The biosynthesis of the alpha,beta-epoxyketone pharmacophore in natural product proteasome inhibitors

在过去的十年中，蛋白酶体已经成为一种有效的治疗靶点。2012年7月，Carfilzomib(Kyprolis®)是一种四肽环氧酮，在美国被批准用于复发和难治性多发性骨髓瘤患者。它是从天然产物环氧米星发展而来的，它含有两个紧邻的强亲电基团的独特药效团：α，β-环氧酮弹头。这个结构基团允许高度特异和不可逆的结合到真核蛋白酶体的凝乳酶样部分。直到最近，关于α，β-环氧酮的生物合成还一无所知。在我们的初步工作中，我们鉴定并异源表达了环氧米星和表观霉素基因簇。值得注意的是，α，β-环氧酮弹头的形成不能从基因簇中推断出来。因此，我们预测在末端聚酮的转化过程中会发生不寻常的生物转化。在这个项目中，我们打算阐明导致α，β-环氧酮的生物合成途径。为此，我们将主要集中在分析异源基因缺失突变。我们还计划进行同位素浓缩前驱体喂养实验。此外，将使用体外生物化学和蛋白质结晶学来研究各个酶的催化机理。除此之外，基因组挖掘揭示了一条同源途径，我们认为这条途径指导了Matlystatin样蛋白抑制剂的形成。为了验证这一发现，我们将分离并异源表达相应的基因簇。这项实验应该成为我们未来以基因组为导向的药物发现活动的概念证明。这项研究建议将使我们深入了解具有治疗意义的一类环氧酮的生物学和生物发生，并将促进产生具有更好性能的蛋白酶体抑制剂。

The proteasome has advanced as a validated therapeutic target over the last decade. In July 2012 Carfilzomib (Kyprolis®) a tetrapeptide epoxyketone was approved for the use in patients with relapsed and refractory multiple myeloma in the USA. It was developed from the natural product epoxomicin comprising a unique pharmacophore of two strongly electrophilic groups in the immediate proximity to each other: the alpha,beta-epoxyketone warhead. This structural group allows a highly specific and irreversible binding to the chymotrypsin-like moiety of the eukaryotic proteasome. Until recently nothing was known about the biosynthesis of the alpha,beta-epoxyketone. In our preliminary work, we identified and heterologously expressed the epoxomicin and the eponemycin gene clusters. Notably, the formation of the alpha,beta-epoxyketone warhead could not be deduced from the gene clusters. We therefore predict that unusual biotransformations take place in the conversion of the terminal polyketide. In this project we intend elucidate the biosynthetic pathway which leads to the alpha,beta-epoxyketone. To this end we will primarily focus on the analysis of heterologous gene deletion mutants. We also plan to perform isotope-enriched precursor feeding experiments. Moreover, the catalytic mechanisms of respective enzymes will be investigated using in vitro biochemistry and protein crystallography. In addition to that, genome mining revealed a homologous pathway which we assume to direct the formation of matlystatin-like protesome inhibitors. To verify this finding, we will isolate and heterologously express the corresponding gene cluster. This experiment should serve as a proof of concept for our future genome-guided drug discovery campaigns. This research proposal will give us deep insides in the biology and biogenesis of the therapeutically important class of epoxyketones and will facilitate the generation of proteasome inhibitors with improved properties.