Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands.

Ligand recognition of the NK1 receptor (substance P receptor) by peptide agonist and non-peptide antagonist has been investigated and compared by the use of fluorescent ligands and spectrofluorometric methods. Analogues of substance P (SP) labeled with the environment-sensitive fluorescent group 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) at either position 3, 8, or 11 or with fluorescein at the Nalpha position were synthesized and characterized. Peptides modified at the alpha-amino group or at positions 3 or 11 conserved a relatively good affinity for NK1 and agonistic properties. Modification at position 8 resulted in an 18, 000-fold decrease in affinity. A fluorescent dansyl analogue of the non-peptide antagonist CP96,345 was prepared and characterized. The quantum yield of fluorescence for dansyl-CP96,345 was much higher than for any of the dansyl-labeled peptides indicating that the micro-environment of the binding site is more hydrophobic for the non-peptide antagonist than for the peptide agonists. Comparison of collisional quenching of fluorescence by the water-soluble hydroxy-Tempo compound showed that dansyl-CP96,345 is buried and virtually inaccessible to aqueous quenchers, whereas dansyl- or fluoresceinyl-labeled peptides were exposed to the solvent. Anisotropy of all fluorescent ligands increased upon binding to NK1 indicating a restricted motional freedom. However, this increase in anisotropy was more pronounced for the dansyl attached to the non-peptide antagonist CP96,345 than for the fluorescent probes attached to different positions of SP. In conclusion, our data indicate that the environment surrounding non-peptide antagonist and peptide agonists are vastly different when bound to the NK1 receptor. These results support recent observations by mutagenesis and cross-linking work suggesting that peptide agonists have their major interaction points in the N-terminal extension and the loops forming the extracellular face of the NK1 receptor. Our data also suggest that neither the C terminus nor the N terminus of SP appears to penetrate deeply below the extracellular surface in the transmembrane domain of the receptor.

Photoaffinity labeling of the human substance P (neurokinin-1) receptor with [3H2]azido-CP-96,345, a photoreactive derivative of a nonpeptide antagonist.

An azido derivative of [3H2](2S, 3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl) methyl)-1-azabicyclo[2.2.2]octon-3-amine (CP-96,345), a potent nonpeptide antagonist of the substance P (SP) (neurokinin-1) receptor, was synthesized and shown to have an affinity for the human SP receptor similar to that of the parent compound, CP-96,345. When Chinese hamster ovary cells expressing the human SP receptor were photolabeled with this compound and analyzed with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, several radioactive bands were observed, including a major band centered at molecular mass 80 kDa, the expected value for the SP receptor expressed in Chinese hamster ovary cells. Only the labeling of the 80-kDa protein was specific: nonradiolabeled CP-96,345 but not its optical enantiomer, CP-96,344 was a potent inhibitor of photoincorporation. SP prevented photolabeling only at concentrations higher than expected from its binding affinity but similar to those shown in a competition binding assay to displace radioiodinated analogue of CP-96,345. Antiserum generated against a synthetic peptide corresponding to the carboxyl terminus of the human SP receptor immunoprecipitated only the 80-kDa photoaffinity labeled protein, confirming that it is the human SP receptor. Interestingly, a second antiserum that was generated against the third extracellular loop of the G protein-coupled receptor no longer immunoprecipitated the receptor when covalently labeled with [3H2]azido-CP-96,345. This result indicates either that attachment of the antagonist modified the antigenic region directly, suggesting involvement of this domain in the binding of CP-96,345, or that the loss of recognition by the antiserum is secondary to a change in conformation induced by the covalent attachment of the antagonist at a different site.

5-Phenyl-3-ureidobenzazepin-2-ones as cholecystokinin-B receptor antagonists.

A series of 5-phenyl-3-ureidobenzazepin-2-one cholecystokinin-B (CCK-B) receptor antagonists was synthesized using Beckmann ring expansion of a suitable 4-phenyl-1-tetralone as a key step. Structure-activity relationship studies revealed the importance of the 5-phenyl group for potent and selective CCK-B affinity. Addition of an 8-methyl substituent and resolution provided the potent (CCK-B IC50 = 0.48 nM) CCK-B antagonist 4. The role of the 5-phenyl group as part of a "privileged structure" for high-affinity receptor antagonism is discussed.

Aza-tricyclic substance P antagonists.

The synthesis and structure-activity relationships of a series of aza-tricyclic analogs of the quinuclidine substance P (SP) antagonist 1 are described. The SP receptor affinity of these compounds was found to vary according to the size of the new ring fused to the quinuclidine and the mode of fusion. Correlations between receptor affinity and (1) the steric bulk of the newly introduced ring fusion and (2) the dihedral angle between the benzhydryl and benzylamino substituents of these aza-tricyclic compounds were explored.

Discovery of CP-96,345 and its characterization in disease models involving substance P.

Studies with CP-96,345, a potent, selective, orally active, nonpeptide NK1 receptor antagonist, have provided considerable insight into SP pharmacology. Rather than being a primary neurotransmitter, SP prolongs the nociception produced by other neurotransmitters. By controlling endothelial permeability, SP plays a major role in inflammation and inflammatory aspects of asthma, possibly by regulating the access of neutrophils to an inflammatory site. These results indicate potential therapeutic applications for SP antagonists in the treatment of chronic pain, inflammation, and inflammatory aspects of asthma, and signal a new era in the clinical management of these important diseases.

Synthesis of a nonpeptide carbon-11 labeled substance P antagonist for PET studies.

CP 96,345 is a nonpeptide high affinity antagonist of the substance P (NK1) receptor. The radiosynthesis of [11C]CP 96,345 suitable for Positron Emission Tomography (PET) applications is described. [11C]CP 96,345 was prepared by O-methylation of a desmethyl precursor via in situ generation of its phenolate salt. The in vivo tissue distribution of [11C]CP 96,345 in guinea pigs (n = 2) at 5 and 30 min was determined. Uptake was low in brain (approximately 0.04% dose/g) and highest (approximately 1-2% dose/g) in the spleen and lungs. The present findings indicate that the use of [11C]CP 96,345 in PET might be more applicable to the study of substance P receptors in peripheral tissues involved with inflammatory disease and arthritis.

The discovery of (2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)methyl]-1- azabicyclo[2.2.2]-octan-3-amine as a novel, nonpeptide substance P antagonisst.

We describe the structure-activity relationship development of a series of quinuclidines which culminated in the first potent, selective, nonpeptide substance P (SP) antagonist, (2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxy-phenyl)methyl]-1- azabicyclo[2.2.2]octan-3-amine, 3 (CP-96,345). Compound 3 is a potent displacer of [3H]SP binding in human IM-9 cells and blocks SP-induced and capsaicin-induced plasma extravasation, as well as SP-induced salivation in the rat in vivo. This compound may both help to further our understanding of the interactions of small molecules with peptide receptors and serve to evaluate the therapeutic potential of a SP antagonist.

Effect of CP-96,345, a nonpeptide substance P receptor antagonist, on salivation in rats.

CP-96,345 [(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl) methyl]-1-azabicyclo[2.2.2]octan-3-amine) antagonism of substance P-stimulated salivation was investigated in pentobarbital-anesthetized rats. Administered either intraperitoneally or orally, CP-96,345 produced dose-dependent inhibition of the sialogogic response elicited by substance P, with a median effective dose of 12-24 mumol/kg (5-10 mg/kg) of body weight, but had no effect on acetylcholine-stimulated salivation. CP-96,345 produced concentration-dependent inhibition of [3H]substance P binding to rat submaxillary gland membranes, with a median effective concentration of 34 +/- 3.6 nM. These biological activities were confined to CP-96,345 in that the 2R,3R enantiomer (CP-96,344) was without effect.

A potent nonpeptide antagonist of the substance P (NK1) receptor.

CP-96,345 [(2S, 3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)- methyl]-1-azabicyclo[2.2.2]octan-3-amine] is a potent nonpeptide antagonist of the substance P (NK1) receptor. CP-96,345 inhibited 3H-labeled substance P binding and was a classical competitive antagonist in the NK1 monoreceptor dog carotid artery preparation. CP-96,345 inhibited substance P-induced salivation in the rat, a classical in vivo bioassay, but did not inhibit NK2, NK3, or numerous other receptors; it is thus a selective NK1 antagonist. This compound may prove to be a powerful tool for investigation of the physiological properties of substance P and exploration of its role in diseases.