Tissue-specific delivery of probes by control of membrane trafficking of endoprot

通过控制内切酶的膜运输实现探针的组织​​特异性递送

• 批准号: 7937894
• 负责人: BLAKE PETERSON
• 金额: $ 50万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937894
• 关键词: Address; Aldehydes; Animal Model; Animals; Area; Binding; Bioinformatics; Biological; Biological Assay; Blood Circulation; C-terminal; Caenorhabditis elegans; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Chemicals; Cholesterol; Cleaved cell; Confocal Microscopy; Data; Development; Drug Delivery Systems; Drug Kinetics; Early Endosome; Endocytosis; Enzymes; Exhibits; Fluorescence Resonance Energy Transfer; Foundations; Genetic; Gonadal structure; Half-Life; Health; Histocompatibility Testing; Human; Image; Improve Access; In Vitro; Individual; Intestines; Iron; Kansas; Laser Scanning Confocal Microscopy; Lead; Libraries; Life; Ligands; Link; Mammalian Cell; Measures; Mediating; Membrane; Membrane Protein Traffic; Methods; Microinjections; Modeling; Modification; Molecular Models; Molecular Probes; Molecular Weight; Muscle; Nature; Nematoda; Neurons; Organism; Pathway interactions; Peptide Hydrolases; Peptide Library; Peptides; Pharmaceutical Preparations; Plasma; Play; Poison; Problem Solving; Process; Property; Protease Inhibitor; Proteins; RNA Interference; Reporting; Research; Role; Screening procedure; Site; Specialist; Structure; Subcutaneous Tissue; System; Technology; Testing; Tissue Model; Tissues; Toxic effect; Transferrin; Transferrin Receptor; Transferrin-Binding Proteins; Translations; Universities; Vertebrates; Work; base; candidate identification; cholesterylamine; dalton; design; fluorescence imaging; fluorophore; genetic manipulation; improved; in vivo; inhibitor/antagonist; member; mimicry; model design; molecular modeling; mutant; novel; novel strategies; protein aminoacid sequence; public health relevance; receptor; tool; uptake

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06): Enabling Technologies and specific Challenge Topic, 06-DK-101: Development of cell-specific delivery systems for therapy and imaging. One of the major unsolved problems in drug delivery is how to transport poorly permeable molecules across membrane barriers and release them in specific cells or tissues. The way that Nature solves this problem is through membrane trafficking; cell impermeable ligands such as the iron-carrying transferrin protein bind cognate receptors, in this case the transferrin receptor, that reside in dynamic membrane trafficking pathways. The pathway occupied by the transferrin receptor involves rapid cycling between the cell surface and early endosomes, delivers transferrin to every cell of vertebrate animals, and represents a remarkable natural delivery vehicle. We previously demonstrated that derivatives of the synthetic membrane anchor N-alkyl-3¿- cholesterylamine exhibit a unique biological activity: when added to mammalian cells, they efficiently engage the membrane trafficking pathway occupied by the transferrin receptor, allowing them to rapidly cycle between the plasma membrane and early endosomes. In this way, N-alkyl-3¿-cholesterylamines can function as small artificial cell surface receptors; when linked to motifs that bind drugs or molecular probes, they can shuttle these compounds into cells and tissues via endocytosis. These unique biological activities result from functional mimicry of free cholesterol, a key component of plasma and endosomal membranes. By binding to the free cholesterol receptor NPC1L1, these compounds engage natural mechanisms that control cellular uptake and membrane trafficking of this critical membrane component. This biological activity enables N-alkyl- 3¿-cholesterylamines to dramatically enhance the volume of distribution (Vd) of linked peptides, drugs, and probes in vivo. Because transferrin is efficiently delivered to all cells in all tissues via the transferrin receptor, the ability of cholesterylamine-linked compounds to efficiently engage the same membrane trafficking pathway offers unprecedented potential to create transformative new tools for drug delivery. This proposal is focused the creation of low molecular weight cholesterylamine-linked peptides (<3000 daltons) that selectively deliver fluorescent molecular probes into specific tissues. The release of these probes in defined tissues will be mediated by tissue-specific endoproteases that cleave specific peptide substrates. To discover these peptide substrates, a "small-but-smart" library comprising 3375 fluorescent cholesterylamine- linked peptides arrayed as 512 pools of eight peptides each will be screened using confocal laser scanning microscopy after microinjection of the nematode C. elegans. The linked N-alkyl-3¿-cholesterylamine moiety will play two roles: it will enhance the Vd of the members of the peptide library, improving access to all tissues in vivo, and it will selectively display peptide substrates to the subset of proteases found on cell surfaces and in the early endosomal system. By flanking members of the peptide library with red and green fluorophores, fluorescence resonance energy transfer (FRET) will allow quantification of in vivo cleavage of members of the peptide library. Cleavage of these peptides by tissue-specific endoproteases will unload cargo in specific tissue types. Probe delivery into six easily identified tissues (neurons, muscle, hypodermis, gonad, intestine, execretory cell) of C. elegans will be quantified based on red, green, and FRET fluorescence images obtained from confocal microscopy. This information, combined with other pharmacokinetic parameters such as Vd, half- life in vivo, and toxicity, will be used to construct quantitative structure-property relationship (QSPR) models. These models are designed to guide the synthesis of non-toxic compounds with optimal tissue-targeting properties. To facilitate the translation of this approach to higher animal models, we will identify specific proteases that process cholesterylamine-linked peptides in C. elegans. Optimized peptide substrates will be converted into protease inhibitors by modification with C-terminal aldehyde and/or other protease inhibitory motifs at putative sites of scissile bonds. These inhibitors will be used to purify and identify proteases from extracts of C. elegans. Further studies using genetic mutants, RNAi methods, and expression of putative proteases in alternative tissues of C. elegans will be used to validate these target proteins. The proposed use of N-alkyl-3¿- cholesterylamines to transport molecular probes across membrane barriers, combined with the use of linked peptide libraries to identify substrates of tissue-specific proteases, represents a fundamentally new approach to tissue-specific drug delivery. PUBLIC HEALTH RELEVANCE: The research proposed here is highly relevant to the advancement of human health. Using small libraries of cholesterylamine-linked fluorescent peptides and high-content screening by confocal microscopy, we propose to identify relatively low molecular weight compounds that selectively release molecular probes in specific tissues of the model organism C. elegans. By identifying lead compounds from these studies, and identifying proteases that mediate tissue-specific release of linked probes, this research will lay the foundation for the development of novel methods for tissue-specific delivery of drugs and imaging agents in higher organisms.

描述（由应用程序提供）：此应用程序解决广泛的挑战领域（06）：启用技术和特定挑战主题，06-DK-101：开发特定细胞的治疗和成像输送系统。药物输送中主要未解决的问题之一是如何在跨膜屏障中运输不可渗透的分子并将其释放在特定的细胞或组织中。大自然解决这个问题的方式是通过膜贩运。细胞不可渗透的配体，例如携带铁的转铁蛋白蛋白结合了同源受体，在这种情况下是转铁蛋白受体，它位于动态膜运输途径中。转铁蛋白受体所占据的途径涉及细胞表面和早期内体之间的快速循环，将转铁蛋白传递到脊椎动物的每个细胞，并代表着一个显着的自然递送载体。我们先前证明了合成膜锚定N-烷基3¿-胆固醇的衍生物表现出独特的生物学活性：当添加到哺乳动物细胞中时，它们有效地吸引了转移蛋白受体占据的膜运输途径，从而使它们能够在血浆膜之间快速循环循环。这样，n-烷基-3¿-色脂氨可以充当小的人造细胞表面受体。当与结合药物或分子问题结合的基序相关时，它们可以通过内吞作用将这些化合物穿梭到细胞和组织中。这些独特的生物学活性是由游离胆固醇的功能模仿引起的，这是血浆和内体机理的关键组成部分。通过与游离胆固醇受体NPC1L1结合，这些化合物具有控制这种关键膜成分的细胞摄取和膜运输的自然机制。这种生物学活性使n-烷基-3。色脂胺可显着增强体内连接的辣椒，药物和问题的分布量（VD）。由于转铁蛋白在始终通过转铁蛋白受体有效地传递到所有细胞，因此胆固醇连接的化合物有效参与相同的膜运输途径的能力提供了前所未有的潜力，可以创建用于药物输送的变革性新工具。该建议集中于产生低分子量胆固醇连接的Petides（<3000 daltons），这些Petides（<3000 daltons）选择性地将荧光分子问题传递到特定的组织中。这些问题在定义的组织中的释放将由组织特异性内螺旋蛋白酶介导，以清除特异性肽底物。为了发现这些肽底物，一个“小但智能”库，完成了3375个荧光胆固醇连接的肽，这些肽是八个petides的512个池，每个petides的池将使用共聚焦激光扫描显微镜进行筛选，并在Microdoction sekemandode C. elecans C. Elegans C. Elegans。链接的N-Alkyl-3¿-cholesterylamine部分将扮演两个角色：它将增强肽库成员的VD，从而提高了体内所有时代的访问，并且将有选择地显示肽底物，以在细胞表面和早期内体系统中建立的蛋白质子集显示到蛋白质的子集。通过具有红色和绿色荧光团肽文库的侧面成员，荧光共振能量转移（FRET）将允许定量肽文库成员的体内切割。通过组织特异性的内螺旋虫对这些宠物的裂解将在特定组织类型中卸载货物。探针递送到六个易于鉴定的组织（神经元，肌肉，性皮，性腺，肠，执行细胞）中，将根据从共聚焦显微镜获得的红色，绿色和FRET荧光图像进行定量。这些信息与其他药代动力学参数相结合，例如VD，体内半衰期和毒性，将用于构建定量结构 - 毛皮关系（QSPR）模型。这些模型旨在指导具有最佳组织靶向特性的无毒化合物的合成。为了促进这种方法转化为更高动物模型的方法，我们将确定特定的蛋白质，这些蛋白质在秀丽隐杆线虫中处理胆固醇连接的肽。优化的肽底物将通过用C末端醛和/或其他蛋白质抑制基序在剪刀键的推定位点进行修饰，将优化的肽底物转化为蛋白酶抑制剂。这些抑制剂将用于纯化和鉴定秀丽隐杆线虫提取物中的蛋白质。使用遗传突变体，RNAi方法和推定蛋白在秀丽隐杆线虫替代组织中的进一步研究将用于验证这些靶蛋白。拟议中N-烷基3¿-胆固醇层在跨膜屏障中运输分子问题，再加上使用连接的肽库来鉴定组织特异性蛋白酶的底物，这是一种从根本上讲，这是一种基本的新方法，用于组织特异性药物递送。 公共卫生相关性：这里提出的研究与人类健康的发展高度相关。我们建议使用共聚焦显微镜的胆固醇连接荧光肽和高含量筛选的小文库，我们建议鉴定相对较低的分子量化合物，这些化合物在模型有机体C. exemans C. elegans的特定组织中有选择地释放分子问题。通过从这些研究中鉴定铅化合物，并确定介导组织特异性问题释放的蛋白酶，这项研究将为开发新型方法​​在高等生物体中开发用于组织特异性药物和成像剂的新方法。