Multi-Wavelength Fluorescence Radical Dosimetry for Real-Time Assessment of Protein Footprinting Radical Yield

用于实时评估蛋白质足迹自由基产量的多波长荧光自由基剂量测定

• 批准号: 10250755
• 负责人: Scot Randy Weinberger
• 金额: $ 25.14万
• 依托单位: GENNEXT TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10250755
• 关键词: Address; Adopted; Adoption; Amino Acids; Antibodies; Antibody Binding Sites; Antineoplastic Agents; Appearance; Award; Behavior; Binding; Binding Sites; Biological Process; Biological Products; Biological Response Modifier Therapy; Blood capillaries; Chemistry; Comparative Study; Dangerousness; Detection; Drug Targeting; Environment; Epitopes; Extracellular Protein; Fluorescence; Fluorine; G-Protein-Coupled Receptors; Generations; Goals; Growth Factor; Higher Order Chromatin Structure; Human; Hydrophobicity; Hydroxyl Radical; Immune response; Industry; Label; Laboratories; Lasers; Lead; Letters; Ligands; Link; Membrane; Membrane Proteins; Modification; Molecular Conformation; Monitor; Monoclonal Antibodies; National Institute of General Medical Sciences; Nature; Patients; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Phosphotransferases; Play; Process; Production; Protein Conformation; Protein Footprinting; Proteins; Proteome; Proteomics; Reaction; Reproducibility; Research; Role; Safety; Signal Transduction; Small Business Innovation Research Grant; Sodium; Solvents; Structure; Surface; Techniques; Technology; Time; Toxin; Transmembrane Domain; Universities; Washington; Work; adverse drug reaction; analytical tool; aqueous; base; detector; dosimetry; druggable target; experimental study; improved; innovation; interest; new technology; novel; pathogen; pathogenic virus; professor; programs; research and development; tool

The GenNext Phase I SBIR submssion entitled “Multi-Wavelength Fluorescence Radical Dosimetry for Real-Time Assessment of Protein Footprinting Radical Yield” 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. We have developed commercial solutions to perform HRPF. Recently a new and valuable footprinting technique that relies upon trifluoromethyl (TFM) radicals created by OH radical attack of aqueous sodium triflinate has been developed that shows great promise when combined with HRPF. TFM protein footprinting (TFMPF) is highly complementary to HRPF, as TFMPF effectively labels amino acid residues that are relatively “silent” to OH radical attack. When used together, HRPF and TFMPF provide substantial coverage and detection of solvent accessible residues, and as such represent a transformative improvement to biopharmaceutical HOS assessment. The practice of TFMPF has been pioneered by Professor Michael Gross of Washington University, St. Louis, Mo. While showing great promise to address unmet challenges in pharmaceutical research, reproducibility for TFMPF is challenged by variability of background scavenging. Collaborating with the Gross laboratory, our work will extend our innovative HRPF radical dosimetry technology to TFMPF. GenNext Technologies is the only company commercializing products for HRPF HOS analysis. Our goal is to convert the combined use of HRPF and TFMPF process from an academic research experiment into a valuable analytical tool. Once simplified and transformed into a robust technique, we envision the facile combination of HRPF and TFMPF to enable: 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 Phase I SBIR提交的题为“多波长荧光 用于实时评估蛋白质足迹自由基产率的自由基剂量学”是 对新的和改进的工具的公认的需要作出反应， 生物药物结构分析（HOS）和膜蛋白靶向研究。 一种新兴的HOS分析技术是羟基自由基足迹法（HRPF）。HRPF 涉及通过与羟基自由基反应对蛋白质外部进行不可逆标记 随后进行MS分析以鉴定蛋白质的外部部分。我们有 开发了商业解决方案来执行HRPF。最近一个新的和有价值的 足迹技术依赖于OH产生的三氟甲基（TFM）自由基 三氟甲磺酸钠水溶液的自由基攻击已被开发，显示出巨大的希望 当与HRPF结合时。TFM蛋白质足迹（TFMPF）是高度互补的 对于HRPF，TFMPF有效地标记相对“沉默”的氨基酸残基， 激进的攻击。如果一起使用，HRPF和TFMPF提供了大量的覆盖面 和溶剂可及残留物的检测，因此代表了一种变革性的 改进生物制药HOS评估。 TFMPF的实践是由Michael Gross教授开创的， 密苏里州圣路易斯市华盛顿大学虽然在解决未实现的问题方面表现出很大的希望， 药物研究的挑战，TFMPF的重现性受到挑战， 背景清除的可变性。与格罗斯实验室合作，我们的工作 将我们创新的HRPF自由基剂量测定技术扩展到TFMPF。GenNext Technologies是唯一一家将HRPF HOS分析产品商业化的公司。 我们的目标是将HRPF和TFMPF工艺的联合使用从一种 将学术研究实验转化为有价值的分析工具。一旦简化， 转换成一个强大的技术，我们设想的HRPF和 TFMPF使：互补位和表位与mAb生物药物相互作用 它们的膜靶点;阐明铅与正构或 变构膜靶点;揭示第二信使信号级联反应， GPCR先导化合物;并检测正构/变构抗- 肿瘤的靶点，如激酶和生长因子。