Direct demonstration of impaired functionality of a purified desensitized beta-adrenergic receptor in a reconstituted system.

Long-term exposure of various cell types to beta-adrenergic agonists such as isoproterenol leads to an attenuated responsiveness ("desensitization") of the adenylate cyclase system to further challenge with these agonists. The turkey erythrocyte model system was used earlier to show that a covalent modification of the receptor (phosphorylation) is associated with this process. The functionality of the "desensitized" beta-adrenergic receptor was assessed by implanting purified beta-adrenergic receptor preparations from control and desensitized turkey erythrocytes into phospholipid mixtures and then fusing them with receptor-deficient cells (Xenopus laevis erythrocytes). Desensitized beta-adrenergic receptors showed a 40 to 50 percent reduction in their ability to couple to the heterologous adenylate cyclase system, comparable to the reduction in their functionality observed in their original membrane environment. These results demonstrate the utility of recently developed receptor reconstitution techniques for assessing the functionality of purified receptors and show a direct link between a covalent modification of a membrane-bound receptor and its impaired functionality in a reconstituted system.

Endogenous proteinases modulate the function of the beta-adrenergic receptor-adenylate cyclase system.

Photoaffinity labeling techniques have recently demonstrated that mammalian beta 1- and beta 2-adrenergic receptors reside on peptides of Mr 62 000-64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of Mr 30 000-55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the beta 2-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of Mr 40 000-55 000 are fully functional with respect to their ability to bind beta-adrenergic antagonist radioligands such as [3H]dihydroalprenolol and beta-adrenergic antagonist photoaffinity reagents such as p-azido-m-[125I]iodobenzylcarazolol. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc- variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of beta-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.

Guanine nucleotides regulate both dopaminergic agonist and antagonist binding in porcine anterior pituitary.

Radiolabelled agonist and antagonist binding to porcine anterior pituitary dopaminergic receptor indicates the existence of two affinity forms of the receptor. These two forms of the receptor are modulated by guanine nucleotides. The agonist high affinity form of the receptor is converted to a low affinity form, whereas the antagonist low affinity form is converted to the high affinity form under the influence of guanine nucleotides. Thus, guanine nucleotides appear to reciprocally modulate the same two forms of the receptor discriminated by both agonists and antagonists. These observations may provide insights into the biochemical mechanism by which dopamine exerts its regulation of prolactin release in the anterior pituitary.

A novel radioiodinated high affinity ligand for the D2-dopamine receptor. Characterization of its binding in bovine anterior pituitary membranes.

A novel high affinity dopaminergic ligand, N-(p-aminophenethyl)spiroperidol, has been synthesized and radioiodinated to a specific radioactivity of 2175 Ci/mmol. Binding of this ligand to bovine anterior pituitary membranes is: rapid (40-60 min to equilibrium at 25 degrees C) and reversible (t1/2 = 1 h at 25 degrees C); saturable and of high affinity (KD approximately 20 pM) and displays a typical D2-dopaminergic specificity. The ligand, which identifies the same number of receptor sites as other tritiated antagonist ligands, can be used in different tissues and preparations to delineate the characteristics of the D2 receptor. Thus, this high affinity, high specific radioactivity ligand (N-(p-amino-m-[125I]iodophenethyl)spiroperidol) represents a tool which until now had not been available for the characterization of the D2-dopamine receptor.

2-Phenyl-3H-imidazo[4,5-b]pyridine-3-acetamides as non-benzodiazepine anticonvulsants and anxiolytics.

A series of 2-phenyl-3H-imidazo[4,5-b]pyridine-3-acetamides were designed and synthesized as non-benzodiazepine anxiolytics based on a molecular disconnection of a typical 1,4-benzodiazepine (BZD). A number of these compounds showed submicromolar potency in a [3H]benzodiazepine binding assay in vitro and good potency in protecting rodents against pentylenetetrazole-induced seizures. Compound 84 appears to be a selective anticonvulsant (pentylenetetrazole) agent when tested against a profile of chemically and electrically induced seizures in mice. In addition, compound 148 appears to be a selective anxiolytic/hypnotic agent on the basis of biochemical and pharmacological characterization. It appears to be a full BZD agonist as assessed by GABA shift ratio and to be effective in punishment and nonpunishment animal models of anxiety. In addition, it shows a lower side-effect profile than diazepam as assessed by rotorod neurotoxicity and potentiation of ethanol-induced sleep time in mice. The chemistry and structure-activity relationships of this series is discussed.

Optical isomers of rocastine and close analogues: synthesis and H1 antihistaminic activity of its enantiomers and their structural relationship to the classical antihistamines.

The enantiomers of 2-[2-(dimethylamino)ethyl]-3,4-dihydro-4-methylpyrido[3,2-f]-1,4- oxazapine-5(2H)-thione (rocastine) and two of its more potent analogues were prepared with an enantiomeric purity of greater than 99.9%. The antihistaminic activity of these compounds was assessed by their ability to block histamine-induced lethality in guinea pigs and to inhibit [3H]mepyramine binding to guinea pig cortex. In this series, compounds having the R configuration at the 2-position are at least 300 times more potent than the S isomers. Conformational analysis and molecular modeling suggest that rocastine can adopt a conformation in which the pyridine ring, ether oxygen, and protonated amine functions are positioned similarly to the corresponding elements of the probable binding conformers of some of the more classical antihistamines. This conformation, boatlike in the oxazepine ring with the side chain quasi-equatorial and folded back toward the ring, is the likely binding conformer at the histamine H1 receptor, and the available structure-activity relationship data is consistent with this interpretation.

Structure-activity relationship studies on 1-[2-(4-Phenylphenoxy)ethyl]pyrrolidine (SC-22716), a potent inhibitor of leukotriene A(4) (LTA(4)) hydrolase.

Leukotriene B(4) (LTB(4)) is a pro-inflammatory mediator that has been implicated in the pathogenesis of a number of diseases including inflammatory bowel disease (IBD) and psoriasis. Since the action of LTA(4) hydrolase is the rate-limiting step for LTB(4) production, this enzyme represents an attractive pharmacological target for the suppression of LTB(4) production. From an in-house screening program, SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) was identified as a potent inhibitor of LTA(4) hydrolase. Structure-activity relationship (SAR) studies around this structural class resulted in the identification of a number of novel, potent inhibitors of LTA(4) hydrolase, several of which demonstrated good oral activity in a mouse ex vivo whole blood assay.

Dopamine receptor of the porcine anterior pituitary gland. Solubilization and characterization.

In the anterior pituitary gland, dopamine controls the release of prolactin from the mammotrophs. The dopamine receptors in the porcine gland have been shown to exist in two different affinity states of equal proportion, one bearing high affinity for agonists and labeled by 3H-agonist-ligands and the other displaying low affinity for agonists. Both forms of the receptor can be labeled by 3H-antagonist-ligands. Dopamine receptors from porcine anterior pituitary membranes can be solubilized with retention of their ability to interact with specific dopaminergic ligands. Treatment of membrane preparations with 1% digitonin resulted in the solubilization of 20-25% of the specific binding sites labeled by [3H] spiroperidol with a specific activity of about 100 fmoles/mg. The receptor was a glycoprotein as assessed by the interaction of these binding sites with agarose-immobilized lectin. [3H]Spiroperidol binding in solubilized preparations was saturable, of high affinity (KD = 570 pM), and to a single class of stereoselective binding sites. Agonist competition for [3H]spiroperidol binding indicated that, whereas the solubilized receptor retained its dopaminergic specificity, the high-affinity interactions of the receptor with agonists present in membranes and sensitive to guanine nucleotides were lost in solubilized preparations. Thus, the KD values calculated from the agonist competition curves for [3H]spiroperidol corresponded to the agonist affinities for the low-affinity state of the receptor documented in membranes. However, high-affinity agonist binding and its sensitivity to guanine nucleotides were preserved when the membrane-bound receptor was prelabeled with the agonist [3H]N-n-propylnorapomorphine prior to solubilization. These results suggest that a component that confers agonist high-affinity binding and guanine nucleotide responsiveness to the receptor is lost during solubilization unless a stable complex is formed with the agonist prelabeled receptor prior to solubilization.

Dopaminergic inhibition of adenylate cyclase correlates with high affinity agonist binding to anterior pituitary D2 dopamine receptors.

Dopamine (DA) and the dopaminergic agonists n-propylnorapomorphine (NPA), 2-amino-6,7-dihydroxytetrahydronaphthalene (ADTN) and apomorphine (APO) inhibit forskolin-stimulated adenylate cyclase activity in a dose-dependent fashion by more than 40% in membrane preparations of the porcine anterior pituitary gland. These agonists exhibit apparent dissociation constants that follow an expected dopaminergic order of potency (NPA greater than ADTN greater than or equal to APO greater than DA). The inhibition is dependent on guanine nucleotides and is reversible by dopaminergic antagonists (spiroperidol greater than (+)-butaclamol much greater than (-)-butaclamol). The potencies of these agonists in inhibiting forskolin-stimulated adenylate cyclase activity correlate with the agonist dissociation constants (KH) for binding to the high affinity receptor state (RH) in porcine anterior pituitary membranes (De Lean et al., Mol. Pharmacol. 1982, 22, 290-297) and the EC50 for inhibition of prolactin release from rat anterior pituitary cells in culture (Caron et al., J. Biol. Chem. 1978, 253, 2244-2253). Furthermore, the intrinsic activities of dopamine and the other agonists for inhibition of forskolin-stimulated adenylate cyclase are similar and correlate well with the ability of these agents to induce a comparable proportion (50%) of the receptor in a high affinity state. Together these data provide additional support for the physiological relevance of the high affinity agonist binding state of the D2 receptor in mediating the decrease in prolactin secretion via attenuation of adenylate cyclase.

AHR-12245: a potential anti-absence drug.

AHR-12245, 2-(4-chlorophenyl)-3H-imadazo[4,5-b]pyridine-3-acetamid, ethosuximide, Na valproate, phenytoin, and clonazepam were evaluated in mice and rats with a battery of well-standardized anticonvulsant test procedures. The results obtained indicate that the anticonvulsant profile of AHR-12245 is similar to that for ethosuximide and clonazepam. AHR-12245 is effective in nontoxic intraperitoneal doses in mice by the maximal electroshock seizure (MES), pentylenetatrazol (s.c. PTZ), bicuculline, and picrotoxin tests but ineffective against strychnine-induced seizures; it is effective after nontoxic oral doses in both mice and rats by the s.c. PTZ test and ineffective by the MES test. The candidate antiepileptic substance was also ineffective against seizures induced in amygdala and corneally kindled rats. The PIs for AHR-12245 by the s.c. PTZ test were 4.5 to 12 times higher than those for the prototype agents, except that for clonazepam when administered orally in mice. The in vitro studies indicate that AHR-12245 is a weak inhibitor of benzodiazepine (BDZ) receptor binding but does inhibit adenosine uptake. These results indicate that AHR-12245 is a relatively nontoxic agent with a profile of anticonvulsant action which suggests it should be useful in generalized absence seizures.

Agonist binding promotes a guanine nucleotide reversible increase in the apparent size of the bovine anterior pituitary dopamine receptors.

Dopamine receptors, solubilized from bovine anterior pituitary membranes with the detergent digitonin, retained a typical dopaminergic specificity for the binding of both agonists and antagonists. The affinities of antagonists for binding to the soluble receptors are virtually identical with those observed with the membrane-bound receptors. The affinities of agonists however, correspond to those for the form of the receptors in the membranes having low affinity for those agonists (De Lean, A., Kilpatrick, B. F., and Caron, M. G. (1982) Mol. Pharmacol. 22, 290-297). Thus, after solubilization, agonist high affinity interactions with the receptor and their sensitivity to modulation by guanine nucleotides are lost. However, high affinity agonist binding and its sensitivity to guanine nucleotides can be preserved if the membrane-bound receptors are prelabeled with the agonist [3H]n-propylapomorphine prior to solubilization. In order to investigate the molecular basis for these changes in the properties of agonist binding, the solubilized receptors were characterized by chromatographic procedures. Using molecular exclusion high pressure liquid chromatography, [3H]n-propylapomorphine-prelabeled receptors elute as an apparent larger molecular species than either unlabeled or antagonist [( 3H]spiroperidol)-pre-labeled receptors. Moreover, incubation of the pooled agonist-prelabeled receptor peak with guanine nucleotides effects a decrease in the apparent size of the receptors such that upon rechromatography they elute in a position coincidental with the 3H-antagonist-pre-labeled receptor peak. Thus, occupancy of the receptors by agonists promotes the formation of a guanine nucleotide-sensitive agonist high affinity form of the receptor which is of larger apparent size presumably due to the association of the receptor with a guanine nucleotide regulatory protein.

A high-throughput assay for cyclic nucleotide phosphodiesterases.

The search for cyclic nucleotide phosphodiesterase inhibitors in large chemical and natural product libraries is limited by assay throughput. A high-throughput assay that can monitor different phosphodiesterase activities would be useful for these inhibitor searches. We have developed a sensitive phosphodiesterase assay which is based on the selective precipitation of the radiolabeled 5'-nucleotide product by zinc and barium. This assay is unique to previous precipitation methods in that it measures product formed, either GMP or AMP. The procedure was adapted to a microtiter plate format for high-throughput, making it possible to perform in excess of 960 assays per day. This method was used to determine accurate enzyme kinetic constants for three different phosphodiesterases. The results agree favorably with literature values. Using the cyclic guanosine 3',5'-monophosphate-specific phosphodiesterase, IC50 values for the inhibitors M&B 22,948 and 3-isobutyl-1-methylxanthine were also determined. Demonstration of appropriate Km and inhibitor IC50 values verifies the ability of this new assay to identify selective cyclic nucleotide phosphodiesterase inhibitors.

Use of procainamide gels in the purification of human and horse serum cholinesterases.

Two large-scale methods based primarily on the use of procainamide-Sepharose gels were developed for the purification of horse and human serum non-specific cholinesterases. With method I, the procainamide-Sepharose 4B gel was used in the first step to handle large volumes of serum. With method II, the procainamide-Sepharose 4B gel was used in the final step to obtain pure enzyme. Although both methods gave electrophoretically pure cholinesterase preparations in good yields, they were significantly more efficient at purifying the horse enzyme than the human enzyme. To study this problem, the relative binding of human and horse cholinesterases to procainamide-, methylacridinium (MAC)-, m-trimethylammoniophenyl (m-PTA)- and p-trimethylammoniophenyl (p-PTA)-Sepharose 4B gels were measured, by using two approaches. In one, binding was measured by a procedure involving equilibration of pure cholinesterase in a small volume of diluted gel slurry (4%, v/v). A partially purified preparation of Electrophorus acetylcholinesterase was included. Pure human cholinesterase bound consistently more tightly to each of the gels than did horse cholinesterase, and the acetylcholinesterase appeared to bind the gels 10-100 times more tightly than did the non-specific cholinesterases. The order of binding for the cholinesterases, beginning with the tightest, was: procainamide-Sepharose 4B, MAC-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. For the acetylcholinesterase the order was: MAC-Sepharose 4B, procainamide-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. The second approach involved passing native sera or partially purified sera fractions through 1 ml test columns of each of the four affinity gels to determine their retention capacity for the cholinesterases. With these impure samples, the MAC-Sepharose 4B gels proved superior to the procainamide-Sepharose 4B gels at retaining human cholinesterase, but the opposite was true for the horse cholinesterase.

Inhibition of two monomeric butyrylcholinesterases from rabbit liver by chlorpromazine and other drugs.

A new form of cholinesterases has been discovered in rabbit liver; the new enzymes are monomeric butyrylcholinesterases (EC 3.1.1.8), mBuChE I and mBuChE II. These enzymes are inhibited reversibly by chlorpromazine in the pharmacologically active concentration range and they exhibit mixed competitive-noncompetitive inhibition patterns. The apparent competitive inhibition constants, Ki with chlorpromazine, are 1.8 x 10(-6) M for mBuChE I and 7.6 x 10(-6) M for mBuChE II, whereas the noncompetitive inhibition constant is 1.1 x 10(-5) M for mBuChE II as determined by spectrophotometric assay with n-butyrylthiocholine iodide substrate. Although inhibition of mBuChE I also exhibited noncompetitive behavior, a binding constant could not be determined. Human serum oligometric butyrylcholinesterase (oBuChE) was employed as a control cholinesterase and also demonstrated mixed inhibition kinetics. The competitive inhibition constant for the oBuChE was 5.5 x 10(-7) M in the low substrate region, whereas the apparent noncompetitive binding constant was 1.6 x 10(-5) M in the activated higher substrate region with n-butyrylthiocholine iodide as the substrate and chlorpromazine as the reversible inhibitor. The presence of a noncompetitive binding component indicates the presence of an operative modifier or allosteric site binding the inhibitor on both mBuChEs and the oBuChE. The inhibition constants were calculated assuming that the enzymes followed simple Michaelian kinetics.

AHR-16303B, a novel antagonist of 5-HT2 receptors and voltage-sensitive calcium channels.

In vivo and in vitro methods were used to characterize AHR-16303B, a novel compound with antagonistic action at 5-HT2 receptors and voltage-sensitive calcium channels. The 5-HT2 receptor-antagonistic properties of AHR-16303B were demonstrated by inhibition of (a) [3H]ketanserin binding to rat cerebral cortical membranes (IC50 = 165 nM); (b) 5-hydroxytryptamine (5-HT)-induced foot edema in rats (minimum effective dose, (MED) = 0.32 mg/kg orally, p.o.); (c) 5-HT-induced vasopressor responses in spontaneously hypertensive rats (SHR) (ID50 = 0.18 mg/kg intravenously (i.v.), 1.8 mg/kg p.o.), (d) 5-HT-induced antidiuresis in rats (MED = 1 mg/kg p.o.), and (e) platelet aggregation induced by 5-HT + ADP (IC50 = 1.5 mM). The calcium antagonist properties of AHR-16303B were demonstrated by inhibition of (a) [3H]nimodipine binding to voltage-sensitive calcium channels on rabbit skeletal muscle membranes (IC50 = 15 nM), (b) KCl-stimulated calcium flux into cultured PC12 cells (IC50 = 81 nM), and (c) CaCl2-induced contractions of rabbit thoracic aortic strips (pA2 = 8.84). AHR-16303B had little or no effect on binding of radioligands to dopamine2 (DA2) alpha 1, alpha 2, H1, 5-HT1 alpha, beta 2, muscarinic M1, or sigma opioid receptors; had no effect on 5-HT3 receptor-mediated vagal bradycardia; and had only minor negative inotropic, chronotropic, and dromotropic effects on isolated guinea pig atria. In conscious SHR, 30 mg/kg p.o. AHR-16303B completely prevented the vasopressor responses to i.v. 5-HT, and decreased blood pressure (BP) by 24% 3 h after dosing.

Four new clerodane diterpenes from the leaves of Casearia guianensis which inhibit the interaction of leukocyte function antigen 1 with intercellular adhesion molecule 1.

Four new clerodane diterpenes, casearinols A and B (1 and 2) and casearinones A and B (3 and 4), were isolated from the leaves of Casearia guianensis. These immunomodulatory compounds have been structurally elucidated primarily on the basis of 2D NMR analysis and spectral data comparison with known compounds. These compounds inhibited the binding of T-cell leukocyte function antigen 1 to intercellular adhesion molecule 1.

AHR-14310C: a potent, long-acting, nonsedating H1-antihistamine that prevents antigen-induced mucus formation in sensitive rats.

AHR-14310C(5-[2-[4-[bis(4-fluorophenyl)hydroxymethyl- 1-piperidinyl]ethyl]-3-methyl]-2-oxazolidinone ethanedioate, hydrochloride salt) displays potent and long-acting antihistaminic activity in guinea pigs and in dog and guinea pig models of immediate hypersensitivity. Given orally 1, 5 or 24 hr before an i.v. histamine challenge, AHR-14310C produced ED50 values of 0.76, 0.22 and 0.58 mg/kg, respectively, in protecting naive guinea pigs from the lethal effects of the histamine challenge. AHR-14310C was also effective when the histamine was administered as an aerosol (1-, 5- and 24-hr ED50 values = 0.69, 0.38 and 1.08 mg/kg, respectively). AHR-14310C also attenuated the anaphylactic responses to aerosolized antigen in sensitive guinea pigs and the skin response to antigen in naturally sensitive dogs. AHR-14310C, at doses in vastly excess of those required to block histamine-induced hypotension, did not alter the electroencephalogram of cats, as did the sedating antihistamine, diphenhydramine. AHR-14310C did not affect the autonomic responses to acetylcholine, isoproterenol, epinephrine or 1,1-dimethyl-4-phenylpiperazinium chloride in dogs. At low doses (0.316-1.0 mg/kg p.o.), AHR-14310C blocked antigen-induced tracheal mucous changes in sensitive rats. AHR-14310C has therapeutic potential in allergic individuals, particularly in asthmatics, where bronchorrhea or mucus plugging is a problem.

Isolation and structure of leukotriene-A4 hydrolase inhibitor: 8(S)-amino-2(R)-methyl-7-oxononanoic acid produced by Streptomyces diastaticus.

The novel amino acid 8(S)-amino-2(R)-methyl-7-oxononanoic acid (1) was isolated from the soil-borne microorganism Streptomyces diastaticus during our screening for inhibitors of leukotriene-A4 hydrolase (LTA4H), a requisite enzyme in the biosynthesis of the potent inflammatory mediator leukotriene-B4 (LTB4). The structure of 1 was determined by detailed spectroscopic analyses and is related to 7-keto-8-aminopelargonic acid (2), a biosynthetic precursor of biotin. The relative potency of 1 (LTA4H IC50 = 0.6 microM) warranted further biological studies.

Rocastine (AHR-11325), a rapid acting, nonsedating antihistamine.

Rocastine [AHR-11325, 2-[2-(dimethylamino)ethyl]-2,3-dihydro-4-methylpyrido-[3,2-f]-1,4- oxazepine-5(4H)-thione (E)-2-butenedioate)] is a rapid-acting, potent, nonsedating antihistamine. In guinea pigs challenged with a lethal dose of histamine, rocastine is as effective [based on 1 hr. oral, protective dose (PD50S)] as brompheniramine, chlorpheniramine, pyrilamine, and promethazine and superior to astemizole, diphenhydramine, terfenadine, and oxatomide. Rocastine has a faster onset of action than does terfenadine; rocastine being as effective with a 15 min pretreatment time (PD50 = 0.13 mg/kg) as it is with a 1 hr pretreatment time (PD50 = 0.12 mg/kg), while the 15 min PD50 of terfenadine (PD50 = 44.0 mg/kg) is 22 times greater than the 1 hr PD50 (PD50 = 1.93 mg/kg). Against aerosolized histamine, rocastine was 7.12 x, 2.63 x, and equipotent to pyrilamine in preventing histamine-induced prostration at pretreatment times of 1,3, and 6 hr, respectively. Rocastine protected guinea pigs from collapse induced by aerosolized antigen; rocastine was approximately 36 x more potent (based on 1 hr PD50) than diphenhydramine and as potent as oxatomide and terfenadine. Rocastine did not alter the EEG of cats at doses in vast excess (150x) of its antihistaminic dose nor did it potentiate yohimbine toxicity in mice. Further, rocastine possesses no anticholinergic, antiadrenergic, or antiserotonergic properties in vitro. Rocastine is a selective, nonsedating, H1-antagonist with a rapid onset of action.