RESUMEN
A potentiometric, spectroscopic (UV-visible, CD and EPR) and electrospray ionization mass spectrometric (ESI-MS) study of Cu(II) binding to the neurokinin A with point mutation (S5A) (ANKA), His-Lys-Thr-Asp-Ala(5)-Phe-Val-Gly-Leu-Met-NH2 and its N-acethyl derivative (Ac-ANKA), Ac-His-Lys-Thr-Asp-Ala(5)-Phe-Val-Gly-Leu-Met-NH2 were carried out. For the ANKA and Ac-ANKA the additional deprotonation was not observed. It suggests that for the tachykinin peptides with C-terminal sequence of neurokinin A for the additional deprotonation the presence of the serine residue is necessary. For the Cu(II)-ANKA 1:2 system at physiological pH 7.4 the CuH2L2 species is present with histamine-like 4N, 2×{NH2,NIm} coordination mode. With increasing pH the deprotonation and coordination of amide nitrogen atoms occur and the CuH-2L, CuH-3L complexes are formed. In pH range 4.5 - 9.5 the dimeric Cu2HL2, Cu2L2 and Cu2H-1L2 species in solution are also present. To elucidate the products of the copper(II)- catalyzed oxidation of the ANKA and Ac-ANKA, the liquid chromatography-mass spectrometry (LC-MS) method and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. In the presence of hydrogen peroxide with 1:1 peptide-H2O2 molar ratio for both peptides the oxidation of the methionine residue to methionine sulfoxide was observed. For the Cu(II)-peptide-hydrogen peroxide in 1:2:2 molar ratio systems oxidations of the histidine residues to 2-oxo-histidines and methionine sulfoxide to methionine sulfone were detected.
Asunto(s)
Alanina/química , Complejos de Coordinación/química , Cobre/química , Neuroquinina A/química , Oligopéptidos/química , Mutación Puntual , Serina/química , Alanina/genética , Animales , Cromatografía Liquida , Histidina/química , Humanos , Peróxido de Hidrógeno/química , Cinética , Espectrometría de Masas , Metionina/análogos & derivados , Metionina/química , Neuroquinina A/síntesis química , Neuroquinina A/genética , Oligopéptidos/síntesis química , Oligopéptidos/genética , Oxidación-Reducción , Protones , Serina/genéticaRESUMEN
A chemical reagent, N-acetyl-cys((succinimidyl-6-(thioacetyl)amino) hexanoate)-ser-arg-arg-ala-ser-val-tyr-amide ("phosite NHS ester"), allowing the introduction of phosphorylation sites into proteins, peptides, or small molecules, has been synthesized and characterized. The phosite reagent enables the enzymatic radiolabeling of any protein, peptide, or small molecule containing a reactive amine using [(32)P] or [(33)P]ATP and protein kinase A. The utility of the reagent has been demonstrated in cytokine and G protein-coupled radioligand receptor binding assays using whole cell and immobilized receptor formats. Use of the reagent does not require genetic manipulation of the target ligand.
Asunto(s)
Proteínas Portadoras/metabolismo , Neuroquinina A/síntesis química , Fosfopéptidos/síntesis química , Fosfoproteínas/síntesis química , Ensayo de Unión Radioligante/métodos , Receptores de Superficie Celular , Receptores de Neuroquinina-2/metabolismo , Animales , Células CHO , Proteínas Portadoras/análisis , Cromatografía Líquida de Alta Presión/métodos , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citocinas/síntesis química , Citocinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Indicadores y Reactivos , Marcaje Isotópico/métodos , Leptina/síntesis química , Leptina/metabolismo , Neuroquinina A/metabolismo , Oligopéptidos , Fosfopéptidos/metabolismo , Fosfoproteínas/metabolismo , Radioisótopos de Fósforo , Fosforilación , Receptores de Leptina , Receptores de Neuroquinina-2/análisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , Espectrometría de Masa de Ion Secundario/métodos , TransfecciónRESUMEN
Steric and electrostatic requirements at position 6 of [Nle(10)]NKA(4-10), a full agonist of NK-2 receptors, for molecular recognition by the receptor were studied. Two series of peptide analogues, (a) p-substituted analogues, [p-X-Phe(6), Nle(10)]NKA(4-10), where X = F, Cl, Br, I, NH(2), NO(2), and (b) [D-Phe(6),Nle(10)]NKA(4-10), [Trp(6),Nle(10)]NKA(4-10), and [Chex-Ala(6),Nle(10)]NKA(4-10), were synthesized, and their biological activity was examined. Competition binding experiments with [(3)H]NKA were performed using cloned human NK-2 receptors expressed in CHO cells. Antagonistic and agonistic properties of the analogues were studied using an in vitro functional assay with hamster tracheal rings. The rank order of potency of agonists was [Nle(10)]NKA(4-10) approximately [p-F-Phe(6),Nle(10)]NKA(4-10) > [p-NH(2)-Phe(6),Nle(10)]NKA(4-10) > [p-Cl-Phe(6),Nle(10)]NKA(4-10) > [p-NO(2)-Phe(6),Nle(10)]NKA(4-10) > [Trp(6),Nle(10)]NKA(4-10). Size and planarity of the aromatic side chain were crucially important for the biological activity, whereas electron-donating and electron-withdrawing properties of the para-substituent were less important. The results favor the hypothesis that weakly polar pi-pi interactions exist between the aromatic group and the receptor.
Asunto(s)
Neuroquinina A/análogos & derivados , Fragmentos de Péptidos/química , Receptores de Neuroquinina-2/agonistas , Receptores de Neuroquinina-2/metabolismo , Animales , Unión Competitiva , Células CHO , Cricetinae , Humanos , Técnicas In Vitro , Masculino , Mesocricetus , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Neuroquinina A/síntesis química , Neuroquinina A/química , Neuroquinina A/metabolismo , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Relación Estructura-Actividad , Tráquea/efectos de los fármacos , Tráquea/fisiologíaRESUMEN
Parallel compound synthesis enables large numbers of individual compounds to be prepared simultaneously using semiautomated techniques. This fast and efficient methodology has an important role to play in accelerating lead optimisation and hence the whole drug discovery process. The potential of this strategy to rapidly optimise chemical leads and provide structure-activity relationship (SAR) information was demonstrated in two therapeutic areas, antiviral agents (herpes simplex virus), and neurokinin-2 receptor antagonists.
Asunto(s)
Antivirales/síntesis química , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Herpesvirus Humano 1/efectos de los fármacos , Preparaciones Farmacéuticas/síntesis química , Receptores de Neuroquinina-2/antagonistas & inhibidores , Animales , Antivirales/farmacología , Unión Competitiva , Células CHO/citología , Células CHO/metabolismo , Chlorocebus aethiops , Cricetinae , ADN Helicasas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/estadística & datos numéricos , Industria Farmacéutica/métodos , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/crecimiento & desarrollo , Espectroscopía de Resonancia Magnética , Neuroquinina A/agonistas , Neuroquinina A/síntesis química , Neuroquinina A/metabolismo , Ensayo de Unión Radioligante , Ratas , Receptores de Neuroquinina-2/análisis , Relación Estructura-Actividad , Células Vero/citología , Ensayo de Placa Viral/métodosAsunto(s)
Contracción Muscular/efectos de los fármacos , Músculo Liso/fisiología , Neuroquinina A/análogos & derivados , Fragmentos de Péptidos/farmacología , Animales , Cricetinae , Técnicas In Vitro , Músculo Liso/efectos de los fármacos , Neuroquinina A/síntesis química , Neuroquinina A/farmacología , Fragmentos de Péptidos/síntesis química , Fenilalanina , Relación Estructura-Actividad , Tráquea/efectos de los fármacos , Tráquea/fisiologíaAsunto(s)
Neuroquinina A/análogos & derivados , Neuroquinina A/química , Oligopéptidos/química , Conformación Proteica , Secuencia de Aminoácidos , Estabilidad de Medicamentos , Datos de Secuencia Molecular , Neuroquinina A/síntesis química , Neuroquinina B/química , Oligopéptidos/síntesis química , Relación Estructura-ActividadRESUMEN
A new radioligand, [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10), based on the selective agonist [Lys5,MeLeu9,Nle10]-NKA(4-10) has been developed. Binding in rat fundus membranes was displaced by NP gamma > NKA > or = [Lys5,MeLeu9,Nle10]-NK(4-10) > neuropeptide K > [Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10) > SP > [Sar9,Met(O2)11]-SP >> senktide, indicating binding to NK-2 receptors. Preliminary studies demonstrated high specific binding in membranes from rat urinary bladder, duodenum and colon. Specific binding in rat brain and lung was negligible, and binding in a range of guinea-pig tissues was no more than 35% specific. These data may indicate species differences in NK-2 receptors.
Asunto(s)
Membrana Celular/metabolismo , Neuroquinina A/análogos & derivados , Neuroquinina A/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de Neuroquinina-2/análisis , Animales , Unión Competitiva , Fundus Gástrico/metabolismo , Cobayas , Radioisótopos de Yodo , Cinética , Masculino , Neuroquinina A/síntesis química , Especificidad de Órganos , Fragmentos de Péptidos/síntesis química , Ensayo de Unión Radioligante , Ratas , Receptores de Neuroquinina-2/metabolismoRESUMEN
The tyrosyl derivative of the tachykinin NK2 selective agonist [Lys5,MeLeu9,Nle10]NKA-(4-10) was iodinated and the product [125I][Lys5,Tyr(I2)2,MeLeu9,Nle10]NKA-(4-10) purified using reverse phase HPLC. The binding characteristics of this novel radioligand were investigated in homogenates of rat gastric fundus. Binding was saturable, reversible and to a single population of high affinity sites of KD 1.3 +/- 0.2 nM (n = 4). Specific binding of [125I][Lys5,Tyr(I2)7,MeLeu9,Nle10]NKA-(4-10) was inhibited by neuropeptide gamma SR 48968 > or = neurokinin A (NKA) > or = [Lys5,MeLeu9,Nle10]NKA-(4-10) > [Lys5,Tyr7,MeLeu9,Nle10] NKA-(4-10) > neuropeptide K > [Lys5,Tyr(I2)7,MeLeu9,Nle10]NKA-(4-10) > MDL 29,913 > [127I]- Bolton-Hunter-NKA > neurokinin B > substance P (SP) >> MEN 10207 > [Sar9,Met(O2)11]SP >> senktide, indicating binding to NK2 receptors. NKA, [Lys5,MeLeu9,Nle10]NKA-(4-10) and [Lys5,Tyr(I2)7,MeLeu9,Nle10]NKA-(4-10) contracted the isolated fundus strip, with pD2 values 7.9, 7.7 and 7.4, respectively. This novel, highly selective radioligand should prove useful in characterisation studies in peripheral tissues.
Asunto(s)
Encéfalo/efectos de los fármacos , Neuroquinina A/análogos & derivados , Fragmentos de Péptidos/metabolismo , Receptores de Neurotransmisores/efectos de los fármacos , Animales , Unión Competitiva , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , Femenino , Fundus Gástrico/efectos de los fármacos , Fundus Gástrico/metabolismo , Masculino , Neuroquinina A/síntesis química , Neuroquinina A/metabolismo , Fragmentos de Péptidos/síntesis química , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores de Neuroquinina-2 , Receptores de Neurotransmisores/metabolismoRESUMEN
A series of analogues of neurokinin A (NKA) has been synthesized and characterized by testing for their abilities, in vitro, to contract guinea pig tracheal smooth muscle or to antagonize NKA-, NKB- and SP-induced contraction of this tissue. Substitution of NKA residues Gly8 or Leu9 by conformationally restricting amino acids produced peptides that were antagonists of NKA action, but the type and specificity of the antagonism depended on the size of the peptide. Thus, while [Ala5, Aib8, Leu10]NKA(2-10) showed no agonism and was a specific, competitive antagonist of NKA, [Ala5, Aib8, Leu10]NKA(4-10) was a noncompetitive antagonist of NKA and substance P (SP) and was itself a weak agonist at concentrations above 10(-7) M.
Asunto(s)
Contracción Muscular/efectos de los fármacos , Músculo Liso/fisiología , Neuroquinina A/análogos & derivados , Neuroquinina A/farmacología , Taquicininas/farmacología , Tráquea/fisiología , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Carbacol/farmacología , Relación Dosis-Respuesta a Droga , Cobayas , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Músculo Liso/efectos de los fármacos , Neuroquinina A/síntesis química , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Relación Estructura-Actividad , Tráquea/efectos de los fármacosAsunto(s)
Neuroquinina A/análogos & derivados , Secuencia de Aminoácidos , Animales , Cobayas , Técnicas In Vitro , Métodos , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Neuroquinina A/síntesis química , Neuroquinina A/química , Relación Estructura-Actividad , Tráquea/efectos de los fármacosAsunto(s)
Neuroquinina A/análogos & derivados , Neuroquinina A/antagonistas & inhibidores , Neuroquinina B/análogos & derivados , Neuroquinina B/antagonistas & inhibidores , Sustancia P/análogos & derivados , Sustancia P/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Cobayas , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Intestinos/fisiología , Datos de Secuencia Molecular , Estructura Molecular , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Músculo Liso/fisiología , Neuroquinina A/síntesis química , Neuroquinina A/farmacología , Neuroquinina B/síntesis química , Neuroquinina B/farmacología , Conformación Proteica , Ratas , Receptores de Neuroquinina-1 , Receptores de Neuroquinina-2 , Receptores de Neuroquinina-3 , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/metabolismo , Relación Estructura-Actividad , Sustancia P/síntesis química , Sustancia P/farmacologíaRESUMEN
By introducing D-Trp in position 6 and 8 along with pyroglutamic acid (Pyr) in position 4 or Nle in position 10 of NKA(4-10) we have obtained selective although weak NK-2 tachykinin receptor antagonists. Similar substitutions, previously reported on the sequence of SP, gave rise to nonselective antagonists presumably for the limited selectivity of the agonist used as template. Further modifications like the addition of a third D-Trp in position 9 gave rise to more potent but less selective antagonists, thus showing that each amino acid substitution can dramatically affect selectivity.
Asunto(s)
Neuroquinina A/síntesis química , Fragmentos de Péptidos/síntesis química , Receptores de Neurotransmisores/efectos de los fármacos , Taquicininas/antagonistas & inhibidores , Triptófano , Secuencia de Aminoácidos , Animales , Cobayas , Técnicas In Vitro , Datos de Secuencia Molecular , Músculo Liso/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Neuroquinina A/farmacología , Fragmentos de Péptidos/farmacología , Ratas , Receptores de TaquicininasRESUMEN
Dimeric analogs of neurokinin A and neurokinin B COOH-terminal heptapeptides were synthesized in order to examine the effect of ligand dimerization on the receptor selection. Dimerization was carried out at the NH2-terminus of peptides with succinic acid, yielding succinyl bis[Asp-Ser-Phe-Val-Gly-Leu-Met-NH2] (D-NKA4-10) and succinyl bis[Asp-Phe-Phe-Val-Gly-Leu-Met-NH2] (D-NKB4-10). In the assay using rat vas deferens (RVD), it was found that the deletion of the NH2-terminal tripeptide from native neurokinin A or B enhances the activity 1.5- to 8-fold, resulting in formation of NK-2 receptor specific ligands NKA4-10 and NKB4-10. When dimeric analogs of these shortened peptides, namely D-NKA4-10 and D-NKB4-10, were examined in RVD and guinea pig ileum (GPI), they were fairly potent in GPI, but not in RVD. Under conditions in which the NK-1 receptors in GPI were desensitized with NK-1 specific substance P methyl ester, dimers reduced the activity drastically, while the corresponding monomers exhibited unchanged activity. These results suggest that dimerization of the COOH-terminal heptapeptide of neurokinins changes the receptor selection of peptides from NK-2 to NK-1, and that the NK-1 receptor has a structure favorable to a dimeric peptide ligand.