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1.
J Med Chem ; 65(15): 10588-10610, 2022 08 11.
Article in English | MEDLINE | ID: mdl-35881556

ABSTRACT

Significant interest in the development of high-affinity ligands for Siglecs exists due to the various therapeutically relevant functions of these proteins. Here, we report a new strategy to develop and design Siglec ligands as disialyl-oligosaccharide mimetics exemplified on Siglec-2 (CD22). We report insights into development of dimeric ligands with high affinity and avidity to cell surface-expressed CD22, assay development, tool compounds, structure activity relationships, and biological data on calcium flux regulation in B-cells. The binding modes of selected ligands have been modeled based on state-of-the-art molecular dynamics simulations on the microsecond timescale, providing detailed views on ligand binding and opening a new perspective on drug design efforts for Siglecs. High-avidity dimeric ligands containing a linker opening the way towards bispecifics are presented as well.


Subject(s)
Receptors, Antigen, B-Cell , Sialic Acid Binding Immunoglobulin-like Lectins , Biomimetic Materials , Humans , Ligands , Oligosaccharides/pharmacology , Sialic Acid Binding Ig-like Lectin 2
2.
Sci Signal ; 15(723): eabf9570, 2022 03.
Article in English | MEDLINE | ID: mdl-35230871

ABSTRACT

The protein tyrosine phosphatase CD45 plays a crucial role in B cell antigen receptor (BCR) signaling by activating Src family kinases. Cd45-/- mice show altered B cell development and a phenotype likely due to reduced steady-state signaling; however, Cd45-/- B cells show relatively normal BCR ligation-induced signaling. In our investigation of how BCR signaling was restored in Cd45-/- cells, we found that the coreceptor CD22 switched from an inhibitory to a stimulatory function in these cells. We disrupted the ability of CD22 to interact with its ligands in Cd45-/- B cells by generating Cd45-/-St6galI-/- mice, which cannot synthesize the glycan ligand of CD22, or by treating Cd45-/- B cells in vitro with the sialoside GSC718, which inhibits ligand binding to CD22. BCR ligation-induced signaling was reduced by ST6GalI deficiency, but not by GSC718 treatment, suggesting that CD22 restored BCR ligation-induced signaling in Cd45-/- mature B cells by altering cellular phenotypes during development. CD22 was required for the increase in the surface amount of IgM-BCR on Cd45-/- B cells, which augmented signaling. Because B cell survival depends on steady-state BCR signaling, IgM-BCR abundance was likely increased by the selective survival of IgM-BCRhi Cd45-/- B cells because of CD22-mediated signaling under conditions of substantially reduced steady-state signaling. Because the amount of surface IgM-BCR is increased on B cells from patients with other BCR signaling deficiencies, including X-linked agammaglobulinemia, our findings suggest that CD22 may contribute to the partial restoration of B cell function in these patients.


Subject(s)
B-Lymphocytes , Receptors, Antigen, B-Cell , Animals , B-Lymphocytes/metabolism , Leukocyte Common Antigens , Lymphocyte Activation , Mice , Receptors, Antigen, B-Cell/metabolism , Sialic Acid Binding Ig-like Lectin 2/genetics , Sialic Acid Binding Ig-like Lectin 2/metabolism , Signal Transduction , src-Family Kinases/metabolism
3.
Chembiochem ; 18(13): 1216-1225, 2017 07 04.
Article in English | MEDLINE | ID: mdl-28374962

ABSTRACT

CD22 is a member of the Siglec family. Considerable attention has been drawn to the design and synthesis of new Siglec ligands to explore target biology and innovative therapies. In particular, CD22-ligand-targeted nanoparticles with therapeutic functions have proved successful in preclinical settings for blood cancers, autoimmune diseases, and tolerance induction. Here we report the design, synthesis and affinity evaluation of a new class of Siglec ligands: namely sialic acid derivatives with a triazole moiety replacing the natural glycoside oxygen atom. In addition, we describe important and surprising differences in binding to CD22 expressed at the cell surface for compounds with distinct valences. The new class of compounds might serve as a template for the design of ligands for other members of the Siglec family and next-generation CD22-ligand-based targeted therapies.


Subject(s)
B-Lymphocytes/drug effects , Glycosides/chemical synthesis , Sialic Acid Binding Ig-like Lectin 2/metabolism , Sialic Acids/chemistry , Triazoles/chemical synthesis , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Binding Sites , Carbohydrate Conformation , Carbohydrate Sequence , Cell Line, Tumor , Drug Design , Enzyme-Linked Immunosorbent Assay , Glycosides/pharmacology , Humans , Immunoglobulin M/chemistry , Ligands , Protein Binding , Sialic Acid Binding Ig-like Lectin 2/antagonists & inhibitors , Sialic Acid Binding Ig-like Lectin 2/chemistry , Triazoles/pharmacology
4.
J Med Chem ; 60(3): 941-956, 2017 02 09.
Article in English | MEDLINE | ID: mdl-28103033

ABSTRACT

Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar Kd values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds.


Subject(s)
Antigens, Differentiation, Myelomonocytic/metabolism , Immune Evasion , Lectins/metabolism , Neoplasms/immunology , Drug Design , Humans , Killer Cells, Natural/immunology , Ligands
5.
ACS Chem Biol ; 9(7): 1444-50, 2014 Jul 18.
Article in English | MEDLINE | ID: mdl-24807582

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.


Subject(s)
B-Lymphocytes/drug effects , Immunologic Factors/chemistry , N-Acetylneuraminic Acid/analogs & derivatives , Sialic Acid Binding Ig-like Lectin 2/immunology , B-Lymphocytes/immunology , Cell Line , Drug Discovery , Humans , Immunologic Factors/pharmacology , Ligands , N-Acetylneuraminic Acid/pharmacology , Receptors, Antigen, B-Cell/immunology , Sialic Acid Binding Ig-like Lectin 2/antagonists & inhibitors
6.
J Biol Chem ; 289(4): 2112-26, 2014 Jan 24.
Article in English | MEDLINE | ID: mdl-24311790

ABSTRACT

MAPK activity is negatively regulated by members of the dual specificity phosphatase (Dusp) family, which differ in expression, substrate specificity, and subcellular localization. Here, we investigated the function of Dusp16/MKP-7 in the innate immune system. The Dusp16 isoforms A1 and B1 were inducibly expressed in macrophages and dendritic cells following Toll-like receptor stimulation. A gene trap approach was used to generate Dusp16-deficient mice. Homozygous Dusp16tp/tp mice developed without gross abnormalities but died perinatally. Fetal liver cells from Dusp16tp/tp embryos efficiently reconstituted the lymphoid and myeloid compartments with Dusp16-deficient hematopoietic cells. However, GM-CSF-induced proliferation of bone marrow progenitors in vitro was impaired in the absence of Dusp16. In vivo challenge with Escherichia coli LPS triggered higher production of IL-12p40 in mice with a Dusp16-deficient immune system. In vitro, Dusp16-deficient macrophages, but not dendritic cells, selectively overexpressed a subset of TLR-induced genes, including the cytokine IL-12. Dusp16-deficient fibroblasts showed enhanced activation of p38 and JNK MAPKs. In macrophages, pharmacological inhibition and siRNA knockdown of JNK1/2 normalized IL-12p40 secretion. Production of IL-10 and its inhibitory effect on IL-12 production were unaltered in Dusp16tp/tp macrophages. Altogether, the Dusp16 gene trap mouse model identifies an essential role in perinatal survival and reveals selective control of differentiation and cytokine production of myeloid cells by the MAPK phosphatase Dusp16.


Subject(s)
Dual-Specificity Phosphatases/immunology , Immunity, Innate/physiology , Interleukin-10/immunology , Interleukin-12/immunology , Macrophages/immunology , Mitogen-Activated Protein Kinase Phosphatases/immunology , Toll-Like Receptors/immunology , Animals , Dual-Specificity Phosphatases/genetics , Dual-Specificity Phosphatases/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/immunology , Extracellular Signal-Regulated MAP Kinases/metabolism , Immunity, Innate/drug effects , Interleukin-10/genetics , Interleukin-10/metabolism , Interleukin-12/biosynthesis , Interleukin-12/genetics , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/metabolism , Mice , Mice, Mutant Strains , Mitogen-Activated Protein Kinase Phosphatases/genetics , Mitogen-Activated Protein Kinase Phosphatases/metabolism , Toll-Like Receptors/agonists , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolism
7.
Eur J Immunol ; 42(10): 2792-802, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22777817

ABSTRACT

CD22 is an inhibitory co-receptor of the B-cell receptor (BCR) on B cells. Since CD22 is ubiquitously expressed in the B-cell lineage and CD22 endocytosis can be triggered efficiently, antibodies and antibody-based immunotoxins against CD22 are used to target B cells both in B-cell lymphomas and leukemias, as well as in autoimmune diseases. CD22 recognizes α2,6-linked sialic acids as endogenous ligands. We have developed new synthetic sialosides as ligands for human CD22. These sialosides bind CD22 on human B cells with high affinity and can efficiently enhance IgM-triggered Ca(2+) signaling. We coupled these sialosides to Pseudomonas exotoxin A to generate a novel CD22 ligand-based immunotoxin. This sialoside-exotoxin-A construct can specifically kill CD22-positive B-cell lymphoma cells. It binds specifically to CD22-positive B-cell lymphoma cells and is dominant over endogenous cis-ligands on the B-cell surface. The sialoside-exotoxin-A construct is efficiently internalized by endocytosis into B-cell lymphoma cell lines. Thus we show the development of a new therapeutic compound for targeting CD22 on human B cells, both for B-cell lymphoma, as well as for B-cell-mediated autoimmune diseases.


Subject(s)
ADP Ribose Transferases/metabolism , Autoimmune Diseases/therapy , B-Lymphocytes/immunology , Bacterial Toxins/metabolism , Exotoxins/metabolism , Immunotoxins/metabolism , Lymphoma, B-Cell/therapy , N-Acetylneuraminic Acid/metabolism , Pseudomonas/immunology , Sialic Acid Binding Ig-like Lectin 2/metabolism , Virulence Factors/metabolism , ADP Ribose Transferases/genetics , Autoimmune Diseases/immunology , B-Lymphocytes/drug effects , Bacterial Toxins/genetics , Calcium Signaling , Cations, Divalent/chemistry , Cell Differentiation , Cell Lineage , Cytotoxicity, Immunologic , Exotoxins/genetics , HL-60 Cells , Humans , Immunoglobulin M/immunology , Immunotoxins/genetics , Lymphoma, B-Cell/immunology , Molecular Targeted Therapy , N-Acetylneuraminic Acid/analogs & derivatives , N-Acetylneuraminic Acid/chemical synthesis , Receptors, Antigen, B-Cell/metabolism , Virulence Factors/genetics , Pseudomonas aeruginosa Exotoxin A
8.
EMBO J ; 30(13): 2705-18, 2011 May 27.
Article in English | MEDLINE | ID: mdl-21623346

ABSTRACT

The quality and quantity of BCR signals impact on cell fate decisions of B lymphocytes. Here, we describe novel gene-targeted mice, which in the context of normal VDJ recombination show hypomorphic expression of immunoglobulin µ heavy chain (µHC) mRNA levels and hence lower pre-BCR and BCR levels. Hypomorphic expression of µHC leads to augmented selection processes at all stages of B-cell development, noticeably at the expansion of pre-B cells, the positive selection of immature B lymphocytes in the bone marrow and the selection of the follicular (FO), marginal zone (MZ) and B1 B-lymphocyte compartment in peripheral lymphoid organs. Immature as well as mature FO and MZ B lymphocytes in the peripheral lymphoid organs express lower levels of the receptor for B-cell activating factor (BAFF). In addition, hypomorphic expression of the BCR favours receptor editing. Together, our results highlight the critical importance of pre-BCR and BCR receptor levels for the normal development of B-lymphocyte subpopulations in the context of intact VDJ recombination and a diverse antibody repertoire.


Subject(s)
B-Lymphocyte Subsets/physiology , Cell Differentiation/immunology , Genes, Immunoglobulin Heavy Chain/physiology , Receptors, Antigen, B-Cell/immunology , Receptors, Antigen, B-Cell/metabolism , Alleles , Animals , Antibody Formation/genetics , Antibody Formation/physiology , B-Cell Activation Factor Receptor/metabolism , B-Lymphocyte Subsets/metabolism , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/physiology , Cell Differentiation/genetics , Cells, Cultured , Genes, Immunoglobulin Heavy Chain/genetics , Immunoglobulin mu-Chains/genetics , Immunoglobulin mu-Chains/metabolism , Lectins/genetics , Lectins/immunology , Lectins/metabolism , Mice , Mice, Transgenic , Precursor Cells, B-Lymphoid/immunology , Precursor Cells, B-Lymphoid/metabolism , Precursor Cells, B-Lymphoid/physiology , Receptors, Antigen, B-Cell/genetics , Sialic Acid Binding Immunoglobulin-like Lectins
9.
J Exp Med ; 208(4): 823-39, 2011 Apr 11.
Article in English | MEDLINE | ID: mdl-21464221

ABSTRACT

By studying mice in which the Nfatc1 gene was inactivated in bone marrow, spleen, or germinal center B cells, we show that NFATc1 supports the proliferation and suppresses the activation-induced cell death of splenic B cells upon B cell receptor (BCR) stimulation. BCR triggering leads to expression of NFATc1/αA, a short isoform of NFATc1, in splenic B cells. NFATc1 ablation impaired Ig class switch to IgG3 induced by T cell-independent type II antigens, as well as IgG3(+) plasmablast formation. Mice bearing NFATc1(-/-) B cells harbor twofold more interleukin 10-producing B cells. NFATc1(-/-) B cells suppress the synthesis of interferon-γ by T cells in vitro, and these mice exhibit a mild clinical course of experimental autoimmune encephalomyelitis. In large part, the defective functions of NFATc1(-/-) B cells are caused by decreased BCR-induced Ca(2+) flux and calcineurin (Cn) activation. By affecting CD22, Rcan1, CnA, and NFATc1/αA expression, NFATc1 controls the Ca(2+)-dependent Cn-NFAT signaling network and, thereby, the fate of splenic B cells upon BCR stimulation.


Subject(s)
B-Lymphocytes/immunology , Calcineurin/physiology , NFATC Transcription Factors/physiology , Signal Transduction/physiology , Spleen/immunology , Animals , Calcium/metabolism , Immunoglobulin Class Switching , Lymphocyte Activation , Mice , Receptors, Antigen, B-Cell/physiology , T-Lymphocytes/physiology
10.
J Immunol ; 184(7): 3665-76, 2010 Apr 01.
Article in English | MEDLINE | ID: mdl-20194721

ABSTRACT

Compartmentalization of the BCR in membrane rafts is important for its signaling capacity. Swiprosin-1/EFhd2 (Swip-1) is an EF-hand and coiled-coil-containing adaptor protein with predicted Src homology 3 (SH3) binding sites that we identified in membrane rafts. We showed previously that Swip-1 amplifies BCR-induced apoptosis; however, the mechanism of this amplification was unknown. To address this question, we overexpressed Swip-1 and found that Swip-1 amplified the BCR-induced calcium flux in WEHI231, B62.1, and Bal17 cells. Conversely, the BCR-elicited calcium flux was strongly attenuated in Swip-1-silenced WEHI231 cells, and this was due to a decreased calcium mobilization from intracellular stores. Complementation of Swip-1 expression in Swip-1-silenced WEHI231 cells restored the BCR-induced calcium flux and enhanced spleen tyrosine kinase (Syk) tyrosine phosphorylation and activity as well as SLP65/BLNK/BASH and phospholipase C gamma2 (PLCgamma2) tyrosine phosphorylation. Furthermore, Swip-1 induced the constitutive association of the BCR itself, Syk, and PLCgamma2 with membrane rafts. Concomitantly, Swip-1 stabilized the association of BCR with tyrosine-phosphorylated proteins, specifically Syk and PLCgamma2, and enhanced the constitutive interaction of Syk and PLCgamma2 with Lyn. Interestingly, Swip-1 bound to the rSH3 domains of the Src kinases Lyn and Fgr, as well as to that of PLCgamma. Deletion of the predicted SH3-binding region in Swip-1 diminished its association and that of Syk and PLCgamma2 with membrane rafts, reduced its interaction with the SH3 domain of PLCgamma, and diminished the BCR-induced calcium flux. Hence, Swip-1 provides a membrane scaffold that is required for the Syk-, SLP-65-, and PLCgamma2-dependent BCR-induced calcium flux.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Intracellular Signaling Peptides and Proteins/immunology , Membrane Microdomains/immunology , Phospholipase C gamma/immunology , Protein-Tyrosine Kinases/immunology , Receptors, Antigen, B-Cell/immunology , Signal Transduction/immunology , Adaptor Proteins, Signal Transducing/metabolism , Animals , Blotting, Western , Calcium-Binding Proteins , Cell Line , Cell Separation , Flow Cytometry , Fluorescent Antibody Technique , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Activation/immunology , Membrane Microdomains/metabolism , Mice , Phospholipase C gamma/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, B-Cell/metabolism , Syk Kinase
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