Fragment Binding to β-Secretase 1 without Catalytic Aspartate Interactions Identified via Orthogonal Screening Approaches
| Autor: | Richard Alexander, Frederik J. R. Rombouts, Daan Van Glabbeek, Michel Carpentier, Alex De Groot, Ann Vos, Gary Tresadern, Martina Palomino-Schätzlein, Joyce Dijkmans, Erna Cleiren, Antonio Pineda-Lucena, Katleen Fierens, Diederik Moechars, Andrés A. Trabanco, Stefan Masure |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification biology Chemistry Stereochemistry General Chemical Engineering Mutant Active site General Chemistry 01 natural sciences Combinatorial chemistry Article 0104 chemical sciences Catalysis lcsh:Chemistry 010404 medicinal & biomolecular chemistry 03 medical and health sciences 030104 developmental biology Förster resonance energy transfer Enzyme lcsh:QD1-999 Heteronuclear molecule biology.protein Surface plasmon resonance Spectroscopy |
| Zdroj: | ACS Omega ACS Omega, Vol 2, Iss 2, Pp 685-697 (2017) |
| ISSN: | 2470-1343 |
| Popis: | 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 15N 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. |
| Databáze: | OpenAIRE |
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