Cellular membrane affinity chromatography kit for drug discovery

项目摘要 天然产物是药理活性化合物的丰富来源。然而，由于技术上的限制， 目前使用的高通量筛选技术与复杂的天然样品不兼容。一 从天然产品中识别新药先导化合物的最大挑战之一是 提取物，这需要大量的时间和努力来发现生物活性的次生代谢物。 传统的筛选方法通常需要通过化学方法从混合物中分离出单个化合物。 分离，然后是去复制和分析，这是耗时、劳动密集型和昂贵的。 高通量筛选技术主要集中在单个化合物的合成库上，而不是 与混合物兼容。相反，蛋白质靶标固定技术可以直接捕获结合。 化合物，消除了分离单个化合物的需要。不幸的是，大部分蛋白质 固定化方法受到化合物与载体表面的显著非特异性结合的影响， 这使得它们不适合于筛选复杂的混合物。此外，大多数被固定的目标 固体表面的蛋白质目前仅限于胞浆蛋白质，如酶或抗体。这是一个 很大的局限性，因为超过50%的现代药物使用膜蛋白作为主要靶点。 细胞膜亲和层析(CMAC)是一种可以用于鉴定 化合物，存在于复杂的基质中，并与固定化的跨膜特别相互作用 蛋白。CMAC柱已被证明在筛选植物提取物和烟雾冷凝物用于新的 针对不同类别的跨膜蛋白的配体。然而，阻止的挑战之一是 CMAC从更广泛的用途来看，是一种相对复杂的CMAC柱制备方案，缺乏易操作的特点 使用捕获和释放层析套件。我们建议开发一种新颖的、可申请专利的CMAC试剂盒 优化了CMAC柱的制备工艺。使用这种新方法使细胞匀浆 带有目标蛋白的膜片段将被固定在IAM.PC.DD2柱上，仅产生 作者：雷吉斯科技公司。使用预填充柱的固定化步骤消除了增溶的需要 以及当前方案中使用的透析步骤。新开发的CMAC试剂盒将加快识别 以跨膜蛋白为靶标的植物化学物质。这一新的分析方法将非常适合于建立 具有药理活性的天然化合物，可能会使用现代筛选平台进行进一步测试。 CMAC也是一种工具，可以取代基于功能细胞的分析来确定某些药效学 候选药物的性质，例如：Kd(结合50%受体的药物浓度)。这个 CMAC试剂盒可以很容易地应用于药物发现实验室，以确定可以进一步 用于治疗某些形式的癌症、神经退行性疾病、慢性疼痛、糖尿病和许多其他疾病 疾病。

Project Summary Natural products are a rich source of pharmacologically active compounds. However, due to technical constrains the currently used high-throughput screening techniques are not compatible with complex natural samples. One of the greatest challenges in the identification of new drug leads from natural products is the complexity of the extracts, that requires enormous amount of time and effort to discover biologically active secondary metabolites. Traditional screening methods often require isolation of individual compounds from mixtures through chemical separation, followed by derepliction and assay analysis, which are time consuming, labor intensive, and costly. High throughput screening techniques mainly focus on synthetic libraries of individual compounds and are not compatible with mixtures. In contrast, protein target immobilization techniques can directly fish out binding compounds eliminating the need to isolate individual compounds. Unfortunately, most of the protein immobilization methodologies suffer from significant non-specific binding of compounds to support surfaces, which makes them not suitable for screening complex mixtures. In addition, the majority of the immobilized target proteins on solid surfaces are currently limited to cytosolic proteins, such as enzymes or antibodies. This is a great limitation, because over 50% of all modern pharmaceuticals use membrane proteins as prime targets. Cellular membrane affinity chromatography (CMAC) is an approach that allows for the identification of compounds, present in complex matrices and specifically interacting with the immobilized transmembrane protein. CMAC columns have proven to be useful in screening plant extracts and smoke condensates for new ligands targeting different classes of transmembrane proteins. However, one of the challenges that prevents CMAC from wider use is the relatively complex preparation protocol of CMAC columns and the lack of easy-to- use catch and release chromatographic kits. We propose the development of a novel and patentable CMAC kit with an optimized preparation protocol of CMAC columns. Using this novel approach homogenized cell membrane fragments with the targeted proteins will be immobilized on IAM.PC.DD2 columns, produced solely by Regis Technologies. The immobilization step using pre-packed columns eliminates the need for solubilization and dialysis steps used in the current protocol. The newly developed CMAC kit will speed up the identification of phytochemicals targeting transmembrane proteins. This novel assay will be ideally suited to build libraries of pharmacologically active natural compounds that further might be tested using modern screening platforms. CMAC is also a tool that can replace functional cell-based assays in determining certain pharmacodynamic properties of drug candidates, for example: Kd (the concentration of drug that binds 50% of the receptors). The CMAC kit can easily be applied in drug discovery laboratories to identify novel drug templates that can be further used to treat certain forms of cancer, neurodegenerative diseases, chronic pain, diabetes and many other illnesses.