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Harnessing Polyketide Assembly Lines for Medicinal Chemistry

利用聚酮化合物装配线进行药物化学

基本信息

批准号：
10651828
负责人：
Adrian Tristan Keatinge-Clay
金额：
$ 31.8万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10651828
关键词：
Acceleration Acyl Carrier Protein Affect Amphotericin Anabolism Anti-Bacterial Agents Antibiotics Antifungal Agents Antineoplastic Agents Azithromycin Bioinformatics Carbon Chemicals Collaborations Communities Complement Complex DNA Development Docking Engineering Ensure Enzymes Escherichia coli Evolution Freedom Future Gatekeeping Gene Transfer Generations Genetic Recombination Goals Human Hybrids Image Immunosuppressive Agents Instruction Investigation Knowledge Libraries Ligation Logic Macrolides Manuals Mass Spectrum Analysis Measures Medicine Methods Mutation Natural Products Nature Pharmaceutical Chemistry Pharmaceutical Preparations Positioning Attribute Reporting Scientist Seminal Sirolimus Surface Synthesis Chemistry Tertiary Protein Structure Testing Update Work Writing analog combinatorial design design,build,test desosamine functional restoration glycosylation hydroxyl group improved migration monomer mutant new technology next generation novel therapeutics picromycin polyketide synthase polyketides success virtual

项目摘要

Nature has provided not only a synthetic machinery that can be used to accelerate the development of medicines (our long-term goal) but also a plethora of examples for how this machinery synthesizes medicines. However, the potential of polyketide assembly lines remains virtually untapped by medicinal chemistry. Beyond manipulating the DNA encoding these synthases and identifying suitable heterologous hosts, an incorrect understanding of the logic of these molecule factories has thwarted their engineering. Over the last several years, bioinformatic evidence has mounted that the modular unit recombined during assembly line evolution differs from the traditional polyketide synthase module that most scientists employ in their designs. Our lab has helped redefine the module such that a gatekeeping ketosynthase (KS) domain is at its most downstream position and has demonstrated that synthases designed with the updated boundary outperform those designed with the traditional boundary. After many design-build-test cycles, we are now able to rapidly engineer pentaketide synthases that produce preparative levels of stereochemically-dense polyketides from E. coli. Our lab is positioned to further our knowledge of assembly line logic as we engineer assembly lines that generate medicinally-relevant products. Through Specific Aim 1 (the bottom-up approach) we will push the substrate tolerance limits of KSs, asking them to accept intermediates with substituents beyond the b-carbon that differ from those they naturally accept. Through 3 ligations with DNA encoding 5 pikromycin modules, 125 pentaketide synthases will be constructed. Mass spectrometry methods, including imaging, will quickly identify struggling synthases. Guided by a bioinformatics/structural study of KS gatekeeping recently completed in our lab, we will predict what mutations will remove bottlenecks in these assembly lines. Gain-in-function mutants will inform future engineering. Through Specific Aim 2 (the top-down approach) pikromycin modules will be combined through 4 ligations to yield 100 heptaketide synthases. The products will be similar to narbonolide, the product of the pikromycin synthase, but with differing combinations of ketide units at the second, third, fifth, and sixth positions. After optimizing synthases as in the first aim, desosamine biosynthesis/transfer genes will be supplied to generate narbomycin analogs. As from the seminal, modular syntheses of macrolides performed by the Andrew Myers lab, we anticipate discovering several new macrolide antibiotics. In Specific Aim 3 (the horizontal approach) a library of 32 hybrid pentaketide synthases will be constructed using modules from the pikromycin and spinosyn assembly lines. We hypothesize that many of these will be inactive due to incompatibilities between KS and acyl carrier protein (ACP) domains at intermodular junctions. An interface repeatedly identified by docking servers for cognate KS and ACP domains will guide KS surface mutations to restore function to inactive synthases. We seek to identify a set of mutations that permit the docking of diverse ACPs, thus facilitating the recombination of all modules and providing access to as much polyketide chemical space as possible.

大自然不仅提供了一种可用于加速药物开发的合成机器， (our长期目标），但也有大量的例子，说明这一机制如何合成药物。然而，在这方面，聚酮化合物装配线的潜力实际上仍未被药物化学开发。超出操纵编码这些酶的DNA并鉴定合适的异源宿主，对这些分子工厂逻辑的理解阻碍了它们的工程化。在过去的几年里，生物信息学证据表明，在装配线进化过程中重组的模块化单元与大多数科学家在他们的设计中使用的传统聚酮合酶模块。我们的实验室帮助重新定义所述模块，使得守门酮合酶（KS）结构域位于其最下游位置，已经证明，使用更新的边界设计的机器人性能优于使用传统边界。经过多次设计-制造-测试循环，我们现在能够快速设计pentaketide 从E.杆菌我们实验室正在定位，以进一步我们的装配线逻辑的知识，因为我们工程师的装配线，医学相关产品。通过具体目标1（自下而上的方法），我们将推动基板 KSs的容忍限度，要求他们接受取代基超过b-碳的中间体，他们自然接受的人。通过与编码5个吡克罗霉素模块的DNA的3个连接，将建造一座城堡。包括成像在内的质谱分析方法将迅速识别出 - 是的在我们实验室最近完成的KS守门的生物信息学/结构研究的指导下，我们将预测哪些突变将消除这些装配线中的瓶颈。功能获得型突变体将告知未来工程通过特定目标2（自上而下的方法），将合并吡克罗霉素模块通过4次连接得到100个七肽聚糖酶。这些产品将类似于纳博纳，但是在第二、第三、第五和第六个位置具有不同的酮化合物单元组合岗位在如第一个目的中优化脱氢酶之后，将提供脱氧葡萄糖胺生物合成/转移基因以产生那博霉素类似物。从大环内酯的开创性的模块化合成开始，安德鲁·迈尔斯实验室，我们预计会发现几种新的大环内酯类抗生素。在具体目标3（水平方法）将使用来自匹克罗霉素的模块构建32个杂合喷他卡肽酶的文库和多杀菌素生产线。我们假设，许多这些将是无效的，由于不兼容之间 KS和酰基载体蛋白（ACP）结构域在模块间连接。一个接口反复标识为同源KS和ACP结构域的对接服务器将引导KS表面突变以恢复失活的功能， - 是的我们试图确定一组允许不同ACP对接的突变，从而促进所有模块的重组，并提供尽可能多的聚酮化合物化学空间。