1. Chemical screening by time-resolved X-ray scattering to discover allosteric probes
- Author
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Brosey, Chris A, Link, Todd M, Shen, Runze, Moiani, Davide, Burnett, Kathryn, Hura, Greg L, Jones, Darin E, and Tainer, John A
- Subjects
Biochemistry and Cell Biology ,Medicinal and Biomolecular Chemistry ,Chemical Sciences ,Biological Sciences ,Biotechnology ,Generic health relevance ,Allosteric Regulation ,Structure-Activity Relationship ,Apoptosis Inducing Factor ,Scattering ,Small Angle ,X-Ray Diffraction ,Humans ,Drug Discovery ,Models ,Molecular ,Kinetics ,Small Molecule Libraries ,Biochemistry & Molecular Biology ,Biochemistry and cell biology ,Medicinal and biomolecular chemistry - Abstract
Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF). By monitoring oxidized and X-ray-reduced AIF states, TR-HT-SAXS leverages structure and kinetics to generate a multidimensional screening dataset that identifies fragment chemotypes allosterically stimulating AIF dimerization. Fragment-induced dimerization rates, quantified with time-resolved SAXS similarity analysis (kVR), capture structure-activity relationships (SAR) across the top-ranked 4-aminoquinoline chemotype. Crystallized AIF-aminoquinoline complexes validate TR-SAXS-guided SAR, supporting this conformational chemotype for optimization. AIF-aminoquinoline structures and mutational analysis reveal active site F482 as an underappreciated allosteric stabilizer of AIF dimerization. This conformational discovery pipeline illustrates TR-HT-SAXS as an effective technology for targeting chemical leads to important macromolecular states.
- Published
- 2024