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

描述(由申请人提供)：本申请涉及广泛的挑战领域(06)：使能技术和具体的挑战主题，06-DK-101：开发用于治疗和成像的特定细胞递送系统。药物输送中尚未解决的主要问题之一是如何将渗透性差的分子穿过膜屏障，并在特定的细胞或组织中释放它们。自然界解决这个问题的方法是通过膜转运；细胞不透水的配体，如携带铁的转铁蛋白蛋白，与同源受体结合，在这种情况下，转铁蛋白受体驻留在动态的膜转运途径中。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，将转铁蛋白输送到脊椎动物的每一个细胞，是一种非凡的自然输送工具。我们先前证明了合成膜锚定N-烷基-3-胆固醇胺的衍生物具有独特的生物活性：当添加到哺乳动物细胞中时，它们有效地参与了转铁蛋白受体占据的膜转运途径，使它们能够在质膜和早期内吞体内之间快速循环。通过这种方式，N-烷基-3-胆固醇胺可以起到人造细胞表面小受体的作用；当连接到结合药物或分子探针的基序时，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。这些独特的生物活性源于对游离胆固醇的功能模拟，游离胆固醇是血浆和内膜的关键成分。通过与游离胆固醇受体NPC1L1结合，这些化合物参与了控制细胞对这一关键膜成分的摄取和膜运输的自然机制。这种生物活性使N-烷基-3-胆固醇胺能够显著提高连接肽、药物和探针在体内的分布体积(Vd)。由于转铁蛋白通过转铁蛋白受体被有效地输送到所有组织中的所有细胞，胆固醇胺连接的化合物有效地参与相同的膜转运途径的能力为创造变革性的新的药物输送工具提供了前所未有的潜力。这项建议的重点是创造低分子质量的胆固醇胺连接的多肽(&lt；3000道尔顿)，选择性地将荧光分子探测器输送到特定的组织。这些探针在特定组织中的释放将由组织特异的内切酶介导，该酶能裂解特定的多肽底物。为了发现这些多肽底物，在显微注射线虫后，将使用共聚焦激光扫描显微镜对3375个荧光胆固醇胺连接多肽的文库进行筛选，这些多肽排列为512个池，每个池8个肽。连接的N-烷基-3-胆固醇胺部分将发挥两个作用：它将增强多肽库成员的Vd，改善体内所有组织的可获得性，以及它将选择性地将多肽底物展示给细胞表面和早期内体系统中发现的蛋白酶子集。荧光共振能量转移(FRET)通过用红色和绿色荧光团在多肽库成员的两侧进行，将允许对多肽库成员的体内切割进行量化。这些多肽被组织特异性内切酶裂解后，将在特定的组织类型中卸载货物。根据共聚焦显微镜获得的红色、绿色和FRET荧光图像，将定量测量进入线虫六个容易识别的组织(神经元、肌肉、皮下组织、性腺、肠道、排泄细胞)的探针。这些信息结合其他药代动力学参数，如Vd、体内半衰期和毒性，将被用于构建定量结构-性质关系(QSPR)模型。这些模型旨在指导合成具有最佳组织靶向性的无毒化合物。为了便于将这种方法转化到更高的动物模型中，我们将识别线虫中处理胆固醇胺连接多肽的特定蛋白酶。优化的多肽底物将通过在剪切键的假定位置上用C-末端醛和/或其他蛋白酶抑制基序修饰来转化为蛋白酶抑制剂。这些抑制剂将用于提纯和鉴定线虫提取物中的蛋白酶。进一步使用遗传突变、RNAi方法和在线虫替代组织中表达假定的蛋白酶的研究将被用来验证这些目标蛋白。建议使用N-烷基-3-胆固醇胺跨膜屏障运输分子探针，结合使用连接的多肽库来识别组织特异性蛋白水解酶的底物，代表了一种组织特异性药物输送的全新方法。 公共卫生相关性：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文库和共聚焦显微镜的高含量筛选，我们建议鉴定在模式生物秀丽线虫的特定组织中选择性释放分子探针的相对低分子量化合物。通过从这些研究中确定先导化合物，并确定介导连接探针的组织特异性释放的蛋白酶，这项研究将为开发在高等生物体中组织特异性给药和显像剂的新方法奠定基础。

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.