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

Abstract

There has been growing interest in the use of rapid and selective separation methods such as highperformance 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 human serum albumin (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 spectroscopy and UV/vis spectroscopy to be stable for up to three weeks at room temperature when in 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.

Analysis of multi-site drug–protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin

“…The glimepiride, tamoxifen, digitoxin, racemic warfarin, and R- warfarin solutions were used within two weeks of preparation [8,58,61]. Previous studies have shown that solutions of L-tryptophan in pH 7.4 phosphate buffer are only stable for a period of 2–9 days, so these solutions were prepared and used within one day of preparation [8,61,62]. …”

Fenretinide inhibits macrophage inflammatory mediators and controls hypertension in spontaneously hypertensive rats via the peroxisome proliferator-activated receptor gamma pathway

“…It is known to contain two major binding sites for the majority of drugs, namely Sudlow site I (also often called the warfarin-binding site)56 and Sudlow site II (also often called the benzodiazepine site)57 (Figure 2E). Sudlow site I, located in subdomain IIA, is a big hydrophobic cavity where interactive associations of drugs bind 55.…”

Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin

“…All solutions and samples containing R- warfarin were used within two weeks of preparation to ensure stable conditions for drug-protein binding measurements [21,22,38]. The L-tryptophan solutions were prepared fresh daily in the same pH 7.4 buffer, as described previously [21,22,39]. These samples were applied or injected at a typical flow rate of 0.50 mL/min, as shown in earlier studies to provide reproducible retention factors and binding capacities for these drugs and related solutes on similar HSA columns [10,18,21,22].…”