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发生和分泌的调节

• 批准号: 10091489
• 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 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091489
• 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/antagonist; 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.

细胞分泌途径中动态平衡的丧失与癌症等人类主要疾病有关， 糖尿病和炎症。分泌型和细胞壁蛋白也是人类感染宿主的关键。 病原菌和真菌以及分泌的蛋白水解酶在生物膜的形成中起着重要作用。大环天然 产品(NPs)Hun-7293(来自真菌)和apratoxins(来自蓝藻)，以及辅酶 据报道，Hun-7293的合成类似物可以在早期阻止共翻译蛋白易位。 具有不同程度选择性的分泌途径，但它们到底是如何影响蛋白质生物发生的 未知。我们发现了一种机制上不同的蛋白质转位抑制剂，CoibamylA(CBA，来自 蓝藻)，导致观察到Hun-7293、蜂毒素和CBA共享一个共同的细胞靶点， Sec61蛋白通道(转运子)，但抑制不同蛋白质的生物合成和分泌。这 提供了发现和使用新的NPs来阐明细胞分泌途径作为一种治疗方法的机会 目标，并提供潜在药物线索的储存库，以加强日益减少的药物管道 新的化学物质。我们计划利用一种涉及天然产品和合成材料的多学科方法 化学、化学生物学、药理学和进化基因组学追求以下三个目标：1) 2)阐明特定的Sec61结合位点和 CBA和两个活性合成类似物的抑制机制；3)利用基因组学工作流程进行进化- 基于对新的真菌NPs功能的预测。在目标1中，现有的NP文库可能富含非极性 将使用U87 MG的初级功能筛选来筛选新的蛋白平衡调节剂 表达Gaussia荧光素酶(Gluc)的细胞及其对Sec61依赖的二级靶点分析 抑制内质网转位。初步数据显示，Gluc分泌试验的命中率为0.3%。在目标2中， 化学遗传学筛选方法将用于确定Sec61功能是如何被CBA干扰的，以下是 通过生物化学和结构生物学来解决内质网转位抑制的机制基础。这个 两个新的CBA类似物的相对选择性分布，并对来自目标1的新NP进行了优先排序，相对于CBA- ApxA和辅酶转运蛋白，将通过无细胞和基于细胞的分析来确定。在目标3中，基因组多样化 将利用真菌的系统发育和生态学在进化的背景下对NPs进行研究。例如，NRPS (腺化)A结构域的系统发育将揭示生物合成基因簇(BGC)的进化关系， 并将用于预测人类细胞的结构、功能，以及生态学和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平衡以满足治疗需求的可行性， 同时避免由于非特异性抑制分泌蛋白生物合成而产生的毒性。预计它也将 为针对蛋白质分泌抑制剂的真菌生产者提供进化和生态学原理。