Chemical screening by time-resolved X-ray scattering to discover allosteric probes.

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.

Applying HT-SAXS to chemical ligand screening.

Chemical probes are invaluable tools for investigating essential biological processes. Understanding how small-molecule probes engage biomolecular conformations is critical to developing their functional selectivity. High-throughput solution X-ray scattering is well-positioned to profile target-ligand complexes during probe development, bringing conformational insight and selection to traditional ligand binding assays. Access to high-quality synchrotron SAXS datasets and high-throughput data analysis now allows routine academic users to incorporate conformational information into small-molecule development pipelines. Here we describe a general approach for benchmarking and preparing HT-SAXS chemical screens from small fragment libraries. Using the allosteric oxidoreductase Apoptosis-Inducing Factor (AIF) as an exemplary system, we illustrate how HT-SAXS efficiently identifies an allosteric candidate among hits of a microscale thermophoresis ligand screen. We discuss considerations for pursuing HT-SAXS chemical screening with other systems of interest and reflect on advances to extend screening throughput and sensitivity.