MEMBRANE PERMEANT PEPTIDES FOR IMAGING CELL FUNCTION

用于细胞功能成像的膜永久肽

• 批准号: 6633498
• 负责人: David Piwnica-Worms
• 金额: $ 33.18万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633498
• 关键词: MCF7 cell; apoptosis; athymic mouse; bioengineering /biomedical engineering; bioimaging /biomedical imaging; cell membrane; chelating agents; chemical conjugate; contrast media; cysteine endopeptidases; cytoplasm; disease /disorder model; enzyme activity; fluorescence microscopy; laboratory rat; membrane permeability; neoplastic cell; peptides; protein sequence; radionuclide imaging /scanning; radionuclides; technetium; tissue /cell culture

Whether apoptotic stimuli arise from the nucleus, cell membrane surface, or the mitochondria, ultimately, the stimuli converge on a process of activation of a family of cysteine proteases known as the caspases (cysteine aspartases). Activation of members of the caspase family have been shown to be necessary for programmed cell death in a number of biological systems and evidence strongly points to caspase-3 as standing at the center of the execution pathway of the cell death program. While existing in a non-active pro-enzyme form in the cytosol of resting cells, caspase-3 is one key "effector" protease when activated. Thus, to monitor the final commitment of tumor cells to death pathways, the need exists to directly quantify the enzymatic activity of caspase-3 in vivo. To meet this challenge, we have designed and synthesized prototype of an entirely new class of peptide based imaging agents that can permeate the cell membrane. Incorporating permeation motifs of viral proteins, these prototypic membrane permeant peptide conjugates are capable of translocating across the cell membrane to the cytosolic compartment. Engineered with substrate recognition sequences and appropriate peptide-based motifs for chelating radionuclides such as technetium-99m (Tc-99m), these agents will enable imaging of caspase-3 activity in vivo. Enzyme-dependent signal amplification should result in high quality enzyme-specific molecular images. In this way, focal retention of radioactivity, a "hot spot", will be generated on tumor images where there exists enzymatically active caspase-3. We propose to define the structural determinants of the viral protein cell membrane permeation sequences for use in peptide imaging conjugates, use a degenerate peptide combinatorial library to determine the preferred recognition sequences of the peptide imaging substrates, define the structural determinants and charge of the chelation moiety of our peptide conjugates that will confer favorable cellular retention properties to the imaging fragments, and perform pre-clinical evaluation of Tc-99m labeled peptide chelation conjugates in order to study the time course of activation of caspase-dependent apoptosis in tumor models grown in nude mice. Studies with these novel imaging tools will assist interrogation of the efficacy of new therapies, including gene therapy and new molecular targeted chemotherapy in cancer.

无论细胞凋亡刺激是来自细胞核、细胞膜表面还是线粒体，最终刺激都会集中在称为半胱天冬酶（半胱氨酸天冬氨酸酶）的半胱氨酸蛋白酶家族的激活过程上。 Caspase 家族成员的激活已被证明是许多生物系统中程序性细胞死亡所必需的，并且有证据强烈表明 caspase-3 处于细胞死亡程序执行途径的中心。 虽然 caspase-3 以非活性酶原形式存在于静息细胞的细胞质中，但激活后是一种关键的“效应”蛋白酶。 因此，为了监测肿瘤细胞对死亡途径的最终承诺，需要直接量化体内 caspase-3 的酶活性。 为了应对这一挑战，我们设计并合成了一种全新的基于肽的成像剂原型，该成像剂可以渗透细胞膜。这些原型膜渗透肽结合物结合了病毒蛋白的渗透基序，能够跨细胞膜易位至胞质隔室。 这些试剂采用底物识别序列和适当的基于肽的基序进行设计，用于螯合锝-99m (Tc-99m) 等放射性核素，能够对体内 caspase-3 活性进行成像。 酶依赖性信号放大应产生高质量的酶特异性分子图像。 这样，在存在酶活性 caspase-3 的肿瘤图像上将产生放射性的焦点保留，即“热点”。我们建议定义用于肽成像缀合物的病毒蛋白细胞膜渗透序列的结构决定因素，使用简并肽组合库来确定肽成像底物的优选识别序列，定义螯合部分的结构决定因素和电荷我们的肽缀合物将为成像片段赋予有利的细胞保留特性，并对 Tc-99m 标记的肽螯合缀合物进行临床前评估，以研究激活的时间过程裸鼠肿瘤模型中 caspase 依赖性细胞凋亡。 使用这些新颖的成像工具进行的研究将有助于研究新疗法的功效，包括基因疗法和新的癌症分子靶向化疗。