An instrument capable of measuring H/D exchange within a single second at a defined temperature will be assembled to test the hypothesis that quantitative data could be obtained about protein conformational changes at a significantly higher temporal resolution. This will be accomplished by development of a novel rapid in-line mixing module capable of determining the deuterium incorporation level after 1 second, in place of the current measuring capability of 30 seconds. The quantitative data collected using the proposed instrument will be especially powerful for determining what changes in protein structure regulate signaling pathways, such as those controlled by kinases and nuclear receptors. Preliminary results indicate that data obtained from the proposed instrument will in the long term simplify and speed discovery of new medicines with greater specificity to allow more individualized treatments for patients suffering from chronic diseases like diabetes, atherosclerosis, and acute conditions related to cancer. ? Specific Aims Phase I of the project encompasses three Specific Aims over a period of 18 months whereupon a clearly defined end-point Milestone represents a significant achievement.
In Specific Aim #1, equipment to achieve rapid mixing for both initiation and quench of the exchange reaction will be assembled and tested.
In Specific Aim #2 the assembled rapid mixing module along with means to control temperature, will be swapped for the 30 sec mixing module of the current H/D exchange system. Finally in Specific Aim #3 the newly assembled instrument, demonstrated to completely mix solutions in one second, will be tested with proteins and protein:ligand complexes with known H/D exchange properties. Phase I end point Milestone: Determine H/D Exchange within 1 sec at a defined temperature Advancement of the project is dependent upon demonstrating determination of H/D exchange within one second using the temperature-controlled rapid mixing system. The commercialization of such an instrument, as proposed in Phase II, will provide a way to more accurately define protein dynamics and protein:protein interactions as needed for drug discovery, basic science, and proteomics research. As the test molecule we chose a representative of the nuclear receptor (NR) family that regulates pathways involved in chronic diseases such as diabetes, cancer and atherosclerosis, and is the target of drugs exhibiting annual sales nearing $13B. ? ? ?
|Coales, Stephen J; E, Sook Yen; Lee, Jessica E et al. (2010) Expansion of time window for mass spectrometric measurement of amide hydrogen/deuterium exchange reactions. Rapid Commun Mass Spectrom 24:3585-92|