RESUMO
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.
Assuntos
Linfócitos B/efeitos dos fármacos , Glicosídeos/síntese química , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Ácidos Siálicos/química , Triazóis/síntese química , Linfócitos B/citologia , Linfócitos B/metabolismo , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Glicosídeos/farmacologia , Humanos , Imunoglobulina M/química , Ligantes , Ligação Proteica , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/química , Triazóis/farmacologiaRESUMO
A fragment library consisting of 3D-shaped, natural product-like fragments was assembled. Library construction was mainly performed by natural product degradation and natural product diversification reactions and was complemented by the identification of 3D-shaped, natural product like fragments available from commercial sources. In addition, during the course of these studies, novel rearrangements were discovered for Massarigenin C and Cytochalasin E. The obtained fragment library has an excellent 3D-shape and natural product likeness, covering a novel, unexplored and underrepresented chemical space in fragment based drug discovery (FBDD).
Assuntos
Produtos Biológicos/química , Citocalasinas/química , Lactonas/química , Bibliotecas de Moléculas Pequenas/química , Compostos de Espiro/química , Produtos Biológicos/síntese química , Cristalografia por Raios X , Citocalasinas/síntese química , Descoberta de Drogas , Lactonas/síntese química , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Compostos de Espiro/síntese químicaRESUMO
Siglecs (sialic acid recognizing immunoglobulin like lectins) are a family of lectins with specificity for sialic acid containing carbohydrates. Synthetic sialic acid derivatives with high affinity proved useful to unravel the biological role of the ligand binding domain, although many of their functions in immunity remain unknown. Here we present design, synthesis, affinity evaluation and molecular modeling of novel 9-N-oxamoyl modified sialosides as Siglec-7 ligands.
Assuntos
Ácido N-Acetilneuramínico/química , Ácidos Neuramínicos/química , Lectinas/metabolismo , Ligantes , Estrutura MolecularRESUMO
Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.
Assuntos
Biologia Computacional , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/química , Simportadores/metabolismo , Azepinas/metabolismo , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Indóis/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos/genética , Projetos Piloto , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Relação Estrutura-Atividade , Simportadores/genéticaRESUMO
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.
Assuntos
ADP Ribose Transferases/metabolismo , Doenças Autoimunes/terapia , Linfócitos B/imunologia , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Imunotoxinas/metabolismo , Linfoma de Células B/terapia , Ácido N-Acetilneuramínico/metabolismo , Pseudomonas/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Fatores de Virulência/metabolismo , ADP Ribose Transferases/genética , Doenças Autoimunes/imunologia , Linfócitos B/efeitos dos fármacos , Toxinas Bacterianas/genética , Sinalização do Cálcio , Cátions Bivalentes/química , Diferenciação Celular , Linhagem da Célula , Citotoxicidade Imunológica , Exotoxinas/genética , Células HL-60 , Humanos , Imunoglobulina M/imunologia , Imunotoxinas/genética , Linfoma de Células B/imunologia , Terapia de Alvo Molecular , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/síntese química , Receptores de Antígenos de Linfócitos B/metabolismo , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
Siglec-7 regulates immune cell activity and is a promising target for immunomodulation. Here, we report the discovery of novel sialic acid derivatives binding to Siglec-7. Synthesis and affinity measurements are complemented by high-quality models of sialoside-Siglec-7 complexes based on molecular dynamics (MD) simulations on the microsecond time scale. We provide details for the predicted binding modes for the new ligands, e.g., that an extension of the carbon backbone leads to a different molecular interaction pattern with the receptor and the nearby water structure than found for known Siglec-7 ligands. Further on, we uncover some shortcomings of the GLYCAM06 and GAFF2 force fields when used for the simulation of sialoside-based glycomimetics. Our results open new opportunities for the rational design of Siglec-7 inhibitors. In addition, we provide strategies on how to use and visualize MD simulations to describe and investigate sialoside-Siglec complexes in general.
Assuntos
Ácido N-Acetilneuramínico , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Proteínas de Transporte , LigantesRESUMO
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.
Assuntos
Receptores de Antígenos de Linfócitos B , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Materiais Biomiméticos , Humanos , Ligantes , Oligossacarídeos/farmacologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
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.
Assuntos
Antígenos de Diferenciação Mielomonocítica/metabolismo , Evasão da Resposta Imune , Lectinas/metabolismo , Neoplasias/imunologia , Desenho de Fármacos , Humanos , Células Matadoras Naturais/imunologia , LigantesRESUMO
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.