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器（我们的长期目标），还有大量关于这种机器如何合成药物的例子。然而，药物化学实际上尚未开发聚酮化合物装配线的潜力。超过操纵编码这些合酶的 DNA 并识别合适的异源宿主，这是不正确的对这些分子工厂逻辑的理解阻碍了他们的工程。在过去的几年里，生物信息证据表明，装配线进化过程中重组的模块化单元不同于大多数科学家在设计中采用的传统聚酮合酶模块。我们的实验室提供了帮助重新定义模块，使守门酮合酶 (KS) 结构域位于其最下游位置，并且已经证明，使用更新边界设计的合酶优于使用更新边界设计的合酶传统边界。经过多次设计-构建-测试周期后，我们现在能够快速设计五肽从大肠杆菌中产生制备水平的立体化学致密聚酮化合物的合酶。我们的实验室是当我们设计生产生成的装配线时，我们的定位是加深我们对装配线逻辑的了解医药相关产品。通过具体目标 1（自下而上的方法），我们将推动基材 KS 的耐受极限，要求它们接受除 b 碳以外具有不同取代基的中间体来自他们自然接受的人。通过与编码 5 个匹克霉素模块、125 个五肽的 DNA 进行 3 次连接将构建合酶。包括成像在内的质谱方法将快速识别陷入困境的患者合酶。在我们实验室最近完成的 KS 守门生物信息学/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）将组合匹克霉素模块通过4次连接产生100个七肽合酶。该产品将类似于 narbonolide，该产品匹克霉素合酶，但在第二、第三、第五和第六位具有不同的酮化物单元组合职位。按照第一个目标优化合酶后，将提供去糖胺生物合成/转移基因生成那博霉素类似物。从开创性的大环内酯类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 中（横向方法）将使用 pikromycin 的模块构建 32 种混合五肽合酶的文库和多杀菌素装配线。我们假设其中许多将由于之间的不兼容性而处于不活动状态模块间连接处的 KS 和酰基载体蛋白 (ACP) 结构域。重复标识的接口同源 KS 和 ACP 域的对接服务器将引导 KS 表面突变将功能恢复至非活动状态合酶。我们寻求鉴定一组允许不同 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。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Assessing and harnessing updated polyketide synthase modules through combinatorial engineering.

通过组合工程评估和利用更新的聚酮合酶模块。

DOI：
发表时间：
2023-07-28
期刊：
影响因子：
0
作者：
Ray, Katherine A;Lutgens, Joshua D;Bista, Ramesh;Zhang, Jie;Desai, Ronak R;Hirsch, Melissa;Miyazawa, Takeshi;Cordova, Antonio;Keatinge
通讯作者：
Keatinge

Boosting titers of engineered triketide and tetraketide synthases to record levels through T7 promoter tuning.

通过 T7 启动子调整，将工程化三酮化合物和四酮化合物合酶的滴度提高至创纪录水平。