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.

凋亡刺激是由细胞核，细胞膜表面还是线粒体产生的，最终，刺激会在激活的半胱氨酸蛋白酶家族的过程中收敛，称为胱天蛋白酶（半胱氨酸缓体）。 caspase家族成员的激活已被证明是许多生物系统中编程细胞死亡必不可少的，并且证据强烈指向Caspase-3，因为站在细胞死亡程序执行途径的中心。 虽然以非活性促酶形式存在于静息细胞的细胞质中，但caspase-3是激活时的一个关键“效应子”蛋白酶。 因此，为了监测肿瘤细胞对死亡途径的最终承诺，存在直接量化caspase-3体内酶促活性的需求。 为了应对这一挑战，我们设计了一个全新的基于肽的成像剂的原型，这些原型可以渗透到细胞膜。这些原型膜胶肽结合物结合了病毒蛋白的渗透基序，能够在细胞膜上易位到胞质室。 这些药物采用底物识别序列和适当的基于肽的基序设计，用于螯合放射性核素，例如Technetium-99M（TC-99M），这些药物将在体内实现caspase-3活性的成像。 酶依赖性信号扩增应导致高质量的酶特异性分子图像。 以这种方式，将在存在酶促活性caspase-3的肿瘤图像上产生放射性的局灶性保留，即“热点”。 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,并对TC-99M标记的肽螯合剂进行临床前评估，以研究裸鼠生长的肿瘤模型中caspase依赖性凋亡的激活时间。 使用这些新型成像工具的研究将有助于询问新疗法的功效，包括基因疗法和新的分子靶向化学疗法。