膜蛋白与膜蛋白组学

膜蛋白是指能够结合或整合到细胞或细胞器的膜上的蛋白质的总称。现在市场上销售的药物80％以上都是作用在膜蛋白上的,膜蛋白是理想的药物靶点,因此,对膜蛋白的研究具有十分重要意义。但由于膜蛋白的溶解、分离、鉴定十分困难,造成膜蛋白组学研究的滞后。本文就膜蛋白的结构,膜蛋白在细胞生理和人类疾病中扮演的重要角色,膜蛋白作为药物靶点的潜力以及膜蛋白组学研究技术作一综述。     

天然抗肿瘤药物筛选方法的研究进展

目前发现许多天然药物具有抗肿瘤作用. 随着细胞生物学、分子药理学和肿瘤药理学研究的发展,针对细胞和分子靶点的天然药物已成为当今抗肿瘤药物研究的重要方向. 在研究过程中,建立合理的抗肿瘤药物筛选方法显得尤为重要. 详细介绍近年来天然抗肿瘤药物的筛选方法,为抗肿瘤药物的研究与开发提供参考.     

膜蛋白与膜蛋白组学

膜蛋白是指能够结合或整合到细胞或细胞器的膜上的蛋白质的总称。现在市场上销售的药物80％以上都是作用在膜蛋白上的,膜蛋白是理想的药物靶点,因此,对膜蛋白的研究具有十分重要意义。但由于膜蛋白的溶解、分离、鉴定十分困难,造成膜蛋白组学研究的滞后。本文就膜蛋白的结构,膜蛋白在细胞生理和人类疾病中扮演的重要角色,膜蛋白作为药物靶点的潜力以及膜蛋白组学研究技术作一综述。     

膜蛋白结构预测的研究进展

综述近年来膜蛋白结构预测的研究进展，分类介绍了α螺旋和β桶状跨膜蛋白结构预测方法之基本原理和应用。作为重要的基因产物之一，跨膜蛋白在生物体中发挥各种重要功能，而膜蛋白偶联受体是大多数药物的作用靶点。基于结构的药物设计是目前药物研发的主要方法，可膜蛋白的三维结构很难采用x-光晶体衍射和核磁共振技术测定，因此应用生物信息学方法预测膜蛋白结构成为当今研究热点。     

以DNA为靶点的含糖基药物的研究进展

目的综述以DNA为作用靶点的含糖类药物研究及其糖基部分在药物活性中的重要作用,为海洋药物的研究提供参考。方法以近几年国内外30篇有代表性的文献以相关文献为依据进行整理、归纳、分析总结。结果与结论药物分子中的糖部分对DNA的识别及选择位点起了关键的作用,含有糖基的抗肿瘤抗生素药物的研究已成为生物化学和医学领域的前沿。同时,对从海洋生物中提取的活性化合物进行糖基化修饰,多渠道寻找活性先导化合物,也是海洋药物以及其他天然药物研究与开发的有效途径。     

分子蒸馏技术在天然药物分离纯化中的应用

目的介绍国内分子蒸馏技术在天然药物分离纯化中的应用状况。方法依据文献资料结合自身的实际工作成果,讨论分子蒸馏在国内天然药物分离纯化中的工艺研究状况及设备研制水平。结果分子蒸馏在国内天然药物分离纯化上得到了越来越广泛的应用,其工艺研究及设备制造水平已逐渐趋向成熟。结论分子蒸馏作为液—液热敏分离及高提纯的专用技术,其在天然药物分离纯化中的应用前景非常看好。     

分子蒸馏技术及其在药学领域的应用

目的介绍分子蒸馏技术在药学领域中的应用进展。方法以国内外相关文献为主要依据,对分子蒸馏技术的原理、特点、设备种类及在药学领域中的应用等方面进行综述。结果归纳了分子蒸馏技术在药学领域中的应用。包括对天然物中功能性脂肪酸、脂溶性微量成分、挥发油等活性物质的提取纯化,及对药物合成粗产物中目标药的富集提纯。结论分子蒸馏技术可在天然药物与合成药物中的活性物质的分离、浓缩与纯化过程中发挥独特功效。     

分子蒸馏技术在天然药物分离纯化中的应用

目的介绍国内分子蒸馏技术在天然药物分离纯化中的应用状况。方法依据文献资料结合自身的实际工作成果．讨论分子蒸馏在国内天然药物分离纯化中的工艺研究状况及设备研制水平。结果分子蒸馏在国内天然药物分离纯化上得到了越来越广泛的应用，其工艺研究及设备制造水平已逐渐趋向成熟。结论分子蒸馏作为液一液热敏分离及高提纯的专用技术，其在天然药物分离纯化中的应用前景非常看好。     

多靶点抗肿瘤天然产物研究进展

天然产物及其衍生物是抗肿瘤药物的重要组成部分.目前在临床上应用的天然产物来源的抗肿瘤药物以传统细胞毒类药物或靶向某一特定靶点的分子靶向药物为主,其应用受限于药物相关不良反应和肿瘤耐药.近年的研究表明,抗肿瘤活性天然产物往往能够靶向肿瘤细胞的多个靶点,影响肿瘤发生发展中的多个过程.由于肿瘤是一种多因素诱发的系统性疾病,多...     

基于全球上市药物数据的抗阿尔茨海默病重定位新药发现

目的从上市药物中寻找潜在的多靶点抗阿尔茨海默病(AD)药物。方法本文利用实验室前期基于机器学习算法建立的抗AD多靶点药物预测平台,对全球上市药物数据库进行了预测和挖掘。结果从全球上市药物数据库中预测出13个至少作用于1个抗AD药物靶点,且可以通过多种作用对抗AD的上市药物,利用Cytoscape构建化合物——靶点网络;并针对上市药物阿戈美拉汀及其预测的多靶点蛋白进行分子对接以验证预测结果,蛋白靶点包括ADORA2A,ACHE,BACE1,PTGS2,MAOB,SIGMAR1和ESR1,阿戈美拉汀均能与以上靶点产生相互作用。结论本研究应用机器学习算法、网络药理学方法及分子对接方法,初步揭示了上市药物数据库中抗AD作用的潜在药物,为上市药物抗AD作用重定位提供了重要信息。     

A reversed phase HPLC assay for the simultaneous quantitation of non-ionic and ionic surfactants in bioprocess intermediates

This report describes a rapid and accurate reversed phase HPLC method for the simultaneous quantitation of multiple surfactants in various bioprocess solution matrices including cell lysates. Separation and quantitation of a mixture of the cationic detergent domiphen bromide from the non-ionic detergent Triton X-100 in crude cell mixtures can be achieved within 15 min using a TSK-gel C18-NPR reversed phase column and an aqueous mobile phase gradient of acetonitrile:water with the reagent PIC-B8 as ion-pairing modifier. The linear dynamic range for quantitation of domiphen bromide (DB) and Triton in this assay extends from 20 to 2000 microM. Linear regression analyses from the standard curve determinations showed an R2 of > or = 0.990. The assay does not show any interferences from proteins or other cellular contaminants such as nucleic acids. The assay has been used to evaluate clearance of these compounds throughout the purification process of an adenovirus-based vaccine candidate, as well as to determine the effects of process changes on detergent clearance.     

Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system

Abstract: The cannabinergic system is present in a variety of organs and tissues that perform a wide range of essential physiologic functions making it an inherently important therapeutic target for drug discovery. In order to augment our knowledge regarding the interactions between cannabinoid receptors (CBs) and their ligands, efficient and effective tools are essential for robust expression and purification of these membrane‐bound proteins. In this report, we describe a suitable method for purification of the human cannabinoid receptor 2 (CB2) to a qualitative and quantitative level sufficient for mass spectral analysis. We utilized a baculovirus expression system, incorporating several epitope tags to facilitate purification and to ameliorate the effect the tags have on CB2 expression and function. Expressed protein encoded by a carboxy (C)‐terminal His‐tagged CB2 construct displayed a B_max value of 9.3 pmol/mg with a K_D of 7.30 nm using [3_H]CP–55⁹⁴⁰, a standard cannabinoid radioligand, and was selected for subsequent purification experiments. Western blot analysis of purified membrane protein yielded several forms of CB2, the most abundant being a 41 kDa peptide. A second protein species was observed with an apparent molecular weight of 46 kDa representing a glycosylated form of CB2. In addition, a CB2 homodimer was also identified. The purified receptor was subjected to mass spectroscopic analysis to confirm its identity and purity. Mass spectra corresponding to the intracellular, extracellular and transmembrane domains were obtained. These experiments exemplify the importance of high‐level expression systems when developing membrane‐bound protein purification strategies. This work will aid in the identification of receptor–ligand binding sites, the characterization of molecular features involved in receptor activation, and the elucidation of the CB2 receptor tertiary structure.     

Neurotoxic esterase: characterization of the solubilized enzyme and the conditions for its solubilization from chicken brain microsomal membranes with ionic, zwitterionic, or nonionic detergents

Neurotoxic esterase (NTE) is a membrane-bound protein found in highest concentration in brain and lymphocytes. The enzyme has no known physiological function, but its organophosphorylation and aging in neural tissue are thought to trigger the pathogenesis of organophosphorus-induced delayed neuropathy (OPIDN). Solubilization of NTE from microsomal membranes from hen or chick brain was studied with ten detergents encompassing ionic, zwitterionic, or nonionic types. Corrected yields of NTE solubilized over a range of [detergent]/[protein] ratios were determined by dividing the activity not sedimenting in detergent at 100,000 g for 60 min at 4 degrees by the activity in the original microsomal fraction with no detergent present. Highest corrected yields were obtained with sodium cholate (44%), Triton X-100 (48%), and nonyl-GPS (57%). Partial loss of NTE activity occurred in the presence of detergent which could be prevented by the inclusion of asolectin in the solubilization preparation. NTE could not be solubilized by omitting detergent or by substituting 2 M NaCl for detergent. Mipafox pI50 values obtained from complete titration curves carried out on NTE solubilized in Triton X-100, sodium cholate, or sodium cholate/asolectin were indistinguishable from the value for native enzyme from brain homogenate. These results indicate that NTE exhibits the properties of an integral membrane protein with lipid dependence. The enzyme can be solubilized in good yield with a variety of detergents with retention of its characteristic differential inhibition by paraoxon and mipafox, a necessary prelude to bulk purification of the enzymatically active protein.     

Characterization of membrane protein reconstitution in LUVs of different lipid composition by fluorescence anisotropy

A major requirement to perform structural studies with membrane proteins is not only to define efficient reconstitution protocols, that assure a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. For this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. In this study we report the reconstitution of OmpF in preformed DMPC and E. coli liposomes using two different techniques for detergent removal: (1) exclusion chromatography and (2) incubation with detergent-adsorbing beads. The incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine OmpF effect on the structural order of membrane lipids. These results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. Furthermore, it is possible to state that although the insertion is directly related to the size distributions of proteoliposomes, it could be efficiently recognized by steady-state fluorescence anisotropy. This technique, more popular among cell biologists, can be a very practical and straightforward alternative to DLS to confirm membrane protein insertion.     

Identification and Quantification of a Problematic Host Cell Protein to Support Therapeutic Protein Development

Monitoring of residual host cell proteins (HCPs) in therapeutic protein is essential to ensure product quality, safety and efficacy. Despite the development of advanced mass spectrometry techniques and optimized workflows, identifying and quantifying all problematic HCPs present at low levels remain challenging. Here, we developed a practical, effective strategy for the identification and quantification of low abundance HCPs, which facilitates the improvement of downstream purification process to eliminate potentially problematic HCPs. A case study of using this strategy to investigate a problematic HCP is presented. Initially, a commonly used native digestion approach coupled with UPLC-MS/MS was applied for HCP profiling, wherein several lipases and proteases were identified in a monoclonal antibody named mAb1 in early stages of purification process development. A highly active lipase, liver carboxylesterase (CES), was found to be responsible for polysorbate 80 degradation. To facilitate process improvement, after the identification of CES, we developed a highly sensitive LC-MS/MS-MRM assay with a lower limit of quantification of 0.05 ppm for routine monitoring of the CES in mAb1 produced through the different processes. This workflow was applied in low-level lipase identification and absolute quantification, which facilitated the investigation of polysorbate degradation and downstream purification improvement to further remove the problematic HCP. The current MRM method increased the sensitivity of HCP quantification by over 10-fold that in previously published studies, thus meeting the needs for quantification of problematic HCPs at sub-ppm to ppb levels during drug development. This workflow could be readily adapted to the detection and quantification of other problematic HCPs present at extremely low levels in therapeutic protein drug candidates.     

Effect of surfactant monomers on chloramphenicol association to an albumin-lecithin complex: a model for modified drug absorption.

The binding of chloramphenicol to an albumin-lecithin complex in the presence or absence of premicellar concentrations of both ionic and non-ionic surfactants has been examined. Long chain, strong ionic detergents, such as sodium dodecyl sulphate or cetyltrimethylammonium bromide, severely perturb protein structure and eventually allow full separation of the complex into lecithin and albumin-detergent complexes. The dissociation process is reversible upon the removal of the detergent by exhaustive dialysis. After the splitting of the complex, the amount of antibiotic associated with the lipid-protein mixture increases. Structural alteration of the albumin-lecithin complex and the increase in the binding of chloramphenicol have an effect on the transfer rate of this antibiotic across an artificial barrier consisting of an aqueous dispersion of the same complex, as observed in a model system. It is suggested that a reversible alteration in membrane structure, and consequently in membrane permeability, might be easily effected, at the molecular level, through a reversible dissociation of structural lipoproteins into their components, operated by premicellar concentrations of ionic surfactants. This represents a tentative picture of the possible events taking place within the membrane and modifying the absorption rate of a drug, when it is associated with surfactants in a pharmaceutical preparation.     

Purification of cytochrome P-450 and NADPH cytochrome p-450 reductase from human liver

Two methods for the purification of cytochromes-P450 from microsomes of human liver are described. Method A: Cyt-P450 were solubilized from microsomes using a non ionic detergent, the Lubrol. The Cyt-P450 were purified by affinity, hydrophobicity followed by ion-exchange chromatography on DEAE-5PW column (HPLC) with an overall yield of 18% and a specific activity of 10 nmole/mg of protein. The recovery of NADPH Cyt-P450 reductase by method A (affinity) is about 60% with a specific activity of 16.2 U.I./mg of protein. Method B: Cyt-P450 were solubilized from microsomes using a zwitterionic detergent, the CHAPS. Cyt-P450 were filtered and separated by chromatofocusing on Mono-P column (HPLC). By this method it was possible to increase strongly the specific activity keeping a yield of 50% of Cyt-P450. Also it was possible to apply this method to small samples of human liver like biopsies (0.5 to 2.5 g).     

NMR-Based Screening of Membrane Protein Ligands

Membrane proteins pose problems for the application of NMR-based ligand-screening methods because of the need to maintain the proteins in a membrane mimetic environment such as detergent micelles: they add to the molecular weight of the protein, increase the viscosity of the solution, interact with ligands non-specifically, overlap with protein signals, modulate protein dynamics and conformational exchange and compromise sensitivity by adding highly intense background signals. In this article, we discuss the special considerations arising from these problems when conducting NMR-based ligand-binding studies with membrane proteins. While the use of ¹³C and ¹⁵N isotopes is becoming increasingly feasible, ¹⁹F and ¹H NMR-based approaches are currently the most widely explored. By using suitable NMR parameter selection schemes independent of or exploiting the presence of detergent, ¹H-based approaches require least effort in sample preparation because of the high sensitivity and natural abundance of ¹H in both, ligand and protein. On the other hand, the ¹⁹F nucleus provides an ideal NMR probe because of its similarly high sensitivity to that of ¹H and the lack of natural ¹⁹F background in biologic systems. Despite its potential, the use of NMR spectroscopy is highly underdeveloped in the area of drug discovery for membrane proteins.     

Progress of drug transport study based on absolute quantitative method for membrane transporter proteins

Knowing the amount of membrane transporter expression in human tissue is one of the key issues in the rational and reliable prediction of pharmacokinetic profiles in humans. Recently, we have developed a simultaneous and highly sensitive method for the absolute quantification of multiple membrane transporter proteins in mammalian tissues. To develop quantitative analysis of high molecular-weight membrane proteins, we have solved problems using proteomics technology as follows: 1) The target proteins are detected via tryptic peptides that can be dissolved and analyzed with LC-MS/MS, while membrane protein is difficult to dissolve. 2) LC-MS/MS in multiple reaction monitoring (MRM) mode produce a highly sensitive and selective response for transporter proteins with low expression by separation from highly abundant molecules. 3) Analyte specificity for each peptide was demonstrated in amino acid sequences using multiple MRM detection. Selection of the peptide probe was very important for highly sensitive analysis with LC-MS/MS. We set criteria for peptide selection using an informatics approach. Peptides without unstable residue, double basic residues, and integral membrane domain were selected as useful probes. The developed method will provide an inclusive assay platform for all transporter proteins expressed in both animal/human tissues and will contribute to progress in drug discovery and development.     

Passive and iontophoretic controlled delivery of salmon calcitonin through artificial membranes

The development of a transdermal delivery system for drug molecules of high molecular weight (peptides or proteins) is nowadays a great scientific and commercial challenge. For these molecules, the passive transport through the skin is generally very low and should be enhanced by the application of the electrical current (a method called iontophoresis). A very important component of a transdermal iontophoretic system is the artificial membrane, which acts as the interface between the drug reservoir and the skin. The optimum membrane should (i) provide an effective drug delivery; (ii) have low electrical resistance and (ii) have low drug adsorption. In this work, the selection of membrane(s) for a transdermal iontophoretic salmon calcitonin (sCT, MW approximately 3500) system is performed. The passive and iontophoretic transport of sCT through porous artificial membranes, the sCT adsorption to them and the electrical resistance of all porous membranes in iontophoretic experiments is studied. The sCT transport through the membranes is compared with that through human skin, and based on the above three criteria the optimum membranes are selected for the sCT transdermal system.