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 ...