Discovery of Multifold Modified Sialosides as Human CD22/Siglec-2 Ligands with Nanomolar Activity on B-Cells

Abstract

Sialic acids are abundant in higher domains of life and lectins recognizing sialosaccharides are heavily involved in the regulation of the human immune system. Modified sialosides are useful tools to explore the functions of those lectins, especially members of the Siglec (sialic acid binding immunoglobulin like lectin) family. Here we report design, synthesis, and affinity evaluation of novel sialoside classes with combined modification at positions 2, 4, and 9 or 2, 3, 4, and 9 of the sialic acid scaffold as human CD22 (human Siglec-2) ligands. They display up to 7.5 × 10(5)-fold increased affinity over αMe Neu5Ac (the minimal Siglec ligand). CD22 is a negative regulating coreceptor of the B-cell receptor (BCR). In vitro experiments with a human B-lymphocyte cell line showed functional blocking of CD22 upon B-cell receptor (BCR) stimulation in the presence of nanomolar concentrations of the novel ligands. The observed increased Ca(2+) response corresponds to enhanced cell activation, providing an opportunity to therapeutically modulate B-lymphocyte responses, e.g., in immune deficiencies and infections.

Structural characterisation of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt’s lymphoma (BL) Daudi cells by NMR spectroscopy

“…The presence of a C-3-hydroxyl group in N -acetylneuraminic acid derivatives has been shown to increase resistance to enzymatic hydrolysis by sialidases33 and, in some cases, may yield slight improvements in binding affinity compared to the unsubstituted parent compound9. As shown in Fig.…”

“…Doxorubicin-loaded liposomes decorated with 9-BPC-Neu5Acα(2,3)Galβ(1,4)Glc that target B cell lymphoma were effective in extending life in a xenograft mouse model, however malignant B cell killing was not complete, likely due to insufficient affinity and selectivity of the siglec ligand 9-BPC-Neu5AcαGalβ(1,4)Glc that binds Siglec-2 expressed on B cells4. Siglec-2 ligands with improved binding affinity have been developed910 however, our group has succeeded in introducing for the first time functionalities at both C-4 and C-9 positions on 2 , 9-biphenylcarboxamido-4- meta -nitrophenyl-carboxamido-Neu5Acα2Me (9-BPC-4- m NPC-Neu5Acα2Me, 3 ) (Fig. 1) using a NMR-based structure guided approach11.…”

Therapeutic Targeting of Siglecs using Antibody- and Glycan-Based Approaches

“…Ligand design has also been facilitated by SAR analysis of sialic acid analog libraries [71,75-81]. Synthetic libraries have been evaluated to identify potential Siglec ligands using, for example, inhibition assays, surface plasmon resonance, and isothermal titration calorimetry.…”

Synthesis and biological evaluation of 9- N -oxamyl sialosides as Siglec-7 ligands

“…We obtained amine 3 by reduction of the corresponding azide 63 under maintenance of the methyl ester. 15 We used HATU to directly couple oxamic acids to 3 because of its general simple activation of carboxylic acids, fast reaction times, high yields and easy deactivation of excess reagent. 64 Interestingly, activation of alkyloxamic acids with HATU proceeded slowly and unreacted HATU was still present after 60 s. Upon addition of 3, the desired compounds and byproduct 13 were obtained (Scheme 1).…”

“…[4][5][6] These substitutions were subsequently modified and combined with other residues to enhance affinity and selectivity within the Siglec group. [7][8][9][10][11][12][13][14][15] Further affinity enhancing substitutions, including a Siglec-7 selective ligand, were found by screening a set of substituted di-and trisaccharides. 8,[16][17][18][19] Previously, high affinity Siglec ligands proved to be very useful to study the biological function of their lectin domain.…”