A new method for high throughput screening has been developed based upon the inclusion of the target receptor in a flowing chromatographic system. In this approach, the receptor is immobilized on a solid support and the support packed into a small column. The test chemicals are passed through the column, over the immobilized target, and the time that it takes for the compounds to pass from the beginning of the column to its end is directly related to the strength of interaction between the target and the compound, i.e. the binding affinity of the ligand-receptor complex. Using this method, complex chemical and biological mixtures can be rapidly sorted between compounds that interact and do not interact with the disease-related target. At the same time, the compounds that bind to the target are themselves rapidly sorted between low, medium and high affinity binders. Thus, the method quickly provides a large amount of data with high information content. We have developed prototypes for the G-protein coupled receptor-based screening using the kappa, mu and delta subtypes of the opioid receptor and various subtypes of the nicotinic receptor. The nicotinic receptor-based columns have been used to screen complex chemical mixtures and have identified competitive agonists and antagonists in the mixture as well as non-competitive inhibitors. A study using non-competitive inhibitors and non-linear chromatography was conducted and demonstrated that the method can be used to identify and characterize the non-competitive inhibitors. Chemometric analysis and molecular modeling have been used to describe and predict the interactions between the nicotinic receptors and non-competitive inhibitors.
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