Ultrahigh Throughput Microscale Mass Spectrometry for Pharmaceutical Prenylation Enzyme Engineering

用于药物异戊二烯化酶工程的超高通量微型质谱分析

• 批准号: 10325565
• 负责人: Adam R. Abate
• 金额: $ 25.38万
• 依托单位: FLUID DISCOVERY INC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-25 至 2022-09-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10325565
• 关键词: Abate; Acids; Address; Anabolism; Biological Assay; Businesses; Cannabinoids; Cells; Characteristics; Chemicals; Chemistry; Collaborations; Computer Assisted; Contract Services; Coupling; Custom; DNA sequencing; Detection; Development; Dimethylallyltranstransferase; Elements; Engineering; Enzymes; Equipment; Evolution; Family; Generations; Goals; Industrialization; Lead; Libraries; Liquid substance; Mass Spectrum Analysis; Methods; Microfluidics; Modification; Molecular; Nanoarray Analytical Device; Natural Products; Optics; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Preparation; Process; Production; Protocols documentation; Provider; Reagent; Recovery; Research; Resolution; Robotics; Sampling; Scanning; Services; Slide; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; System; Techniques; Technology; Testing; Time; Variant; Vertebral column; Work; analytical method; base; cannabigerolic acid; catalyst; cost; cost effectiveness; design; direct application; drug discovery; drug modification; drug production; improved; innovation; instrument; novel; physical property; prenylation; product development; programs; quantum; research and development; screening; synthetic biology; tool

Project Summary Biocatalyst can be key tools in the synthesis of natural product-based pharmaceuticals and as powerful components of the pharmaceutical chemist’s drug modification kit. For example, the aromatic prenyltransferase NphB has utility in the synthesis of common prenylated compounds or derived backbones include cannabinoids, alpha acids, beta acids, phenylpropanoids, naphterpins, and marinones. The same enzyme also has utility as a general tool for compound functionalization. Our partner, BioMediCan has developed a whole cell biocatalysis platform based on this enzyme for production of natural and nonnatural rare cannabinoids. However, for all the attractive qualities and uses of enzyme- based or whole-cell biocatalysis, NphB as well as many other enzymes have not evolved with the characteristics required for industrial or pharmaceutical goals. These limitations arise because of the limitations of conventional analytical methods and the narrow detection capabilities of the current suite of ultrahigh-throughput microfluidic techniques. To develop novel high efficient platform and to improve the NphB biocatalyst, Fluid Discovery will engineer a microfluidic based biocatalysis sample preparation system with capacities of high throughput sample processing and low cost and enable its seamless coupling with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for high sensitivity and general applicability. This method, called μMALDI-TOF, will not only allow Fluid Discovery and BioMediCan to take the first steps towards becoming a key provider of novel compounds and enzymatic tools, but also provide a quantum leap in biocatalyst discovery and engineering through a highly scalable and universal screening platform. Our Phase I goal is to establish and demonstrate the capacity of this platform on engineering NphB in whole cell biocatalysis. In Phase II, we will continue our work via engineering NphB for improving and expanding the current whole-cell biocatalyst platform and initiate contracted service to early access synthetic biology companies. Ultimately, we plan to leverage the generality of our product for applications in the generation of biocatalysts for either green chemistry drug production or as tools for the molecular diversification that underlies lead compound modification and optimization.

项目摘要 生物催化剂可以是合成基于天然产品的药物的关键工具，并作为功能强大的组件 药物化学家的药物修饰套件。例如，芳族前转移酶NPHB具有实用性 普通原型化合物或衍生的骨干的合成包括大麻素，α酸，β酸， 苯丙烷，萘和Marinones。相同的酶也具有实用性作为化合物的一般工具 功能化。我们的合作伙伴BioMedican基于此酶开发了一个全细胞生物催化平台 天然和非天然稀有大麻素的产生。但是，对于酶的所有吸引力和用途 基于或全细胞生物催化，NPHB以及许多其他酶尚未随着特征而发展 工业或药物目标所需的。这些限制是由于常规的局限性 分析方法和超高直率微流体的当前套件的狭窄检测能力 技术。为了开发新型的高效平台并改善NPHB生物催化剂，流体发现将设计 基于微流体的生物催化样品制备系统，具有高吞吐量样品处理能力 低成本，并使其无缝耦合与基质辅助激光解吸/电离（MALDI）质量 光谱法具有高灵敏度和一般适用性。这种称为μmaldi-tof的方法不仅允许流体 发现和生物医学是迈出成为新颖化合物和酶促的关键提供商的第一步 工具，但还通过高度可扩展和通用的生物催化剂发现和工程提供了量子飞跃 筛选平台。我们的I阶段目标是建立和演示该平台在工程上的能力 全细胞生物催化中的NPHB。在第二阶段，我们将通过工程NPHB继续我们的工作，以改善和 扩展当前的全细胞生物催化剂平台，并启动合同服务以早期访问合成生物学 公司。最终，我们计划利用我们的产品的一般性用于生成的应用 用于绿色化学药物的生产或作为基础铅分子多样化的工具的生物催化剂 复合修改和优化。