Investigating Trends in Ligand Binding to Human Serum Albumin by in silico and Fluorescence Assays

KUMARASIRI, M.; LLARRULL, L.I.; ODANIEL, P.; YAMAGUCHI, T.; HESEK, D.; CHANG, M.

Notre Dame, Indiana

Jornada; Chemistry-Biology-Biochemistry Interface Retreat; 2009

University of Notre Dame

Human Serum Albumin (HSA) is the most abundant protein in human blood plasma. It has two drug binding sites with one having a slightly larger cavity than the other. HSA is known to bind strongly to a diverse list of drugs, reducing the active drug concentrations. Thus, identifying structural features that promote binding is crucial for the drug design process. Here, we investigate the binding ability of several types of ligands to HSA. We performed in silico docking and scoring to identify binding trends of ligands. By using a set of 20 compounds, we found that binding to the larger binding site in HSA is preferable. The list of compounds was then extended to 72 by including derivatives of the scored 20 compounds as suggested by the trends in scoring. Based on many docking and scoring attempts, we identified several structural features that cause stronger ligand binding to HSA. A selected subset of compounds was then used to determine the dissociation constants to HSA experimentally. The dissociation constants of different non-fluorescent compounds for HSA were evaluated by the analysis of the displacement of the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS). The assays were carried out in a 96-well format and the intensity of fluorescence measurements at the maximum of the ANS emission spectrum was compared for the different compounds. The dissociation constant for each compound (Kd) was estimated using a single-point displacement assay, as described by Epps et al (Epps, D. E.; Raub, T. J.; Kezdy, F. J. Biochemistry 1995, 227, 342-350). The trends in the relative Kd values obtained from the ANS displacement assay correlate well with the trends observed in docking and scoring.