Evaluation of indole‐based probes for high‐throughput screening of drug binding to human serum albumin: Analysis by high‐performance affinity chromatography

There has been growing interest in the use of rapid and selective separation methods such as high‐performance affinity chromatography (HPAC) or affinity capillary electrophoresis (ACE) for the characterization of drug–protein interactions. L ‐Tryptophan is commonly used in these and other methods as a site‐selective probe for examining the binding of small solutes and drugs at Sudlow site II on the protein HSA. However, solutions of L ‐tryptophan can be unstable and are generally prepared fresh daily for these studies. In this report, HPAC was used to examine other indole compounds as possible replacements for L ‐tryptophan as a site‐selective probe for use in the high‐throughput screening of drug binding to HSA; the implications of these results in the use of such compounds in ACE were also considered. The probe candidates that were tested included indole‐3‐acetic acid, indole‐3‐carboxylic acid, indole‐3‐butyric acid, indole‐3‐propionic acid, indole‐3‐methanol, 3‐acetylindole, and 3‐methylindole. All of these compounds were found by 1 H NMR and UV–Vis spectroscopy to be stable for up to 3 wk at room temperature when kept in a pH 7.4, 0.067 M phosphate buffer. The binding of these compounds was examined by using columns that contained immobilized HSA. 3‐Acetylindole was found to be the best candidate in this group for use as an alternative probe to L ‐tryptophan for Sudlow site II. This probe had the same binding site and a similar affinity to L ‐tryptophan but was more stable in aqueous solution, making it suitable for high‐throughput screening of drug–HSA binding in both HPAC and ACE.