Dynamically-resolved structural studies of proteins from fungal and bacterial pathogens

摘要 耐药性和脱敏是一个日益受到全球关注的问题。为了克服这些治疗 问题，药物发现管道必须利用所有可用的结构信息来 产生新的、有效的和安全的疗法。目前的药物化学努力通常会使 使用静态的低温蛋白质结构，它不整合已知的动力学 管理蛋白质的功能。从结构-活动关系到动态-活动关系将 使我们追求药物发现的方式多样化，为更先进的疗法打开大门。至 获得这些知识后，所提议的项目将进行时间分辨的结构研究 来自真菌和细菌病原体的与医学相关的蛋白质。尽管具有挑战性，但时间- 分辨结构生物学在过去的十年中已经成熟，使其适用于更广泛的范围 蛋白质靶标。这项研究将打破方法障碍，迅速获得动态- 活性关系，根据这些关系，我们将确定动力学如何与蛋白质功能相关。 将使用互补的生化和生物物理研究来明确解决 蛋白质亚稳中间体。采用光笼和低温诱捕等方法 Trigger同步样本中的蛋白质功能，利用PI的工作和专业知识 以及现有的设施，包括一个专门的有机合成实验室。 将实现两个初步目标。第一个为金黄色葡萄球菌Curis 3-脱氧-D-阿拉伯庚磺酸盐7- 磷酸合成酶(DAHPS)是真菌生存所必需的一种酶，因为它催化了 芳香氨基酸的生物合成。它被认为是目前很有希望的药物靶点。 抗真菌药物，它是动态变构调节的。第二个目标是B。 假腮腺，一种被认为对氧化应激反应负责的蛋白质， 机制仍不清楚。这两个项目都将使用总体方法来确定目标 将生成对动态活动关系的新见解并提供结构的流水线 短命的中间体。除了这笔赠款中提议的尖端工作外，这些 这些发现将推动一项专注于利用动态信息的长期研究计划 以推动新的药物设计。 这项拨款建议的基础工作将填补我们在蛋白质机制方面的知识空白。 以及它与动力学的关系，并将为新的药物发现方法铺平道路。这个 拟议的时间分辨实验具有挑战性，但由于多学科的合作而成为可能 PI的专业知识以及强有力的合作，推动了丰富的科学讨论和 畅通无阻地接触到美国和国外的尖端技术和设施。

Summary Drug resistance and desensitization is a growing global concern. To overcome these therapeutic issues, drug discovery pipelines have to make use of all available structural information to generate novel, potent and safe therapeutics. Current medicinal chemistry efforts typically make use of static, cryo-temperature protein structures, which do not integrate the dynamics known govern protein function. Advancing from structure-activity to dynamic-activity relationships will diversify the way we pursue drug discovery, opening doors to more advanced therapeutics. To obtain this knowledge, the projects proposed will pursue time-resolved structural studies of medically-relevant proteins from fungal and bacterial pathogens. Although challenging, time- resolved structural biology has matured over the last decade making it amenable to a wider range of protein targets. This research will break methodological barriers to swiftly obtain dynamic- activity relationships, from which we will determine how dynamics correlate to protein function. Complementary biochemical and biophysical studies will be used to unambiguously resolve protein metastable intermediates. Methods such as photocaging and cryo-trapping are used to trigger synchronize protein function in the sample, making use of the work and expertise of the PI as well as the available facilities, including a dedicated organic synthesis laboratory. Two initial targets will be pursued. The first one, C. Auris 3-Deoxy-D-arabinoheptulosonate 7- phosphate Synthase (DAHPS) is an enzyme essential for fungal survival as it catalysis a step in the biosynthesis of aromatic amino-acids. It is considered a promising drug target to current antifungals and it is dynamically allosterically regulated. The second target is rubrerythrin from B. pseudomallei, a protein believed to be responsible for oxidative stress responses for which the mechanism is still unclear. Both projects will be targeted using an overarching methodological pipeline which will generate new insights into dynamic-activity relationships and provide structures of short-lived intermediates. Beyond the cutting-edge work proposed in this grant, these discoveries will fuel a long-term research program focusing on harnessing dynamic information to drive novel drug design. The fundamental work proposed in this grant will fill gaps in our knowledge of protein mechanism and its relationship to dynamics, and will pave the way to novel drug discovery approaches. The proposed time-resolved experiments are challenging but made possible by the multi-disciplinary expertise of the PI as well as strong collaborations that drive fertile scientific discussions and unobstructed access to cutting edge technology and facilities in the USA and abroad.