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: 10506915
• 负责人: Lukasz Michal Ciesla
• 金额: $ 2.68万
• 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/10506915
• 关键词: Address; Adoption; Affinity Chromatography; Alabama; Antibodies; Artificial Membranes; Binding; 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; National Center for Complementary and Integrative Health; 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.

项目摘要 天然产品是药理学活性化合物的丰富来源。但是，由于技术约束 当前使用的高通量筛选技术与复杂的天然样品不兼容。一 从天然产物鉴定新药铅的最大挑战是 提取物，需要大量的时间和精力来发现生物活性的二级代谢产物。 传统的筛选方法通常需要从化学的混合物中隔离单个化合物 分离，然后进行剥离和测定分析，这些分析是耗时，劳动力密集和昂贵的。 高通量筛选技术主要集中于单个化合物的合成库，而不是 与混合物兼容。相比之下，蛋白质靶标技术可以直接捕捞结合 化合物消除了分离单个化合物的需求。不幸的是，大多数蛋白质 固定方法具有显着的非特异性结合，以支持表面， 这使得它们不适合筛选复杂的混合物。另外，大多数固定目标 目前，固体表面上的蛋白质仅限于胞质蛋白，例如酶或抗体。这是一个 极大的局限性，因为超过50％的现代药物使用膜蛋白作为主要靶标。 细胞膜亲和力色谱法（CMAC）是一种允许鉴定的方法 化合物，以复杂的矩阵存在，并特别与固定的跨膜相互作用 蛋白质。事实证明，CMAC色谱柱可用于筛选植物提取物和新的烟雾冷凝物 靶向不同类别的跨膜蛋白的配体。但是，阻止的挑战之一 更广泛使用的CMAC是CMAC色谱柱的相对复杂的准备方案，缺乏易于易用的 使用捕获和释放色谱套件。我们提出了一种新颖且可专利的CMAC套件的开发 使用CMAC列的优化准备协议。使用这种新型方法均质细胞 具有靶蛋白的膜片段将被固定在IAM.pc.dd2列上，仅产生 由Regis Technologies。使用预包装列的固定步骤消除了溶解的需求 当前协议中使用的透析步骤。新开发的CMAC套件将加快识别 靶向跨膜蛋白的植物化学物质。这种小说测定法非常适合建造 可以使用现代筛选平台进行进一步测试的药理学活性天然化合物。 CMAC也是一种工具，可以替代基于功能细胞的测定法以确定某些药效学 例如，候选药物的特性：KD（结合50％受体的药物浓度）。这 CMAC套件可以轻松地应用于药物发现实验室，以识别可以进一步的新型药物模板 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。

项目成果

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

细胞膜亲和层析柱鉴定与固定化原肌球蛋白激酶受体 B 相互作用的特殊植物代谢物

• DOI: 10.3791/63118
• 发表时间: 2022
• 期刊: Journal of Visualized Experiments
• 作者: Arituluk, Zekiye Ceren;Adhikari, Bishnu;Maitra, Urmila;Goodman, Caroline;Ciesla, Lukasz M.
• 通讯作者: Ciesla, Lukasz M.