A Genomic Approach to Discovering Novel Cathepsin Inhibitors from Cyanobacteria

项目摘要 创伤性脑损伤是全世界和美国发病率和死亡率的主要原因；1，2 然而，目前还没有FDA批准的药物来减轻继发性心脏病的破坏性后果 在最初的神经元损伤之后，由复杂的神经病理引起的损伤。组织蛋白酶B与L 代表有希望的药物靶点，作为这些半胱氨酸蛋白酶在动物中的敲除和抑制研究 模型显示行为和神经认知后遗症的改善。3-7显然需要新的 治疗颅脑损伤后继发性损伤的治疗选择，组织蛋白酶B和L抑制剂 医药方面的希望。FDA批准的药物中有60%以上来自天然产品或受天然产品启发 (NPS).8已知蓝藻产生具有一系列生物活性的NPs，包括作为蛋白酶的活性 9，10个NPs由巨型酶生物合成，这些巨型酶被编码为离散的基因组包，称为 生物合成基因簇(BGC)。11我们假设基因组方法可以用来增强药物 蓝藻通过三个目标发现新型组织蛋白酶B和L抑制剂的努力 下面。首先，将开发一种创新的基于药效团的基因组挖掘管道(目标1)。在这 目的，从虚拟对接实验中推导出的药效团和已知的组织酶抑制剂将用于 预测负责产生所需药效团的酶结构域。这种反向生物合成 预测将被用于建立生物信息学模型，以询问已测序的蓝藻基因组以找到 候选人BGC。最感兴趣的BGC将被识别、描绘和产生化合物 通过培养或异源表达(目标2)。使用完整的反生物合成预测 鸡酰胺A，一种表现出纳米分子组织蛋白L抑制作用的化合物，我们将寻找BGC 在斯克里普斯的基因组库中或在新的蓝藻集合中。来自AIM 1的其他BGC将是 也是在这一目标下发展起来的。如果BGC不是按构成表达的，或者如果BGC不与 可培养的生物体，将追求异源表达(目标3)。化合物分离与分子 接下来将建立网络，以分析和注释所生产的天然产品的化学空间。 化合物将在生物测试中进行测试，以评估组织蛋白酶B和L对纯化酶以及在 神经细胞和神经胶质细胞研究。已确定的药物需求与发现的药物之间的差距 新的生物活性分子可以通过本论文中提出的创新的多学科方法连接起来。 申请。拟议的项目将作为斯克里普斯NIH F32 NRSA奖学金的一部分进行 加州大学圣地亚哥分校海洋研究所，由William Gerwick教授和William Gerwick教授共同赞助 维维安·胡克和一组合作者将培训这位博士后进行虚拟对接实验， 生物信息学、异源基因表达和组织蛋白酶相关生物测定。

Project Abstract Traumatic brain injury (TBI) is a leading cause of morbidity and mortality world-wide and in the United States;1,2 however, there are no FDA-approved medications to ameliorate the devastating consequences of secondary injury caused by complex neuropathological cascades following the initial neuronal insult. Cathepsin B and L represent promising drug targets, as knock-out and inhibition studies of these cysteine proteases in animal models reveal improvement in behavioral and neurocognitive sequelae.3–7 There is a clear need for new therapeutic options to treat secondary injury following TBI, and cathepsin B and L inhibitors have pharmaceutical promise. Over 60% of FDA-approved drugs are derived from or inspired by natural products (NPs).8 Cyanobacteria are known to produce NPs with a range of bioactivity, including activity as protease inhibitors.9,10 NPs are biosynthesized by megaenzymes that are encoded as discrete genomic packages called biosynthetic gene clusters (BGCs).11 We hypothesize that a genomic approach can be used to enhance drug discovery efforts from cyanobacteria for novel cathepsin B and L inhibitors through the three aims outlined below. First, an innovative pharmacophore-based genome mining pipeline will be developed (Aim 1). In this aim, pharmacophores deduced from virtual docking experiments and known cathepsin inhibitors will be used to predict enzymatic domains responsible for the creation of the desired pharmacophore. This retrobiosynthetic prediction will be used to make bioinformatic models to interrogate sequenced cyanobacterial genomes to find candidate BGCs. The BGCs of highest interest will be identified, delineated, and the compounds produced through either cultivation or heterologous expression (Aim 2). Using a full retrobiosynthetic prediction for gallinamide A, a compound that demonstrates nanomolar cathepsin L inhibition, we will search for the BGC within the genomic library at Scripps or in new cyanobacterial collections. Additional BGCs from Aim 1 will be developed in this aim as well. If the BGC is not constitutively expressed or if the BGC is not associated with a cultivatable organism, heterologous expression will be pursued (Aim 3). Compound isolation and molecular networking to analyze and annotate the chemical space of the natural products produced will follow. Compounds will be tested in bioassays to evaluate cathepsin B and L activity on purified enzymes as well as in neuronal and glial cellular studies. The gap between the identified pharmaceutical need and the discovery of novel bioactive molecules can be bridged by the innovative multidisciplinary approach presented in this application. The proposed project will be carried out as part of an NIH F32 NRSA Fellowship at Scripps Institution of Oceanography, UC San Diego, under the co-sponsorship of Professors William Gerwick and Vivian Hook and a team of collaborators that will train the postdoctoral fellow in virtual docking experiments, bioinformatics, heterologous gene expression, and cathepsin-related bioassays.