Development of High Affinity Selective VIP1 Receptor Agonists

Gourlet, P., A. Vandermeers, P. Vertongen, J. Rathe, P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. Development of high affinity selective VIP₁ receptor agonists. Peptides 18(10) 1539–1545, 1997.—The biological effects of VIP are mediated by at least two VIP receptors: the VIP₁ and the VIP₂ receptors that were cloned in rat, human and mice. As the mRNA coding for each receptor are located in different tissues, it is likely that each receptor modulates different functions. It is therefore of interest to obtain selective agonists for each receptor subtype. In the present work, we achieved the synthesis of two VIP₁ receptor selective agonists derived from secretin and GRF. [R¹⁶]chicken secretin had IC₅₀ values of binding of 1, 10,000, 20, and 3000 nM for the rat VIP₁-, VIP₂-, secretin- and PACAP receptors, respectively. This peptide, however, had a weaker affinity for the human VIP₁ receptor (IC₅₀ of 60 nM). The chimeric, substituted peptide [K¹⁵,R¹⁶,L²⁷]VIP(1-7)/GRF(8-27) had IC₅₀ values of binding of 1, 10,000, 10,000 and 30,000 nM for the rat VIP₁-, VIP₂-, secretin- and PACAP receptors, respectively. Furthermore, its also showed an IC₅₀ of 0.8 nM for the human VIP₁ receptor and a low affinity for the human VIP₂ receptor. It is unlikely that this GRF analogue interacted with a high affinity to the pituitary GRF receptors as it did not stimulate rat pituitary adenylate cyclase activity. The two described analogues stimulated maximally the adenylate cyclase activity on membranes expressing each receptor subtype.     

Affinity probes for the GABA-gated chloride channel: 5e-tert-Butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes with photoactivatable, fluorescent, biotin, agarose and protein substituents

Affinity probes for the noncompetitive blocker or picrotoxinin site of the γ-aminobutyric acid (GABA)-gated chloride channel were designed for four types of applications: photoaffinity reagents to covalently label the binding site; fluorescent probes for receptor analysis; biotinylated compounds and agarose/sepharose conjugates for affinity chromatography; ligand-protein/enzyme conjugates for immunoassay. These 5e-tert-butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes were optimized by structure-activity studies for potency as inhibitors of ³H ethynylbicycloorthobenzoate binding to bovine brain membranes, measured as the concentration for 50% inhibition (IC₅₀). Preferred compounds are 5e-(CH₃)₃CCH(CH₂S)₂CH-2e-C₆H₄-4-CCCH₂OCH₂C(O)R, wherein R confers the following properties and 1C₅₀ values: R = SCH₂CH₂SCH₂C(O)C₆H₄-4-N₃, photo-affinity, 9 nM; R = NHCH₂CH₂NHC(O)C₆H₂-2-OH,5-1,4-N₃, photoaffinity, 105 nM; R = SCH₂CH₂S-4-benzofurazan-7-NO₂, fluorescent, 13 nM; R = SCH₂CH₂SCH₂-5-fluorescein, fluorescent, 27 nM; R = NHCH₂CH₂NH[C(O)(CH₂)₅NH]₂-biotin, affinity chromatography, 190 nM. The most potent photoaffinity ligand (IC₅₀ 9 nM) was labeled at 7 Ci mmol⁻¹ by reacting the appropriate thiol with ³H 4-azidophenacyl bromide (obtained by alumina-catalyzed tritium exchange of its enolizable hydrogens). The first steps have been taken in using the NCB site for affinity chromatography of the GABA_A receptor in CHAPS-solubilized bovine brain membranes with the dithiane-biotin probe and an avidin-acrylic bead system or with an analogous dithiane-agarose/sepharose column eluting with GABA or dithiane as above (R = OH). A protein conjugate of a related dithiane-monosulfone elicited production of specific antisera in rabbits. These findings illustrate the diversity and utility of new affinity probes prepared in the alkynylphenyldithiane series.     

Multiple interactions of the Asp(2.61(98)) side chain of the gonadotropin-releasing hormone receptor contribute differentially to ligand interaction

Mutation of Asp(2.61(98)) at the extracellular boundary of transmembrane helix 2 of the gonadotropin-releasing hormone (GnRH) receptor decreased the affinity for GnRH. Using site-directed mutagenesis, ligand modification, and computational modeling, different side chain interactions of Asp(2.61(98)) that contribute to high-affinity binding were investigated. The conservative Asp(2. 61(98))Glu mutation markedly decreased the affinity for a series of GnRH analogues containing the native His(2) residue. This mutant showed smaller decreases in affinity for His(2)-substituted ligands. The loss of preference for His(2)-containing ligands in the mutant receptor shows that Asp(2.61(98)) determines the specificity for His(2). Analysis of the affinities of a series of position 2-substituted ligands suggests that a hydrogen bond forms between Asp(2.61(98)) and the delta NH group of His(2) and that Asp(2. 61(98)) forms a second hydrogen bond with the ligand. Substitution of Asp(2.61(98)) with an uncharged residue further decreased the affinity for all ligands and also decreased receptor expression. Computational modeling indicates an intramolecular ionic interaction of Asp(2.61(98)) with Lys(3.32(121)) in transmembrane helix 3. The uncharged, Lys(3.32(121))Gln mutation also markedly decreased agonist affinity. The modeling and the similar phenotypes of mutants with uncharged substitutions for Asp(2.61(98)) or Lys(3.32(121)) are consistent with the presence of this helix 2-helix 3 interaction. These studies support a dual role for Asp(2.61(98)): formation of an interhelical interaction with Lys(3.32(121)) that contributes to the structure of the agonist binding pocket and an interaction with His(2) of GnRH that helps stabilize agonist complexing.     

Dopamine receptors in human foetal brains: Characterization, regulation and ontogeny of [H]spiperone binding sites in striatum

Eighteen corpora striata from normal human foetal brains ranging in gestational age from 16 to 40 weeks and five from post natal brains ranging from 23 days to 42 years were analysed for the ontogeny of dopamine receptors using [³H]spiperone as the ligand and 10 mM dopamine hydrochloride was used in blanks. Spiperone binding sites were characterized in a 40-week-old foetal brain to be dopamine receptors by the following criteria: (1) It was localized in a crude mitochondrial pellet that included synaptosomes; (2) binding was saturable at 0.8 nM concentration; (3) dopaminergic antagonists spiperone, haloperidol, pimozide, trifluperazine and chlorpromazine competed for the binding with IC₅₀ values in the range of 0.3–14 nM while agonists—apomorphine and dopamine gave IC₅₀ values of 2.5 and 10 μM, respectively suggesting a D₂ type receptor.

Epinephrine and norepinephrine inhibited the binding much less efficiently while mianserin at 10 μM and serotonin at 1 mM concentration did not inhibit the binding. Bimolecular association and dissociation rate constants for the reversible binding were 5.7 × 10⁸ M⁻¹ min⁻¹ and 5.0 × 10⁻² min⁻¹, respectively. Equilibrium dissociation constant was 87 pM and the K_D obtained by saturation binding was 73 pM.

During the foetal age 16 to 40 weeks, the receptor concentration remained in the range of 38–60 fmol/mg protein or 570–1080 fmol/g striatum but it increased two-fold postnatally reaching a maximum at 5 years Significantly, at lower foetal ages (16–24 weeks) the [³H]spiperone binding sites exhibited a heterogeneity with a high (K_D, 13–85 pM) and a low (K_D, 1.2–4.6 nM) affinity component, the former accounting for 13–24% of the total binding sites. This heterogeneity persisted even when sulpiride was used as a displacer. The number of high affinity sites increased from 16 weeks to 24 weeks and after 28 weeks of gestation, all the binding sites showed only a single high affinity.

GTP decreased the agonist affinity as observed by dopamine competition of [³H]spiperone binding in 20-week-old foetal striata and at all subsequent ages. GTP increased IC₅₀ values of dopamine 2 to 4.5 fold and Hill coefficients were also increased becoming closer to one suggesting that the dopamine receptor was susceptible to regulation from foetal life onwards.     

Structure-activity relationships of adenosines with heterocyclic N6-substituents

Two series of N⁶-substituted adenosines with monocyclic and bicyclic N⁶ substituents containing a heteroatom were synthesized in good yields. These derivatives were assessed for their affinity ([³H]CPX), potency, and intrinsic activity (cAMP accumulation) at the A₁ adenosine receptor in DDT₁ MF-2 cells. In the monocyclic series, the N⁶-tetrahydrofuran-3-yl and thiolan-3-yl adenosines (1 and 26, respectively) were found to possess similar activities, whereas the corresponding selenium analogue 27 was found to be more potent. A series of nitrogen containing analogues showed varying properties, N⁶-((3R)-1-benzyloxycarbonylpyrrolidin-3-yl)adenosine (30) was the most potent at the A₁AR; IC₅₀ = 3.2 nM. In the bicyclic series, the effect of a 7-azabicyclo[2.2.1]heptan-2-yl substituent in the N⁶-position was explored. N⁶-(7-Azabicyclo[2.2.1]heptan-2-yl)adenosine (38) proved to be a reasonably potent A₁ agonist (K_i = 51 nM, IC₅₀ = 35 nM) while further substitution on the 7″-nitrogen with tert-butoxycarbonyl (31, IC₅₀ = 2.5 nM) and 2-bromobenzyloxycarbonyl (34, IC₅₀ = 9.0 nM) gave highly potent A₁AR agonists.     

Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

Interaction of amitriptyline with muscarinic receptor subtypes in the rat brain

The affinity of amitriptyline for muscarinic receptors in rat brain areas was studied using autoradiographic techniques including image analysis. As shown by competitive inhibition of [³H]-l-quinuclidinyl benzilate binding, amitriptyline was found to be a potent inhibitor of muscarinic receptors throughout the rat brain. Muscarinic receptors in the external layers of the cortex displayed a high affinity for amitriptyline (IC₅₀ = 65.8 ± 2.1 nM), while the hippocampal regions had somewhat lower affinities (e.g. IC₅₀ = 96.3 ± 3.4 nM). Amitriptyline bound with lower affinity in the thalamus and various midbrain regions, such as the paraventricular nucleus of the thalamus and the superior colliculus, which had IC₅₀ values of 112 ± 6.8 and 117 ± 32.6 nM, respectively. Other midbrain regions displayed higher affinities, for example, the substantia nigra had an IC₅₀ value of 62.8 ± 0.9 nM. The data show that amitriptyline binds with high affinity to muscarinic receptors with a modest subtype selectivity that is unlike that of either pirenzepine or AF-DX 116. In addition, amitriptyline at concentrations of 10 nM-1 μM antagonized the oxotremorine-induced inhibition of acetylcholine release in cortical nerve endings, demonstrating activity at M₂ autoreceptors.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Pharmacological actions of Y-24180, a new specific antagonist of Platelet Activating Factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of ³H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC₅₀ 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC₅₀ values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC₅₀ 0.84 nM) was more potent than WEB 2086 (IC₅₀ 4.21 nM) and etizolam (IC₅₀ 998 nM). Y-24180, WEB 2086 and etizolam displaced ³H-PAF binding from the washed-platelets of rabbits with an IC₅₀ value of 3.50, 9.35 and 29.5 nM, respective- ly. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180(1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced ³H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 μ M. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Stereoselective synthesis of a novel 2-aza-7-oxabicyclo[3.3.0]octane as neurokinin-1 receptor antagonist

A novel neurokinin-1 receptor antagonist, (±)-(1R^*,3S^*,4S^*,5S^*)-4-[(N-(2-methoxy-5-trifluoromethoxybenzyl)amino]-3-phenyl-2-aza-7-oxabicyclo[3.3.0]octane (1), was synthesized stereoselectively using Padwa’s intramolecular 1,3-dipolar cycloaddition methodology as the key step. Compound (±)-1 showed high affinity for the NK-1 receptors in human IM-9 cells with an IC₅₀ value of 0.22 nM. This new structural scaffold demonstrated significant in vivo antagonistic activity in the guinea pig ureter capsaicin-induced plasma extravasation model with an ED₅₀ value of 1–10 mg/kg, po.     

Identification of secretin,vasoactive intestinal peptide and glucagon binding sites: from chimaeric receptors to point mutations

We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln(3) side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp(3)]glucagon as well as by glucagon.     

In Vitro Properties of a High Affinity Selective Antagonist of the VIP1 Receptor

Gourlet, P., P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. In vitro properties of a high affinity selective antagonist of the VIP₁ receptor. Peptides 18(10) 1555–1560, 1997.—A selective high affinity VIP₁ receptor antagonist [Acetyl-His¹, D-Phe², Lys¹⁵, Arg¹⁶, Leu¹⁷] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP₁ receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP₁-, rat VIP₂- or the human VIP₂- recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP₁ receptor. The IC₅₀ values of ¹²⁵I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP₁-, rat VIP₂-, human VIP₁- and human VIP₂ receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP₁ receptor mediated stimulation of adenylate cyclase activity. The K_i values were respectively of 15 ± 5 nM and 2 ± 1 nM for the rat and human VIP₁ receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP₁ receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.     

5,7-Diphenyl-3-ureidohexahydroazepin-2-ones as Cholecystokinin-B receptor ligands

A series of 5,7-diphenyl-3-ureidohexahydroazepin-2-one cholecystokinin-B (CCK-B) receptor antagonists was synthesized using Beckmann ring expansion of a suitable 2,4-diphenylcyclohexanone as a key step. SAR studies revealed the importance of the 5-aryl group for high and selective CCK-B receptor affinity, as illustrated in compound (−)-10i (CCK-B IC₅₀ = 6.8 nM).     

[H]lysergic acid diethylamide (LSD): differential agonist and antagonist binding properties at 5-HT receptor subtypes in rat brain

[³H]Lysergic acid diethylamide (LSD) in the presence of 40 nM ketanserin labeled the 5-HT_1A receptor subtype in rat hippocampal membranes. In the presence of guanosine triphosphate (GTP), the B_max and affinity of [³H]LSD binding to the 5-HT_1A binding site were significantly decreased. [³H]LSD in the presence of 40 nM WB4101 labeled the 5-HT₂ receptor subtype in homogenates of rat frontal cortex. In contrast to the effect on [³H]LSD binding to the 5-HT_1A binding site, GTP produced no significant effect on either the B_max or the K_D of [³H]LSD binding to the 5-HT₂ binding site. Competition of 5-HT for [³H]LSD binding to the 5-HT₂ binding site was best described by a computer-derived model assuming two binding sites. In the presence of GTP, the 5-HT competition curve was shifted significantly to the right with an approx. 3-fold increase in the IC₅₀. These binding characteristics are consistent with [³H]LSD acting as an antagonist at the 5-HT₂ receptor which has multiple affinity states for agonists and is coupled to a guanine nucleotide regulatory subunit. Thus, [³H]LSD has binding characteristics consistent with it acting as an agonist at the 5-HT_1A receptor subtype but as an antagonist at the 5-HT₂ receptor subtype in rat brain.     

Two basic residues of the h-VPAC1 receptor second transmembrane helix are essential for ligand binding and signal transduction

We mutated the vasoactive intestinal peptide (VIP) Asp(3) residue and two VPAC(1) receptor second transmembrane helix basic residues (Arg(188) and Lys(195)). VIP had a lower affinity for R188Q, R188L, K195Q, and K195I VPAC(1) receptors than for VPAC(1) receptors. [Asn(3)] VIP and [Gln(3)] VIP had lower affinities than VIP for VPAC(1) receptors but higher affinities for the mutant receptors; the two basic amino acids facilitated the introduction of the negatively charged aspartate inside the transmembrane domain. The resulting interaction was necessary for receptor activation. 1/[Asn(3)] VIP and [Gln(3)] VIP were partial agonists at VPAC(1) receptors; 2/VIP did not fully activate the K195Q, K195I, R188Q, and R188L VPAC(1) receptors; a VIP analogue ([Arg(16)] VIP) was more efficient than VIP at the four mutated receptors; and [Asn(3)] VIP and [Gln(3)] VIP were more efficient than VIP at the R188Q and R188L VPAC(1) receptors; 3/the [Asp(3)] negative charge did not contribute to the recognition of the VIP(1) antagonist, [AcHis(1),D-Phe(2),Lys(15),Arg(16),Leu(27)] VIP ()/growth hormone releasing factor (8-27). This is the first demonstration that, to activate the VPAC(1) receptor, the Asp(3) side chain of VIP must penetrate within the transmembrane domain, in close proximity to two highly conserved basic amino acids from transmembrane 2.     

Mapping structural determinants within third intracellular loop that direct signaling specificity of type 1 corticotropin-releasing hormone receptor

The type 1 corticotropin-releasing hormone receptor (CRH-R1) influences biological responses important for adaptation to stressful stimuli, through activation of multiple downstream effectors. The structural motifs within CRH-R1 that mediate G protein activation and signaling selectivity are unknown. The aim of this study was to gain insights about important structural determinants within the third intracellular loop (IC3) of the human CRH-R1α important for cAMP and ERK1/2 pathways activation and selectivity. We investigated the role of the juxtamembrane regions of IC3 by mutating amino acid cassettes or specific residues to alanine. Although simultaneous tandem alanine mutations of both juxtamembrane regions Arg(292)-Met(295) and Lys(311)-Lys(314) reduced ligand binding and impaired signaling, all other mutant receptors retained high affinity binding, indistinguishable from wild-type receptor. Agonist-activated receptors with tandem mutations at the proximal or distal terminal segments enhanced activation of adenylyl cyclase by 50-75% and diminished activation of inositol trisphosphate and ERK1/2 by 60-80%. Single Ala mutations identified Arg(292), Lys(297), Arg(310), Lys(311), and Lys(314) as important residues for the enhanced activation of adenylyl cyclase, partly due to reduced inhibition of adenylyl cyclase activity by pertussis toxin-sensitive G proteins. In contrast, mutation of Arg(299) reduced receptor signaling activity and cAMP response. Basic as well as aliphatic amino acids within both juxtamembrane regions were identified as important for ERK1/2 phosphorylation through activation of pertussis toxin-sensitive G proteins as well as G(q) proteins. These data uncovered unexpected roles for key amino acids within the highly conserved hydrophobic N- and C-terminal microdomains of IC3 in the coordination of CRH-R1 signaling activity.     

Substituted piperidin-2-one biphenyltetrazoles as angiotensin II antagonists

A novel series of substituted piperidine-2-ones has been identified as antagonists of angiotensin II. These compounds showed high affinity for the receptor in bovine adrenal cortex binding assays with IC₅₀'s as low as 20nM. They are potent inhibitors of angiotensin II induced contractions in rabbit aortic rings, with pA₂ values as high as 9. A number of these compounds are also orally active as antihypertensives in spontaneously hypertensive rat preparations.     

Protease-activated receptor-2 (PAR-2): structure-function study of receptor activation by diverse peptides related to tethered-ligand epitopes

Protease-activated receptor-2 (PAR-2) is a tethered-ligand, G-protein-coupled receptor that is activated by proteolytic cleavage or by small peptides derived from its cleaved N-terminal sequence, such as SLIGRL-NH2. To assess specific PAR activity, we developed an immortalized murine PAR-1 (-/-) cell line transfected with either human PAR-2 or PAR-1. A "directed" library of more than 100 PAR agonist peptide analogues was synthesized and evaluated for PAR-2 and PAR-1 activity to establish an in-depth structure-function profile for specific action on PAR-2. The most potent agonist peptides (EC50 = 2-4 microM) had Lys at position 6, Ala at position 4, and pFPhe at position 2; however, these also exhibited potent PAR-1 activity (EC50 = 0.05-0.35 microM). We identified SLIARK-NH2 and SL-Cha-ARL-NH2 as relatively potent, highly selective PAR-2 agonists with EC50 values of 4 microM. Position 1 did not tolerate basic, acidic, or large hydrophobic amino acids. N-Terminal capping by acetyl eliminated PAR-2 activity, although removal of the amino group reduced potency by just 4-fold. At position 2, substitution of Leu by Cha or Phe gave equivalent PAR-2 potency, but this modification also activated PAR-1, whereas Ala, Asp, Lys, or Gln abolished PAR-2 activity; at position 3, Ile and Cha were optimal, although various amino acids were tolerated; at position 4, Ala or Cha increased PAR-2 potency 2-fold, although Cha introduced PAR-1 activity; at position 5, Arg or Lys could be replaced successfully by large hydrophobic amino acids. These results with hexapeptide C-terminal amides that mimic the native PAR-2 ligand indicate structural modes for obtaining optimal PAR-2 activity, which could be useful for the design of PAR-2 antagonists.     

Mutagenesis of a Single Amino Acid in the Rat μ-Opioid Receptor Discriminates Ligand Binding

Abstract: To investigate the role of Asp¹¹⁴ in the cloned rat μ-opioid receptor for ligand binding, the charged amino acid was mutated to an asparagine to generate the mutant μ receptor D114N. The wild-type μ receptor and the D114N mutant were then stably expressed in human embryonic kidney 293 cells, and the binding affinities of a series of opioids were investigated. The μ-selective agonists [ d -Ala²,MePhe⁴,Gly-ol⁵]enkephalin and morphine and the endogenous peptides Met-enkephalin and β-endorphin exhibited greatly reduced affinities for the D114N mutant compared with the wild-type μ receptor, as did the potent synthetic agonist etorphine. In contrast to the full agonists, the partial agonists buprenorphine and nalorphine and the antagonists diprenorphine and naloxone bound with similar affinities to the wild-type and D114N mutant μ receptors. The reduced affinities of the full agonists for the D114N mutant did not involve an uncoupling of the receptor from G proteins because methadone and etorphine stimulated the D114N μ receptors to inhibit adenylyl cyclase. Although the Asp¹¹⁴ to Asn¹¹⁴ mutation reduced full-agonist binding, mutation of His²⁹⁷ to Asn²⁹⁷ in the μ receptor did not but, in contrast, did reduce binding affinity of the partial agonist buprenorphine and the antagonist diprenorphine. These results indicate that some partial agonists and antagonists may have different determinants for binding to the μ receptor than do the prototypical full agonists.