title: Human recombinant beta-secretase immobilized enzyme reactor for fast hits' selection and characterization from a virtual screening library.
abstract: In the present work, a human recombinant BACE1 immobilized enzyme reactor (hrBACE1-IMER) has been applied for the sensitive fast screening of 38 compounds selected through a virtual screening approach. HrBACE1-IMER was inserted into a liquid chromatograph coupled with a fluorescent detector. A fluorogenic peptide substrate (M-2420), containing the β-secretase site of the Swedish mutation of APP, was injected and cleaved in the on-line HPLC-hrBACE1-IMER system, giving rise to the fluorescent product. The compounds of the library were tested for their ability to inhibit BACE1 in the immobilized format and to reduce the area related to the chromatographic peak of the fluorescent enzymatic product. The results were validated in solution by using two different FRET methods. Due to the efficient virtual screening methodology, more than fifty percent of the selected compounds showed a measurable inhibitory activity. One of the most active compound (a bis-indanone derivative) was characterized in terms of IC(50) and K(i) determination on the hrBACE1-IMER. Thus, the hrBACE1-IMER has been confirmed as a valid tool for the throughput screening of different chemical entities with potency lower than 30μM for the fast hits' selection and for mode of action determination.
authors.affiliation: Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: De Simone
authors.forename: Francesca
authors.initials: F
authors.lastname: Mancini
authors.forename: Sandro
authors.initials: S
authors.lastname: Cosconati
authors.forename: Luciana
authors.initials: L
authors.lastname: Marinelli
authors.forename: Valeria
authors.initials: V
authors.lastname: La Pietra
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.forename: Vincenza
authors.initials: V
authors.lastname: Andrisano
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors*; D019149:Bioreactors; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors*; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D012680:Sensitivity and Specificity; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D054852:Small Molecule Libraries; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human