Salinosporamide Biosynthesis and Engineering

说明(申请人提供)：盐孢子胺A是一种有效的不可逆转的蛋白酶体抑制剂，目前正处于Ib期人类临床试验中，用于治疗多发性骨髓瘤和其他癌症。这种海洋细菌天然产物基于其β-内酰胺-内酯药效基团，具有独特的作用机制，不同于FDA批准的唯一蛋白酶体抑制剂--硼酸盐硼替佐米多肽。在最后的支撑期，我们建立了盐孢子胺组装的生物合成基础，并在卤代反应、预苯酸盐生物化学和聚酮前体供应方面发现了一些新的酶反应。这些基本知识的翻译使我们能够通过基因工程合理地设计新的盐孢酰胺类似物进行生物学评价。这项工作有助于确定盐孢酰胺类抗癌药物的构效关系。尽管到目前为止我们取得了重大进展，但由于盐孢子胺从新的分子构建块中史无前例地组装而成，我们仍然对其如何生物合成只有一个粗略的了解。许多问题仍然存在，而新的机会已经浮出水面，以回应这一正在进行的研究计划中的发现。酶发现、合成生物学、化学酶合成、基因组挖掘和蛋白酶体生物化学方面的机会非常适合这一天然产物生物合成计划。为了实现本申请中概述的广泛目标，我们提出了一个涉及五个具体目标的多学科项目。首先，我们计划从功能和结构上描述SALC 酮合成酶及其在盐孢酰胺β-内酰胺内酯核心形成中的关键生物合成作用。其次，我们将应用SALC的功能，以合成的酰氨基酸硫酯为基础，利用重组盐孢酰胺生物合成酶，建立一种简化的化学酶促合成盐孢酰胺衍生物的方法。第三，我们的目标是对合成盐孢子胺新氨基酸残基环己烯丙氨酸的生物合成酶进行功能表征，环己烯丙氨酸是其强大的蛋白酶体结合亲和力的关键。第四，我们将从功能上描述专职蛋白酶体--Sali亚基及其在热带葡萄球菌对盐孢酰胺的自身抗性中所扮演的角色。第五，我们计划开发新的基于巴豆酰-辅酶A还原酶的表达系统，用于工程合成具有卤化(氟和氯)侧链和支化侧链的新的聚酮合成酶延伸单元，用于设计新的聚酮分子。

DESCRIPTION (provided by applicant): Salinosporamide A is a potent irreversible proteasome inhibitor presently in phase Ib human clinical trials for the treatment of multiple myeloma and other cancers. This marine bacterial natural product has a distinctive mechanism of action based on its ?-lactam-?-lactone pharmacophore that differs from the only FDA-approved proteasome inhibitor, the peptide boronate bortezomib. During the last period of support, we established the biosynthetic foundation of salinosporamide assembly and discovered a number of novel enzymatic reactions in halogenation, prephenate biochemistry, and polyketide precursor supply. Translation of this basic knowledge allowed us to rationally design through genetic engineering new salinosporamide analogues for biological evaluation. This work helped determine the structure-activity relationships within the salinosporamide family of anticancer agents. Despite our significant progress to date, we still only have a cursory understanding of how salinosporamide is biosynthesized due to its unprecedented assembly from novel molecular building blocks. Numerous questions remain, while new opportunities have surfaced in response to discoveries made in this ongoing research program. Opportunities in enzyme discovery, synthetic biology, chemoenzymatic synthesis, genome mining, and proteasome biochemistry are uniquely suited for this natural product biosynthetic program. To accomplish the broad goals outlined in this application, we propose a multidisciplinary project involving five specific aims. First, we plan to functionally and structurally characterize the SalC ketosynthase and its key biosynthetic role in the formation of the ?-lactam-?-lactone core of salinosporamide. Second, we will apply the function of SalC to develop a streamlined chemoenzymatic synthesis of salinosporamide derivatives based on a focused library of ?-lactam-?-lactones from synthetic acylamino acid thioesters with recombinant salinosporamide biosynthetic enzymes. Third, we aim to functionally characterize the biosynthetic enzymes responsible for the synthesis of salinosporamide's novel amino acid residue, cyclohexenylalanine, which is paramount to its potent proteasome binding affinity. Fourth, we will functionally characterize the dedicated proteasome ?-subunit SalI and its hypothesized role in S. tropica self-resistance against salinosporamide. And fifth, we plan to develop new crotonyl-CoA reductase-based expression systems for the engineered biosynthesis of new polyketide synthase extender units with halogenated (fluorine and chlorine) and branched side chains for the design of new polyketide molecules.