Computational and experimental approaches assess the interactions between bovine β-lactoglobulin and synthetic compounds of pharmacological interest

Extending a previous investigation, the ability of binding to the model calycin β-lactoglobulin (BLG) was evaluated both in silico and in vitro for several fluorine-containing (semi-)synthetic molecules of pharmacological and pharmaceutical interest (antibiotics, vastatins, steroid drugs). Simulation procedures included molecular docking according to a Montecarlo-simulated annealing protocol and molecular dynamics; heteronuclear NMR and denaturant gradient gel electrophoresis were the selected experimental techniques. For the tested drugs, ranking of the binding affinity was consistently assessed by computation and by experiment. The affinity for BLG increased in the sequence: 5-fluorosalycilic acid < dexamethasone ≪ sulindac = norfloxacin < fluvastatin. The computed Ki for fluorosalycilate was in the order of 10−4 M; accordingly, in a molecular dynamics simulation the chemical diffused out of the BLG calyx in less than 2 ns, and no evidence of binding was found by NMR or electrophoresis. Conversely, the Ki for fluvastatin and norfloxacin were in the order of 10−7 and 10−6 M, similar to the affinity for BLG by natural ligands, such as retinoids and long-chain fatty acids. Moreover fluvastatin was found still bound to the protein after 5 ns of molecular dynamics simulation. Interaction of fluvastatin and norfloxacin with BLG was made evident by changes in chemical shift and dynamic parameters in the 19F NMR spectra and in effective urea concentration and cooperativity features in denaturant gradient gel electrophoresis. Such findings prove BLG may act as a drug carrier accepting in its cavity molecules of different bulk, rigidity and hydrophobicity.