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 这项研究对特殊关注通知（NOSI）不响应于21-006“基本科学 互补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“开发目标和不靶向的生物信息学方法来识别活动 天然产物混合物中的成分。” 天然产品（NP）的结构阐明仍然是NP发现运动中的关键限制步骤。 i）缺乏足够数量的材料来引起结构阐明的困难 分析方法（例如核磁共振（NMR）光谱和X射线晶体学）； ii）内在 NP的物理特性和iii）NMR功能在确定相对配置方面的限制。 X射线 晶体学仍然是明确结构确定的金标准，包括分配 立体化学。但是，新分离的NP的X射线晶体学分析通常受到挫败 数量不足，无法提供足够大的晶体，以用于单晶衍射或固态差的较差 即使有足够的材料，也排除了大型原始晶体的形成。鉴于这些 挑战，我们设想了最近报道的冷冻电子显微镜（冷冻）模态的应用 微晶电子衍射（微晶）可能导致NP发现领域的垂直进步 对这种NOSI的反应，因为最近已证明微型提供了从中提供明确的结构 结构复杂化合物的亚微米尺寸晶体，未产生大晶体 适用于X射线分析。 在此提案中，我们旨在利用冷冻/微型方法来解决结构中的主要瓶颈 阐明（部分）纯化的NP和化学复杂的NP混合物。我们假设我们可以进步 通过开发和优化高通量平台技术的NP研究领域 识别复杂混合物中的NP，并产生一种新型分子的衍射特征，以整合到 生物信息学方法。为了评估这一假设，我们将执行三个具体目标：1） 求解（部分）纯化的NP的固定结构； 2）开发一个基于高通量的微型平台 复合发现； 3）解决复杂NP混合物的结构测定中的主要瓶颈。为了 所有目的，我们将利用一个由三个NP藏品组成的独一无二和广泛的组（化学库 提取物和部分纯化的馏分）源自植物，海洋生物和丝状真菌。我们 由于这些研究的结果，预期NP结构识别速度和准确性的进步， 加速了药物相关的NPS的发现率，以改善人类健康。