Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The the most potent compound, 25 (FM26), displayed submicromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and cocrystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt.

Understanding how emerging influenza viruses recognize host cells is critical in evaluating their zoonotic potential, pathogenicity, and transmissibility between humans. The surface of the influenza virus is covered with hemagglutinin (HA) proteins that can form multiple interactions with sialic acid-terminated glycans on the host cell surface. This multivalent binding affects the selectivity of the virus in ways that cannot be predicted from the individual receptor–ligand interactions alone. Here, we show that the intrinsic structural and energetic differences between the interactions of avian- or human-type receptors with influenza HA translate from individual site affinity and orientation through receptor length and density on the surface into virus avidity and specificity. We introduce a method to measure virus avidity using receptor density gradients. We found that influenza viruses attached stably to a surface at receptor densities that correspond to a minimum number of approximately 8 HA–glycan interactions, but more interactions were required if the receptors were short and human-type. Thus, the avidity and specificity of influenza viruses for a host cell depend not on the sialic acid linkage alone but on a combination of linkage and the length and density of receptors on the cell surface. Our findings suggest that threshold receptor densities play a key role in virus tropism, which is a predicting factor for both their virulence and zoonotic potential.

The fucosylation of glycans leads to diverse structures and is associated with many biological and disease processes. The exact determination of fucoside positions by tandem mass spectrometry (MS/MS) is complicated because rearrangements in the gas phase lead to erroneous structural assignments. Here, we demonstrate that the combined use of ion‐mobility MS and well‐defined synthetic glycan standards can prevent misinterpretation of MS/MS spectra and incorrect structural assignments of fucosylated glycans. We show that fucosyl residues do not migrate to hydroxyl groups but to acetamido moieties of N‐acetylneuraminic acid as well as N‐acetylglucosamine residues and nucleophilic sites of an anomeric tag, yielding specific isomeric fragment ions. This mechanistic insight enables the characterization of unique IMS arrival‐time distributions of the isomers which can be used to accurately determine fucosyl positions in glycans.

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The fucosylation of glycans leads to diverse structures and is associated with many biological and disease processes. The exact determination of fucoside positions by tandem mass spectrometry (MS/MS) is complicated because rearrangements in the gas phase lead to erroneous structural assignments. Here, we demonstrate that the combined use of ion‐mobility MS and well‐defined synthetic glycan standards can prevent misinterpretation of MS/MS spectra and incorrect structural assignments of fucosylated glycans. We show that fucosyl residues do not migrate to hydroxyl groups but to acetamido moieties of N‐acetylneuraminic acid as well as N‐acetylglucosamine residues and nucleophilic sites of an anomeric tag, yielding specific isomeric fragment ions. This mechanistic insight enables the characterization of unique IMS arrival‐time distributions of the isomers which can be used to accurately determine fucosyl positions in glycans.

Protein O-glycosylation is one of the most diverse post-translational modifications. A critical step in the analysis of Oglycomes is the release of glycans from glycoconjugates. Current release methods rely mainly on β-elimination, which can result in peeling reactions and loss of base-sensitive functionalities leading to misidentification of glycans. To address this challenge, welldefined synthetic glycopeptides were used to establish a robust workflow for the oxidative release of O-glycans suitable for glycomics. Treatment of O-glycopeptides with neutralized hypochlorite resulted in the selective formation of lactic/glycolic acid glycosides, thereby retaining unique information of the parent amino acid (serine/threonine) that is lost by β-elimination. It locks the glycan in a closed ring configuration, thereby preventing peeling, and furthermore, the carboxylate of the anomeric tag promotes ionization in negative ion mode mass spectrometry, thereby increasing signal intensities. Labile modifications such as sialic acids, sulfates, and acetyl esters are maintained during the release procedure. The promise of the approach was demonstrated by the analysis of O-glycans from bovine submaxillary mucin, which identified mono-and di-O-acetylated sialoglycans as well as previously undetected tri-O-acetylated and sulfated glycans. The use of well-defined glycopeptide standards made it also possible to identify reaction intermediates, which in turn allowed us to postulate a reaction mechanism for oxidative O-glycan release under neutral conditions.

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