Modulation of Protein Biogenesis and Secretion by Natural Product Translocon Ligands

细胞分泌途径中稳态的丧失与主要的人类疾病如癌症， 糖尿病和炎症。分泌蛋白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酶在生物膜形成中起作用。大环天然 产品（NP）匈奴-7293（来自真菌）和apratoxins（来自蓝细菌），以及cotransin 据报道，匈奴-7293的合成类似物可在肿瘤的早期阶段阻断共翻译蛋白质易位， 分泌途径具有不同程度的选择性，但它们究竟如何影响蛋白质生物合成， 未知我们发现了一种机制上不同的蛋白质易位抑制剂，coibamide A（CbA）， 蓝细菌），已经导致观察到匈奴-7293、黄曲霉毒素和CbA共享共同的细胞靶点， Sec 61蛋白通道（translocon），但抑制不同蛋白质的生物合成和分泌。这 提出了机会，发现和使用新的纳米粒子，以阐明细胞分泌途径作为一种治疗 目标，并提供一个潜在的药物线索库，以加强日益减少的制药管道， 新的化学物质。我们计划利用涉及天然产品和合成产品的多学科方法， 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）阐明特异性Sec 61结合位点， 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- 基于新真菌纳米粒功能的预测。在目标1中，现有的NP文库可能富含非极性 将使用U87 MG中的初级功能筛选来筛选缩肽的新蛋白质抑制调节剂 工程化以表达Gaussia荧光素酶（Gluc）的细胞和Sec 61依赖性的基于二级靶点的测定 抑制ER易位。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Sec 61功能如何被CbA干扰， 通过生物化学和结构生物学的方法来解决ER易位抑制的机制基础。的 两种新的CbA类似物的比较选择性概况，以及相对于CbA，来自目标1的优先化的新NP。 将在无细胞和基于细胞的试验中测定ApxA和共转运蛋白。在目标3中，基因组多样化 将在进化背景下使用真菌的生殖发育学和生态学来研究纳米颗粒。例如，NRPS （腺苷酸化）A-结构域同源性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和功能，以及生态学和NP多样性之间的相关性。 这种多方面的方法将揭示靶向细胞蛋白质稳态用于治疗需求的可行性， 同时避免了由于分泌蛋白生物合成的非特异性抑制而引起的毒性。预计还将 为靶向蛋白分泌抑制剂的真菌生产者提供进化和生态学原理。

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.