Sialyltransferases catalyze transfer of sialic acid to glycoconjugates in the Golgi, generating cell-surface sialoglycans that modulate cell–cell and immune interactions. Among these enzymes, ST6GAL1 (α-2,6-sialyltransferase 1) is highlighted for its importance in regulating sialoglycan-mediated signaling and its relevance to cancer biology. Many reported β-galactoside ST6GAL1 inhibitors are derivatives of CMP‑Neu5Ac and are highly polar, which limits passive membrane permeability and complicates cellular application. The noncarbohydrate inhibitor JFD 00458 similarly exhibits poor membrane permeability.
To address this limitation, the authors designed analogs of JFD 00458 in which the sulfonic-acid group was replaced by sulfonic-acid bioisosteres, with particular focus on sulfonylurea replacements. The goal was to increase passive membrane permeability while retaining ST6GAL1 inhibitory activity and direct target engagement.
An expanded series of JFD 00458 analogs was prepared incorporating different sulfonic-acid bioisosteres. The chemical modifications preserved the substituted phenoxy phenyl core present in the parent scaffold while varying the linker and aromatic substituents attached through the sulfonylurea or alternative bioisosteric motifs. The source summarizes an expanded set of sulfonylurea-containing compounds and highlights representative analogs (including compounds labeled 6d and 6e) that showed notable functional differences.
Passive membrane permeability was evaluated using the parallel artificial membrane permeability assay (PAMPA). Incorporation of sulfonic-acid bioisosteres, and in particular the sulfonylurea motif, improved passive permeability compared with the parent JFD 00458. The best-performing analog in the PAMPA reached log(P_app [cm/s]) = -4.3, demonstrating a measurable increase in passive permeation for the optimized sulfonylureas. The report frames these permeability data as evidence that sulfonylureas can serve as membrane-permeable bioisosteres for sulfonic acid in this inhibitor class.
An expanded series of sulfonylurea analogs displayed improved inhibitory potency against ST6GAL1 in biochemical assays. The most potent inhibitor reported in the series had an IC50 of 1.9 µM. Potency varied across the series and correlated with structural features of the sulfonylurea-linked aromatic substituents, with certain substitutions associated with superior inhibition.
To confirm direct target engagement, microscale thermophoresis (MST) was used to measure binding between ST6GAL1 and selected inhibitors. MST returned similar apparent dissociation constants for JFD 00458 and the optimized sulfonylureas, with apparent Kd values reported at approximately 22–25 µM. These MST data indicate that the sulfonylurea replacements maintain measurable binding to ST6GAL1 despite improving permeability and, in some cases, biochemical potency.
Saturation transfer difference (STD) NMR experiments were performed to define ligand portions that contact ST6GAL1. STD NMR identified the substituted phenoxy phenyl core as a common ST6GAL1-contacting epitope across JFD 00458 and its analogs. In the more potent sulfonylurea analogs (notably compounds 6d and 6e), closer contacts were observed from the sulfonylurea-linked aromatic substituents, and these closer contacts correlated with higher inhibitory potency within the series. The combined STD NMR and potency data support a structure–activity relationship in which both the conserved phenoxy phenyl core and the nature of the sulfonylurea-linked substituents contribute to inhibition.
The study establishes sulfonylureas as viable membrane-permeable sulfonic-acid bioisosteres within a JFD 00458-based ST6GAL1 inhibitor scaffold. Replacement of the sulfonic acid with sulfonylurea motifs led to improved passive permeability (best PAMPA log(P_app) = -4.3) and produced compounds with improved biochemical potency (best IC50 = 1.9 µM) while preserving direct ST6GAL1 binding (apparent Kd ≈ 22–25 µM by MST). STD NMR localized critical enzyme–ligand contacts to the substituted phenoxy phenyl core and to sulfonylurea-linked aromatic substituents in the most active analogs, informing structure–activity relationships.
These results provide a framework for further chemical optimization of ST6GAL1 inhibitors with enhanced membrane permeability and retained target engagement. The source does not report cell-based functional assays, in vivo efficacy, toxicity, or clinical translation; such studies would be required to assess biological activity beyond biochemical inhibition and passive permeability.