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.

大自然不仅提供了可用于加速药物开发的合成机制（我们的长期目标），这也是该机械如何合成药物的众多例子。然而，聚酮化合物组装线的潜力实际上仍未被药物化学所开利。超过操纵编码这些合酶并识别合适异源宿主的DNA，不正确对这些分子工厂的逻辑的理解阻碍了他们的工程。在过去的几年中，生物信息学证据已经安装在装配线演化期间的模块化单元与大多数科学家在设计中采用的传统聚酮合成酶模块。我们的实验室帮助了重新定义模块，以使靶向酮合酶（KS）域处于最下游的位置，已经证明，使用更新的边界设计的合成酶优于设计的合成酶传统边界。经过许多设计建造测试周期之后，我们现在能够快速设计pentaketide 从大肠杆菌中产生立体密度聚酮化合物的制剂水平的合成酶。我们的实验室是当我们设计生成的装配线时与药物相关的产品。通过特定的目标1（自下而上的方法），我们将推动基板 KSS的公差限制，要求他们接受与B碳之外的取代基的中间体他们自然接受的人。通过3个编码5个pikromycin模块的DNA绑扎，125五烷基合成酶将被构造。包括成像在内的质谱方法将迅速识别苦难合酶。在最近在我们的实验室完成的KS Gatekeeping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。收益功能突变体将告知未来的工程。通过特定的AIM 2（自上而下的方法）将合并pikromycin模块通过4个诱饵产生100个七生合酶。产品将类似于Narbonolide，该产品是产品 pikromycin合酶的，但在第二，第三，第五和第六位置。在优化合成酶之后，如第一个目标中，将提供异丝胺生物合成/转移基因产生毒素类似物。从开创性的，大环体的模块化合成。安德鲁·迈尔斯（Andrew Myers）实验室，我们预计会发现几种新的大花环抗生素。在特定目标3（水平方法）将使用来自pikromycin的模块构建32个杂种五晶酸合成酶的库和旋转组装线。我们假设，由于之间的许多不兼容 ks和酰基载体蛋白（ACP）结构域在跨型连接处。一个反复识别的接口对接用于同源KS和ACP域的服务器将指导KS表面突变以恢复功能至不活跃合酶。我们试图确定一组允许对接ACP的突变，从而促进重组所有模块，并提供尽可能多的聚酮化学空间的访问。