RESUMO
Libiguins are limonoids with highly potent sexual activity enhancing effects, originally isolated from the Madagascarian Meliaceae species Neobeguea mahafalensis, where they exist in only minute quantities. Their low natural abundance has hampered mapping of their biological effects. Here we describe an approach to the semisynthesis of libiguin A and its close analogues 1-3 starting from phragmalin, which is a limonoid present in high amounts in a commercially cultivated Meliaceae species, Chukrasia tabularis, allowing the preparation of libiguins in appreciable quantities.
Assuntos
Limoninas/síntese química , Limoninas/química , Limoninas/isolamento & purificação , Meliaceae/química , Conformação MolecularRESUMO
In a screening programme directed towards the discovery of drugs that could enhance sexual activity, we found that a decoction of the root bark of Neobeguea mahafalensis displayed an extraordinarily high potency and remarkably long duration in augmenting sexual activity in male rodents. Bioassay-guided fractionation led to the isolation of two pharmacoactive constituents, which turned out to be novel 1,8,9-orthoacetate phragmalin limonoids that we named libiguins A and B, each with a C-16/30 δ-lactone ring. Chemical structures were established by the interpretation of their 1D and 2D NMR data. In vivo pharmacological tests showed that starting with a treatment from 0.004-0.4 mg/kg/day for three consecutive days, over a 3-h sampling period, these limonoids induced a long-lasting augmentation of frequency and sustainment of mounting behaviour in male rodents, with an effect lasting for up to 11 days post-treatment. Libiguin A proved to be markedly more potent than libiguin B. This report is the first of limonoids having such an effect, and the findings could lead to novel therapies for the treatment of sexual dysfunction. Moreover, the results can serve as an opening to elucidate the central physiological control of mating behaviour, which is still not well mapped out.
Assuntos
Afrodisíacos/farmacologia , Limoninas/farmacologia , Meliaceae/química , Extratos Vegetais/farmacologia , Comportamento Sexual/efeitos dos fármacos , Animais , Afrodisíacos/isolamento & purificação , Limoninas/química , Limoninas/isolamento & purificação , Masculino , Camundongos , Estrutura Molecular , Casca de Planta , Extratos Vegetais/química , Raízes de Plantas , RatosRESUMO
Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-nitroprop-1-enylbenzene to diethyl malonate. Following the chromatographic separation of the methyl erythro- and threo-4-nitro-3R- and 3S-phenylpentanoate diastereoisomers, target compounds were obtained by their reductive cyclisation into 5-methyl-4-phenylpyrrolidin-2-one enantiomers and the attachment of the acetamide group to the heterocyclic nitrogen. Experiments with electrically stimulated rat vas deference contractions induced by the PRE-084, an agonist of sigma-1 receptor, showed that (4R,5S)- and (4R,5R)-2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamides with an R-configuration at the C-4 chiral centre in the 2-pyrrolidone ring were more effective positive allosteric modulators of sigma-1 receptor than were their optical antipodes.
Assuntos
Acetamidas/síntese química , Acetamidas/farmacologia , Receptores sigma/química , Acetamidas/química , Regulação Alostérica , Animais , Ratos , Ratos Wistar , Estereoisomerismo , Relação Estrutura-Atividade , Receptor Sigma-1RESUMO
In this article, we describe the characteristic (15)N and (1)HN NMR chemical shifts and (1)J((15)N-(1)H) coupling constants of various symmetrically and unsymmetrically substituted 1,4-dihydropyridine derivatives. The NMR chemical shifts and coupling constants are discussed in terms of their relationship to structural features such as character and position of the substituent in heterocycle, N-alkyl substitution, nitrogen lone pair delocalization within the conjugated system, and steric effects.
Assuntos
Di-Hidropiridinas/química , Espectroscopia de Ressonância Magnética , Teoria Quântica , Isótopos de NitrogênioRESUMO
Chiral, nonracemic o-aminobenzylamines were prepared in a highly diastereoselective reduction of atropisomeric N-tert-butanesulfinylketimines. The ortho-substituent ensures the distinct reactivity of atropisomers 4d-f. The free energy of activation for atropisomerization of sulfinylimines 4d-f in THF-d(8) was determined by NMR methods to range from 70.8 to 97.9 kJ/mol.
RESUMO
The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase.
Assuntos
Proteínas de Bactérias/química , DnaB Helicases/química , Zinco/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , DnaB Helicases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
The diastereotopy of the methylene protons at positions 2 and 6 in 1,4-dihydropiridine derivatives with various substituents has been investigated. NMR spectroscopy and quantum chemistry calculations show that the CH···O intramolecular hydrogen bond is one of the factors amplifying the chemical shift differences in the 1H-NMR spectra.
Assuntos
Di-Hidropiridinas/química , Di-Hidropiridinas/síntese química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
Chiral, nonracemic 1,3-diamines were prepared in a highly diastereoselective reduction of diaryl N-tert-butanesulfinylketimines. Correlation between facial selectivity of the reduction and E or Z geometry of the starting ketimines suggests involvement of a cyclic transition state for the reduction. The ortho-substituent controls the geometry of N-tert-butanesulfinylketimines in the solid state and provides additional stabilization of the cyclic transition state.
RESUMO
Carnitine acetyltransferase (CrAT; EC 2.3.1.7) catalyzes the reversible transfer of acetyl groups between acetyl-coenzyme A (acetyl-CoA) and L-carnitine; it also regulates the cellular pool of CoA and the availability of activated acetyl groups. In this study, biochemical measurements, saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, and molecular docking were applied to give insights into the CrAT binding of a synthetic inhibitor, the cardioprotective drug mildronate (3-(2,2,2-trimethylhydrazinium)-propionate). The obtained results show that mildronate inhibits CrAT in a competitive manner through binding to the carnitine binding site, not the acetyl-CoA binding site. The bound conformation of mildronate closely resembles that of carnitine except for the orientation of the trimethylammonium group, which in the mildronate molecule is exposed to the solvent. The dissociation constant of the mildronate CrAT complex is approximately 0.1 mM, and the K(i) is 1.6 mM. The results suggest that the cardioprotective effect of mildronate might be partially mediated by CrAT inhibition and concomitant regulation of cellular energy metabolism pathways.
Assuntos
Fármacos Cardiovasculares/farmacologia , Carnitina O-Acetiltransferase/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Metilidrazinas/farmacologia , Animais , Sítios de Ligação , Biocatálise , Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/metabolismo , Carnitina O-Acetiltransferase/química , Carnitina O-Acetiltransferase/metabolismo , Columbidae , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Espectroscopia de Ressonância Magnética , Metilidrazinas/química , Metilidrazinas/metabolismo , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Two molecules of indole derivative, e.g. indole-5-carboxylic acid, reacted with one molecule of thiol, e.g. 1,2-ethanedithiol, in the presence of trifluoroacetic acid to yield adducts such as 3-[2-(2-amino-5-carboxyphenyl)-1-(2-mercaptoethylthio)ethyl]-1Hindole-5-carboxylic acid. Parallel formation of dimers, such as 2,3-dihydro-1H,1'H-2,3'-biindole-5,5'-dicarboxylic acid and trimers, such as 3,3'-[2-(2-amino-5-carboxyphenyl) ethane-1,1-diyl]bis(1H-indole-5-carboxylic acid) of the indole derivatives was also observed. Reaction of a mixture of indole and indole-5-carboxylic acid with 2-phenylethanethiol proceeded in a regioselective way, affording 3-[2-(2-aminophenyl)-1-(phenethylthio)ethyl]-1H-indole-5-carboxylic acid. An additional product of this reaction was 3-[2-(2-aminophenyl)-1-(phenethylthio)ethyl]-2,3-dihydro-1H,1'H-2,3'-biindole-5'-carboxylic acid, which upon standing in DMSO-d6 solution gave 3-[2-(2-aminophenyl)-1-(phenethylthio)ethyl]-1H,1'H-2,3'-biindole-5'-carboxylic acid. Structures of all compounds were elucidated by NMR, and a mechanism for their formation was suggested.
Assuntos
Indóis/química , Compostos de Sulfidrila/química , Dimerização , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
[This corrects the article DOI: 10.1098/rsos.180088.].
RESUMO
1,4-Dihydropyridine (1,4-DHP) derivatives have been synthesized and characterized by 1H, 13C, 15N nuclear magnetic resonance (NMR) spectroscopy, secondary proton/deuterium 13C isotope shifts, variable temperature 1H NMR experiments and quantum-chemical calculation. The intramolecular hydrogen bonds NHâ¯O=C and CHâ¯O=C in these compounds were established by NMR and quantum-chemical studies The downfield shift of the NH proton, accompanied by the upfield shift of the 15N nuclear magnetic resonance signals, the shift to the higher wavenumbers of the NH stretching vibration in the infrared spectra and the increase of the 1J(15N,1H) values may indicate the shortening of the N-H bond length upon intramolecular NHâ¯O=C hydrogen bond formation.
RESUMO
The human Wnt-binding protein Wnt-inhibitory factor-1 (WIF-1) comprises an N-terminal WIF module followed by five EGF-like repeats. Here we report the three-dimensional structure of the WIF domain of WIF-1 determined by NMR spectroscopy. The fold consists of an eight-stranded beta-sandwich reminiscent of the immunoglobulin fold. Residual detergent (Brij-35) used in the refolding protocol was found to bind tightly to the WIF domain. The binding site was identified by intermolecular nuclear Overhauser effects observed between the WIF domain and the alkyl chain of the detergent. The results point to a possible role of WIF domains as a recognition motif of Wnt and Drosophila Hedgehog proteins that are activated by palmitoylation.
Assuntos
Proteínas de Transporte/química , Espectroscopia de Ressonância Magnética , Proteínas Repressoras/química , Proteínas Wnt/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog , Humanos , Dados de Sequência Molecular , Polidocanol , Polietilenoglicóis/metabolismo , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Proteínas Wnt/metabolismoRESUMO
Mature lung surfactant protein C (SP-C) corresponds to residues 24-58 of the 21 kDa proSP-C. A late processing intermediate, SP-Ci, corresponding to residues 12-58 of proSP-C, lacks the surface activity of SP-C, and the SP-Ci alpha-helical structure does not unfold in contrast to the metastable nature of the SP-C helix. The NMR structure of an analogue of SP-Ci, SP-Ci(1-31), with two palmitoylCys replaced by Phe and four Val replaced by Leu, in dodecylphosphocholine micelles and in ethanol shows that its alpha-helix vs. that of SP-C is extended N-terminally. The Arg-Phe part in SP-Ci that is cleaved to generate SP-C is localized in a turn structure, which is followed by a short segment in extended conformation. Circular dichroism spectroscopy of SP-Ci(1-31) in microsomal or surfactant lipids shows a mixture of helical and extended conformation at pH 6, and a shift to more unordered structure at pH 5. Replacement of the N-terminal hexapeptide segment SPPDYS (known to constitute a signal in intracellular targeting) of SP-Ci with AAAAAA results in a peptide that is mainly unstructured, independent of pH, in microsomal and surfactant lipids. Addition of a synthetic dodecapeptide, corresponding to the propeptide part of SP-Ci, to mature SP-C results in slower aggregation kinetics and altered amyloid fibril formation, and reduces the surface activity of phospholipid-bound SP-C. These data suggest that the propeptide part of SP-Ci prevents unfolding by locking the N-terminal part of the helix, and that acidic pH results in structural disordering of the region that is proteolytically cleaved to generate SP-C.
Assuntos
Proteína C Associada a Surfactante Pulmonar/química , Animais , Dicroísmo Circular , Estabilidade de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Micelas , Microscopia Eletrônica , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Precursores de Proteínas/ultraestrutura , Proteína C Associada a Surfactante Pulmonar/metabolismo , Proteína C Associada a Surfactante Pulmonar/ultraestrutura , Coelhos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , SuínosRESUMO
A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10 degrees C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG=2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min(-1) measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E.coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Inteínas/fisiologia , Dobramento de Proteína , Adenosina Trifosfatases/genética , Amidas/química , Sequência de Aminoácidos , Dicroísmo Circular , Ciclização , DNA Helicases/genética , DnaB Helicases , Entropia , Escherichia coli/enzimologia , Escherichia coli/genética , Inteínas/genética , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Desnaturação Proteica , Estrutura Terciária de Proteína , Prótons , Espectrometria de Massas por Ionização por Electrospray , Termodinâmica , Ureia/farmacologiaRESUMO
Lunasin is a 43 amino acid peptide with anti-cancer, antioxidant, anti-inflammatory and cholesterol-lowering properties. Although the mechanism of action of lunasin has been characterized to some extent, its exact three-dimensional structure as well as the function of the N-terminal sequence remains unknown. We established a novel method for the production of recombinant lunasin that allows efficient isotope labeling for NMR studies. Initial studies showed that lunasin can exist in a reduced or oxidized state with an intramolecular disulfide bond depending on solution conditions. The structure of both forms of the peptide at pH 3.5 and 6.5 was characterized by CD spectroscopy and multidimensional NMR methods. The data indicate that lunasin belongs to the class of intrinsically disordered proteins. The analysis of secondary structure propensities indicates the presence of two helical regions and an extended (beta strand) conformation at the C-terminus. We hypothesize that the transient secondary structure elements could be stabilized upon interaction with the histones H3 and H4. The newly discovered redox properties of lunasin could explain its antioxidant and anti-inflammatory activity.
Assuntos
Antioxidantes/química , Proteínas Intrinsicamente Desordenadas/química , Peptídeos/química , Proteínas de Plantas/química , Acetilação , Sequência de Aminoácidos/genética , Anticarcinógenos/química , Anticarcinógenos/uso terapêutico , Antioxidantes/uso terapêutico , Dissulfetos/química , Histonas/química , Histonas/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Neoplasias/tratamento farmacológico , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Conformação Proteica em alfa-Hélice , Proteínas de Soja/química , Proteínas de Soja/metabolismoRESUMO
The cyclization of arylalkynes under selenobromination conditions, combined with an acid-induced 3,2-aryl shift, was elaborated as a general synthetic pathway for the preparation of polyhydroxy-2- and -3-arylbenzo[b]selenophenes from the same starting materials. The redox properties, free-radical-scavenging ability, and cytotoxicity against malignant cell lines (MCF-7, MDA-MB-231, HepG2, and 4T1) of the synthesized compounds were explored, and the obtained results were used to consider the structure-activity relationships (SARs) in these compounds. Consequently, the structural features that were responsible for the highly potent peroxyl-radical-scavenging activity were established.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Compostos Organosselênicos/química , Compostos Organosselênicos/farmacologia , Antineoplásicos/síntese química , Antioxidantes/síntese química , Derivados de Benzeno/síntese química , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclização , Humanos , Neoplasias/tratamento farmacológico , Compostos Organosselênicos/síntese química , Oxirredução/efeitos dos fármacosRESUMO
SOCS3 is a negative regulator of cytokine signalling that inhibits Janus kinase-signal transduction and activator of transcription (JAK-STAT) mediated signal tranduction by binding to phosphorylated tyrosine residues on intracellular subunits of various cytokine receptors, as well as possibly the JAK proteins. SOCS3 consists of a short N-terminal sequence followed by a kinase inhibitory region, an extended SH2 domain and a C-terminal suppressor of cytokine signalling (SOCS) box. SOCS3 and the related protein, cytokine-inducible SH2-containing protein, are unique among the SOCS family of proteins in containing a region of mostly low complexity sequence, between the SH2 domain and the C-terminal SOCS box. Using NMR, we assigned and determined the secondary structure of a murine SOCS3 construct. The SH2 domain, unusually, consists of 140 residues, including an unstructured insertion of 35 residues. This insertion fits the criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry. Instead, we propose that the PEST sequence has a functional role unrelated to phosphotyrosine binding, possibly mediating efficient proteolytic degradation of the protein. The latter half of the kinase inhibitory region and the entire extended SH2 subdomain form a single alpha-helix. The mapping of the true SH2 domain, and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close to the SOCS box for phosphotyrosine binding.
Assuntos
Estrutura Secundária de Proteína , Proteínas Supressoras da Sinalização de Citocina/química , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfotirosina/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA. In this work the 3D structure of the R3H domain from human Smubp-2 was determined by NMR spectroscopy. It is the first 3D structure determination of an R3H domain. The fold presents a small motif, consisting of a three-stranded antiparallel beta-sheet and two alpha-helices, which is related to the structures of the YhhP protein and the C-terminal domain of the translational initiation factor IF3. The similarities are non-trivial, as the amino acid identities are below 10%. Three conserved basic residues cluster on the same face of the R3H domain and could play a role in nucleic acid recognition. An extended hydrophobic area at a different site of the molecular surface could act as a protein-binding site. A strong correlation between conservation of hydrophobic amino acids and side-chain solvent protection indicates that the structure of the Smubp-2 R3H domain is representative of R3H domains in general.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli , Ressonância Magnética Nuclear Biomolecular , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fator de Iniciação 3 em Procariotos/química , Estrutura Terciária de Proteína , Alinhamento de Sequência , Soluções , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L.