A Genomic Approach to Discovering Novel Cathepsin Inhibitors from Cyanobacteria

项目摘要 创伤性脑损伤（TBI）是全球和美国发病率和死亡率的主要原因; 1，2 然而，没有FDA批准的药物来改善继发性 在最初的神经元损伤后由复杂的神经病理级联反应引起的损伤。组织蛋白酶B和L 代表有前途的药物靶点，因为在动物中对这些半胱氨酸蛋白酶的敲除和抑制研究 模型揭示了行为和神经认知后遗症的改善。3 -7显然需要新的 治疗TBI后继发性损伤的治疗选择，组织蛋白酶B和L抑制剂具有 制药承诺超过60%的FDA批准的药物是来自或灵感来自天然产品 8已知蓝细菌产生具有一系列生物活性的NP，包括作为蛋白酶的活性。 NPs由巨酶生物合成，巨酶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用于增强药物的生物合成。 通过概述的三个目标，从蓝藻中发现新型组织蛋白酶B和L抑制剂的努力 下面首先，将开发基于药效团的创新基因组挖掘管道（目标1）。在这 目的，从虚拟对接实验和已知的组织蛋白酶抑制剂推导的药效团将用于 预测负责产生所需药效团的酶结构域。这种逆生物合成的 预测将被用来制作生物信息学模型，以询问测序的蓝藻基因组， 候选BGC。最感兴趣的BGC将被识别，描绘，并产生化合物 通过培养或异源表达（Aim 2）。使用完整的逆生物合成预测， gallinamide A，一种表现出纳摩尔组织蛋白酶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文库中或在新的蓝藻收藏中。目标1的其他BGC将 也是在这个目标下发展起来的。如果BGC不是组成型表达的，或者如果BGC不与一个或多个基因相关， 为了获得可培养的生物体，将追求异源表达（目的3）。化合物分离和分子 随后将建立网络，分析和注释所生产的天然产品的化学空间。 将在生物测定中测试化合物以评价组织蛋白酶B和L对纯化的酶的活性，以及在生物测定中评价组织蛋白酶B和L对纯化的酶的活性。 神经元和神经胶质细胞研究。确定的药物需求和发现的药物之间的差距 新的生物活性分子可以通过本报告中提出的创新的多学科方法来桥接。 应用程序.拟议的项目将作为美国国立卫生研究院F32 NRSA奖学金的一部分在斯克里普斯进行 海洋学研究所，加州大学圣地亚哥分校，共同赞助的教授威廉格威克和 Vivian Hook和一组合作者将在虚拟对接实验中培训博士后， 生物信息学、异源基因表达和组织蛋白酶相关的生物测定。

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.