In-Cell Radical Dosimetry (ICRD) for improved in vivo Fast Photo-oxidation of Proteins Hydroxyl Radical Protein Footprinting

细胞内自由基剂量测定 (ICRD)，用于改善蛋白质体内快速光氧化羟基自由基蛋白质足迹

• 批准号: 10009765
• 负责人: Scot Randy Weinberger
• 金额: $ 26.15万
• 依托单位: GENNEXT TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10009765
• 关键词: Acids; Address; Adopted; Adoption; Antibodies; Antibody Binding Sites; Antineoplastic Agents; Appearance; Award; Behavior; Binding; Biological Assay; Biological Process; Biological Products; Biological Response Modifier Therapy; Blood capillaries; Cell Separation; Cells; Comparative Study; Complex; Crowding; Cytoplasmic Protein; DNA-Protein Interaction; Dangerousness; Data Analyses; Detection; Detergents; Diffusion; Drug Targeting; Environment; Epitopes; Fluorescence; G-Protein-Coupled Receptors; Goals; Growth Factor; Higher Order Chromatin Structure; Human; Hydroxyl Radical; Immune response; In Vitro; Individual; Industry; Kinetics; Label; Laboratories; Lasers; Lead; Letters; Ligands; Link; Lipids; Liquid substance; Maryland; Membrane; Membrane Proteins; Membrane Transport Proteins; Methods; Molecular Conformation; Monoclonal Antibodies; Morphologic artifacts; National Institute of General Medical Sciences; Nature; Nuclear Proteins; Organism; Pathogenicity; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Phosphotransferases; Play; Positioning Attribute; Process; Production; Protein Conformation; Protein Footprinting; Proteins; Proteome; Proteomics; Pump; Reaction; Reproducibility; Research; Role; Safety; Signal Transduction; Small Business Innovation Research Grant; Structure; Styrenes; Surface; System; Techniques; Technology; Toxin; Ultraviolet Rays; Universities; Vero Cells; Work; adverse drug reaction; analytical tool; base; computerized data processing; copolymer; detector; dosimetry; drug candidate; experimental study; fluidity; improved; in vivo; innovation; interest; irradiation; light scattering; maleic acid; member; mimetics; nanodisk; new technology; oxidation; particle; programs; protein complex; protein function; reconstitution; research and development; tool; unilamellar vesicle

The GenNext Phase I SBIR submssion entitled “In-Cell Radical Dosimetry (ICRD) for improved in vivo FPOP HRPF” is responsive to the ackowledged need for new and improved tools for higher order structural analysis (HOS) of biopharmaceuticals and membrane protein target studies. An emerging HOS analysis technique is hydroxyl radical foot-printing (HRPF). HRPF involves the irreversible labeling of a protein's exterior by reaction with hydroxyl radicals with subsequent MS analysis to identify the outer portions of the protein. The most widely used method for generating OH radicals employs a quick burst of UV light, and is appropriately called fast photochemical oxidation of proteins (FPOP).We have developed commercial solutions to perform in vitro FPOP. The practice of applying the results of in vitro structural experiments to authentic in vivo behavior has been brought into question. Macromolecular crowding within a cell limits diffusion, thus altering reaction kinetics, association rates of proteins, and protein-DNA interactions. These effects are not observed while performing in vitro studies. Because of in vitro shortcomings, there has been recent desire to extend the use of FPOP to whole cells in an in vivo manner [13]. The practice of in vivo or in-cell FPOP (IC-FPOP) has been pioneered by Dr. Lisa Jones of the University of Maryland [13, 26-28]. While showing great promise to address unmet challenges in pharmaceutical research, reproducibility for IC-FPOP is challenged by variability of intracellular background scavenging and cell-to-cell isolation irreproducibility. Collaborating with the Jones laboratory, our work will extend our innovative in vitro radical dosimetry technology to in vivo radical scavenging. GenNext Technologies is the only company commercializing products for FPOP HOS analysis. Our goal is to convert the IC-FPOP process from an academic research experiment into a valuable analytical tool. Once simplified and transformed into a robust technique, we envision IC-FPOP to enable cell-based assays to: paratope and epitope the interaction of mAb biopharmaceuticals with their membrane targets; elucidate the dynamics of lead binding to orthosteric or allosteric membrane targets; to reveal secondary messenger signaling cascades of GPCR lead compounds; and to detect the impact of orthosteric / allosteric anti-neoplastics upon targets such as kinases and growth factors.

GenNext 第一阶段 SBIR 提交的题为“In-Cell Radical Dosimetry” （ICRD）改进体内 FPOP HRPF”是对公认的需求的回应 用于生物制药高阶结构分析 (HOS) 的新工具和改进工具 和膜蛋白靶点研究。一种新兴的 HOS 分析技术是羟基 激进足迹（HRPF）。 HRPF 涉及蛋白质的不可逆标记 通过与羟基自由基反应和随后的 MS 分析来识别外部 蛋白质的外部部分。最广泛使用的产生 OH 自由基的方法 采用快速爆发的紫外光，被适当地称为快速光化学 蛋白质氧化 (FPOP)。我们开发了商业解决方案来执行 体外 FPOP。体外结构实验结果应用的实践 真实的体内行为已受到质疑。大分子拥挤 细胞内限制扩散，从而改变反应动力学、蛋白质的结合率， 以及蛋白质-DNA 相互作用。在体外进行时未观察到这些效应 研究。由于体外实验的缺点，最近人们希望扩展 以体内方式使用 FPOP 处理全细胞 [13]。 Lisa 博士开创了体内或细胞内 FPOP (IC-FPOP) 的实践 马里兰大学的琼斯 [13, 26-28]。在展现出巨大希望的同时 解决药物研究中未满足的挑战，IC-FPOP 的重现性 受到细胞内背景清除和细胞间分离的可变性的挑战 不可再现性。与琼斯实验室合作，我们的工作将扩展我们的 创新的体外自由基剂量测定技术可清除体内自由基。下一代 Technologies 是唯一一家将 FPOP HOS 分析产品商业化的公司。 我们的目标是将 IC-FPOP 过程从学术研究实验转变为 一个有价值的分析工具。一旦简化并转化为一种强大的技术，我们 设想 IC-FPOP 使基于细胞的检测能够： 互补位和表位相互作用 mAb 生物制药及其膜靶点；阐明铅的动力学 与正构或变构膜靶标结合；揭示第二信使 GPCR 先导化合物的信号级联；并检测正位/的影响 针对激酶和生长因子等靶标的变构抗肿瘤药物。