respectively. Benzomalvins B (2) and C (3) were only weakly active. The structures of these compounds were determined by spectroscopic methods including MS measurements and NMR analysis.

WIN 66306, a new neurokinin antagonist produced by an Aspergillus species: fermentation, isolation and physico-chemical properties.

WIN 66306 (1a), a cyclic peptide containing a novel amino acid, was isolated as a neurokinin antagonist from an Aspergillus species, labelled SC230. Conditions that maximized the production of 1a were developed, leading also to production of the related compound WIN 68577 (2) and rosellichalasin (3). Both 2 and 3 were more active in the rat NK1 than in the human NK1 receptor binding assay, while 1a was more active at the human receptor with an inhibitor affinity constant of 7 microM.

WIN 64821, a new competitive antagonist to substance P, isolated from an Aspergillus species: structure determination and solution conformation

Two new diketopiperazine dimers, WIN 64821 (1a) and WIN 64745 12), were isolated from an Aspergillus culture originally isolated from soil and their structures established on the basis of chemical and spectroscopic evidence. The dimer 1a has C1 symmetry with each of two equivalent monomeric subunits biosynthetically constructed from one phenylalanine and one tryptophan residue. Dimer 1a is a competitive antagonist to substance P (SP) at the human NK1 receptor with an inhibitor affinity constant (K i ) of 230±30 nM against [ 125 I]SP in human astrocytoma cells

Quinuclidine based NK-1 antagonists 2: determination of the absolute stereochemical requirements

The relative and absolute stereochemical requirements for high affinity binding to the human NK-1 receptor are defined for a series of quinuclidine ethers. Whilst the S stereochemistry at C-3 is essential for high afifnity, surprising both epimers at C-2 are active.

Characterization of the binding of [ 125I-iodo-histidyl,methyl-Phe 7] neurokinin B to the neurokinin-3 receptor

We have characterized the binding of [ 125I-iodo-histidyl, methyl Phe 7]neurokinin B ( 125I-NKB) to the human neurokinin-3 (NK3) receptor. 125I-NKB specifically binds to the NK3 receptor expressed in CHO cells with a Kd of 0.2 nM. The ligand displays little crossreactivity with the human NK1 and NK2 receptors. The binding of 125I-NKB to the human NK3 receptor and to rat cortex membranes is inhibited by neurokinin B with IC 50 of 1.5 nM and 4 nM, respectively. In contrast, 350- to 500-fold higher concentrations of substance P and neurokinin A are required to inhibit binding to either receptor preparation. The data suggest that 125I-NKB is a high affinity, selective ligand for the human and rat NK3 receptor.

Thapsigargin blocks the mobilisation of intracellular calcium caused by activation of human NK1 (long) receptors expressed in chinese hamster ovary cells

Neurokinin receptors can couple to several second messenger systems including phospholipase C and intracellular calcium mobilisation. Using FURA-2 microspectrofluorimetry, this study examines the mobilisation of calcium in CHO cells which had been stably transfected with the long isoform of the human NK1 receptor. Substance P caused a concentration-dependent rise in inositol phosphate production which was correlated with a reversible increase in intracellular calcium levels. The mobilisation of calcium was reduced by the removal of extracellular calcium from the bathing solution, and almost totally abolished by depletion of intracellular calcium pools with 1 μM thapsigargin. These data suggest that the transduction mechanism associated with NK1 (long) receptor activation can utilise both intracellular and extracellular calcium pools in this expression system.

Baculovirus expression of human NK 2 receptor

Baculovirus expression of human NK 2 receptor

Characterization of a human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG.

The human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [3H]substance P binding sites were present on cell homogenates, whereas [3H]neurokinin A or [3H]-senktide binding sites were absent. The binding was saturable and reversible. The binding of [3H]substance P was inhibited by very low concentrations of [L-Pro9]substance P and [Sar9,Met(O2)11]substance P; septide was approximately 1,000-fold less potent. The most potent peptide antagonist was trans-4-hydroxy-1-(1H-indol-3-ylcarbonyl)-L-prolyl-N-methyl-N-(phe nylmethyl)-L- tyrosineamide. The rank order of potency for the nonpeptide antagonists was (S,S)-CP 96,345 > (+/-)-CP 96,345 > (+/-)-2-chlorobenzylquinuclidinone > (R,R)-CP 96,345 > RP 67580 > RP 68651. In [3H]-inositol-labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK1 receptor agonists for stimulating inositol phosphate production and for inhibiting [3H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK1 receptor.