Skip to main content
[Preprint]. 2024 Oct 12:2024.10.11.617879. [Version 1] doi: 10.1101/2024.10.11.617879

Fig. 3 |. Development of a Siglec-7/9 degrader via the discovery of high-affinity and selective Siglec-7/9 ligands.

Fig. 3 |

a, The synthesis of sulfamide linked α2–6 or α2–3 9-amino-Neu5Ac-LacNAc (9-amino-Neu5Ac-Galβ1–4GlcNAc) derivatives using SuFEx click chemistry, in which 9-amine tagged Neu5Ac is subjected to reaction with a library of iminosulfur oxydifluorides to form diversified sulfamide-linked Neu5Ac mimetics. SuFEx, Sulfur (VI) fluoride exchange; Neu5Ac, N-acetylneuraminic acid; Gal, galactose; GlcNAc, N-acetylglucosamine. b, The structures of a library of sulfamide-linked Neu5Ac-LacNAc derivatives. c, Measurement of Siglec-7 binding affinity of the synthetic Neu5Ac ligands (shown in b) installed on the cell surface by staining with Siglec-7Fc determined by MFI using flow cytometry. MFI, mean fluorescence intensity. d, An ELISA assay for Siglec cross-binding assessment of HRP-functionalized SigL tetramers. ELISA, enzyme-linked immunosorbent assay. Data are mean ± s.d. Two-tailed unpaired Student’s t-test. ns, not significant (d). e, Biolayer interferometry (BLI) assay for determination of the binding affinity KD affiliated with rate constants of kon (association), koff (dissociation) for (8bio)4-SA and (9bio)4-SA tetramers and anti-Siglec-7/-9 antibodies towards Siglec-7 and Siglec-9, respectively. f, Schematic presentation of rapid internalization and recycling of Siglec-7/-9 in myeloid cells upon treatment with (SigLbio)4-SA tetramer. g, Schematic presentation of the rationale for degrading Siglec-7/-9 through the incorporation of M6P into the (SigLbio)4-SA tetramer, which targets and delivers Siglec-7/-9 to the lysosomes for degradation upon recognition by the M6P receptor (M6PR). h, Analysis of cell-surface Siglec-7 levels in Siglec-7+ U937-derived macrophages after treatment with (8bio)4-SA with or without M6P conjugation for 1 hour at various doses. i, Analysis of cell-surface Siglec-9 levels in Siglec-9+ U937-derived macrophages after treatment with (8bio)4-SA with or without M6P conjugation for 1 hour at various doses. j,k, West blot analysis of Siglec-7 and -9 in Siglec-7/-9 KO, WT and R-mutant U937-derived macrophages after treatment with 30 nM (8bio)4-SA-M6P4 (Sig7/9de) for 24 hours in comparison with ctrs (treated with PBS, (8bio)4-SA or SA-M6P4, and Siglec-KO cells), as wells as in the presence of lysosome inhibitor BafA1 or proteasome inhibitor MG132. l, Microscopic confirmation of the degradation of Siglec-7 and -9 in Siglec-expressing U937-derived macrophages, respectively. Scale bar, 10 μm.