Open AccessFragment Binding to β-Secretase 1 without Catalytic Aspartate Interactions Identified via Orthogonal Screening Approaches
Author(s) -
Frederik Rombouts,
Richard Alexander,
Erna Cleiren,
Alex De Groot,
Michel Carpentier,
Joyce Dijkmans,
Katleen Fierens,
Stefan Masure,
Diederik Moechars,
Martina PalominoSchätzlein,
Antonio PinedaLucena,
Andrés A. Trabanco,
Daan Van Glabbeek,
Ann Vos,
Gary Tresadern
Publication year - 2017
Publication title -
acs omega
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.779
H-Index - 40
ISSN - 2470-1343
DOI - 10.1021/acsomega.6b00482
Subject(s) - surface plasmon resonance , heteronuclear molecule , heteronuclear single quantum coherence spectroscopy , förster resonance energy transfer , chemistry , stereochemistry , enzyme , mutant , nuclear magnetic resonance spectroscopy , crystallography , combinatorial chemistry , fluorescence , biochemistry , materials science , nanotechnology , physics , quantum mechanics , nanoparticle , gene
An approach to identify β-secretase 1 (BACE1) fragment binders that do not interact with the catalytic aspartate dyad is presented. A ThermoFluor (thermal shift) and a fluorescence resonance energy transfer enzymatic screen on the soluble domain of BACE1, together with a surface plasmon resonance (SPR) screen on the soluble domain of BACE1 and a mutant of one catalytic Asp (D32N), were run in parallel. Fragments that were active in at least two of these assays were further confirmed using one-dimensional NMR (WaterLOGSY) and SPR binding competition studies with peptidic inhibitor OM99-2. Protein-observed NMR (two-dimensional 15 N heteronuclear single-quantum coherence spectroscopy) and crystallographic studies with the soluble domain of BACE1 identified a unique and novel binding mode for compound 12 , a fragment that still occupies the active site while not making any interactions with catalytic Asps. This novel approach of combining orthogonal fragment screening techniques, for both wild-type and mutant enzymes, as well as binding competition studies could be generalized to other targets to overcome undesired interaction motifs and as a hit-generation approach in highly constrained intellectual property space.