Modulation of Protein Biogenesis and Secretion by Natural Product Translocon Ligands

天然产物易位子配体对蛋白质生物合成和分泌的调节

• 批准号: 10357568
• 负责人: JANE E ISHMAEL
• 金额: $ 38.86万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10357568
• 关键词: Binding; Binding Sites; Biochemistry; Biogenesis; Biological Assay; Biology; Bioluminescence; Cell Wall; Cells; Cellular Assay; Cellular Membrane; Chemicals; Chronic Disease; Client; Complex; Cyanobacterium; Data; Depsipeptides; Development; Diabetes Mellitus; Disease; Ecology; Eukaryotic Cell; Evolution; Extracellular Protein; Future; Gene Cluster; Genes; Genome; Genomics; Goals; Homeostasis; Human; Infection; Inflammation; Inflammatory; Knowledge; Libraries; Ligands; Luciferases; Malignant Neoplasms; Membrane; Membrane Proteins; Metabolism; Microbial Biofilms; Natural Products; Nerve Degeneration; Pathway interactions; Pattern; Peptide Hydrolases; Peptide Library; Peptide Signal Sequences; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phenotype; Phylogeny; Play; Protein Biosynthesis; Protein Import; Protein Secretion; Protein translocation; Proteins; Proteome; Reporter; Reporting; Role; Structure; Synthesis Chemistry; Testing; Therapeutic; Therapeutic Agents; Toxic effect; Wood material; Work; analog; apratoxin; base; cellular engineering; cellular targeting; comparative; cytotoxic; fungus; human disease; inhibitor; interdisciplinary approach; mycolactone; novel therapeutics; pathogenic bacteria; pathogenic fungus; proteostasis; screening; secretory protein; structural biology; therapeutic target

Loss of homeostasis in the cellular secretory pathway is implicated in major human diseases such as cancer, diabetes and inflammation. Secreted and cell-wall proteins are also critical for host infection by human pathogenic bacteria and fungi, and secreted proteases play a role in biofilm formation. Macrocyclic natural products (NPs) HUN-7293 (from fungi) and the apratoxins (from cyanobacteria), as well as the cotransin synthetic analogs of HUN-7293, are reported to block cotranslational protein translocation at an early stage of the secretory pathway with varying degrees of selectivity, but exactly how they influence protein biogenesis is unknown. Our discovery of a mechanistically distinct inhibitor of protein translocation, coibamide A (CbA, from cyanobacteria), has led to the observation that HUN-7293, apratoxins and CbA share a common cellular target, the Sec61 protein channel (translocon), yet inhibit the biogenesis and secretion of different proteins. This presents the opportunity to discover and use new NPs to elucidate the cellular secretory pathway as a therapeutic target, and to provide a reservoir of potential drug leads to reinforce the dwindling pharmaceutical pipelines of new chemical entities. We plan to utilize a multidisciplinary approach involving natural products and synthetic chemistry, chemical biology, pharmacology and evolutionary genomics to pursue the following three aims: 1) Expand and define the class of NPs that target proteostasis; 2) Elucidate the specific Sec61 binding site and inhibitory mechanism of CbA and two active synthetic analogs; 3) Utilize a genomics workflow for evolution- based prediction of new fungal NPs’ function. In Aim 1, existing NP libraries likely to be rich in non-polar depsipeptides will be screened for new proteostasis modulators using a primary functional screen in U87MG cells engineered to express Gaussia luciferase (Gluc) and a secondary target-based assay for Sec61-dependent inhibition of ER translocation. Preliminary data give a hit rate of 0.3% for the Gluc secretion assay. In Aim 2, chemogenetic screening approaches will be used to determine how Sec61 function is perturbed by CbA, followed by biochemistry and structural biology to resolve the mechanistic basis for ER translocation inhibition. The comparative selectivity profile of two new CbA analogs, and prioritized new NPs from aim 1, relative to CbA- ApxA and cotransin, will be determined in cell-free and cell-based assays. In Aim 3, the genomic diversification of NPs will be investigated in an evolutionary context using phylogeny and ecology of fungi. For example, NRPS (Adenylation) A-domain phylogenies will reveal evolutionary relationships of biosynthetic gene clusters (BGCs), and will be used to predict structure, function in human cells, and correlation between ecology and NP diversity. This multidimensional approach will reveal the feasibility of targeting cellular proteostasis for therapeutic needs, while avoiding toxicities due to non-specific inhibition of secretory protein biosynthesis. It is also to expected to provide evolutionary and ecological rationale for targeting fungal producers of protein secretion inhibitors.

在癌症等主要人类疾病中隐含细胞分泌途径中体内平衡的丧失， 糖尿病和炎症。分泌和细胞壁蛋白对于人类的宿主感染也至关重要 致病细菌和真菌，分泌的蛋白酶在生物膜形成中起作用。大环天然 产品（NPS）HUN-7293（来自真菌）和Apratoxins（来自蓝细菌）以及Cotransin 据报道，HUN-7293的合成类似物在早期的早期阶段阻止了惯性蛋白易位 具有不同程度的选择性的秘书途径，但它们的影响恰恰是如何影响蛋白质生物发生 未知。我们发现了蛋白质易位的机械不同抑制剂CoIbamide A（CBA，来自 蓝细菌）导致观察到HUN-7293，ARPATOXOXIN和CBA具有共同的细胞靶标， SEC61蛋白通道（转运）却抑制了不同蛋白质的生物发生和分泌。这 为发现和使用新的NP阐明细胞秘书途径的机会是一种疗法 目标，并提供潜在药物的储层，以加强逐渐减少的药物管道 新化学实体。我们计划利用涉及天然产品和合成的多学科方法 化学，化学生物学，药物和进化基因组学以追求以下三个目的：1） 扩展和定义靶向蛋白抑制剂的NP类别； 2）阐明特定的Sec61结合位点和 CBA和两个主动合成类似物的抑制作用； 3）利用基因组学工作流进行进化 - 基于新真菌NPS功能的预测。在AIM 1中，现有的NP库可能会富含非极性 将使用U87mg中的主要功能屏幕筛选Depepeptides的新蛋白杀菌调节剂 设计用于表达高斯荧光素酶（GLUC）的细胞和依赖Sec61的次级靶标测定 抑制ER易位。初步数据给出了GLUC分泌测定法的命中率为0.3％。在AIM 2中， 化学发生筛查方法将用于确定CBA如何扰动SEC61的功能 通过生物化学和结构生物学来解决ER转运抑制的机理基础。这 相对于CBA- APXA和Cotransin将在无细胞和基于细胞的测定中确定。在AIM 3中，基因组多样化 NPS将在进化环境中使用真菌的系统发育和生态学进行研究。例如，NRP （腺苷酸化）A域系统发育将揭示生物合成基因簇（BGC），，，， 并将用于预测人类细胞中的结构，功能以及生态学与NP多样性之间的相关性。 这种多维方法将揭示靶向细胞蛋白抑制的可行性，以满足治疗需求， 同时避免由于非特异性抑制分泌蛋白生物合成而引起的毒性。这也是期望的 为靶向蛋白质分泌抑制剂的真菌生产者提供进化和生态原理。