Structures of the Inhibitory Receptor Siglec-8 in Complex with a High-Affinity Sialoside Analogue and a Therapeutic Antibody

Abstract

Human sialic acid binding immunoglobulin-like lectin-8 (Siglec-8) is an inhibitory receptor that triggers eosinophil apoptosis and can inhibit mast cell degranulation when engaged by specific monoclonal antibodies (mAbs) or sialylated ligands. Thus, Siglec-8 has emerged as a critical negative regulator of inflammatory responses in diverse diseases, such as allergic airway inflammation. Herein, we have deciphered the molecular recognition features of the interaction of Siglec-8 with the mAb lirentelimab (2C4, under clinical development) and with a sialoside mimetic with the potential to suppress mast cell degranulation. The three-dimensional structure of Siglec-8 and the fragment antigen binding (Fab) portion of the anti-Siglec-8 mAb 2C4, solved by Xray crystallography, reveal that 2C4 binds close to the carbohydrate recognition domain (V-type Ig domain) on Siglec-8. We have also deduced the binding mode of a high-affinity analogue of its sialic acid ligand (9-N-napthylsufonimide-Neu5Ac, NSANeuAc) using a combination of NMR spectroscopy and X-ray crystallography. Our results show that the sialoside ring of NSANeuAc binds to the canonical sialyl binding pocket of the Siglec receptor family and that the high affinity arises from the accommodation of the NSA aromatic group in a nearby hydrophobic patch formed by the Nterminal tail and the unique G−G′ loop. The results reveal the basis for the observed high affinity of this ligand and provide clues for the rational design of the next generation of Siglec-8 inhibitors. Additionally, the specific interactions between Siglec-8 and the Nlinked glycans present on the high-affinity receptor FcεRIα have also been explored by NMR.

Tumor Carbohydrate Associated Antigen Analogs as Potential Binders for Siglec‐7

“…Moreover, these studies can be a starting point for the design and synthesis of high-affinity ligands of therapeutic relevance, that may prevent cancer cells from evading the immune system. [23,24,25]…”

“…Thus, here we describe the molecular recognition of two STn derivatives by Siglec‐7, providing information regarding the binding affinities and the ligands’ accommodation into the Siglec‐7 binding site. Moreover, these studies can be a starting point for the design and synthesis of high‐affinity ligands of therapeutic relevance, that may prevent cancer cells from evading the immune system [23,24,25] …”

Synthesis and Binding Mode Predictions of Novel Siglec-7 Ligands

“…Previous studies investigating Siglec selectivity indicate that selectivity for individual Siglecs can be achieved by modifications at position-9. 35,40,42,44,59 In addition, detailed investigations of the molecular interactions of selected, modified ligands, like a recent report for a Siglec-8 ligand, 60 provide the indispensable basis to further rationalize Siglec ligand design toward high affinity while maintaining individual Siglec selectivity. Further on, we analyzed the rotational conformation of the glycerol chain of the ligand because it obviously plays an important role in receptor binding.…”

“…Ligand selectivity over other α-sialosides binding receptors, especially over closely related ones of the CD33-related Siglec subgroup, is important. Previous studies investigating Siglec selectivity indicate that selectivity for individual Siglecs can be achieved by modifications at position-9. ,,,, In addition, detailed investigations of the molecular interactions of selected, modified ligands, like a recent report for a Siglec-8 ligand, provide the indispensable basis to further rationalize Siglec ligand design toward high affinity while maintaining individual Siglec selectivity.…”

Tetra‐ and Hexavalent Siglec‐8 Ligands Modulate Immune Cell Activation

“…The question remains, whether the mimetics 3 and 34 occupy the same binding pocket as their natural counterpart 6′‐sulfo‐sLe x ( 1 , PDB ID: 2N7B) [16] and the recently published 9‐ N ‐naphthylsulfonamide‐Neu5Ac derivative (PDB ID: 7QUI) [32] . Since both, glycomimetic 3 and 34 exhibit the same pharmacophore as the two compounds mentioned above, that is, the same molecular features necessary for molecular recognition, it can be assumed that they occupy the Siglec‐8 binding site identically.…”