In vitro biosynthesis of molecular warheads from hydroxamate- and β-lactone-containing protease inhibitors

含异羟肟酸和β-内酯的蛋白酶抑制剂分子弹头的体外生物合成

• 批准号: 419817603
• 负责人: Professor Dr. Leonard Kaysser
• 金额: --
• 依托单位: Lehrstuhl für Pharmazeutische Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/419817603?language=en
• 关键词: vitro; biosynthesis; molecular; warheads; hydroxamate

Small-molecule protease inhibitors from natural sources have inspired the development of numerous pharmaceutical agents. In many cases, these natural products exhibit their inhibitory properties by warhead-mediated binding to the active site of target enzymes. In this research program we are planning to reconstitute the biosynthesis of the warhead moieties of two well-studied compound classes: the hydroxamate metalloproteinase inhibitors and the ß-lactone proteasome inhibitors. In a DFG-funded project we recently found that the carbon skeleton of the N-hydroxy-2-pentyl-succinamic acid warhead of actinonin and matlystatin is assembled by an unprecedented variation of the ethylmalonyl-CoA pathway. This involves the reductive carboxylation of octenoyl-CoA followed by epimerization and 1,2-rearrangement of the hexylmalonyl-CoA product. Moreover, we showed that the formation of 2-carboxy-3-alkyl ß-lactone warhead of belactosin and cystargolide is reminiscent of leucine biosynthesis featuring the aldol reaction of acetyl-CoA with an alpha-ketoacid and the stereo-specific isomerisation of the 2-alkylmalate product to 3-alkylmalate. Thus, both pathways represent unique adaptions of central biochemical processes to the secondary metabolism in bacteria with high potential for synthetic metabolic engineering. In the current project we want to individually reconstitute the enzymatic reactions in the biosynthesis of N-hydroxy-2-pentyl-succinamic acid in vitro and get a detailed understanding of the 1,2-rearrangement reaction of (2R)-hexylmalonyl-CoA by mechanistic studies and protein crystallography. We plan to evaluate key enzymatic reactions in the construction of the 2-carboxy-3-alkyl ß-lactone scaffold for their biotechnological capabilities. Moreover, we will investigate the formation of the ß-lactone ring of the cystargolides and belactosins by in vitro biochemistry with the prospect to identify the first bona fide β-lactone synthetase in natural product biosynthesis. The proposed research project will give us detailed mechanistic insights into two unique biosynthetic pathways in the bacterial secondary metabolism. This information will allow us to implement synthetic biology strategies for the design and production of tailor-made non-natural derivatives of these pharmaceutically interesting compound classes and facilitate the discovery of novel protease inhibitors by genome mining.

来自天然来源的小分子蛋白酶抑制剂激发了许多药剂的开发。在许多情况下，这些天然产物通过弹头介导的与靶酶的活性位点的结合而表现出其抑制特性。在这项研究计划中，我们计划重建两个充分研究的化合物类别的弹头部分的生物合成：异羟肟酸金属蛋白酶抑制剂和β-内酯蛋白酶体抑制剂。在DFG资助的项目中，我们最近发现放线菌素和matlystatin的N-羟基-2-戊基-琥珀酰胺酸弹头的碳骨架是由乙基丙二酰辅酶A途径的前所未有的变化组装的。这涉及辛烯酰-CoA的还原羧化，随后是己基丙二酰-CoA产物的差向异构化和1，2-重排。此外，我们表明，belactosin和cystargolide的2-羧基-3-烷基α-内酯弹头的形成使人想起亮氨酸生物合成，其特征在于乙酰辅酶A与α-酮酸的羟醛缩合反应和2-烷基苹果酸产物到3-烷基苹果酸的立体特异性异构化。因此，这两种途径都代表了中央生化过程对细菌次级代谢的独特适应，具有很高的合成代谢工程潜力。在本项目中，我们希望在体外单独重构N-羟基-2-戊基-琥珀酰胺酸生物合成中的酶促反应，并通过机理研究和蛋白质晶体学详细了解（2 R）-己基丙二酰-CoA的1，2-重排反应。我们计划评估2-羧基-3-烷基β-内酯支架构建中的关键酶促反应的生物技术能力。此外，我们将通过体外生物化学研究球花内酯和半乳糖苷的β-内酯环的形成，前景鉴定天然产物生物合成中第一个真正的β-内酯合成酶。拟议的研究项目将为我们提供详细的机械见解，在细菌次级代谢中的两个独特的生物合成途径。这些信息将使我们能够实施合成生物学策略，用于设计和生产这些药学上有趣的化合物类的定制非天然衍生物，并通过基因组挖掘促进新型蛋白酶抑制剂的发现。