RESUMEN

Selective muscarinic agonists could be useful in the treatment of neurological disorders such as Alzheimer's disease, schizophrenia, and chronic pain. Many muscarinic agonists have been developed, yet most exhibit at best limited functional selectivity for a given receptor subtype perhaps because of the high degree of sequence homology within the putative binding site, which appears to be buried within the transmembrane domains. Bivalent compounds containing essentially two agonist pharmacophores within the same molecule were synthesized and tested for receptor binding affinity and muscarinic agonist activity. A series of bis-1,2,5-thiadiazole derivatives of 1,2,5,6-tetrahydropyridine linked by an alkyloxy moiety exhibited very high affinity (K(i) < 1 nM) and strong agonist activity. The degree of activity depended on the length of the linking alkyl group, which could be replaced by a poly(ethylene glycol) moiety, resulting in improved water solubility, binding affinity, and agonist potency.

Asunto(s)

Agonistas Muscarínicos/síntesis química , Piridinas/síntesis química , Tiadiazoles/síntesis química , Unión Competitiva , Línea Celular , Diseño de Fármacos , Humanos , Ligandos , Modelos Moleculares , Agonistas Muscarínicos/química , Agonistas Muscarínicos/farmacología , Fosfatidilinositoles/metabolismo , Estructura Terciaria de Proteína , Piridinas/química , Piridinas/farmacología , Ensayo de Unión Radioligante , Receptor Muscarínico M1 , Receptor Muscarínico M3 , Receptor Muscarínico M5 , Receptores Muscarínicos/metabolismo , Solubilidad , Relación Estructura-Actividad , Tiadiazoles/química , Tiadiazoles/farmacología , Transfección

RESUMEN

Cholinergic neurons degenerate in Alzheimer's disease, resulting in cognitive impairments and memory deficits, and drug development efforts have focused on selective M1 muscarinic agonists. 5-(3-Ethyl-1,2,4- oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine trifluoroacetic acid (CDD-0102) stimulates M1 muscarinic receptors in rat brain [Messer, W.S., Jr., Abuh, Y.F., Liu, Y., Periyasamy, S., Ngur, D.O., Edgar, M.A., El-Assadi, A.A., Sbeih, S., Dunbar, P.G., Roknich, S., Rho, T., Fang, Z., Ojo, B., Zhang, H., Huzl, J.J., III, Nagy, P.I., 1997a. J. Med. Chem. 40, 1230-1246.] and improves memory function in rats with lesions of the basal forebrain cholinergic system. Moreover, CDD-0102 exhibits oral bioavailability, few side effects and low toxicity, and thus represents a viable candidate for clinical studies. Despite the development of functionally selective agonists such as xanomeline and CDD-0102, there is room for improvements in ligand affinity and selectivity. The high degree of amino acid homology within transmembrane domains has hindered the development of truly selective agonists. Site-directed mutagenesis, biochemical and molecular modeling studies have identified key amino acid residues such as Thr192 and Asn382 in the binding of agonist to M1 receptors [Huang, X.P., Nagy, P.I., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1999. Br. J. Pharmacol. 126, 735-745.]. Recent work has implicated residues at the top of transmembrane domain VI in the binding of muscarinic agonists and activation of M1 receptors [Huang, X.P., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1998. J. Pharmacol. Exp. Ther. 286, 1129-1139.]. Thus, residues such as Ser388 represent molecular targets for the further development of agonists with improved M1 receptor affinity, selectivity and activity.

Asunto(s)

Enfermedad de Alzheimer/tratamiento farmacológico , Agonistas Muscarínicos/síntesis química , Piridinas/síntesis química , Receptores Muscarínicos/efectos de los fármacos , Tiadiazoles/síntesis química , Enfermedad de Alzheimer/genética , Animales , Diseño de Fármacos , Inyecciones Intraperitoneales , Ligandos , Masculino , Modelos Moleculares , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/uso terapéutico , Mutagénesis Sitio-Dirigida , Piridinas/farmacología , Piridinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M1 , Receptores Muscarínicos/química , Receptores Muscarínicos/genética , Tiadiazoles/farmacología , Tiadiazoles/uso terapéutico

RESUMEN

Fatty acylated dipeptides homologous to Gi alpha N-termini affect ligand binding to muscarinic acetylcholine receptors. Myristylglycine-serine containing dipeptides decrease antagonist binding at both M1 and M2 muscarinic receptors. Palmitate on the serine analogous to native palmitoylated cysteine affords dipeptide which selectively decreases the number of high affinity agonist binding sites at M2 but not M1 receptor.

Asunto(s)

Dipéptidos/farmacología , Ácidos Grasos/química , Antagonistas Muscarínicos/farmacología , Receptores Muscarínicos/metabolismo , Acilación , Animales , Sitios de Unión , Células CHO , Cricetinae , Dipéptidos/química , Ligandos , Antagonistas Muscarínicos/química

RESUMEN

Transmembrane domain VI of muscarinic acetylcholine receptors plays an important role in ligand binding and receptor function. A human M(1) (HM(1)) mutant receptor, HM(1)(S388Y, T389P), displayed significantly enhanced agonist potency, binding affinity, and G protein coupling. The mutations are located at the top of transmembrane domain VI and about two helical turns above Tyr381 and Asn382, which are important for ligand binding and receptor function. To determine the functional role of individual mutations of Ser388Tyr and Thr389Pro, we created stable A9 L cell lines expressing HM(1)(S388Y) or HM(1)(T389P) receptors. In phosphatidylinositol hydrolysis assays, muscarinic agonists showed greater potency at the HM(1)(S388Y) and HM(1)(S388Y, T389P) mutants compared with the wild-type and HM(1)(T389P) receptors. Acetylcholine demonstrated 105-fold higher potency at HM(1)(S388Y) receptors than at HM(1)(T389P) receptors. Choline (30 microM, the concentration found in Dulbecco's modified Eagle's medium) exhibited 90% stimulation at HM(1)(S388Y) receptors but was inactive at HM(1)(T389P) receptors. In ligand binding experiments, mutation of Ser388Tyr resulted in significantly increased agonist binding affinity. In contrast, mutation of Thr389Pro did not change agonist binding affinity but rendered multiple agonist binding sites, and the high-affinity binding was sensitive to GTP analogs. These results demonstrate that the Ser388Tyr mutation is responsible for enhanced agonist potency and binding affinity, whereas the Thr389Pro mutation alters G protein interactions. The data suggest that Ser388 and Thr389 are potential targets for modulation of agonist binding and G protein coupling.

Asunto(s)

Receptores Muscarínicos/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Línea Celular , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Agonistas Muscarínicos/farmacología , Mutación , Prolina/metabolismo , Conformación Proteica , Ensayo de Unión Radioligante , Receptor Muscarínico M1 , Receptores Muscarínicos/química , Receptores Muscarínicos/genética , Serina/metabolismo , Treonina/metabolismo , Tirosina/metabolismo

RESUMEN

Pharyngeal pumping is essential for nematode feeding and survival and is dramatically stimulated by serotonin (5-HT). In the present study, a cDNA pool was prepared from poly A + RNA isolated from pharynxes dissected from the parasitic nematode, Ascaris suum, and was used as a template for RT-PCR with degenerate primers designed from sequences conserved in 5-HT receptors from a variety of sources. A putative 5-HT receptor cDNA (AS1) was identified which exhibited most identity to the 5-HT2 family of receptors. AS1 was 1925 nucleotides, did not appear to be trans-spliced and contained a 3' untranslated region of 127 nucleotides with a polyadenylation signal (ATTAAA) and a short poly A+ tail. The coding region predicted a protein of 532 amino acids with a molecular weight of 60 176. When AS1 was transiently expressed in COS-7 cells, isolated membranes exhibited the high affinity, saturable binding of [125I]LSD. More importantly, [125I]LSD binding was inhibited by 5-HT, but not other biogenic amines, supporting the identification of AS1 as a 5-HT receptor. Additional cDNAs identical, in part, to AS1 were also identified. AS1deltaIV lacked a predicted 42 amino acids at the carboxy terminus of the third intracellular loop, while AS2 and AS3 contained different COOH-termini, regions implicated in G-protein coupling in other heptahelical receptors. A portion of the gene (5htn) encoding AS1 also was cloned and sequenced. This genomic fragment was about 10 kb, contained the entire AS1 open reading frame and included eight exons and seven introns. From this analysis, it appears that these different AS cDNAs were generated by alternative-splicing, AS1deltaIV from the deletion of exon IV, and AS2 and AS3 from the use of alternative sites within exon VII as 5' splice acceptor sites for exon VIII. Using RT-PCR and primers specific for each of the isoforms, AS1 -3 appeared to be expressed in pharynx, while only AS1 and AS2 were present in body wall muscle. More importantly, the deletion of exon IV appeared to be associated exclusively with AS1 in pharynx and AS2 in muscle.

Asunto(s)

Empalme Alternativo , Ascaris suum/genética , Receptores de Serotonina/genética , Secuencia de Aminoácidos , Animales , Ascaris suum/metabolismo , Células COS , Clonación Molecular , ADN Complementario/genética , ADN de Helmintos/genética , Dietilamida del Ácido Lisérgico/metabolismo , Datos de Secuencia Molecular , Músculos/metabolismo , Especificidad de Órganos , Faringe/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo

RESUMEN

Conserved amino acids, such as Thr in transmembrane domains (TM) V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/or receptor activation. In order to determine the functional roles of Thr192 and Asn382 in human M1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala. HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala)] were stably expressed in A9 L cells. The Kd values for 3H-(R)-QNB and Ki values for other classical muscarinic antagonists were similar at HM1(WT) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating PI hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors. The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M1 receptors. Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor.

Asunto(s)

Asparagina/fisiología , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Receptores Muscarínicos/efectos de los fármacos , Treonina/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Asparagina/química , Sitios de Unión , Unión Competitiva , Células CHO , Línea Celular , Cricetinae , Humanos , Modelos Moleculares , Agonistas Muscarínicos/química , Agonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/química , Antagonistas Muscarínicos/metabolismo , Fosfatidilinositoles/metabolismo , Pirenzepina/farmacología , Quinuclidinil Bencilato/metabolismo , Ensayo de Unión Radioligante , Receptor Muscarínico M1 , Receptores Muscarínicos/química , Receptores Muscarínicos/metabolismo , Escopolamina/farmacología , Treonina/química , Tritio

RESUMEN

A mutant human m5 receptor containing the mutations of Ser465 to Tyr and Thr466 to Pro showed constitutive activity. By replacing the equivalent Ser388 with Tyr and Thr389 with Pro, we created a mutant human m1 (Hm1) receptor with comparable double mutations. The mutant receptor, Hm1(Ser388Tyr, Thr389Pro), was stably expressed in A9 L cells and displayed enhanced responses to classical muscarinic agonists with significantly increased potencies. Choline, a normal component of growth media, showed an efficacy comparable to acetylcholine and carbachol at Hm1(Ser388Tyr, Thr389Pro) receptors. Methylcarbachol, a selective nicotinic agonist, exhibited partial agonist activity at human m1 wild-type receptors and full agonist activity at Hm1(Ser388Tyr, Thr389Pro) receptors. l-Hyoscyamine inhibited the activities of choline and methylcarbachol. Muscarinic antagonists displayed small reductions in binding affinities, although muscarinic agonists showed greatly increased binding affinities for Hm1(Ser388Tyr, Thr389Pro) receptors. All agonists, including choline and methylcarbachol, showed multiple affinity states at Hm1(Ser388Tyr, Thr389Pro) receptors in the absence of GppNHp. The high affinity binding sites for acetylcholine, arecoline and choline were shifted in the presence of GppNHp. These results suggest that Hm1(Ser388Tyr, Thr389Pro) is conformationally favorable for agonist binding and receptor activation.

Asunto(s)

Agonistas Muscarínicos/farmacología , Receptores Muscarínicos/química , Secuencia de Aminoácidos , Línea Celular , Humanos , Datos de Secuencia Molecular , Agonistas Muscarínicos/metabolismo , Mutación , Fosfatidilinositoles/metabolismo , Conformación Proteica , Quinuclidinil Bencilato/metabolismo , Receptor Muscarínico M1 , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Relación Estructura-Actividad

RESUMEN

Previous studies identified several novel tetrahydropyrimidine derivatives exhibiting muscarinic agonist activity in rat brain. Such compounds might be useful in treating cognitive and memory deficits associated with low acetylcholine levels, as found in Alzheimer's disease. To determine the molecular features of ligands important for binding and activity at muscarinic receptor subtypes, the series of tetrahydropyrimidines was extended. Several active compounds were examined further for functional selectivity through biochemical studies of muscarinic receptor activity using receptor subtypes expressed in cell lines. Several amidine derivatives displayed high efficacy at m1 receptors and lower activity at m3 receptors coupled to phosphoinositide (PI) metabolism in A9 L cells. Four ligands, including 1b, 1f, 2b, and 7b, exhibited marked functional selectivity for m1 vs m3 receptors. Compound 1f also exhibited low activity at m2 receptors coupled to the inhibition of adenylyl cyclase in A9 L cells. Molecular modeling studies also were initiated to help understand the nature of the interaction of muscarinic agonists with the m1 receptor using a nine amino model of the m1 receptor. Several important interactions were identified, including interactions between the ester moiety and Thr192. Additional interactions were found for oxadiazoles and alkynyl derivatives with Asn382, suggesting that enhanced potency and selectivity may be achieved by maximizing interactions with Asp105, Thr192, and Asn382. Taken together, the data indicate that several amidine derivatives display functional selectivity for m1 muscarinic receptors, warranting further evaluation as therapeutic agents for the treatment of Alzheimer's disease. In addition, several amino acid residues were identified as potential binding sites for m1 agonists. These data may be useful in directing efforts to develop even more selective m1 agonists.

Asunto(s)

Agonistas Muscarínicos/síntesis química , Pirimidinas/química , Receptores Muscarínicos/metabolismo , Animales , Arecolina/farmacología , Encéfalo/metabolismo , Carbacol/farmacología , Línea Celular , Modelos Moleculares , Agonistas Muscarínicos/química , Agonistas Muscarínicos/metabolismo , Fosfatidilinositoles/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Quinuclidinil Bencilato/metabolismo , Ratas , Receptor Muscarínico M1 , Receptor Muscarínico M2 , Receptor Muscarínico M3 , Relación Estructura-Actividad

RESUMEN

As part of a continuing effort aimed at the development of selective, efficacious, and centrally active m1 muscarinic agonists for the treatment of Alzheimer's disease, a series of amide and hydrazide amidine derivatives (2a-e and 3b-d) was synthesized and examined for muscarinic agonist activity. Preliminary biochemical studies indicated that 2b, 2d, and 3d bound to muscarinic receptors in rat brain and stimulated phosphoinositide (PI) metabolism in rat cerebral cortex. Compounds 2b and 2d were also highly efficacious at m1 muscarinic receptors expressed in cultured A9 L cells. Molecular modeling studies suggest slightly different modes of interaction with m1 receptors for the ester and amide derivatives. Also, hydrogen-bond formation with a Thr residue may be important for m1 muscarinic agonist potency. The data suggest that the amide moiety can replace the ester group found in muscarinic agonists and provide further support for the utility of amidine derivatives in the development of efficacious m1 agonists.

Asunto(s)

Amidinas/química , Receptores Muscarínicos/metabolismo , Amidinas/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Cinética , Modelos Moleculares , Quinuclidinil Bencilato/metabolismo , Ratas , Receptor Muscarínico M1

RESUMEN

Cobra venom cytotoxins (CTX) have been shown to disrupt cells as different as immunocytes, skeletal myocytes, erythrocytes and tumor cells. Nevertheless, even subpopulations of tumor cells are differentially susceptible to CTX by an order of magnitude. In the present study, our objective was to compare CTX-specific binding with cytolytic potency for two disparate cell types in vitro. We investigated the lytic activity of cytotoxin-III from Naja naja atra (NNA, fraction D) using heart cells and human leukemic T-cells (CEM cells). For both cell types, 50% cytolysis, assessed by tetrazolium dye conversion, occurred with microM concentrations of toxin (EC50 = 2.2 microM). We examined the binding of radiolabeled CTX III to both heart cells and CEM cells and found the apparent dissociation constant (KDapp) to be 0.69 microM and 0.75 microM, for CEM and heart cells respectively. The Bmax for the CEM cells was 1.0 fmoles/cell and that for heart cells was 5.2 fmoles/cell, both exhibiting positive cooperativity between the sites (Hill coefficients 1.4, T-cells; 1.6, heart). Relatively modest dissociation constants plus high numbers of binding sites per cell are consistent with a model of CTX binding to plasma membranes by interaction with phospholipids in the bilayer. Our results suggest that the lytic activity of this cytotoxin follows its binding to a population of sites on the cells in a cooperative fashion.

Asunto(s)

Citotoxinas/metabolismo , Venenos Elapídicos/metabolismo , Miocardio/metabolismo , Linfocitos T/metabolismo , Animales , Sitios de Unión , Línea Celular , Citotoxinas/toxicidad , Venenos Elapídicos/toxicidad , Corazón/efectos de los fármacos , Humanos , Cinética , Unión Proteica , Linfocitos T/efectos de los fármacos

RESUMEN

At least five subtypes of muscarinic acetylcholine receptors are expressed in various mammalian tissue preparations. The following experiment, through the use of direct binding assays (using tritiated quinuclidinyl benzilate), competitive binding assays (using tritiated quinuclidinyl benzilate and unlabeled pirenzepine or AF-DX 116), and autoradiographic techniques, examined whether two of these five putative muscarinic acetylcholine receptor subtypes can be found in avian brain. Accordingly, autoradiographic mapping of pirenzepine-sensitive (M1-like) and AF-DX 116-sensitive (M2-like) muscarinic acetylcholine receptor subtypes in the pigeon telencephalon was conducted. Although both ligands bound throughout the brain, most telencephalic regions, including the archistriatum, the neostriatum, and basal ganglia structures like lobus paraolfactorius, nucleus accumbens, and paleostriatum, showed a higher density of M1-like sites. The exception to this finding was the nucleus basalis which appeared as a region where M2-like sites predominated. Moreover, the telencephalic region with the largest ratio of M1-like to M2-like sites was the lateral portion of the parahippocampus; a characteristic shared with the mammalian dentate gyrus. The findings reported here are generally consistent with previous reports of mammalian M1/M2 receptor distributions.

Asunto(s)

Receptores Muscarínicos/metabolismo , Telencéfalo/metabolismo , Animales , Autorradiografía , Unión Competitiva , Mapeo Encefálico , Columbidae , Receptores Muscarínicos/clasificación , Telencéfalo/fisiología

RESUMEN

A series of alkoxy-1,2,5-thiadiazole derivatives of arecoline was synthesized in an effort to develop M1 muscarinic agonists. The 3-butenyloxy, 2-butynyloxy, cyclopropylmethyloxy, and hexyloxy derivatives stimulated phosphoinositide turnover through muscarinic receptors in the rat hippocampus. The dose-response curves of 2-butynyloxy, cyclopropylmethyloxy and hexyloxy compound together was the same as the response of each separately. Pirenzepine was somewhat more potent than AF-DX 116 for inhibiting the responses produced by low concentrations of thiadiazole derivatives. The data suggest that the cyclopropylmethyloxy-TZTP derivative is functionally a selective M1 agonist. Molecular mechanics calculations indicate that the anti form of the 1,2,5-thiadiazole derivatives of arecoline may be active at M1 receptors.

Asunto(s)

Arecolina/química , Agonistas Muscarínicos/farmacología , Fosfatidilinositoles/metabolismo , Tiadiazoles/farmacología , Animales , Arecolina/análogos & derivados , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Masculino , Antagonistas Muscarínicos/farmacología , Parasimpatolíticos/farmacología , Pirenzepina/análogos & derivados , Pirenzepina/farmacología , Ratas , Ratas Endogámicas , Receptores Muscarínicos/efectos de los fármacos , Tiadiazoles/química

RESUMEN

Inhibition and inactivation of two presynaptic cholinergic "markers", choline acetyltransferase and high affinity choline transporter, has been investigated using inhibitors designed with a redox-reactive catechol tethered to a quaternary ammonium group. Two quaternary ammonium alkyl-substituted catechols, 3[(trimethylammonio)methyl]catechol (TMC, 1) and N,N-dimethylepinephrine (catecholine, 2) were shown to bind weakly and noncompetitively to bovine choline acetyltransferase yet inactivated the enzyme in a time course consistent with the involvement of early intermediates in the spontaneous oxidation of these catechols. Both agents also inhibited high-affinity choline uptake. The time course of TMC and catecholine spontaneous oxidation-dependent inactivation of high affinity choline uptake sites was slower than, if it occurred at all, the spontaneous degradation of measurable choline transport in synaptosomes. When compared with inhibition of uptake of other neurotransmitters, it was shown that catecholine demonstrated more selectivity than TMC toward inhibition of choline transport. Km (microM) and Vmax (pmol/min per mg of protein) were measured for high affinity transport of choline, dopamine, and serotonin and were observed to be Km = 2.04 +/- 0.31, Vmax = 22 +/- 1; Km = 1.4, Vmax = 53; and Km = 0.15, Vmax = 23, respectively, in good agreement with published literature values. Ki's (mM) for catecholine and TMC, calculated from experimentally determined IC50's, were for catecholine 0.13 +/- 0.06, 0.53 +/- 0.09, and 0.39 +/- 0.10, and for TMC 0.06 +/- 0.03, 0.09 +/- 0.03, and 0.09 +/- 0.08, for choline, dopamine, and serotonin transport, respectively. In vivo studies using catecholine suggest that this compound impairs learning ability associated with long-term memory. Thus, catecholine represents a lead compound in a potential series of redox-reactive choline analogs, which may become useful irreversible antagonists of the critical cholinergic macromolecular targets underlying cholinergic hypofunction in disorders such as Alzheimer's disease.

Asunto(s)

Catecoles/síntesis química , Catecoles/farmacología , Colina/metabolismo , Fibras Colinérgicas/efectos de los fármacos , Epinefrina/análogos & derivados , Animales , Transporte Biológico/efectos de los fármacos , Encéfalo/enzimología , Bovinos , Colina O-Acetiltransferasa/antagonistas & inhibidores , Fibras Colinérgicas/metabolismo , Epinefrina/síntesis química , Epinefrina/farmacología , Técnicas In Vitro , Masculino , Oxidación-Reducción , Desempeño Psicomotor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Sinapsis , Sinaptosomas/enzimología

RESUMEN

A series of 5-(3-alkyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidines+ ++ (7a-h) was synthesized for biological evaluation as selective agonists for M1 receptors coupled to phosphoinositide (PI) metabolism in the central nervous system. Each ligand bound with high affinity to muscarinic receptors from rat brain as measured by inhibition of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) binding. 5-(3-Methyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine+ ++ trifluoroacetate (CDD-0098-J;7a) displayed high affinity (IC50 = 2.7 +/- 0.69 microM) and efficacy at muscarinic receptors coupled to PI metabolism in the rat cortex and hippocampus. Increasing the length of the alkyl substituent increased affinity for muscarinic receptors yet decreased activity in PI turnover assays. The hippocampal PI response of 7a was blocked by lower concentrations of pirenzepine (8) or by higher concentrations of either AF-DX 116 (9) or p-fluorohexahydrosiladifenidol (10), suggesting that at low concentrations 7a selectively stimulates PI turnover through M1 receptors.

Asunto(s)

Oxadiazoles/síntesis química , Parasimpaticomiméticos/síntesis química , Pirimidinas/síntesis química , Animales , Sitios de Unión , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Oxadiazoles/química , Oxadiazoles/farmacología , Parasimpaticomiméticos/química , Parasimpaticomiméticos/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Quinuclidinil Bencilato/metabolismo , Ratas , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Relación Estructura-Actividad

RESUMEN

The binding of the muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), which has been suggested as an M3-selective antagonist in peripheral tissues, was examined through quantitative autoradiographic techniques in brain. The ability of 4-DAMP to displace [3H](R)-quinuclindinyl benzilate (QNB) binding to rat brain sections was compared with the known distribution of M1 and M2 muscarinic receptor subtypes as measured previously with pirenzepine and AF-DX 116 (Messer et al., 1989a). 4-DAMP displayed a high affinity for [3H](R)-QNB binding sites in rat brain sections. Analysis of 4-DAMP binding to various brain regions revealed heterogeneous binding profiles, suggesting an interaction with multiple receptor sites. Quantification of the autoradiograms indicated that 4-DAMP bound with the highest affinity to muscarinic receptors in the midline thalamus (IC50 values < 30 nM), and had a slightly lower affinity for hippocampal receptors (IC50 values between 30 and 46 nM). 4-DAMP also displayed a lower affinity for cortical receptors with IC50 values between 30 and 50 nM. The binding profile of the putative M3 muscarinic antagonist did not exhibit a marked selectivity for any single region of brain. The data suggest that whereas 4-DAMP may be selective for M3 receptors in peripheral tissues, it has limited selectivity in the CNS. Minimum energy conformations for 4-DAMP were calculated using molecular mechanics calculations. 4-DAMP displayed two global minimum energy conformations, differing in the relative position of the piperidine ring with respect to the aromatic rings. The minimum energy conformations of 4-DAMP were compared with conformations generated for pirenzepine (Messer et al., 1989a). The lowest energy conformation of 4-DAMP was superimposable on the lowest energy conformation of pirenzepine (RMS = 0.297 A). It is suggested that the conformations available to 4-DAMP permit binding to several muscarinic receptors in the CNS.

Asunto(s)

Encéfalo/metabolismo , Piperidinas/química , Piperidinas/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Autorradiografía , Unión Competitiva , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Conformación Molecular , Pirenzepina/química , Pirenzepina/metabolismo , Quinuclidinil Bencilato/metabolismo , Ratas , Distribución Tisular

RESUMEN

A series of arecoline derivatives was utilized to assess steric and electronic effects important for activating muscarinic receptors in the CNS. Arecoline derivatives in which the methyl ester moiety was replaced by hexyloxy-1,2,5-oxadiazole (2b), hexyloxythiophene (3b) or hexyloxypyrazine (4b) were compared with the hexyloxy-1,2,5-thiadiazole compound (1b) (Hexyloxy-TZTP), known from previous work to be active as an M1/M3 partial agonist. MNDO calculations showed that the N-S bonds of the alkoxythiadiazole ring were highly polarized with the ability to form H-bonds to the N's. On the other hand, the smaller oxadiazole had lower polarities in the N-O bonds and reduced ability to form H-bonds, the thiophene was of comparable size to the thiadiazole and had large C-S bond polarities without the H-bond capability and the pyrazine had limited ability to form H-bonds. The compounds were compared with respect to their abilities to stimulate phosphoinositide (Pl) turnover in the hippocampus of the rat brain. 1b was more active than 2b-4b for stimulating the Pl turnover response. The data suggest that the ability to form H-bonds is an important factor for the ability of 1 to stimulate M1 muscarinic receptors in the CNS.

Asunto(s)

Arecolina/farmacología , Encéfalo/fisiología , Fosfatidilinositoles/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Animales , Arecolina/análogos & derivados , Enlace de Hidrógeno , Técnicas In Vitro , Ratas , Receptores Muscarínicos/fisiología , Estereoisomerismo , Relación Estructura-Actividad

RESUMEN

The activities of the enantiomers of BM-5 were examined to measure muscarinic cholinergic selectivity in the central nervous system. Autoradiographic studies assessed the ability of each enantiomer to inhibit the binding of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) to muscarinic receptors in the rat brain. (+)-(R)-BM-5 inhibited [3H]-(R)-QNB binding to rat brain sections at concentrations below 1.0 microM, while 100-fold higher concentrations of (-)-(S)-BM-5 were required for comparable levels of inhibition. Analysis of the autoradiograms indicated that both stereoisomers had a similar distribution of high affinity binding sites. Each enantiomer displayed higher affinity for muscarinic receptors in the superior colliculi and lower affinity for receptors in the cerebral cortex and hippocampus. (+)-(R)-BM-5 and oxotremorine inhibited adenylyl cyclase activity in the cerebral cortex with efficacies comparable to that for acetylcholine. (+)-(R)-BM-5 was 26-fold more potent than (-)-(S)-BM-5 in inhibiting adenylyl cyclase. Oxotremorine-M and carbamylcholine stimulated phosphoinositide turnover in the cerebral cortex. Oxotremorine had lower activity and (+)-(R)-BM-5 was essentially inactive at comparable concentrations. The difference in activity of the two enantiomers indicates a remarkable stereochemical selectivity for muscarinic receptors. The stereoselectivity index is comparable for both the autoradiographic assays (48) and measures of adenylyl cyclase activity (26) in the cerebral cortex.

Asunto(s)

Oxotremorina/análogos & derivados , Parasimpaticomiméticos/metabolismo , Pirrolidinas/metabolismo , Receptores Muscarínicos/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Autorradiografía , Encéfalo/metabolismo , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Cinética , Parasimpaticomiméticos/química , Pirrolidinas/química , Ratas , Estereoisomerismo

RESUMEN

Multiple intrahippocampal injections of gallamine impair performance of a representational memory task in rats. The binding of [3H]-(-)-quinuclidinyl benzilate (QNB) to rat brain sections was measured to determine if changes in receptor binding were associated with the deleterious effects of gallamine. [3H]-(-)-QNB binding to sections taken from gallamine-injected animals was compared with binding in saline-injected control animals. Autoradiographic analyses indicated an increase in [3H]-(-)-QNB binding sites within all layers of the cerebral cortex and in the superior colliculus in gallamine-treated animals as compared to saline-injected controls. Significant increases were noted in cortical layers IV and V (P less than 0.025) in gallamine-treated animals. No significant changes (P greater than 0.05) in the number of binding sites were observed in the hippocampus, neostriatum or various thalamic nuclei. The ability of unlabeled pirenzepine, gallamine and carbamylcholine to inhibit 0.2 nM [3H]-(-)-QNB binding also was measured to determine changes in the distribution of receptor subtypes. No significant changes were observed in any brain region for the binding of the selective antagonists pirenzepine and gallamine or the agonist carbamyl-choline. Although other possibilities are considered, the data suggest that an increase in the number of muscarinic receptors may contribute to the observed behavioral deficits associated with long-term gallamine treatment.

Asunto(s)

Trietyoduro de Galamina/farmacología , Hipocampo , Receptores Muscarínicos/efectos de los fármacos , Animales , Autorradiografía , Encéfalo/anatomía & histología , Carbacol/farmacología , Trietyoduro de Galamina/administración & dosificación , Inyecciones , Masculino , Pirenzepina/farmacología , Quinuclidinil Bencilato/farmacología , Ratas , Escopolamina/farmacología

RESUMEN

To develop an animal model for testing muscarinic agonists, we examined the effects of cholinergic lesions with the ethylcholine aziridinium ion (AF64A) on two types of memory tasks. The tasks provided a distinction between representational and dispositional memory that could be measured in a single paradigm. Young, male Long-Evans rats were trained in a modified T-maze to learn both a discrimination task and a paired-run alternation task. Once animals learned the tasks, they were administered either saline or AF64A (5 nmol into each hippocampus) via stereotaxic technique. One week following surgery, saline-treated animals exhibited comparable performances (P greater than 0.2) on both the discrimination task (90.0 +/- 2.6% correct) and the alternation task (79.5 +/- 5.7%). In contrast, animals treated with AF64A showed a significant impairment of performance (P less than 0.005) on the alternation task (56.1 +/- 1.7%) as compared to the discrimination task (81.6 +/- 5.0%). Performance of the alternation task was significantly lower for AF64A-treated animals than for controls (P less than 0.02). AF64A-treated animals subsequently injected with pilocarpine (1.0 mg/kg, i.p.) showed moderate improvements in performance on the alternation task, while performance on the discrimination task remained unaffected. Immunocytochemical studies of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) immunoreactivity indicated a loss of ChAT-positive cells in the septal region in AF64A-injected animals while TH-positive cells in the ventral tegmental area were unaffected by the treatment. The data suggest that AF64A can be used to produce selective lesions of the septohippocampal cholinergic system, which plays a greater role in representational memory than in dispositional memory.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Hipocampo/fisiología , Memoria/fisiología , Sistema Nervioso Parasimpático/fisiología , Animales , Aziridinas/antagonistas & inhibidores , Conducta Animal/efectos de los fármacos , Colina/análogos & derivados , Colina/antagonistas & inhibidores , Colina O-Acetiltransferasa/metabolismo , Hipocampo/anatomía & histología , Hipocampo/enzimología , Masculino , Bloqueantes Neuromusculares/farmacología , Sistema Nervioso Parasimpático/anatomía & histología , Sistema Nervioso Parasimpático/enzimología , Parasimpaticomiméticos/farmacología , Fosfatidilinositoles/metabolismo , Pilocarpina/farmacología , Ratas , Receptores Muscarínicos/efectos de los fármacos , Técnicas Estereotáxicas , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

Pilocarpine was tested biochemically in vitro for its ability to stimulate phosphoinositide (PI) turnover in the hippocampus (M1/M3 responses) where it displayed 35% of the maximal carbachol response with an EC50 value of 18 microM, and low-Km GTPase in the cortex (M2 response), where it had 50% of the maximal carbachol response with an EC50 value of 4.5 microM. Behaviorally, pilocarpine was able to restore deficits in a representational memory task (sensitive to M1 antagonists) produced by intrahippocampal injections of AF64A. Twenty-three low-energy conformations of protonated pilocarpine were generated using the program MacroModel. The data indicate that pilocarpine is a partial agonist at both M1 and M2 muscarinic receptors in the CNS. Behaviorally, with respect to the memory task, M1 effects of pilocarpine apparently predominate. It also is conceivable that different conformations of pilocarpine are active as agonists at different muscarinic receptor subtypes.

Asunto(s)

Encéfalo/efectos de los fármacos , Pilocarpina/farmacología , Receptores Muscarínicos/efectos de los fármacos , Animales , Encéfalo/metabolismo , Aprendizaje Discriminativo/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Masculino , Modelos Moleculares , Conformación Molecular , Fosfatidilinositoles/metabolismo , Ratas , Receptores Muscarínicos/clasificación