ReviewMechanistic considerations in high-throughput screening
Section snippets
Defining the rate of approach to equilibrium
For the purposes of this review we shall define receptor–ligand binding interactions to mean the bimolecular interactions between a biological macromolecule (most typically a protein) and another molecule (usually a small-molecular-weight ligand, but also peptides, other proteins, nucleic acids, etc.). The interaction of a receptor with a ligand is defined by the equilibrium illustrated in Scheme 1.
Ligand binding is described by the second order rate constant kon and ligand dissociation by the
Enzyme activity assays
The binding of inhibitors to enzymes is another example of receptor–ligand binding interactions. However, because enzymes are catalysts, the detection of inhibitor binding is most often best accomplished by measuring the diminution of enzymatic activity that is brought about by inhibitory molecules. Thus, some consideration must be given to the type of chemical reaction and the mechanistic details of catalysis by the enzyme.
Postscreening evaluation of hits
Once an HTS campaign is completed and hits have been identified, these hits are next evaluated by several criteria. First, hits are typically evaluated with regard to compound purity and structural tractability. Compounds that, by virtue of their chemical structure, are deemed highly reactive are generally excluded from further evaluation. Likewise, other chemical and physical criteria, such as aqueous solubility, log P, molecular weight, etc., may be used to discard hits that are deemed
Acknowledgements
The author thanks Dr. Ross L. Stein, Dr. Walter DeWolf, Dr. Zhihong Lai, and Dr. David L. Pompliano for many helpful suggestions and Dr. Mark Namchuk for sharing unpublished information and insights.
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