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.

细胞分泌途径稳态的丧失与癌症、癌症等主要人类疾病有关。 糖尿病和炎症对于人类宿主感染也至关重要。 病原菌和真菌以及分泌的蛋白酶在生物膜形成中发挥作用。 产品 (NP) HUN-7293（来自真菌）和 apratoxins（来自蓝藻），以及 cotransin 据报道，HUN-7293 的合成类似物可在早期阶段阻断共翻译蛋白易位 具有不同程度选择性的分泌途径，但它们到底如何影响蛋白质生物发生是 我们发现了一种机制独特的蛋白质易位抑制剂，coibamide A（CbA，来自）。 蓝细菌），导致观察到 HUN-7293、apratoxins 和 CbA 具有共同的细胞靶标， Sec61 蛋白通道（易位子），但抑制不同蛋白的生物发生和分泌。 提供了发现和使用新纳米粒子来阐明细胞分泌途径作为治疗方法的机会 目标，并提供潜在药物先导储备，以加强日益减少的药物管道 我们计划利用涉及天然产物和合成的多学科方法。 化学、化学生物学、药理学和进化基因组学以追求以下三个目标：1） 扩展并定义针对蛋白质稳态的 NP 类别；2) 阐明特定的 Sec61 结合位点并 CbA 和两种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- 基于对新真菌 NP 功能的预测 在目标 1 中，现有 NP 库可能富含非极性。 将使用 U87MG 中的主要功能筛选来筛选缩肽，以寻找新的蛋白质稳态调节剂 细胞被设计为表达 Gaussia 荧光素酶 (Gluc) 以及基于 Sec61 依赖性的二级靶标测定 在目标 2 中，初步数据显示 Gluc 分泌测定的命中率为 0.3%。 化学遗传学筛选方法将用于确定 Sec61 功能如何受到 CbA 的干扰，然后 通过生物化学和结构生物学解析ER易位抑制的机制基础。 相对于 CbA- 两种新 CbA 类似物的比较选择性概况，以及目标 1 中新 NP 的优先顺序 ApxA 和 cotransin 将在无细胞和基于细胞的检测中进行测定 在目标 3 中，基因组多样化。 将使用真菌的系统发育学和生态学在进化背景下研究 NPs。 （腺苷酸化）A 域系统发育将揭示生物合成基因簇 (BGC) 的进化关系， 并将用于预测人体细胞的结构、功能以及生态学和纳米颗粒多样性之间的相关性。 这种多维方法将揭示针对治疗需求靶向细胞蛋白质稳态的可行性， 同时避免由于分泌蛋白生物合成的非特异性抑制而产生的毒性。 为靶向蛋白质分泌抑制剂的真菌生产者提供进化和生态学原理。