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Burkholderia ambifaria is an opportunistic respiratory pathogen belonging to the Burkholderia cepacia complex, a collection of species responsible for the rapidly fatal cepacia syndrome in cystic fibrosis patients. A fucose-binding lectin identified in the B. ambifaria genome, BambL, is able to adhere to lung tissue, and may play a role in respiratory infection. X-ray crystallography has revealed the bound complex structures for four fucosylated human blood group epitopes (blood group B, H type 1, H type 2, and Lex determinants). The present study employed computational approaches, including docking and molecular dynamics (MD), to extend the structural analysis of BambL-oligosaccharide complexes to include four additional blood group saccharides (A, Lea, Leb, and Ley) and a library of blood-group-related carbohydrates. Carbohydrate recognition is dominated by interactions with fucose via a hydrogen-bonding network involving Arg15, Glu26, Ala38, and Trp79 and a stacking interaction with Trp74. Additional hydrogen bonds to non-fucose residues are formed with Asp30, Tyr35, Thr36, and Trp74. BambL recognition is dominated by interactions with fucose, but also features interactions with other parts of the ligands that may modulate specificity or affinity. The detailed computational characterization of the BambL carbohydrate-binding site provides guidelines for the future design of lectin inhibitors.

We have discovered that a small cationic molecule, GW4869, is cytotoxic to a subset of myeloma cell lines and primary myeloma plasma cells. Biochemical analysis revealed that GW4869 binds to anionic phospholipids such as phosphatidylserine - a lipid normally confined to the intracellular side of the cell membrane. However, interestingly, phosphatidylserine was expressed on the surface of all myeloma cell lines tested (n = 12) and 9/15 primary myeloma samples. Notably, the level of phosphatidylserine expression correlated well with sensitivity to GW4869. Inhibition of cell surface phosphatidylserine exposure with brefeldin A resulted in resistance to GW4869. Finally, GW4869 was shown to delay the growth of phosphatidylserine-high myeloma cells in vivo. To the best of our knowledge, this is the first example of using a small molecule to target phosphatidylserine on malignant cells. This study may provide the rationale for the development of phosphatidylserine-targeting small molecules for the treatment of surface phosphatidylserine-expressing cancers.

Euonymus europaeus lectin (EEL) is a carbohydrate-binding protein derived from the fruit of the European spindle tree. EEL was first identified for its erythrocyte agglutinating properties and specificity for B and H blood groups. However, a detailed molecular picture of the structural basis of carbohydrate recognition by EEL remains to be developed. In this study, we performed fluorescence titrations of a range of carbohydrates against EEL. Binding of EEL to a wide range of carbohydrates was observed, including a series of blood group-related carbohydrates, mannosides, chitotriose and sialic acid. Affinity was strongest for carbohydrates with H-related structures and the B trisaccharide. A homology model of EEL was produced from templates identified using the HHPred server, which employs hidden Markov models (HMMs) to identify templates. The HMM approach identified that the best templates for EEL were proteins featuring a ricin B-like (R-type) fold. Separate templates were used to model the core and binding site regions of the lectin. Through the use of constrained docking and spatial comparison with a template ligand, binding modes for the carbohydrate ligands were predicted. A relationship between the experimental binding energies and the computed binding energies of the selected docked poses was determined and optimized. Collectively, our results suggest that EEL utilizes a single site for recognition of carbohydrates terminating in a variety of monosaccharides.

Carbohydrate-protein recognition is vital to many processes in health and disease. In particular, elucidation of the structural basis of carbohydrate binding is important to the development of oligosaccharides and oligosaccharide mimetics as vaccines for infectious diseases and cancer. Computational structural techniques are valuable for the study of carbohydrate-protein recognition due to the challenges associated with experimental determination of carbohydrate-protein complexes. AutoMap is a computer program that we have developed to study protein-ligand recognition. AutoMap determines the interactions taking place in a set of highly ranked poses obtained from molecular docking and processes these to identify the protein residues most likely to be involved in interactions. In this protocol, we describe the use of AutoMap and illustrate its suitability for studying antibody recognition of the Lewis Y tetrasaccharide, which is a potential cancer vaccine antigen.

Molecular docking is a computational method for predicting the placement of ligands in the binding sites of their receptor(s). In this review, we discuss the methodological developments that occurred in the docking field in 2012 and 2013, with a particular focus on the more difficult aspects of this computational discipline. The main challenges and therefore focal points for developments in docking, covered in this review, are receptor flexibility, solvation, scoring, and virtual screening. We specifically deal with such aspects of molecular docking and its applications as selection criteria for constructing receptor ensembles, target dependence of scoring functions, integration of higher-level theory into scoring, implicit and explicit handling of solvation in the binding process, and comparison and evaluation of docking and scoring methods.

Monoclonal antibodies represent the most successful class of biopharmaceuticals for the treatment of cancer. Mechanisms of action of therapeutic antibodies are very diverse and reflect their ability to engage in antibody-dependent effector mechanisms, internalize to deliver cytotoxic payloads, and display direct effects on cells by lysis or by modulating the biological pathways of their target antigens. Importantly, one of the universal changes in cancer is glycosylation and carbohydrate-binding antibodies can be produced to selectively recognize tumor cells over normal tissues. A promising group of cell surface antibody targets consists of carbohydrates presented as glycolipids or glycoproteins. In this review, we outline the basic principles of antibody-based targeting of carbohydrate antigens in cancer. We also present a detailed structural view of antibody recognition and the conformational properties of a series of related tissue-blood group (Lewis) carbohydrates that are being pursued as potential targets of cancer immunotherapy.

The aim of docking is to accurately predict the structure of a ligand within the constraints of a receptor binding site and to correctly estimate the strength of binding. We discuss, in detail, methodological developments that occurred in the docking field in 2010 and 2011, with a particular focus on the more difficult, and sometimes controversial, aspects of this promising computational discipline. The main developments in docking in this period, covered in this review, are receptor flexibility, solvation, fragment docking, postprocessing, docking into homology models, and docking comparisons. Several new, or at least newly invigorated, advances occurred in areas such as nonlinear scoring functions, using machine-learning approaches. This review is strongly focused on docking advances in the context of drug design, specifically in virtual screening and fragment-based drug design. Where appropriate, we refer readers to exemplar case studies. Copyright © 2013 John Wiley & Sons, Ltd.

Naturally occurring antibodies (i.e., those existing without known exogenous anti¬gen stimulation) are fundamental in the response to foreign organisms and function in both innate and adaptive immune responses. Indeed, natural preformed antibodies in a recipient are the first immunological barrier for clinical transplantation. To over¬come the chronic shortage of organs available for transplantation, the practice of live donation across the ABO blood group barrier is now performed in many countries (Nydegger et al. 2005; Wu et al. 2003). Furthermore, pig-to-human xenotransplanta¬tion is also being investigated as a viable solution. However, without intervention, the recipient’s natural antibodies would cause hyperacute rejection of the graft. These natural antibodies recognize carbohydrate epitopes on the donor tissue (A or B blood group antigens for allotransplantation; Galα(1-3)Gal [αGal] for xenotransplantation), which are synthesized by closely related members of family 6 glycosyltransferases. In this chapter, we highlight the biochemical, genetic, and immunological features of these clinically important carbohydrate antigens from the enzymes responsible for their synthesis to their molecular interactions with the immune system.

Modified gangliosides may be overexpressed in certain types of cancer, thus, they are considered a valuable target in cancer immunotherapy. Structural knowledge of their interaction with antibodies is currently limited, due to the large size and high flexibility of these ligands. In this study, we apply our previously developed site mapping technique to investigate the recognition of cancer-related gangliosides by anti-ganglioside antibodies. The results reveal a potential ganglioside-binding motif in the four antibodies studied, suggesting the possibility of structural convergence in the anti-ganglioside immune response. The structural basis of the recognition of ganglioside-mimetic peptides is also investigated using site mapping and compared to ganglioside recognition. The peptides are shown to act as structural mimics of gangliosides by interacting with many of the same binding site residues as the cognate carbohydrate epitopes. These studies provide important clues as to the structural basis of immunological mimicry of carbohydrates.

The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Serratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, R(a), R(d), and R(e) rough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria.

Carbohydrate antigens recognized by “natural” or preformed and elicited antibodies are central to transplantation/transfusion rejection across ABO blood group and species (xenotransplantation) barriers and are also promising candidates for cancer immunotherapy (Ramsland 2005). The key carbohydrate determinants (epitopes) recognized by antibodies are synthesized by a series of intracellular glycosyltransferases and are expressed on the surface of cells as glycolipids and glycoproteins. Often the minimal carbohydrate epitopes are located at the terminal end of more complex oligosaccharide chains, which result in these epitopes being displayed at a wide range of surface densities and contexts (e.g., glycolipids or glycoproteins). For example, many tumor-associated carbohydrate antigens are broadly expressed at very high densities on the cell surface of primary and metastatic tumors, but the same carbohydrates occur at much lower levels and are typically restricted to a few cell types in healthy tissues (Scott and Renner 2001; Ezzelarab et al. 2005; Kobata and Amano 2005; Cazet et al. 2010). Thus, antibodies with similar specificities for individual carbohydrate epitopes can display different and often selective cell-binding profiles, based on the unique presentation of the carbohydrates on the target cells.In this chapter, we discuss the structural glycobiology of the key antibody–carbohydrate interactions important in xenotransplantation and cancer immunotherapy. Progress in both fields is reliant on further mechanistic knowledge of the underlying complex biosynthetic pathways and the structural basis for antibody recognition of isolated and cellular forms of the target carbohydrates. Recent progress in bioinformatics, synthesis of complex carbohydrates and analysis of their interactions with proteins [reviewed in (Seeberger 2008; Taylor and Drickamer 2009; Frank and Schloissnig 2010)] is facilitating the experimental determination and interpretation of three-dimensio

Modified gangliosides may be overexpressed in certain types of cancer, thus, they are considered a valuable target in cancer immunotherapy. Structural knowledge of their interaction with antibodies is currently limited, due to the large size and high flexibility of these ligands. In this study, we apply our previously developed site mapping technique to investigate the recognition of cancer-related gangliosides by anti-ganglioside antibodies. The results reveal a potential ganglioside-binding motif in the four antibodies studied, suggesting the possibility of structural convergence in the anti-ganglioside immune response. The structural basis of the recognition of ganglioside-mimetic peptides is also investigated using site mapping and compared to ganglioside recognition. The peptides are shown to act as structural mimics of gangliosides by interacting with many of the same binding site residues as the cognate carbohydrate epitopes. These studies provide important clues as to the structural basis of immunological mimicry of carbohydrates.

Structures of protein–carbohydrate complexes can be investigated experimentally by x-ray crystallography or nuclear magnetic resonance (NMR) (Chapters 1 and 2). However, the crystallization of these complexes is often complicated by the inherent flexibility of carbohydrates, whereas NMR is more appropriate for the determination of conformation of bound carbohydrates rather than the nature of the interactions occurring with a protein. Computational molecular modeling techniques offer attractive alternatives for the study of protein–carbohydrate interactions. These techniques have been widely used in conformational studies of carbohydrates, as well as in the investigation of carbohydrate–protein recognition. Notably, molecular dock¬ing can provide insight into protein–ligand interactions in systems that are difficult to study experimentally.

Carbohydrate–antibody interactions mediate many cellular processes and immune responses. Carbohydrates expressed on the surface of cells serve as recognition elements for particular cell types, for example, in the ABO(H) blood group system. Antibodies that recognize host-incompatible ABO(H) system antigens exist in the bloodstream of all individuals (except AB individuals), preventing bloodtransfusion and organ transplantation between incompatible donors and recipients. A similar barrier exists for cross-species transplantation (xenotransplantation), in particular for pig-to-human transplantation. All humans express antibodies against the major carbohydrate xenoantigen, Galα(1,3)Gal (αGal), preventing successful xenotransplantation. Although antibody binding sites are precisely organized so as to selectively bind a specific antigen, many antibodies recognize molecules other than their native antigen. A range of peptides have been identified that can mimic carbohydrates and inhibit anti-αGal antibodies. However, the structural basis of how the peptides achieved this was not known. Previously, we developed an in silico method which we used to investigate carbohydrate recognition by a panel of anti-αGal antibodies. The method involves molecular docking of carbohydrates to antibodies and uses the docked carbohydrate poses to generate maps of the antibody binding sites in terms of prevalent hydrogen bonding and van der Waals interactions. We have applied this method to investigate peptide recognition by the anti-αGal antibodies. It was found that the site maps of the peptides and the carbohydrates were similar, indicating that the peptides interact with the same residues as those involved in carbohydrate recognition. This study demonstrates the potential for “design by mapping” of anti-carbohydrate antibody inhibitors.

We have synthesised an extensive series of URB602 analogues as inhibitors of monoacylglycerol lipase (MAGL), which is the major enzyme responsible for metabolising the endocannabinoid 2-arachidonylglycerol. The recently identified crystal structure of MAGL was used in the design strategy and revealed three possible binding sites for URB602 and the proposed analogues. A test series of carbamate analogues were docked into the identified sites to predict the most favourable binding location. The synthesised analogues of URB602 explored the biological effects of isosteric replacement, ring size and substitution, para substitution of the biphenyl moiety and the incorporation of a bicyclic element. The compounds were tested for their ability to inhibit human MAGL. The carbamate analogue 16 displayed the most significant inhibitory activity, reducing MAGL activity to 26% of controls at 100 μM compared to 73% for the parent compound URB602.

Introduction: The existence of specific carbohydrates on the surface of a wide range of cells provides the opportunity for the development of highly targeted therapeutic agents. The potential applications of such agents are diverse, and include vaccines against pathogenic microorganisms, cancer and HIV, and anti-rejection agents for organ transplantation. However, the use of carbohydrates as either therapeutic agents or immunogens is frequently problematic, as they are often rapidly metabolized and poorly immunogenic. Therefore, the search for carbohydrate-mimetic agents is of considerable therapeutic value, for the potential of such agents to both interfere with carbohydrate–protein interactions and to generate carbohydrate-specific immune responses. Areas covered: The review discusses recent examples of carbohydrate-mimetic peptides with regard to the structural and functional aspects of mimicry and the implications of peptide mimicry for application in therapeutics. The reader will gain knowledge of the various mechanisms of peptide carbohydrate mimicry, and the potential importance of these mechanisms in targeted therapeutic design. Expert opinion: Peptide carbohydrate mimicry is manifested by distinct mechanisms, any one of which may be relevant to specific protein targets. As structural information becomes available for a wider variety of systems, the questions about mimicry will be more effectively addressed.

Recognition of pathogen-associated carbohydrates by a broad range of carbohydrate-binding proteins is central to both adaptive and innate immunity. A large functionally diverse group of mammalian carbohydrate-binding proteins are lectins, which often display calcium-dependent carbohydrate interactions mediated by one or more carbohydrate recognition domains. We report here the application of molecular docking and site mapping to study carbohydrate recognition by several lectins involved in innate immunity or in modulating adaptive immune responses. It was found that molecular docking programs can identify the correct carbohydrate-binding mode, but often have difficulty in ranking it as the best pose. This is largely attributed to the broad and shallow nature of lectin binding sites, and the high flexibility of carbohydrates. Site mapping is very effective at identifying lectin residues involved in carbohydrate recognition, especially with cases that were found to be particularly difficult to characterize via molecular docking. This study highlights the need for alternative strategies to examine carbohydrate–lectin interactions, and specifically demonstrates the potential for mapping methods to extract additional and relevant information from the ensembles of binding poses generated by molecular docking.

Docking is a computational technique that places a small molecule (ligand) in the binding site of its macromolecular target (receptor) and estimates its binding affinity. This review addresses methodological developments that have occurred in the docking field in 2009, with a particular focus on the more difficult, and sometimes controversial, aspects of this promising computational discipline. These developments aim to address the main challenges of docking: receptor representation (such aspects as structural waters, side chain protonation, and, most of all, flexibility (from side chain rotation to domain movement)), ligand representation (protonation, tautomerism and stereoisomerism, and the effect of input conformation), as well as accounting for solvation and entropy of binding. This review is strongly focused on docking advances in the context of drug design, specifically in virtual screening and fragment-based drug design.

Carbohydrates are notoriously flexible molecules. However, they have an important role in many biochemical processes as specific ligands. Understanding how carbohydrates are recognized by other biological macromolecules (usually proteins) is therefore of considerable scientific value. Interfering with carbohydrate-protein interactions is a potentially useful strategy in combating a range of disease states, as well as being of critical importance in facilitating allo- and xenotransplantation. We have devised an in silico protocol for analyzing carbohydrate-protein interactions. In this study, we have applied the protocol to determine the structures of aGal-terminating carbohydrate antigens in complex with a panel of xenoreactive antibodies. The most important feature of the binding modes is the fixed conformation of the Galß(1,4)Glc/GlcNAc linkage across all of the binding modes. The preferred conformation of the terminal Gala(1,3)Gal linkage varies depending on the antibody binding site topography, although it is possible that some of the antibodies studied recognize more than one Gala(1,3)Gal conformation. The binding modes obtained indicate that each antibody uses distinct mechanisms in recognizing the target antigens. © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Free ? L chains (F?LCs) are expressed on the surface of myeloma cells and are being assessed as a therapeutic target for the treatment of multiple myeloma. Despite its clinical potential, the mechanism by which F?LCs interact with membranes remains unresolved. In this study, we show that F?LCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound F?LCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of F?LCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated F?LCs exposed on the plasma membrane after exocytosis. Although it is well known that protein aggregates bind membranes, this is only the second example of an aggregate being found on the surface of cells that also secrete the protein in its native form. We postulate that many other aggregation-prone proteins may associate with cell membranes by similar mechanisms. Copyright© 2010 by The American Association of Immunologists, Inc.

Antibody-carbohydrate interactions play central roles in stimulating adverse immune reactions. The most familiar example of such a process is the reaction observed in ABO-incompatible blood transfusion and organ transplantation. The ABO blood groups are defined by the presence of specific carbohydrates expressed on the surface of red blood cells. Preformed antibodies in the incompatible recipient (i.e., different blood groups) recognize cells exhibiting host-incompatible ABO system antigens and proceed to initiate lysis of the incompatible cells. Pig-to-human xenotransplantation presents a similar immunological barrier. Antibodies present in humans recognize carbohydrate antigens on the surface of pig organs as foreign and proceed to initiate hyperacute xenograft rejection. The major carbohydrate xenoantigens all bear terminal Gala(1,3)Gal epitopes (or aGal). In this study, we have developed and validated a site mapping technique to investigate protein-ligand recognition and applied it to antibody-carbohydrate systems. This site mapping technique involves the use of molecular docking to generate a series of antibody-carbohydrate complexes, followed by analysis of the hydrogen bonding and van der Waals interactions occurring in each complex. The technique was validated by application to a series of antibody-carbohydrate crystal structures. In each case, the majority of interactions made in the crystal structure complex were able to be reproduced. The technique was then applied to investigate xenoantigen recognition by a panel of monoclonal anti-aGal antibodies. The results indicate that there is a significant overlap of the antibody regions engaging the xenoantigens across the panel. Likewise, similar regions of the xenoantigens interact with the antibodies. © 2009 Elsevier Ltd. All rights reserved.

Cell surface glycoproteins play vital roles in cellular homeostasis and disease. Antibody recognition of glycosylation on different cells and pathogens is critically important for immune surveillance. Conversely, adverse immune reactions resulting from antibody-carbohydrate interactions have been implicated in the development of autoimmune diseases and impact areas such as xenotransplantation and cancer treatment. Understanding the nature of antibody-carbohydrate interactions and the method by which saccharides fit into antibody binding sites is important in understanding the recognition process. In silico techniques offer attractive alternatives to experimental methods (X-ray crystallography and NMR) for the study of antibody-carbohydrate complexes. In particular, molecular docking provides information about protein-ligand interactions in systems that are difficult to study with experimental, techniques. Before molecular docking can be used to investigate antibody-carbohydrate complexes, validation of an appropriate docking method is required. In this study, four popular docking programs, Glide, AutoDock, GOLD, and FlexX, were assessed for their ability to accurately dock carbohydrates to antibodies. Comparison of top ranking poses with crystal structures highlighted the strengths and weaknesses of these programs. Rigid docking, in which the protein conformation remains static, and flexible docking, where both the protein and ligand are treated as flexible, were compared. This study has revealed that generally molecular docking of carbohydrates to antibodies has been performed best by Glide. © 2009 American Chemical Society.

Mutations of peptides to generate altered peptide ligands, capable of switching immune responses from T helper 1 (Th1) to T helper 2 (Th2), are promising candidates for the immunotherapy of autoimmune diseases such as multiple sclerosis (MS). We synthesized two mutant peptides from myelin basic protein 87-99 (MBP87-99), an immunodominant peptide epitope identified in MS. Mutations of residues K91 and P96, known to be critical T-cell receptor (TCR) contact sites, resulted in the mutant peptides [R91, A96]MBP87-99 and [A 91, A96]MBP87-99. Immunization of mice with these altered peptide ligands emulsified in complete Freund's adjuvant induced both interferon-? (IFN-?) and interleukin-4 (IL-4) responses compared with only IFN-? responses induced to the native MBP 87-99 peptide. It was of interest that [R91, A 96]MBP87-99 conjugated to reduced mannan induced 70% less IFN-? compared with the native MBP87-99 peptide. However, [A91, A96]MBP87-99 conjugated to reduced mannan did not induce IFN-?-secreting T cells, but elicited very high levels of interleukin-4 (IL-4). Furthermore, antibodies generated to [A91, A96]MBP87-99 peptide conjugated to reduced mannan did not cross-react with the native MBP87-99 peptide. By molecular modelling of the mutant peptides in complex with major histocompatibility complex (MHC) class II, I-As, novel interactions were noted. It is clear that the double-mutant peptide analogue [A91, A96]MBP 87-99 conjugated to reduced mannan is able to divert immune responses from Th1 to Th2 and is a promising mutant peptide analogue for use in studies investigating potential treatments for MS. © 2009 Blackwell Publishing Ltd.

Transplantation of organs across species (xenotransplantation) is being considered to overcome the shortage of human donor organs. However, unmodified pig organs undergo an antibody-mediated hyperacute rejection that is brought about by the presence of natural antibodies to Gala(1,3)Gal, which is the major carbohydrate xenoantigen. Genetic modification of pig organs to remove most of the Gala(1,3)Gal epitopes has been achieved, but the human immune system may still recognize residual lipid-linked Gala(1,3) Gal carbohydrates, new (cryptic) carbohydrates or additional non-Gala(1,3) Gal carbohydrate xenoantigens. The structural basis for lectin and antibody recognition of Gala(1,3)Gal carbohydrates is starting to be understood and is discussed in this review. Antibody binding to Gala(1,3)Gal carbohydrates is predicted to primarily involve end-on insertion of the terminal aGal residue, but it is possible that groove-type binding can occur, as for some lectins. It is likely that similar antibody and lectin recognition will occur with other non-Gala(1,3)Gal xenoantigens, which potentially represent new barriers for pig-to-human xenotransplantation. © 2009 Informa UK Ltd All rights reserved.

As part of an anti-psychotic drug discovery program, we report the crystal structure of the title compound, C(24)H(23)ClN(4)O. The mol-ecule has a tricyclic framework with a characteristic buckled V-shaped pyridobenzoxazepine unit, with the central seven-membered heterocycle in a boat configuration. The piperazine ring displays a chair conformation with the 2-phenyl-ethyl substituent assuming an equatorial orientation. There are two crystallographically independent, but virtually identical, mol-ecules in the asymmetric unit.

Multiple sclerosis (MS) is an autoimmune demyelinating disease mediated primarily by CD4+ T cells. The design of peptide mutants of disease-associated myelin epitopes to alter immune responses offers a promising avenue for the treatment of MS. We designed and synthesized a number of peptide analogs by mutating the principal TCR contact residue based on MBP83-99 epitope and these peptides were conjugated to reduced mannan. Immune responses were diverted from Th1 to Th2 in SJL/J mice and generated antibodies which did not cross-react with native MBP protein. Peptide [Y91]MBP83-99 gave the best cytokine and antibody profile and constitutes a promising candidate peptide for immunotherapy of MS. Structural alignment of existing crystal structures revealed the peptide binding motif of I-As. Molecular modeling was used to identify H-bonding and van der Waals interactions between peptides and MHC (I-As). © 2008 Elsevier Ltd. All rights reserved.

Despite the enormous clinical importance for xenotransplantation, very little is known about the 3D structural basis for natural antibody recognition of the major carbohydrate xenoantigen, its derivatives and their peptide counterparts. Fundamentally, understanding the nature of peptide-carbohydrate cross-reactivity is necessary to allow a rational design of useful inhibitors. To satisfy this need, we have initiated a project to investigate the structural aspects of antibody recognition of carbohydrate xenoantigens and their peptide mimics by molecular docking. We aim to analyse critical ligand-protein interactions with a focus on identifying the degree of structural carbohydrate mimicry exhibited by peptide ligands. In this paper, we present docking simulations of complexes between a prototypical xenoreactive monoclonal antibody and two ligands: the major carbohydrate xenoantigen, terminal galactose-(1,3)-galactose epitope [Gal(1,3)Gal] and a peptide inhibitor Galpep1 (DAHWESWL).

A number of treatment options are available to multiple sclerosis patients, however this needs to be improved. Herein, we designed and synthesized a number of peptides by mutating principal TCR contact residues based on MBP83-99 peptide epitope. Immunization of SJL/J mice with MBP83-99 and mutant [A91]MBP83-99, [E91]MBP83-99, [F91]MBP83-99, [Y91]MBP83-99, and [R91, A96]MBP83-99 peptides, induced IFN-?, and only [R91, A96]MBP83-99 mutant peptide was able to induce IL-4 secretion by T cells. T cells against the native MBP83-99 peptide cross-reacted with all peptides except [Y91]MBP83-99 and [R91,A96]MBP83-99. The double mutant [R91, A96]MBP83-99 was able to antagonize IFN-? production in vitro by T cells against the native MBP83-99 peptide. Antibodies generated to [R91, A96]MBP83-99 did not cross-react with whole MBP protein. Molecular modeling between peptide analogs and H2 I-As demonstrated novel interactions. The [R91, A96]MBP83-99 double mutant peptide analog is the most promising for further therapeutic studies. © 2008 Elsevier B.V. All rights reserved.