An instrument to measure protein amide H/D exchange within a second

描述（由申请方提供）：将组装一台能够在规定温度下在一秒钟内测量H/D交换的仪器，以检验以下假设：可以在显著更高的时间分辨率下获得有关蛋白质构象变化的定量数据。这将通过开发一种新型的快速在线混合模块来实现，该模块能够在1秒后确定氘掺入水平，而不是30秒的电流测量能力。使用拟议仪器收集的定量数据对于确定蛋白质结构的哪些变化调节信号通路（例如由激酶和核受体控制的信号通路）将特别强大。初步结果表明，从长远来看，从拟议的仪器中获得的数据将简化和加速具有更高特异性的新药的发现，从而为患有糖尿病、动脉粥样硬化和与癌症相关的急性疾病等慢性疾病的患者提供更个性化的治疗。 具体目标 该项目的第一阶段包括三个具体目标，为期18个月，其中明确界定的终点里程碑代表着一项重大成就。在具体目标#1中，将组装并测试用于实现交换反应启动和淬灭的快速混合的设备。在具体目标#2中，将组装的快速混合模块沿着温度控制装置替换为当前H/D交换系统的30秒混合模块。最后，在特定目标#3中，将使用具有已知H/D交换特性的蛋白质和蛋白质：配体复合物对新组装的仪器进行测试，该仪器已证明可在一秒钟内完全混合溶液。阶段I终点里程碑：在规定的温度下在1秒内测定H/D交换项目的进展取决于使用温控快速混合系统在1秒内测定H/D交换。如第二阶段所提出的，这种仪器的商业化将提供一种更准确地定义蛋白质动力学和蛋白质：药物发现，基础科学和蛋白质组学研究所需的蛋白质相互作用的方法。作为测试分子，我们选择了核受体（NR）家族的代表，该家族调节涉及糖尿病、癌症和动脉粥样硬化等慢性疾病的通路，并且是年销售额接近130亿美元的药物的目标。

DESCRIPTION (provided by applicant): 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.