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

GenNext阶段I SBIR提交题为“细胞内自由基剂量学” 用于改善体内FPOP HRPF的(ICRD)“回应了人们对 生物药物高阶结构分析的新工具和改进工具 以及膜蛋白靶标研究。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分析来鉴定 蛋白质的外部部分。最广泛使用的产生OH自由基的方法 利用快速爆发的紫外光，被恰当地称为快速光化学 蛋白质氧化(FPOP)。我们已经开发了商业解决方案来执行 体外FPOP。将体外结构实验结果应用于临床的实践 真实的体内行为已经受到了质疑。大分子拥挤 在细胞内限制扩散，从而改变反应动力学，蛋白质的结合率， 以及蛋白质与DNA的相互作用。这些效应在体外实验中没有观察到。 学习。由于体外培养的缺陷，最近人们一直希望延长 以活体方式将FPOP用于整个细胞[13]。 体内或细胞内FPOP(IC-FPOP)的实践是由Lisa博士开创的 马里兰大学的琼斯[13，26-28]。在展现出巨大的希望的同时 解决制药研究中尚未满足的挑战，IC-FPOP的重复性是 面临细胞内背景清除和细胞间分离的多样性的挑战 不可再生性。通过与琼斯实验室的合作，我们的工作将扩展我们的 创新的体外自由基剂量学技术，用于体内清除自由基。下一代 科技公司是唯一一家将FPOP HOS分析产品商业化的公司。 我们的目标是将IC-FPOP工艺从学术研究实验转化为 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能够实现基于细胞的分析：对位和表位相互作用 单抗生物药物及其膜靶标；阐明铅的动力学 与正构膜或变构膜靶标结合；揭示第二信使 GPCR先导化合物的信号级联；并检测正构体/ 变构抗肿瘤作用靶点，如激酶和生长因子。

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.