Development and validation of a high-throughput MicroED-driven platform technology for natural product discovery

摘要 本研究是对特殊利益(NOSI)NOT-AT-21-006《基础科学》的响应 研究补充和综合健康方法，包括天然产品或精神和身体 干预措施“目的”是开发有针对性和无针对性的生物信息学方法，以识别活跃的 天然产品混合物中的成分。 天然产物(NP)的结构阐明在NP发现活动中仍然是一个关键的限速步骤。 结构阐明的困难可能源于：i)缺乏足够数量的材料用于传统 分析方法(如核磁共振波谱和X射线结晶学)；二)本征 NP的物理性质，以及III)在确定相对构型时核磁共振能力的限制。X射线 结晶学仍然是明确结构确定的金标准，包括赋值 立体化学。然而，新分离的NPs的X射线结晶学分析经常受到 没有足够的量提供足够大的晶体来进行单晶衍射或固态性能差 即使有足够的材料，也无法形成大的、原始的晶体。考虑到这些 挑战，我们展望了最近报道的冷冻电子显微镜(CryoEM)模式的应用 微晶电子衍射(MicroED)可以直接导致NP发现领域的垂直进步 作为对这一观点的回应，正如MicroED最近被证明从 结构复杂的化合物的亚微米大小的晶体，未能产生大晶体 适用于X射线分析。 在这份提案中，我们的目标是利用CryoEM/MicroED方法来解决结构中的主要瓶颈 说明(部分)提纯的NP和化学上复杂的NP混合物。我们假设我们可以取得进展 NP领域的研究通过开发和优化高通量平台技术来 识别复杂混合物中的NPs，并产生一种新的分子衍射学特征，用于集成到 生物信息学方法。为了评估这一假设，我们将实现三个具体目标：1)使用MicroED来 解决顽固(部分)纯化的纳米粒子的结构；2)开发基于MicroED的高通量平台 化合物的发现；3)解决了复杂NP混合物结构确定的主要瓶颈。为 所有目标，我们将利用一个由三个NP收藏组成的独一无二的庞大小组(化学图书馆 提取物和部分纯化的部分)来自植物、海洋生物和丝状真菌。我们 预计由于这些研究，NP结构鉴定的速度和准确性会有所提高， 加快发现对改善人类健康至关重要的药理上相关的核蛋白。

ABSTRACT This study is responsive to the Notice of Special Interest (NOSI) NOT-AT-21-006 “Fundamental Science Research on Complementary and Integrative Health Approaches, Including Natural Products or Mind and Body Interventions” objectives to “Develop targeted and untargeted bioinformatic approaches to identify active components in a natural product mixture.” Structural elucidation of natural products (NPs) remains a critical rate-limiting step in NP discovery campaigns. Difficulties in structural elucidation can arise from i) the lack of sufficient quantities of material for traditional analytical methods (e.g. nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography); ii) intrinsic physical properties of the NP, and iii) limitations of NMR capabilities in determining relative configuration. X-ray crystallography remains the gold-standard for unambiguous structural determination, including the assignment of stereochemistry. However, X-ray crystallographic analysis of newly-isolated NPs is often thwarted by insufficient quantities to provide crystals large enough for single-crystal diffraction or poor solid-state properties that preclude the formation of large, pristine crystals even when sufficient material is available. Given these challenges, we envision that application of the recently reported cryo-electron microscopy (CryoEM) modality micro-crystal electron diffraction (MicroED) could lead to vertical advances in the field of NP discovery directly responsive to this NOSI, as MicroED has recently been demonstrated to provide unambiguous structures from sub-micron-sized crystals of structurally complex chemical compounds that had failed to yield large crystals suitable for X-ray analysis. In this proposal, we aim to leverage a CryoEM/MicroED approach to resolving major bottlenecks in the structure elucidation of (partially) purified NPs and chemically complex NP mixtures. We hypothesize that we can advance the field of NP research through development and optimization of a high-throughput platform technology to identify NPs in complex mixtures and yield a novel diffractomics signature of molecules for integration into bioinformatics approaches. To evaluate this hypothesis, we will carry out three specific aims: 1) Use MicroED to solve structures of recalcitrant (partially) purified NPs; 2) Develop a high-throughput MicroED-based platform for compound discovery; and 3) Resolve major bottlenecks in structure determination of complex NP mixtures. For all aims, we will leverage a one-of-a-kind and expansive group of three NP collections (chemical libraries of extracts and partially purified fractions) derived from plants, marine organisms, and filamentous fungi. We anticipate advancement in the speed and accuracy of NP structural identification as a result of these studies, accelerating the rate of discovery of pharmacologically relevant NPs key to the improvement of human health.