Soerge Kelm

The Ligand-binding Domain of CD22 Is Needed for Inhibition of the B Cell Receptor Signal, as Demonstrated by a Novel Human CD22-specific Inhibitor Compound

CD22 is a B cell–specific transmembrane protein of the Siglec family. It binds specifically to α2,6-linked sialic acid (Sia) residues, which are also present on glycoproteins on the B cell surface. CD22 acts as a negative regulator in B cell receptor–mediated signaling by recruitment of Src homology 2 domain–containing tyrosine phosphatase (SHP)-1 to its intracellular tail. To analyze how ligand-binding of CD22 influences its intracellular signaling domain, we designed synthetic sialosides as inhibitors for the lectin domain of CD22. One of these compounds inhibited binding of human CD22-Fc to target cells over 200-fold better than Sia and was highly selective for human CD22. When Daudi cells or primary B cells were stimulated with anti-immunoglobulin (Ig)M in presence of this sialoside inhibitor, a higher Ca2+ response was observed, similar to CD22-deficient B cells. Accordingly, a lower tyrosine-phosphorylation of CD22 and SHP-1 recruitment was demonstrated in presence of the sialoside. Thus, by interfering with ligand binding of CD22 on the B cell surface, we have shown for the first time that the lectin domain of CD22 has a direct, positive influence on its intracellular inhibitory domain. Also, we have developed a novel low molecular weight compound which can enhance the response of human B cells.

The structure of siglec-7 in complex with sialosides: leads for rational structure-based inhibitor design

Siglecs (sialic acid binding Ig-like lectins) are transmembrane receptors for sialylated glycoconjugates that modulate cellular interactions and signalling events in the haematopoietic, immune and nervous systems. Siglec-7 is a structural prototype for the recently described family of immune inhibitory CD33-related siglecs and is predominantly expressed on natural killer cells and monocytes, as well as subsets of CD8 T-cells. Siglec-specific inhibitors are desired for the detection of masked and unmasked forms of siglecs, to aid in dissection of signalling pathways and as tools to investigate siglecs as potential therapeutic targets. As a first step towards this end, we present the crystal structure of siglec-7 in complex with a sialylated ligand, the ganglioside analogue DSLc4 [alpha(2,3)/alpha(2,6) disialyl lactotetraosyl 2-(trimethylsilyl)ethyl], which allows for a detailed description of the binding site, required for structure-guided inhibitor design. Mutagenesis and binding assays were used to demonstrate a key structural role for Lys131, a residue that changes conformation upon sialic acid binding. Differences between the binding sites of siglec family members were then exploited using alpha-methyl Neu5Ac (N-acetylneuraminic acid) as a basic scaffold. A co-crystal of siglec-7 in complex with the sialoside inhibitor, oxamido-Neu5Ac [methyl alpha-9-(amino-oxalyl-amino)-9-deoxy-Neu5Ac] and inhibition data for the sialosides gives clear leads for future inhibitor design.

Low Molecular Weight Antagonists of the Myelin-Associated Glycoprotein: Synthesis, Docking, and Biological Evaluation

The injured adult mammalian central nervous system is an inhibitory environment for axon regeneration due to specific inhibitors, among them the myelin-associated glycoprotein (MAG), a member of the Siglec family (sialic-acid binding immunoglobulin-like lectin). In earlier studies, we identified the lead structure 5, which shows a 250-fold improved in vitro affinity for MAG compared to the tetrasaccharide binding epitope of GQ1balpha (1), the best physiological MAG ligand described so far. By modifying the 2- and 5-position, the affinity of 5 could be further improved to the nanomolar range (-->19a). Docking studies to a homology model of MAG allowed the rationalization of the experimental binding properties. Finally, pharmacokinetic parameters (stability in the cerebrospinal fluid, logD and permeation through the BBB) indicate the drug-like properties of the high-affinity antagonist 19a.

Kinetic and thermodynamic properties of MAG antagonists

Paraplegia is caused by injuries of the central nervous system (CNS) and especially young people suffer from these severe consequences as, for example, the loss of motor functions. The lack of repair of the injured nerve strands originates from the inhibitory environment for axon regeneration in the CNS. Specific inhibitory proteins block the regrowth of nerve roots. One of these neurite outgrowth inhibitors is the myelin-associated glycoprotein (MAG), which is a member of the Siglec family (sialic acid-binding immunoglobulin-like lectin). In previous studies, we identified potent small molecule MAG antagonists. In this communication, we report new neuraminic acid derivatives modified in the 4- and 5-position, and the influence of various structural modifications on their kinetic and thermodynamic binding properties.

Consistent Bioactive Conformation of the Neu5Acα(2→3)Gal Epitope Upon Lectin Binding

The injured adult mammalian central nervous system has no capacity for axon regeneration, predominantly due to specific inhibitors expressed on residual myelin and on astrocytes recruited to the injury site. Several of these inhibitory proteins have been identified, including the myelin-associated glycoprotein (MAG). 8] MAG is a transmembrane glycoprotein that belongs to a family of sialic acid-binding immunoglobulin-like lectins, the so-called siglecs. There are two classes of welldefined axonal targets of MAG on the surface of neurons: sialylated glycans, specifically the gangliosides GD1a and GT1b, 13] and proteins of the NgR family. 15] Although the relative roles of gangliosides and NgRs as MAG ligands have yet to be resolved, in some systems MAG inhibition is completely reversed by sialidase treatment, suggesting that MAG uses sialylated glycans as its major axonal ligands. Therefore, potent glycan inhibitors of MAG may be a valuable therapeutic approach to enhance axon regeneration. The native carbohydrate ligand with the highest affinity to MAG is the ganglioside GQ1ba. As a starting point for our search for MAG antagonists, data about the minimal binding epitope of 1 and its bioactive conformation are required, since the most abundant solution conformation does not necessarily represent the bound conformation. Recently, the MAG-affinity of partial structures of GQ1ba (1), namely derivatives of tetrasaccharide 2 and trisaccharide 5, was clearly correlated with their ability to reverse MAG-mediated inhibition of axon outgrowth (Scheme 1). 19] Both saccharides 2 and 5 contain a flexible aACHTUNGTRENNUNG(2!3)-glycosidic linkage between the sialic acid (Neu5Ac) and the central galactose (Gal) residue, and it is unknown which of the solution conformations is recognized by the receptor protein. The objective of this study is to analyze the conformation of these aACHTUNGTRENNUNG(2!3)-glycosidic linkages when bound to MAG. It is known that conformational preorganization of ligands may significantly improve binding affinities, and this information is crucial for the design of potent antagonists. A prominent example is conformationally preorganized E-selectin antagonists based on the bioactive conformation of sialyl Lewis. 21] In general, carbohydrate–protein interactions are characterized by exchange reactions that are fast on the NMR chemical shift and relaxation timescales. Therefore, transferred NOE (trNOE) experiments are ideally suited for the analysis of bioactive conformations of protein-bound carbohydrates. Flexible glycosidic linkages have drawn special attention since different bound conformations are possible. Here, we analyze the bioactive conformations of Neu5AcaACHTUNGTRENNUNG(2!3)GalbACHTUNGTRENNUNG(1!3) ACHTUNGTRENNUNG[Neu5Ac a ACHTUNGTRENNUNG(2! 6)]GalNAc (2), Neu5Aca ACHTUNGTRENNUNG(2!3)Galb ACHTUNGTRENNUNG(1!3) ACHTUNGTRENNUNG[Neu5AcaACHTUNGTRENNUNG(2!6)]Gal (3), Neu5AcaACHTUNGTRENNUNG(2!3)GalbACHTUNGTRENNUNG(1!3)GalNAc (5) and GalbACHTUNGTRENNUNG(1!3) [Neu5Aca ACHTUNGTRENNUNG(2!6)]Gal (6), as well as the tetrasaccharide mimic 4 when bound to MAG (Scheme 1; for the syntheses of 2, 3 and 4 see the Supporting Information). This study complements an accompanying paper that describes the analysis of the binding epitopes of trisaccharide 5 and tetrasaccharide 2 when bound to MAG using saturation transfer difference (STD) NMR. From the change in the pattern of specific interglycosidic NOEs between tetrasaccharide 2 free in solution (Figures 1 A and C) and in the bound form (Figures 1 B and D, namely the disappearance of the NOEs H3’’–H3ax’’’ and H3’’–H3eq’’’), it is concluded that the terminal aACHTUNGTRENNUNG(2!3)-linked Neu5Ac residue is bound in a minus-gauche orientation, as it has been observed before for sialyl Lewis binding to E-selectin. The thorough quantitative analysis of the bioactive conformations of the structurally related ganglioside derivatives 2 to 5 (see the Supporting Information) reveals that the “sialyl Lewis-type binding mode” is a common theme among the carbohydrate-protein interactions studied (Table 1, see also Table S7). Interestingly, binding of trisaccharide 6 was too weak to give sizeable trNOEs (Supporting Information), supporting the assertion that an aACHTUNGTRENNUNG(2!3)-linked Neu5Ac residue is absolutely required for MAG binding. 29] Based on a full relaxation and exchange matrix analysis of the trNOE data employing the program CORCEMA, 31] we deduced a docking model (Supporting Information) for the interaction of the ganglioside derivatives with MAG. A complete set of NOESY spectra of oligosaccharides 2 to 5 in the absence and presence of MAG is found in the Supporting Information (Figures S1 and S2). As an example, Figure 1 [a] Dr. O. Schwardt, Dr. G.-P. Gao, Prof. Dr. B. Ernst Institute of Molecular Pharmacy, University of Basel Klingelbergstrasse 50, 4056 Basel (Switzerland) Fax: (+ 41) 61-267-15-52 E-mail : beat.ernst@unibas.ch [b] Dr. A. Bhunia, Dr. A. J. Benie, Prof. Dr. T. Peters Institute of Chemistry, University of Luebeck Ratzeburger Allee 160, 23538 Luebeck (Germany) [c] Dr. M. Hricovini Institute of Chemistry, Slovak Academy of Science 84238 Bratislava (Slovakia) [d] H. G thje, Prof. Dr. S. Kelm Department of Physiological Biochemistry, University of Bremen 28334 Bremen (Germany) Supporting information for this article is available on the WWW under http ://www.chembiochem.org or from the author: inter alia : full relaxation matrix calculations and docking procedures.

Binding Epitopes of Gangliosides to their Neuronal Receptor, Myelin‐Associated Glycoprotein, from Saturation Transfer Difference NMR

The myelin-associated glycoprotein (MAG) inhibits neurite outgrowth in the central nervous system. Certain gangliosides can block this action of MAG and can thus stimulate the regrowth of severed neurons. In this study we analyzed the binding epitopes of fragments of the ligand with the highest binding affinity to MAG, that is, the ganglioside GQ1ba. Specifically, we studied the binding of three trisaccharides and one tetrasaccharide to the MAG chimera FcMAGd1!3 [8] using saturation transfer difference (STD) NMR. Trisaccharide SiaaACHTUNGTRENNUNG(2!3)GalbACHTUNGTRENNUNG(1!3)GalNAc (2) and the tetrasaccharide, SiaaACHTUNGTRENNUNG(2!3) GalbACHTUNGTRENNUNG(1!3)ACHTUNGTRENNUNG[SiaaACHTUNGTRENNUNG(2!6)]GalNAc (3), are fragments of GQ1ba, whereas the trisaccharide SiaaACHTUNGTRENNUNG(2!3) GalbACHTUNGTRENNUNG(1!3)Gal (1) is a close mimetic. For reference purposes, the commercially available sialyllactose SiaaACHTUNGTRENNUNG(2!3) GalbACHTUNGTRENNUNG(1!4)Glc was also included in the study to obtain its KD value. Understanding the detailed binding mechanisms is essential for developing efficient inhibitors of this interaction. We determined the binding epitopes with (C,H) HSQC STD NMR spectra and 1D STD spectra whenever the HSQC data were ambiguous (see the Supporting Information). The strongest STD effect was on the N-acetyl group at position 5 of the 2!3-linked sialic acid for trisaccharides 1 and 2 (Scheme 1). Therefore, the STD effect of this group was set to 100 % in each spectrum. This can be explained by a hydrophobic interaction with the protein. Also, the glycerol side chain of the 2!3-linked sialic acid showed high STD effects, thus indicating that the region of H-6 to H-8 is involved in binding. The patch from H-4 to H-6 of the central galactosyl residue also shows strong STD contacts probably originating from hydrophobic ACHTUNGTRENNUNGinteractions. The carboxy function of the aACHTUNGTRENNUNG(2!3)-linked sialic acid is believed to interact with Arg118, and the proximity of this part of the oligosaccharide to the carboxy group makes an interaction likely. The low STD values for the protons of the first galactose or galactosamine residues indicate that no significant contribution to binding originates from these residues. Scheme 1. Binding epitopes of the three ligands obtained from the integration of signals in the STD HSQC. The strongest signal (N-acetyl group, left) was scaled to 100 %, and all other signals were related to it. The signal intensity was divided in three categories : black for 31–100 % and mid gray for 1!30 %, while overlapping signals are coded in light gray and designated by , meaning that analysis was only possible for the sum of the signals. The absolute STD values of the N-acetyl groups were 22 % for 1, 24 % for 2 and 16 % for 3.

SIGLEC-4 (MAG) Antagonists: From the Natural Carbohydrate Epitope to Glycomimetics

Siglec-4, also known as myelin-associated glycoprotein (MAG), is a member of the siglec (sialic acid-binding immunoglobulin-like lectins) family. MAG binds with high preference to sialic acids α(2-3)-linked to D-galactose. Although the involvement and relevance of its sialic acid binding activity is still controversial, it could be demonstrated that interactions of MAG with sialylated gangliosides play an important role in axon stability and regeneration. In this article we describe in detail our current understanding of the biological role and the carbohydrate specificity of siglec-4. Furthermore, this review compiles the intensive research efforts leading from the identification of the minimal oligosaccharide binding epitope in gangliosides via micromolar oligosaccharide mimics to the development of small molecular weight and more drug-like sialic acid derivatives binding with low nanomolar affinities. Such compounds will be useful to elucidate MAGs biological functions, which are currently not fully understood.