Developing NMR and Biophysical Strategies to Study Protein-Ligand Interactions and Reveal Interesting Compound Properties

有一种广泛的观点认为，研究配体与大分子的结合应该简单明了。然而，在实践中，可用的工具很少，而且会受到构件的影响。此外，绑定涉及许多事件，其中一些事件尚未完全揭示。这项研究计划旨在开发能够阐明各种结合事件的策略，并揭示小分子配体的相关自由态性质。这项研究将在我的实验室进行基于片段的配体发现(FBLD)工作的背景下进行，在那里将系统地评估数千种化合物与各种蛋白质类型的结合。简而言之，FBLD将涉及筛选片段化合物(~120-300Da)的文库(例如~1000-8000个成员)，以寻找结合剂(Kd~100-1000um)，然后进行后续表征。 我们将首先根据理论和实验属性使用优先级过滤器建立化合物库。化学信息学将用于对具有期望的计算出的物理化学性质、官能团多样性和三维性的片段进行排序。将采用新的核磁共振和生物物理图谱方法来识别不溶化合物，并表征那些在自由态作为单独分子翻滚的化合物，以及那些自组装成聚集体或纳米实体的化合物。自组装的化合物将在后续研究中广泛表征，以建立结构-聚集体-性质关系，并探索用于纳米生物技术目的的设计机会。 然后将对这些文库进行筛选，以确定与蛋白质靶标结合的化合物。将开发一套全面的核磁共振筛选技术，通过核磁共振共振位移(HSQC，DLB，19F核磁共振)、弛豫(CPMG，NOESY)、饱和(STD)和扩散(DOSY)的变化来检测结合。这些和其他生物物理方法将有助于揭示和阐明各种类型的自由态性质和结合模式(例如，专一性、化学计量、混杂、在不同时间尺度上的稳定性)。对产生的大量数据进行评估将需要软件的协助和开发。然后将建立后续的生物物理和结构-活性关系，以最好地表征结合模式并确定与性质的相关性。还将实施新的方案，以便在各种缓冲液、与生物相关的介质和基于细胞的环境中进行有效的筛选，并筛选非典型靶标，如内在无序的蛋白质。 总之，这项研究将建立新的策略，揭示和开发基本的化学性质，并定义新的FBLD方法，以提高我们对定义蛋白质-配体相互作用的分子性质的理解。

There is a widely held view that the study of ligands binding to macromolecules should be simple and straightforward. In practice, however, the available tools are sparse and compromised by artifacts. Moreover, binding involves many events, some of which have yet to be fully revealed. This research program aims to develop strategies to enable the elucidation of various binding events and to expose relevant free-state properties of small-molecule ligands. The research will be executed within the context of mounting fragment-based ligand discovery (FBLD) efforts in my laboratory where the binding of thousands of compounds to a variety of protein types will be systematically evaluated. Briefly, FBLD will involve the screening of libraries (e.g. ~1000-8000 members) of fragment compounds (~120-300 Da) in search for binders (Kd ~ 100-1000 uM) that will then be subjected to follow-up characterization. We will begin by building libraries of compounds using prioritization filters based on theoretical and experimental attributes. Cheminformatics will serve to rank fragments that have desirable calculated physicochemical properties, functional group diversity and three-dimensionality. New NMR and biophysical profiling methods will be implemented to identify insoluble compounds and characterize those that tumble as lone molecules in the free-state versus those that self-assemble into aggregates or nano-entities. Compounds that self-assemble will be extensively characterized in follow-up studies to establish structure-aggregate-property relationships and to explore design opportunities for nano-biotechnology purposes. Theses libraries will then be screened to identify compounds that bind to protein targets. A set of comprehensive NMR screening techniques will be developed to detect binding via changes in NMR resonance shifts (HSQC, DLB, 19F NMR), relaxation (CPMG, NOESY), saturation (STD) and diffusion (DOSY). These and other biophysics methods will help to reveal and elucidate the various types of free-state properties and binding modes (e.g. specificity, stoichiometries, promiscuity, stability at various time-scales). The evaluation of large amounts of data generated will require the assistance and development of software. Follow-up biophysical and structure-activity relationships will then be established to best characterize the binding modes and identify correlations with properties. New protocols will also be implemented to enable efficient screening in various buffers, biologically-relevant media, and cell-based environments, and to screen atypical targets such as intrinsically disordered proteins. In summary, this research will establish new strategies, expose and exploit fundamental chemical properties, and define new FBLD methods to improve our understanding of the molecular properties that define protein-ligand interactions.