Interrogation of Microbial Natural Product Methyltransferases

项目摘要/摘要 多重耐药细菌的流行导致了开发新的和未得到满足的需求的迫切需要。 抗菌药物。靶向细菌天然产物的生物合成已经成为一种很有前途的途径，因为 天然产物作为毒力因子、信号因子和微生物战剂的作用。然而，为了 到目前为止，还没有针对这些生物合成途径中的酶的药物进入临床。因此，系统化 对关键生物合成酶的研究对于充分了解它们在毒力中的个体作用是必要的。 要素生产。 S-腺苷-L-蛋氨酸依赖的甲基转移酶普遍存在于生命的各个领域 并修饰一系列不同的底物，包括蛋白质、核酸和天然产物。在真核生物中， 甲基转移酶活性调节细胞过程，如表观基因调控和神经元 因此，这些甲基转移酶得到了广泛的研究，并已被证实为 抗癌靶标。相比之下，天然产物甲基转移酶仍未被探索为治疗性的“焦油”。 尽管它们在致病毒力因子的生物合成和药剂学上具有重要作用 相关化合物。在生物合成过程中，天然产物甲基转移酶识别 共价连接到载体蛋白上的；因此，成功的甲基转移是底物识别的函数。 甲基转移酶和载体蛋白之间的识别和适当的蛋白质-蛋白质相互作用。交通-- 这些蛋白质-蛋白质相互作用的相似性质使得靶向甲基转移酶特别具有挑战性， 然而，阐明辅因子-蛋白质、底物-蛋白质和蛋白质-蛋白质识别的分子基础将 为推进这些努力迈出了重要的一步。 在这里，我们的总体目标是识别和设计关键的甲基转移酶相互作用在生物合成- 非核糖体肽和聚酮衍生毒力因子结合有机合成的SIS研究 先进的分子生物学和结构生物学。我们建议(目标1)开发小分子以实现分子间相互作用。 门辅助因子和底物甲基转移酶活性，(目标2)捕获甲基转移酶载体的设计工具 蛋白质相互作用，和(目标3)开发选择性活性为基础的探针细菌天然产物甲基- 细菌蛋白质组中用于检测和鉴定的转移酶。这项工作的洞察力将是广泛的- 在基于结构的靶向细菌反式甲基转移酶药物的设计中提供了关键的第一步，具有重要意义 发酵酶，促进烷基的组合生物合成具有精确区域的非天然产物， 立体和化学选择性，以及推进改变生物和物理-化学的药物化学努力- 化学酶法合成特权药效团的化学性质。

PROJECT SUMMARY/ABSTRACT The increased prevalence of multi-drug resistant bacteria has led to an urgent unmet need to develop new an- timicrobial drugs. Targeting bacterial natural product biosynthesis has emerged as a promising avenue, due to the role of natural products as virulence factors, signaling factors, and agents of microbial warfare. However, to date, no drugs targeting enzymes in these biosynthetic pathways have reached the clinic. Hence, systematic investigations into key biosynthetic enzymes are necessary to understand fully their individual roles in virulence factor production. S-adenosyl-L-methionine (SAM)-dependent methyltransferases are ubiquitous throughout all domains of life and modify a diverse array of substrates, including proteins, nucleic acids, and natural products. In eukaryotes, methyltransferase activity modulates cellular processes such as epigenetic gene regulation and neuronal communication; consequently, these methyltransferases are extensively studied and have been validated as anticancer targets. In contrast, natural product methyltransferases have yet to be explored as therapeutic “tar- gets”, in spite of their important role in the biosynthesis of pathogenic virulence factors and pharmaceutically relevant compounds. During biosynthesis, natural product methyltransferases recognize substrates that are covalently tethered to a carrier protein; hence, successful methyl transfer is a function of both substrate recog- nition and proper protein-protein interactions between the methyltransferase and the carrier protein. The tran- sient nature of these protein-protein interactions makes targeting methyltransferases particularly challenging, yet elucidating the molecular basis of cofactor-protein, substrate-protein, and protein-protein recognition would offer a significant step towards advancing these efforts. Herein, our overall goal is to identify and engineer critical methyltransferase interactions during the biosynthe- sis of non-ribosomal peptide and polyketide-derived virulence factors by combining organic synthesis, ad- vanced molecular biology, and structural biology. We propose to (aim 1) develop small molecules to interro- gate cofactor and substrate methyltransferase activity, (aim 2) design tools to capture methyltransferase-carrier protein interactions, and (aim 3) develop selective activity-based probes for bacterial natural product methyl- transferases for detection and identification in the bacterial proteome. The insights from this work will be broad- ly significant by providing critical first steps in structure-based design of drugs targeting bacterial methyltrans- ferases, fostering combinatorial biosynthetic efforts of alkyl groups into unnatural products with precise regio-, stereo-, and chemoselectivity, and advancing medicinal chemistry efforts that alter biological and physico- chemical properties of privileged pharmacophores through chemoenzymatic synthesis.