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

用于天然产物发现的高通量 MicroED 驱动平台技术的开发和验证

• 批准号: 10618979
• 负责人: Hosea Martin Nelson
• 金额: $ 63.21万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-06 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10618979
• 关键词: Acceleration; Algorithms; Animals; Anti-Bacterial Agents; Antifungal Agents; Automobile Driving; Bacteria; Bioinformatics; Biological; Cardiovascular Diseases; Chemicals; Code; Collection; Complementary Health; Complex; Complex Mixtures; Computer software; Crude Extracts; Cryoelectron Microscopy; DNA Microarray Chip; Data; Data Analyses; Data Collection; Deposition; Development; Disease; Disparate; Drug usage; Engineering; Eubacterium; Face; Goals; Health; Human; Human Resources; Image; Infection; Integrative Medicine; Intervention; Location; Malignant Neoplasms; Microarray Analysis; Modality; Modernization; Molds; NMR Spectroscopy; Natural Products; Natural Products Chemistry; Nuclear Magnetic Resonance; Nutrient; Organism; Pain; Pain Measurement; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacopoeias; Plants; Population; Process; Property; Protista; Reporting; Research; Roentgen Rays; Safety; Sales; Sampling; Science; Scientist; Site; Source; Speed; Structure; Time; Transmission Electron Microscopy; Validation; X ray diffraction analysis; X-Ray Crystallography; analytical method; atorvastatin; bioactive natural products; economic impact; electron diffraction; fungus; high throughput technology; hypocholesterolemia; improved; innovation; interest; marine; marine organism; microbial; mind/body; nano; novel; open source; pharmacokinetics and pharmacodynamics; pharmacologic; physical property; signature molecule; small molecule libraries; solid state; stereochemistry; submicron; synergism; technology platform; tool

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.

抽象的 本研究响应特别兴趣通知 (NOSI) NOT-AT-21-006“基础科学 补充和综合健康方法的研究，包括天然产品或身心 干预措施”的目标是“开发有针对性和无针对性的生物信息学方法来识别活性 天然产品混合物中的成分。” 天然产物 (NP) 的结构解析仍然是 NP 发现活动中关键的限速步骤。 结构阐明方面的困难可能是由于 i) 缺乏足够数量的传统材料 分析方法（例如核磁共振（NMR）光谱学和X射线晶体学）； ii) 内在的 NP 的物理性质，以及 iii) NMR 能力在确定相对构型方面的局限性。 X射线 晶体学仍然是明确结构测定的黄金标准，包括分配 立体化学。然而，新分离的纳米颗粒的 X 射线晶体学分析常常受到以下因素的阻碍： 数量不足，无法提供足够大的晶体用于单晶衍射或固态特性较差 即使有足够的材料，也无法形成大的原始晶体。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）可能直接导致纳米粒子发现领域的垂直进步 响应此 NOSI，因为 MicroED 最近已被证明可以提供明确的结构 结构复杂的化合物的亚微米尺寸晶体，未能产生大晶体 适用于X射线分析。 在本提案中，我们的目标是利用 CryoEM/MicroED 方法来解决结构中的主要瓶颈 阐明（部分）纯化的纳米颗粒和化学复杂的纳米颗粒混合物。我们假设我们可以前进 NP研究领域通过开发和优化高通量平台技术来 识别复杂混合物中的纳米颗粒，并产生新的分子衍射组学特征，以便整合到 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 化合物发现； 3）解决复杂纳米粒子混合物结构测定的主要瓶颈。为了 为了实现所有目标，我们将利用由三个 NP 集合组成的独一无二的、广泛的组（化学库） 提取物和部分纯化的级分）源自植物、海洋生物和丝状真菌。我们 预计这些研究的结果将提高 NP 结构识别的速度和准确性， 加快药理学相关纳米颗粒的发现速度，这对于改善人类健康至关重要。