title: Surface plasmon resonance, fluorescence, and circular dichroism studies for the characterization of the binding of BACE-1 inhibitors.
abstract: The mechanism of action underlying β-secretase 1 (BACE-1) inhibition was characterized by a surface plasmon resonance (SPR) method using primary amino groups to immobilize OM99-2, a well-known highly potent peptidic BACE-1 inhibitor, on the carboxyl groups of the dextran layer of a sensor chip. The diluted BACE-1 was mixed with buffer or the test compound and the mixture was flushed through the chip. BACE-1 binding to the immobilized peptide inhibitor was quantified. This SPR method was used to identify BACE-1 inhibitor binding sites and the mechanism of action (competitive/noncompetitive) and to validate findings of fluorescence resonance energy transfer (FRET) inhibition studies. To support this, a multimethodological approach (circular dichroism and fluorescence spectroscopy) was applied in parallel to FRET inhibition studies to characterize the binding modes of peptidic and nonpeptidic BACE-1 inhibitors. Circular dichroism spectroscopy served to correlate the conformation of BACE-1 with enzymatic activity and to monitor secondary structure changes upon ligand binding. In a complementary approach, direct fluorescence spectroscopy was used to characterize different BACE-1 inhibitor binding sites. The influence of pH and inhibitors on BACE-1 secondary structure was also elucidated. This multimethodological approach was applied to identify binding modes of bis(7)-tacrine and myricetin in comparison with well-known peptidic inhibitors.
authors.affiliation: Department of Drug Discovery and Development, Italian Institute of Technology, Genoa, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: De Simone
authors.forename: Francesca
authors.initials: F
authors.lastname: Mancini
authors.forename: Feliciana
authors.initials: F
authors.lastname: Real Fernàndez
authors.forename: Paolo
authors.initials: P
authors.lastname: Rovero
authors.forename: Carlo
authors.initials: C
authors.lastname: Bertucci
authors.forename: Vincenza
authors.initials: V
authors.lastname: Andrisano
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D020349:Surface Plasmon Resonance
chemical_list: D004791:Enzyme Inhibitors; C414614:OM99-2; D009842:Oligopeptides; D053829:Amyloid Precursor Protein Secretases