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.
Conrad
Mandi L
ML
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.
Moser
Annette C
AC
Hage
David S
DS
D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods; D014364:Tryptophan / Q000031:analogs & derivatives
D007211:Indoles; D012709:Serum Albumin; D014364:Tryptophan