Biosynthesis and bioengineering of epoxyketone proteasome inhibitors

环氧酮蛋白酶体抑制剂的生物合成及生物工程

• 批准号: 2590889
• 金额: --
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2590889
• 关键词: Biosynthesis; bioengineering; epoxyketone; proteasome; inhibitors

"Epoxyketones are an important class of bacterial nonribosomal peptides that target the proteolytic -subunit of the proteasome. They inspired the development of carfilzomib, an epoxyketone approved in 2012 for the treatment of multiple myeloma. Oprozimib, a second-generation orally available anticancer epoxyketone, and KZR-616, an epoxyketone that selectively targets the immune proteasome, are currently in phase II clinical trials. All three are manufactured via chemical synthesis, which is costly and unsustainable. We aim to develop cheaper and more sustainable biocatalytic approaches for epoxyketone production.We have investigated the biosynthesis of TMC-86A and eponemycin, two closely related epoxyketones produced by Actinobacteria. The biosynthetic gene clusters for these metabolites have been cloned using transformation-associated recombination (TAR) in yeast and expressed in a heterologous host. TAR-based methods have been used to create in-frame deletions in each of the biosynthetic genes and characterisation of the metabolites accumulated in each of the mutants has provided extensive insights into the nature and order of each of the biosynthetic steps. We have also shown using in vitro biochemical methods that the epoxyketone pharmacophore of these metabolites is assembled from the -dimethyl--keto acid product of a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) by an unusual trifunctional flavin-dependent decarboxylase-dehydrogenase-monooxygenase (epoxyketone synthase). This enzyme has been shown to accept several analogues of the natural substrate.In this project, we aim to determine the X-ray crystal structure of the epoxyketone synthase, providing a basis for rational engineering to further broaden its substrate tolerance, and develop a mutasynthesis approach for the production of epoxyketones that are structurally related to the compounds currently undergoing clinical trials."

环氧酮是一类重要的细菌非核糖体多肽，以蛋白酶体的蛋白水解亚单位为靶标。他们启发了carfilzomib的开发，这是一种环氧酮，于2012年被批准用于治疗多发性骨髓瘤。Oprozimib是第二代口服抗癌环氧酮，KZR-616是一种选择性靶向免疫蛋白酶体的环氧酮，目前处于II期临床试验。这三种燃料都是通过化学合成来制造的，这既昂贵又不可持续。我们的目标是开发更便宜和更可持续的环氧酮生产生物催化方法。我们研究了由放线杆菌产生的两种密切相关的环氧酮-TMC-86A和依诺霉素的生物合成。这些代谢物的生物合成基因簇已在酵母中利用转化相关重组(TAR)克隆并在异源宿主中表达。基于TAR的方法已被用于在每个生物合成基因中创建框内缺失，并且对每个突变体中积累的代谢物的表征为深入了解每个生物合成步骤的性质和顺序提供了广泛的见解。我们还用体外生化方法证明，这些代谢物的环氧酮药效团是由混合非核糖体肽合成酶-聚酮合成酶(NRPS-PKS)的-二甲基酮酸产物通过一个不寻常的三功能黄素依赖的脱羧酶-脱氢酶-单加氧酶(环氧酮合成酶)组装而成的。这种酶已经被证明可以接受几种天然底物的类似物。在这个项目中，我们的目标是确定环氧酮合成酶的X射线晶体结构，为进一步扩大其对底物的耐受性提供合理的工程基础，并开发一种突变合成方法来生产结构上与目前正在进行临床试验的化合物相关的环氧酮。