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

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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.