RESUMO
Permeation of drugs across lipid bilayers is a key factor in dictating how effective they will be. In vivo, the issue is compounded by the presence of drug-exporter proteins such as P-glycoprotein. However, despite intense effort, exactly what controls permeation and susceptibility to export is still poorly understood. In this work we examine two well-studied drugs for which interaction with P-glycoprotein has been studied before: amitriptyline, a known substrate and clozapine, which is not a substrate. Extensive MD simulations, including potential of mean force (PMF) profiles of the compounds in all possible protonation states, reveal that the preferred location of the compounds in different bilayers in different protonation states is remarkably similar. For both molecules in charged states, there is a substantial barrier to crossing the bilayer. Clozapine however, shows an energetic barrier to movement across the bilayer even in a protonation state that results in an uncharged molecule. For amitriptyline there is only a very small barrier of approximately 1.3 kcal mol(-1). Further analysis revealed that the conformational and orientational behavior of the two compounds was also similar, with the sidechain interacting with the lipid headgroups. This effect was much stronger if the sidechain was charged (protonated). These interactions with lipid bilayers were confirmed by NMR ROESY experiments. The results are discussed in terms of their potential interactions with export proteins like P-glycoprotein.
Assuntos
Clozapina/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Dinâmica Molecular , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Prótons , TermodinâmicaRESUMO
Drug export from cells is a major factor in the acquisition of cellular resistance to antimicrobial and cancer chemotherapy, and poses a significant threat to future clinical management of disease. Many of the proteins that catalyse drug efflux do so with remarkably low substrate specificity, a phenomenon known as multidrug transport. For these reasons we need a greater understanding of drug recognition and transport in multidrug pumps to inform research that attempts to circumvent their action. Structural and computational studies have been heralded as being great strides towards a full elucidation of multidrug recognition and transport. In this review we summarise these advances and ask how close we are to a molecular understanding of this remarkable phenomenon.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/química , Animais , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/química , Transporte Biológico , Farmacorresistência Bacteriana , Resistencia a Medicamentos Antineoplásicos , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent KD values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Cardiolipinas/metabolismo , Resistência a Múltiplos Medicamentos , Espectrometria de Massas/métodos , Nucleotídeos/metabolismo , Preparações Farmacêuticas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Ligação Competitiva , Transporte Biológico , Cardiolipinas/química , Análise por Conglomerados , Ciclosporina/química , Ciclosporina/metabolismo , Detergentes/química , Detergentes/metabolismo , Humanos , Imunossupressores/química , Imunossupressores/metabolismo , Cinética , Lipídeos/química , Lipídeos/classificação , Modelos Moleculares , Conformação Molecular , Nucleotídeos/química , Preparações Farmacêuticas/química , Preparações Farmacêuticas/classificação , Ligação Proteica , Conformação ProteicaRESUMO
By far the most studied multidrug resistance protein is P-glycoprotein. Despite recent structural data, key questions about its function remain. P-glycoprotein (P-gp) is flexible and undergoes large conformational changes as part of its function and in this respect, details not only of the export cycle, but also the recognition stage are currently lacking. Given the flexibility, molecular dynamics (MD) simulations provide an ideal tool to examine this aspect in detail. We have performed MD simulations to examine the behaviour of P-gp. In agreement with previous reports, we found that P-gp undergoes large conformational changes which tended to result in the nucleotide-binding domains coming closer together. In all simulations, the approach of the NBDs was asymmetrical in agreement with previous observations for other ABC transporter proteins. To validate the simulations, we make extensive comparison to previous cross-linking data. Our results show very good agreement with the available data. We then went on to compare the influence of inhibitor compounds bound with simulations of a substrate (daunorubicin) bound. Our results suggest that inhibitors may work by keeping the NBDs apart, thus preventing ATP-hydrolysis. On the other hand, repeat simulations of daunorubicin (substrate) in one particular binding pose suggest that the approach of the NBDs is not impaired and that the structure would be still be competent to perform ATP hydrolysis, thus providing a model for inhibition or substrate transport. Finally we compare the latter to earlier QSAR data to provide a model for the first part of substrate transport within P-gp.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Ligantes , Camundongos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade por SubstratoAssuntos
Reagentes de Ligações Cruzadas/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Ácidos Cetoglutáricos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/química , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Ácidos Cetoglutáricos/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
2-Oxoglutarate-dependent nucleic acid demethylases are of biological interest because of their roles in nucleic acid repair and modification. Although some of these enzymes are linked to physiology, their regulatory roles are unclear. Hence, there is a desire to develop selective inhibitors for them; we report studies on AlkB, which reveal it as being amenable to selective inhibition by small molecules. Dynamic combinatorial chemistry linked to mass spectrometric analyses (DCMS) led to the identification of lead compounds, one of which was analyzed by crystallography. Subsequent structure-guided studies led to the identification of inhibitors of improved potency, some of which were shown to be selective over two other 2OG oxygenases. The work further validates the use of the DCMS method and will help to enable the development of inhibitors of nucleic acid modifying 2OG oxygenases both for use as functional probes and, in the longer term, for potential therapeutic use.
Assuntos
Cisteína/análogos & derivados , Proteínas de Escherichia coli/antagonistas & inibidores , Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/antagonistas & inibidores , Piridinas/síntese química , Domínio Catalítico , Técnicas de Química Combinatória , Cristalografia por Raios X , Cisteína/síntese química , Cisteína/química , Ensaios Enzimáticos , Proteínas de Escherichia coli/química , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Ácidos Cetoglutáricos/química , Oxigenases de Função Mista/química , Modelos Moleculares , Ligação Proteica , Piridinas/química , Quinolinas/síntese química , Quinolinas/química , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
The efficacy of gamma-secretase inhibitors in vivo has, to date, been generally assessed in transgenic mouse models expressing increased levels of amyloid-beta (Abeta) peptide thereby allowing the detection of changes in Abeta production. However, it is not clear whether the in vivo potency of gamma-secretase inhibitors is independent of the level of amyloid precursor protein expression. In other words, does a gamma-secretase inhibitor have the same effect in nontransgenic physiological animals versus transgenic overexpressing animals? In the present study, an immunoassay has been developed which can detect Abeta(40) in the rat brain, where concentrations are much lower than those seen in transgenic mice such as Tg2576 (c. 0.7 and 25 nM, respectively) and in cerebrospinal fluid (CSF, c. 0.3 nM). Using this immunoassay, the effects of the gamma-secretase inhibitor LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide] were assessed and robust dose-dependent reductions in rat brain and CSF Abeta(40) levels were observed with ID(50) values of 1.3 mg/kg for both brain and CSF. These values were comparable with those calculated for LY-411575 in transgenic mice. Time course experiments using LY-411575 demonstrated comparable temporal reductions in rat brain and CSF Abeta(40), further suggesting these two pools of Abeta are related. Accordingly, when all the data for the dose-response curve and time course were correlated, a strong association was observed between the brain and CSF Abeta(40) levels. These data demonstrate the utility of the rat as a novel approach for assessing the effects of gamma-secretase inhibitors on central nervous system Abeta(40) levels in vivo.