Leveraging biophysical approaches for the discovery of modulators targeting enzymes in the ubiquitination pathway

The discovery of small molecules that modulate protein function relies not only on functional screening but also on a detailed molecular understanding of ligand recognition. Biophysical techniques such as fluorescence anisotropy, differential scanning fluorimetry (DSF), X-ray crystallography, and saturation transfer difference (STD) NMR provide complementary insights into binding specificity, protein stability, and structural features, enabling the distinction between true interactions and artifacts and facilitating mechanistic characterization. These approaches are particularly valuable for complex targets, such as the ubiquitination system, which relies on precisely regulated protein-protein interactions within an enzymatic cascade involving E1, E2, and E3 enzymes, thereby controlling essential cellular processes including protein degradation and signal transduction. UBE2A, a ubiquitin-conjugating enzyme (E2), occupies a central position in this pathway. Pathogenic variants in UBE2A are linked to intellectual disability, while its overexpression correlates with poor prognosis in breast and liver cancers. Despite its biological and clinical relevance, selective chemical modulators of UBE2A remain poorly characterized. Here, we employed an integrated biophysical platform to identify and characterize modulators of UBE2A. We recapitulated the ubiquitination cascade in vitro using recombinant human UBA1, UBE2A, and ubiquitin produced in Escherichia coli. Ubiquitin was fluorescently labeled with fluorescein isothiocyanate. Exploiting the differential hydrodynamic sizes of free mono-ubiquitin versus ubiquitin-bound E1/E2 complexes in a fluorescence anisotropy readout, we monitored ubiquitination activity and screened a 20,000-compound ChemBridge CNS library in a 384-well format. Hits were validated through concentration-response analysis and counter-screening against UBA1. For hit validation, we employed orthogonal biophysical techniques. DSF was used to assess ligand-induced changes in UBE2A thermal stability, revealing thermal shifts for selected compounds consistent with protein-ligand interactions. X-ray crystallography provided structural information on the potential binding mode of a selected compound, which showed a corresponding electron density near the catalytic Cys88. STD-NMR confirmed direct solution-state binding for another compound, with saturation transfer effects detected across multiple proton resonances. Collectively, this orthogonal biophysical pipeline, spanning functional activity, stability changes, structural context, and solution-state binding, provides a robust framework for characterizing UBE2A ligand interactions and reliably discriminating true binders from assay artifacts.

This work was supported by CAPES (001), FAPESP (2024/07830-3), and PRONAS/PCD (25000.015040/2019-03).