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: 7817256
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7817256
• 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（由申请人提供）：本申请解决了广泛的挑战领域（06）：实现技术和特定挑战主题，06-DK-101：开发用于治疗和成像的细胞特异性递送系统，这是药物中未解决的主要问题之一。传递是如何将渗透性差的分子穿过膜屏障并将其释放到特定的细胞或组织中，大自然解决这个问题的方法是通过细胞不渗透性的配体，例如携带铁的配体。转铁蛋白蛋白结合同源受体，在本例中为转铁蛋白受体，其存在于动态膜运输途径中。转铁蛋白受体占据的途径涉及细胞表面和早期内体之间的快速循环，将转铁蛋白递送至脊椎动物的每个细胞，并代表。我们之前证明了合成膜锚定N-烷基-3的衍生物。 - 胆固醇胺表现出独特的生物活性：当添加到哺乳动物细胞中时，它们有效地参与转铁蛋白受体占据的膜运输途径，使它们能够在质膜和早期内体之间快速循环，N-烷基-3¿ -胆固醇胺可以充当小型人工细胞表面受体；当与结合药物或分子探针的基序连接时，它们可以通过内吞作用将这些化合物运送到细胞和组织中，这些独特的生物活性来自游离胆固醇的功能模拟。通过与游离胆固醇受体 NPC1L1 结合，这些化合物参与控制这种关键膜成分的细胞摄取和膜运输的自然机制，这种生物活性使 N-烷基- 3¿ -胆固醇胺可显着增强连接的肽、药物和探针的体内分布体积 (Vd) 由于转铁蛋白通过转铁蛋白受体有效地递送至所有组织中的所有细胞，因此胆固醇胺连接的化合物能够有效地与转铁蛋白结合。相同的膜运输途径为创建用于药物输送的变革性新工具提供了前所未有的潜力。该提案的重点是选择性地创建低分子量胆固醇胺连接肽（<3000 道尔顿）。将荧光分子探针递送到特定组织中，这些探针在特定组织中的释放将由切割特定肽底物的组织特异性内切蛋白酶介导。在显微注射线虫 C 后，将使用共焦激光扫描显微镜对排列为 512 个 8 个肽的连接肽进行筛选。连接的 N-烷基-3¿ -胆固醇胺部分将发挥两个作用：它将增强肽库成员的Vd，改善体内所有组织的进入，并且它将选择性地将肽底物展示给细胞表面和早期内体中发现的蛋白酶子集通过在肽库成员的两侧添加红色和绿色荧光团，荧光共振能量转移 (FRET) 将能够对肽库成员的体内裂解进行定量。这些肽通过组织特异性内切蛋白酶将货物卸载到特定组织类型中，将根据红色、绿色、和从共焦显微镜获得的 FRET 荧光图像，该信息与 Vd、体内半衰期和毒性等其他药代动力学参数相结合，将用于构建定量分析。结构-性质关系（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，以促进这种方法转化为高等动物模型，我们将鉴定处理胆固醇胺的特定蛋白酶。线虫中的优化肽底物将通过在 C 端醛和/或其他蛋白酶抑制基序的假定位点进行修饰而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酶，使用基因突变体、RNAi 方法和假定的蛋白酶在秀丽隐杆线虫替代组织中的表达的进一步研究将用于验证这些目标蛋白。建议使用 N-烷基-3¿ - 使用胆固醇胺跨膜屏障运输分子探针，结合使用连接的肽库来识别组织特异性蛋白酶的底物，代表了一种全新的组织特异性药物输送方法。 公共健康相关性：这里提出的研究与人类健康的进步高度相关，利用胆固醇胺连接的荧光肽的小型文库和通过共聚焦显微镜进行高内涵筛选，我们建议鉴定选择性释放分子探针的相对低分子量的化合物。通过从这些研究中鉴定先导化合物，并鉴定介导连接探针的组织​​特异性释放的蛋白酶，这项研究将为模型生物体秀丽隐杆线虫的特定组织中的研究奠定基础。开发在高等生物中组织特异性递送药物和显像剂的新方法。