RESUMO
Binding of chemicals to the estrogen receptor (ER) is known to be a key mode of action of endocrine disruption effects. In this study, combined quantitative structure-activity relationship (QSAR) models from discriminant and multilinear regression (MLR) analyses, termed a two-step model, were developed. These were used to predict the binding potency to human ERalpha of four chemical groups, namely alkylphenols, phthalates, diphenylethanes and benzophenones. These groups are considered to be important chemical classes of ER-binders. The descriptors investigated were calculated following the simulation of docking between the receptor and ligand. Discriminant analysis in the first step of a two-step model was applied to distinguish binders from non-binders. It had a concordance, following leave-one-out (LOO), of greater than 87% for all chemical classes. Binders were defined as chemicals whose IC50 was reliably measured in a competitive binding assay. The MLR analysis in the second step was performed for the quantitative prediction of the binding affinity of chemicals that were previously discriminated as binders. The q2 values for alkylphenols and diphenylethanes were 0.75 and 0.74, respectively. However good MLR relationships were not obtained for phthalates and benzophenones as the observed binding affinities of chemicals in these categories were weak and in a too narrow range.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Ligantes , Relação Quantitativa Estrutura-Atividade , Derivados de Benzeno/química , Derivados de Benzeno/metabolismo , Benzofenonas/química , Benzofenonas/metabolismo , Análise Discriminante , Modelos Lineares , Modelos Químicos , Fenóis/química , Fenóis/metabolismo , Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismoRESUMO
The affinity-labeling technique is an extremely important method in receptor biochemistry. The 3-nitro-2-pyridinesulfenyl (Npys) group, attached to a mercapto group, can react only with a free thiol group (the beta-mercapto group of cysteine residue) of the target receptor molecules, forming a disulfide bond. This disulfide bonding is mediated through the thiol-disulfide exchange reaction. Unlike other labeling methods, the approach utilizing such chemically activated thiol-containing ligands is able to reproduce an unlabeled protein by treatment with dithiothreitol, a reducing reagent. This provides several unique aspects for the studies elucidating the structure-function relationships between the peptide and the receptor. Based on the SNpys affinity technique, we have achieved the discriminative disulfide-bonding affinity labeling of the three different subtypes of opioid receptors: mu, delta and kappa. This article reviews our novel affinity techniques in the in vitro receptor biochemistry.
Assuntos
Dissulfetos/análise , Receptores Opioides/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatografia de Afinidade/métodos , Clonagem Molecular , Simulação por Computador , Cisteína , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacocinética , Cinética , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Receptores Opioides/classificação , Receptores Opioides/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
We identified a novel metabolic system of morphine in the opium poppy (Papaver somniferum L.). In response to stress, morphine is quickly metabolized to bismorphine consisting of two morphine units, followed by accumulation in the cell wall. This bismorphine binds predominantly to pectins, which possess high galacturonic acid residue contents, through ionical bonds. Our newly developed method using artificial polysaccharides demonstrated that bismorphine bridges are formed between the two amino groups of bismorphine and the carboxyl groups of galacturonic acid residues, resulting in cross-linking of galacturonic acid-containing polysaccharides to each other. The ability of bismorphine to cross-link pectins is much higher than that of Ca2+, which also acts as a cross-linker of these polysaccharides. Furthermore, we confirmed that cross-linking of pectins through bismorphine bridges leads to resistance against hydrolysis by pectinases. These results indicated that production of bismorphine is a defense response of the opium poppy. Bismorphine formation is catalyzed by anionic peroxidase that pre-exists in the capsules and leaves of opium poppies. The constitutive presence of morphine, together with bismorphine-forming peroxidase, enables the opium poppy to rapidly induce the defense system.
Assuntos
Morfina/metabolismo , Papaver/metabolismo , Plantas Medicinais , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Morfina/química , Derivados da Morfina , Polissacarídeos/químicaRESUMO
Two phospholipases A2 (PLA2s) were purified from the venom of Trimeresurus flavoviridis (Crotalinae) inhabiting Tokunoshima island, Japan, and named PLA-A and PLA-B in the order of elution on a cation-exchange column. Lipolytic activities of PLA-A and PLA-B toward mixed micelles and liposomes were substantially lower than that of PLA2 (an [Asp49]PLA2) which had been isolated from the same venom. Both PLA-A and PLA-B consisted of 122 amino acids and contained aspartate at position 49 (the numbering according to the aligned sequences of PLA2s in Fig. 8), thus belonging to an [Asp49]PLA2 subgroup. PLA-A and PLA-B were identical in sequence with an exception at position 79. PLA-B contained Asn-Gly at positions 79 and 80 which are located in the beta-sheet region. On the other hand, PLA-A had beta-Asp-Gly and alpha-Asp-Gly in high and low proportion, respectively, at the corresponding positions which were produced from Asn-Gly through the base-catalyzed formation and hydrolysis of the succinimide type intermediate. Thus, PLA-A is derived from PLA-B. PLA-B is similar in sequence to PL-X, which had been purified from the venom of T. flavoviridis inhabiting Amami-Oshima island, Japan, and to PL-X', whose cDNA had been cloned from Tokunoshima T. flavoviridis venom gland, rather than PLA2. PLA-B showed strong edema-inducing activity, while PLA-A exhibited rather lower activity. The sequence around position 79 which constitutes a beta-turn segment seems to be crucial for edema-inducing activity. Phylogenetic tree of Tokunoshima T. flavoviridis venom PLA2 isozymes indicated that PLA-B and PL-X' diverged from PLA2 after branching of [Asp49]PLA2 forms and [Lys49]PLA2 forms.
Assuntos
Evolução Biológica , Venenos de Crotalídeos/enzimologia , Edema/induzido quimicamente , Fosfolipases A/análise , Fosfolipases A/toxicidade , Trimeresurus/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Edema/patologia , Eletroforese em Gel de Poliacrilamida , Lipossomos , Micelas , Dados de Sequência Molecular , Fosfolipases A2 , FilogeniaRESUMO
One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8-9 and 12-13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor's acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6-7, 10-11, or 14-15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [Arg-Lys(6-7)]- and [Arg-Lys(10-11)]nociceptins exhibited weak activities (6-9 and 60-90% of nociceptin, respectively) both in the receptor binding assay and in the [(35)S]GTPgammaS binding functional assay. In contrast, [Arg-Lys(14-15)]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTPgammaS functional assay). [Arg-Lys(14-15)]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure-activity studies have suggested that the incorporated Arg-Lys(14-15) interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues.
Assuntos
Arginina/química , Lisina/química , Peptídeos Opioides/química , Peptídeos Opioides/farmacologia , Sequências Repetitivas de Aminoácidos , Sequência de Aminoácidos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Dados de Sequência Molecular , Peptídeos Opioides/síntese química , Peptídeos Opioides/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , NociceptinaRESUMO
In the ligand/receptor interaction, the side chain phenyl group of phenylalanine (Phe) is involved in a so-called hydrophobic interaction, in which the Phe-phenyl group functions as a p element or merely as a hydrophobic element. The thrombin receptor-tethered ligand SFLLRNP consists of the Phe-2 residue essential for receptor activation. In order to explore the molecular characteristics of this Phe-2-phenyl group, a complete set of S/Phe/LLRNP peptides comprising six different difluorophenylalanine isomers [(F(2))Phe] was newly synthesized and assayed to evaluate their ability to induce the aggregation of human platelets. The assay results clarified several important structural elements to conclude that Phe-2-phenyl of S/Phe/LLRNP is in the edge-to-face CH/pi interaction with the receptor aromatic group, utilizing the Phe-phenyl edge along with adjacent benzene hydrogens at positions (2-3) or (5-6). It was also found that the fluorine atom at position 4 increases the acidity of the hydrogen mainly at its ortho position, resulting in a reinforcement of the CH/pi interaction and thus in an enhancement of biological activity. The H-->F replacement in the benzene ring was found to provide an effective structural examination to the Phe residue; i.e., to identify the hydrogens in the CH/pi interaction, and to strengthen the CH/pi interaction.
Assuntos
Derivados de Benzeno/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Receptores de Trombina/agonistas , Derivados de Benzeno/farmacologia , Humanos , Técnicas In Vitro , Ligantes , Fenilalanina/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Receptores de Trombina/química , Relação Estrutura-AtividadeRESUMO
A clottable protein coagulogen of the horseshoe crab Tachypleus tridentatus is proteolytically converted into an insoluble coagulin gel through non-covalent self-polymerization. Here we identified binding sites for the polymerization. A tryptic fragment, derived from the coagulin polymer chemically cross-linked by a bifunctional cross-linker, was isolated. Amino acid sequence analysis indicated that the fragment consists of two peptides cross-linked between Lys(85) and Lys(156). The two lysine residues are oppositely located at the head and tail regions of the elongated molecule separated by a much greater distance than the length of the cross-linker, which suggests that the cross-linking occurs intermolecularly. Based on the x-ray structural analysis, exposure of a hydrophobic cove on the head in response to the release of peptide C has been postulated (Bergner, A., Oganessyan, V., Muta, T., Iwanaga, S., Typke, D., Huber, R., and Bode, W. (1996) EMBO J. 15, 6789-6797). An octapeptide containing Tyr(136), which occupies the tail end of coagulin, was found to inhibit the polymerization. Replacement of Tyr(136) of the peptide with Ala resulted in loss of the inhibitory activity. These results indicated that the polymerization of coagulin proceeds through the interaction between the newly exposed hydrophobic cove on the head and the wedge-shaped hydrophobic tail.
Assuntos
Tromboplastina/química , Tromboplastina/metabolismo , Alanina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Reagentes de Ligações Cruzadas/farmacologia , Eletroforese em Gel de Poliacrilamida , Caranguejos Ferradura , Cinética , Lisina/química , Modelos Moleculares , Dados de Sequência Molecular , Biossíntese Peptídica , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Tromboplastina/antagonistas & inibidores , Fatores de Tempo , Tirosina/química , Raios XRESUMO
Conventional chromatographic analysis showed that phospholipase A(2) (PLA(2)) isoenzymes of the venom of Trimeresurus flavoviridis (Habu snake) of Okinawa island are profoundly different in composition from those of T. flavoviridis of Amami-Oshima and Tokunoshima islands. The most striking feature was that myotoxic [Lys(49)]PLA(2) isoenzymes, called BPI and BPII, which are expressed abundantly in the venoms of Amami-Oshima and Tokunoshima T. flavoviridis, are missing from the venom of Okinawa T. flavoviridis. Northern blot analysis of Okinawa T. flavoviridis venom-gland mRNA species showed the absence of BPI and BPII mRNA species. Analysis by single-stranded conformational polymorphism-PCR of venom-gland mRNA species of T. flavoviridis from three islands, with reference to five DNA species each encoding different PLA(2) isoenzymes from Tokunoshima T. flavoviridis venom gland, also suggested that BPI and BPII mRNA species are not expressed in Okinawa T. flavoviridis venom gland. In contrast, genomic Southern blot analysis with a variety of probes showed that only the bands corresponding to the upstream and downstream regions of the genes for BPI and/or BPII can be detected in Okinawa T. flavoviridis. These results suggested that the genes for BPI and BPII in Okinawa T. flavoviridis genome had been inactivated to form pseudogenes. Differently from Amami-Oshima and Tokunoshima T. flavovirdis genomic DNAs, PCR amplification of the segments of BPI and BPII genes between the 5' moiety of second exon and the middle portion of second intron failed for Okinawa T. flavoviridis genomic DNAs. In sequence analysis of the two segments involving polymorphism between BPI and BPII genes, which are located in first exon and third exon, respectively, only one base was detected at the polymorphic positions for pseudogene in Okinawa T. flavoviridis genome. Based on these facts, it became evident for pseudogene that the upstream region of BPI gene down to the 5' moiety of second exon and the downstream region of BPII gene starting from the middle portion of second intron are in a linked form with a possible insertion. Such observations suggest that venom-gland genes for PLA(2) isoenzymes in T. flavoviridis snakes isolated for one to two million years have evolved independently. Their evolution is regional and seems, from several lines of consideration and observation, to be adaptive to the environment.
Assuntos
Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/genética , Evolução Molecular , Fosfolipases A/genética , Trimeresurus/genética , Animais , Southern Blotting , Dosagem de Genes , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Japão , Modelos Genéticos , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Polimorfismo Conformacional de Fita Simples , Pseudogenes/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
pGlu-Asn-Cys (Cys)-Pro-Arg-Gly-NH(2) (AVP(4-9)), a major metabolite C-terminal fragment of Arginine(8)-vasopressin (AVP), improves the disruption of the learning and memory, and is a far more potent in the mnemonic function than AVP. In this study, we pharmacologically characterized its putative binding site and mechanism of intracellular signaling. Radioligand binding assay showed that [35S]AVP(4-9) could detect specific binding sites in the rat hippocampus membrane preparations, and the binding site was specifically displaced by AVP(4-9) but not by either V(1) or V(2) antagonists. Furthermore, [35S]AVP(4-9) could not detect the cloned rat V(1a), V(1b) and V(2) vasopressin receptors. Even at a low doses (10-100 pM), AVP(4-9) caused an increase in both inositol(1,4, 5)-trisphosphate (Ins(1,4,5)P(3)) and intracellular calcium concentrations ([Ca(2+)](i)) in rat hippocampal cells. The AVP(4-9)-induced [Ca(2+)](i) increase was partially inhibited by the absence of Ca(2+) or by Ca(2+)-channel blocker, suggesting that AVP(4-9) caused the [Ca(2+)](i) increase via release from intracellular calcium store as well as influx from extracellular calcium. For the first time, this study provides evidence to show that AVP(4-9) activates Ins(1,4,5)P(3)/[Ca(2+)](i) pathway through a novel type of receptor in rat hippocampus, which might be potentially important in improving the mnemonic function.
Assuntos
Arginina Vasopressina/metabolismo , Arginina Vasopressina/farmacologia , Hipocampo/química , Hipocampo/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Animais , Ligação Competitiva , Cálcio/farmacocinética , Bloqueadores dos Canais de Cálcio/farmacologia , Inositol 1,4,5-Trifosfato/análise , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Memória/fisiologia , Nicardipino/farmacologia , Nifedipino/farmacologia , Ensaio Radioligante , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Radioisótopos de Enxofre , ômega-Conotoxinas/farmacologiaRESUMO
In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.
Assuntos
Evolução Molecular , Glândulas Exócrinas/química , Fosfolipases A/química , Venenos de Serpentes/química , Sequência de Aminoácidos , Southern Blotting , Clonagem Molecular , Fragmentação do DNA , Biblioteca Gênica , Immunoblotting , Hibridização In Situ , Isoenzimas/química , Isoenzimas/genética , Dados de Sequência Molecular , Fosfolipases A/genética , Fosfolipases A2 , Venenos de Serpentes/genéticaRESUMO
A key target of environmental chemicals, so-called endocrine disruptors, is the nuclear receptors which function as a transcription factor essential for the specific gene expression. In recent years, a number of chemicals that bind to the estrogen receptor, a member of nuclear receptors, have been identified. However, these chemicals bear little structural resemblance to natural estrogens, and thus it is important particularly for assessment and prediction of xenoestrogens to evaluate the structural essentials for binding to the receptor. In this review, together with the recent research topics in the field, we describe the current knowledge of methodology of competitive binding assays for estrogen receptors and the results of subsequent structure-activity studies.
Assuntos
Poluentes Ambientais/metabolismo , Estrogênios/metabolismo , Ensaio Radioligante/métodos , Receptores de Estrogênio/metabolismo , Xenobióticos/metabolismo , Ligação Competitiva , Relação Estrutura-Atividade , Fatores de TranscriçãoRESUMO
Xenoestrogen dialkyl phthalates, C(6)H(4)(COOC(n)H(m))(2), lack the phenolic hydroxyl group that is an essential structural component of the steroid A ring of 17 beta-estradiol. In order to examine whether dialkyl phthalates imitate the steroid structure, we have synthesized a series of 4-hydroxyl derivatives of dialkyl phthalates. The compounds were examined for their ability to displace [(3)H]17 beta-estradiol from the recombinant human estrogen receptor, which was expressed on Sf9 cells using the vaculovirus expression system. Dialkyl 4-hydroxyl phthalates were found to exhibit several-fold higher binding affinities compared to phthalates without the 4-hydroxyl group. From the analyses of receptor binding modes of dialkyl phthalates with and without the 4-hydroxyl group, it was deduced that the phthalic benzene ring mimics the steroid A ring. A biphasic binding curve observed for dicyclohexyl phthalate was also depicted by its 4-hydroxyl derivative, but it increased binding affinity only at the high affinity binding site. These data suggest that the phthalate benzene moiety recognizes the core of the estrogen receptor binding site and the hydrophobic interaction of the dialkyl moiety substantiates the binding characteristics of the phthalates. The present data indicate that even chemicals with slight structural analogy and weak receptor affinity can perturb the endocrine system when administered in high concentrations.
Assuntos
Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismo , Receptores de Estrogênio/metabolismo , Sítios de Ligação , Ligação Competitiva , Estradiol/metabolismo , Cinética , Mimetismo Molecular , Ácidos Ftálicos/toxicidade , Relação Quantitativa Estrutura-Atividade , Trítio , Xenobióticos/química , Xenobióticos/metabolismo , Xenobióticos/toxicidadeRESUMO
An antagonist specific for the thrombin receptor is expected to be a remedy for thrombosis. Structure-activity studies of thrombin receptor-tethered ligand SFLLRNP have revealed the importance of the Phe-2-phenyl group in receptor recognition and the replacement of the Phe-2 by para-fluorophenylalanine [(p-F)Phe] was found to enhance its activity [Nose, T. et al. (1993) Biochem. Biophys. Res. Commun. 193, 694-699]. In order to obtain a small sized antagonist, a series of (p-F)Phe derivatives was designed and synthesized novel structural elements essential for receptor interactions being introduced at both the N and C-termini. beta-Mercaptopropionyl (betaMp) or its derivative activated by S-3-nitro-2-pyridinesulphenyl (Npys) was introduced at the N-terminus, and phenylmethyl amines were coupled to the C-terminus. All compounds were inactive when assayed for human platelet aggregation, indicating that they are not agonists. beta-Mercaptopropionyl derivatives were also inactive as antagonists. However, Npys-containing analogs were found to inhibit the agonist activity of SFLLRNP. In particular, SNpys-betaMp-(p-F)Phe-NH-R [R = -CH(C6H5)2 and -CH2-CH-(C6H5)2] potently suppressed platelet aggregation. The results suggested that (p-F)Phe can be used as a structural core to construct an effective antagonist conformation.
Assuntos
Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/farmacologia , Receptores de Trombina/antagonistas & inibidores , p-Fluorfenilalanina/análogos & derivados , Amidas/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Fragmentos de Peptídeos/farmacologia , Receptores de Trombina/agonistas , Relação Estrutura-AtividadeRESUMO
A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.
Assuntos
Marcadores de Afinidade/química , Dinorfinas/química , Dinorfinas/metabolismo , Receptores Opioides/química , Receptores Opioides/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/citologia , Membrana Celular/metabolismo , Cisteína , Dissulfetos/química , Cobaias , Dados de Sequência Molecular , Ratos , Homologia de Sequência de Aminoácidos , Compostos de Sulfidrila/químicaRESUMO
The blood-brain barrier (BBB) transport and metabolism of a novel arginine-vasopressin fragment 4-9 [AVP(4-9), isoelectric point; (pI) = 9.2] analog, that is, cationic AVP(4-9) (C-AVP(4-9), PI = 9.8), were examined in vivo and in vitro. At 45 min after an i.v. administration to mice, the cerebrum-to-plasma concentration ratios of (35)S-labeled AVP(4-9) and (125)I-labeled C-AVP(4-9) were 0.103 and 0.330 ml/g cerebrum, respectively, and the BBB permeation clearances were 1.47 x 10(-4) and 3.10 x 10(-4) ml/min/g cerebrum, respectively. In the in vitro study using mouse brain capillary endothelial cells immortalized by SV40 infection (MBEC4), the acid-resistant binding values of (35)S-labeled AVP(4-9) and (125)I-labeled C-AVP(4-9) to MBEC4 at 120 min were 0.93 and 1.95 microliter/mg protein (as the cell/medium ratios), respectively. (35)S-labeled AVP(4-9) showed two-phase saturable acid-resistant binding, and its half-saturation constants (K(D)) were 3.8 nM (high affinity) and 45.7 microM (low affinity). (125)I-labeled C-AVP(4-9) showed single-phase saturable acid-resistant binding, with a K(D) value of 16.4 microM. The acid-resistant binding of (125)I-labeled C-AVP(4-9) was significantly dependent on temperature and medium osmolarity. The acid-resistant binding of (125)I-labeled C-AVP(4-9) was inhibited by dancylcadaverine, phenylarsine oxide (endocytosis inhibitors), 2,4-dinitrophenol (a metabolic inhibitor), and AVP(4-9), poly(L-lysine), and protamine (cationic substances), but not by poly(L-glutamic acid) (an anionic peptide) and the V(1) and V(2) vasopressin receptor antagonists. In addition, the conversion of C-AVP(4-9) to AVP(4-9) in the cerebral homogenate was confirmed by HPLC and mass spectrometry. The present results demonstrate that C-AVP(4-9) is transported through the BBB more effectively than AVP(4-9), via absorptive-mediated endocytosis, and that C-AVP(4-9) is converted to the neuroactive parent peptide, AVP(4-9), in the cerebrum.
Assuntos
Arginina Vasopressina/farmacocinética , Barreira Hematoencefálica , Fragmentos de Peptídeos/farmacocinética , Animais , Arginina Vasopressina/química , Arginina Vasopressina/metabolismo , Biotransformação , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/metabolismo , Capilares/metabolismo , Capilares/fisiologia , Cátions , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Endocitose/efeitos dos fármacos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos ICR , Concentração Osmolar , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Prolil Oligopeptidases , Ligação Proteica , Artéria Renal/metabolismo , Serina Endopeptidases/metabolismo , TemperaturaRESUMO
In a computer modeling of transmembrane domains of human thrombin receptor, Lys-158 was found near the ligand binding site. To capture this basic residue, analogs of peptide ligand containing a series of acidic amino acids were synthesized and assayed for human platelet aggregation, and Ser-(p-F)Phe-Aad(= alphaaminoadipic acid)-Leu-Arg-Asn-Pro-NH2 was found to be a potent antagonist.
Assuntos
Antitrombinas/química , Receptores de Trombina/antagonistas & inibidores , Antitrombinas/síntese química , Antitrombinas/metabolismo , Sítios de Ligação , Simulação por Computador , Desenho de Fármacos , Humanos , Lisina/metabolismo , Modelos Moleculares , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/metabolismo , Receptores de Trombina/química , Receptores de Trombina/metabolismo , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
A novel type of conformationally restricted peptides with the structure of H-D-Xaa-Phe-NH-CH2-C6H5 has been developed as inhibitors of serine proteinase chymotrypsin. The D-Xaa-alkyl and Phe-phenyl groups resulted in a formation of the hydrophobic core due to the side-chain-side-chain CH/pie interaction. Their spatial proximity was evidenced by 400 MHz 1H-nmr measurements, observing large upfield shifts of proton signals of D-Xaa-alkyl and nuclear Over-hauser effect (NOE) enhancements between the D-Xaa-alkyl and Phe-phenyl groups. This conformational restriction brought by CH/pie interaction produced an inhibitory structure, in which the C-terminal amide-benzyl group fits the chymotrypsin S1 site and the hydrophobic core binds to the S2 site. The inhibitory conformation was demonstrated crystallographically for the complex between the dipeptide H-D-Leu-Phe-NH-CH2-C6H4(p-F) and gamma-chymotrypsin. Detailed structure-activity studies have substantiated the structure of dipeptides in the active center of the enzyme.
Assuntos
Quimotripsina/química , Quimotripsina/metabolismo , Dipeptídeos/química , Inibidores de Serina Proteinase/química , Sítios de Ligação , Cristalografia por Raios X , Dipeptídeos/síntese química , Dipeptídeos/farmacologia , Desenho de Fármacos , Espectroscopia de Ressonância Magnética , Conformação Proteica , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologiaRESUMO
Octyl- and nonylphenols in the environment have been proposed to function as estrogens. To gain insight into their structural essentials in binding to the estrogen receptor, a series of phenols with saturated alkyl groups at the para position, HO-C6H4-CnH2n+1 (n = 0-12), were examined for their ability to displace [3H]17beta-estradiol in the recombinant human estrogen receptor, which was expressed in Sf9 cells using the vaculovirus expression system. All tested para-alkylphenols were found to bind fully to the estrogen receptors in a dose-dependent manner. The interaction of alkylphenols with the receptor became stronger with increase in the number of the alkyl carbons and the activity was maximized with n = 9 of nonylphenol. Phenol (n = 0) exhibited weak but full binding to the receptor, whereas anisole with a protected phenolic hydroxyl group was completely inactive. Also, alkanes such as n-octane, 2,2, 4-trimethylpentane corresponding to tert-octane, and n-nonane exhibited no binding. The results indicate that the binding of para-alkylphenols to the estrogen receptor is due to the effect of covalent bonding of two constituents of the phenol and alkyl groups, which correspond to the A-ring and hydrophobic moiety of the steroid structure, respectively. When alkylphenols were examined for their receptor binding conformation by 1H-NMR measurements and ab initio molecular orbital calculations, it was suggested that nonbranched alkyl groups are in an extended conformation, while branched alkyl groups are in a folded conformation. These results suggest that branched and nonbranched alkyl moieties of alkylphenols interact differently with the lipophilic ligand binding cavity of the estrogen receptor when compared to the binding of 17beta-estradiol.
Assuntos
Fenóis/metabolismo , Receptores de Estrogênio/metabolismo , Cromatografia Gasosa , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Fenóis/química , Ligação Proteica , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Dialkyl phthalates have been suggested to function as xenoestrogen. To explore the structural essentials, a series of ring and alkyl-chain isomers of dialkyl phthalates C6H4(COOCnHm)2 were examined for their ability to displace [3H]17beta-estradiol in the recombinant human estrogen receptor expressed on Sf9 vaculovirus. Compounds with an alkyl chain of more than C3 (n = 3) exhibited a distinct full receptor binding in a dose-dependent manner. When the ring isomers of C3-diallyl (-CH2-CH=CH2) derivatives, namely diallyl phthalate, diallyl isophthalate, and diallyl terephthalate, were examined, the ortho isomer of diallyl phthalate was most potent to bind to the estrogen receptor. The interaction with the estrogen receptor was optimized with dibutyl phthalates of C4. The conformational studies by 1H-NMR measurements and ab initio molecular orbital calculations have suggested that the structure mimics the interface of steroid A and B/C rings of 17beta-estradiol. Dicyclohexyl phthalate bound to the estrogen receptor with a biphasic binding curve, suggesting the compound discriminates two different receptor conformations.