Cellular membrane affinity chromatography kit for drug discovery

用于药物发现的细胞膜亲和层析试剂盒

• 批准号: 10325006
• 负责人: Lukasz Michal Ciesla
• 金额: $ 22.39万
• 依托单位: REGIS TECHNOLOGIES INC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10325006
• 关键词: Address; Adoption; Affinity Chromatography; Alabama; Antibodies; Artificial Membranes; Binding; Biological; Biological Assay; Buffers; Cell membrane; Cell physiology; Cells; Cellular Assay; Cellular Membrane; Characteristics; Chemicals; Chemistry; Column Chromatography; Complex; Complex Mixtures; Consumption; Data; Data Analyses; Development; Diabetes Mellitus; Dialysis procedure; Drug Targeting; Enzymes; Fishes; G-Protein-Coupled Receptors; Goals; Immobilization; Individual; Integral Membrane Protein; Ion Channel; Laboratories; Lead; Libraries; Ligands; Malignant Neoplasms; Membrane Proteins; Methodology; Methods; Modernization; Natural Product Drug; Natural Products; Nature; Neurodegenerative Disorders; Neurotrophic Tyrosine Kinase Receptor Type 2; Particle Size; Performance; Pharmaceutical Preparations; Pharmacodynamics; Pharmacologic Substance; Pharmacology; Phase; Phytochemical; Plant Extracts; Preparation; Procedures; Process; Property; Protein Tyrosine Kinase; Proteins; Protocols documentation; Research; Research Personnel; Resources; Sampling; Smoke; Solid; Source; Speed; Surface; Techniques; Technology; Temperature; Testing; Time; Universities; Validation; base; chronic pain; commercialization; cost; design; drug candidate; drug discovery; high throughput screening; improved; innovation; interest; new technology; novel; novel strategies; novel therapeutics; particle; pressure; prevent; programs; receptor; scaffold; screening; tool

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.

项目概要 天然产物是药理活性化合物的丰富来源。但由于技术限制 目前使用的高通量筛选技术与复杂的天然样品不兼容。一 从天然产物中鉴定新药先导物的最大挑战是其复杂性 提取物，需要大量的时间和精力来发现具有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，然后是去复制和化验分析，这些过程耗时、劳动密集且成本高昂。 高通量筛选技术主要集中于单个化合物的合成库，而不是 与混合物相容。相比之下，蛋白质靶标固定技术可以直接捞出结合 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 固定方法受到化合物与支持表面的显着非特异性结合的影响， 这使得它们不适合筛选复杂的混合物。此外，大部分固定目标 固体表面上的蛋白质目前仅限于胞质蛋白质，例如酶或抗体。这是一个 很大的局限性，因为超过 50% 的现代药物都使用膜蛋白作为主要靶标。 细胞膜亲和层析 (CMAC) 是一种可以鉴定 存在于复杂基质中并与固定跨膜发生特异性相互作用的化合物 蛋白质。 CMAC 色谱柱已被证明可用于筛选植物提取物和烟气冷凝物中的新物质 靶向不同类别跨膜蛋白的配体。然而，阻碍的挑战之一 CMAC应用广泛的原因是CMAC柱的制备方案相对复杂，且缺乏易于操作的方法。 使用捕获和释放色谱套件。我们建议开发一种新颖且可申请专利的 CMAC 套件 具有优化的 CMAC 柱制备方案。使用这种新颖的方法匀浆细胞 带有目标蛋白的膜片段将被固定在 IAM.PC.DD2 柱上，该柱单独生产 由瑞吉斯技术公司提供。使用预装柱的固定步骤消除了溶解的需要 以及当前方案中使用的透析步骤。新开发的CMAC套件将加速识别 针对跨膜蛋白的植物化学物质。这种新颖的检测方法非常适合构建 具有药理活性的天然化合物，可以使用现代筛选平台进一步进行测试。 CMAC 也是一种可以替代基于功能细胞的测定法来确定某些药效学的工具 候选药物的特性，例如：Kd（结合 50% 受体的药物浓度）。这 CMAC 试剂盒可以轻松应用于药物发现实验室，以识别可进一步开发的新型药物模板 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。