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

摘要 本研究响应特别关注通知（NOSI）NOT-AT-21-006“基础科学 研究补充和综合健康方法，包括天然产品或身心 制定有针对性和无针对性的生物信息学方法， 天然产物混合物中的成分。” 天然产物（NPs）的结构解析仍然是NP发现活动中的关键限速步骤。 结构解析中的困难可能源于i）缺乏足够量的用于传统结构解析的材料， 分析方法（例如核磁共振（NMR）光谱和X射线晶体学）; ii）内在 NP的物理性质，和iii）在确定相对构型中NMR能力的限制。x射线 晶体学仍然是明确的结构确定的黄金标准，包括分配 立体化学。然而，新分离的纳米颗粒的X射线晶体学分析经常受到以下因素的阻碍： 不足以提供足够大的晶体用于单晶衍射或固态性能差 即使当有足够的材料可用时，这也阻止了大的原始晶体的形成。鉴于这些 挑战，我们设想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发现领域的纵向进展 响应于这种NOSI，因为MicroED最近已经被证明可以提供明确的结构， 结构复杂的化合物的亚微米大小的晶体， 适用于X射线分析。 在这项提案中，我们的目标是利用CryoEM/MicroED方法来解决结构中的主要瓶颈 （部分）纯化的NP和化学复杂的NP混合物的解析。我们假设我们可以 NP研究领域通过开发和优化高通量平台技术， 识别复杂混合物中的NP并产生用于整合到 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， 解析柠檬酸盐（部分）纯化的NP的结构; 2）开发基于MicroED的高通量平台， 化合物发现; 3）解决复杂NP混合物结构确定中的主要瓶颈。为 所有的目标，我们将利用一个独一无二的和广泛的三个NP集合组（化学图书馆， 提取物和部分纯化的级分）。我们 作为这些研究的结果，预期NP结构鉴定的速度和准确性的进步， 加快发现对改善人类健康至关重要的与癌症相关的纳米粒的速度。

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.