The influence and manipulation of acid/base properties in drug discovery.

There is a growing awareness of the importance of acid/base properties in medicinal chemistry research. In many drug classes, ionisable groups are present that make critical interactions with the receptor and are essential for potency. Yet the presence of these groups may cause problems with oral bioavailability, pharmacokinetics, or toxicity. Manipulating pKa values during drug development or applying pro-drug techniques are strategies that can overcome potential deficits in a variety of these areas. Knowledge of drug ionisation states coupled with a consideration of pH-specific cellular environments can be used advantageously to target chemoresistance. As modern drug research ventures into drug candidates that exceed the rule of 5, such exploration requires an understanding of drug acid/base properties and how these factors affect ADMET characteristics.

Virtual Screening Against Carbohydrate-Binding Proteins: Evaluation and Application to Bacterial Burkholderia ambifaria Lectin.

Bacterial adhesion to human epithelia via lectins constitutes a therapeutic opportunity to prevent infection. Specifically, BambL (the lectin from Burkholderia ambifaria) is implicated in cystic fibrosis, where lectin-mediated bacterial adhesion to fucosylated lung epithelia is suspected to play an important role. We employed structure-based virtual screening to identify inhibitors of BambL-saccharide interaction with potential therapeutic value. To enable such discovery, a virtual screening protocol was iteratively developed via 194 retrospective screening protocols against 4 bacterial lectins (BambL, BC2L-A, FimH, and LecA) with known ligands. Specific attention was given to the rigorous evaluation of retrospective screening, including calculation of analytical errors for enrichment metrics. The developed virtual screening workflow used crystallographic constraints, pharmacophore filters, and a final manual selection step. The protocol was applied to BambL, predicting 15 active compounds from virtual libraries of approximately 7 million compounds. Experimental validation using fluorescence polarization confirmed micromolar inhibitory activity for two compounds, which were further characterized by isothermal titration calorimetry and surface plasmon resonance. Subsequent testing against LecB from Pseudomonas aeruginosa demonstrated binding specificity of one of the hit compounds. This report demonstrates the utility of virtual screening protocols, integrating ligand-based pharmacophore filtering and structure-based constraints, in the search for bacterial lectin inhibitors.

Molecular Simulations of Carbohydrates with a Fucose-Binding Burkholderia ambifaria Lectin Suggest Modulation by Surface Residues Outside the Fucose-Binding Pocket.

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.

The cationic small molecule GW4869 is cytotoxic to high phosphatidylserine-expressing myeloma cells.

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.

Antibody recognition of aberrant glycosylation on the surface of cancer cells.

Carbohydrate-binding antibodies and carbohydrate-based vaccines are being actively pursued as targeted immunotherapies for a broad range of cancers. Recognition of tumor-associated carbohydrates (glycans) by antibodies is predominantly towards terminal epitopes on glycoproteins and glycolipids on the surface of cancer cells. Crystallography along with complementary experimental and computational methods have been extensively used to dissect antibody recognition of glycan epitopes commonly found in cancer. We provide an overview of the structural biology of antibody recognition of tumor-associated glycans and propose potential rearrangements of these targets in the membrane that could dictate the complex biological activities of these antibodies against cancer cells.

Antibody-Carbohydrate Recognition from Docked Ensembles Using the AutoMap Procedure.

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.

Structural biology of antibody recognition of carbohydrate epitopes and potential uses for targeted cancer immunotherapies.

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.

Novel adenosine A(2A) receptor ligands: a synthetic, functional and computational investigation of selected literature adenosine A(2A) receptor antagonists for extending into extracellular space.

Growing evidence has suggested a role in targeting the adenosine A2A receptor for the treatment of Parkinson's disease. The literature compounds KW 6002 (2) and ZM 241385 (5) were used as a starting point from which a series of novel ligands targeting the adenosine A2A receptor were synthesized and tested in a recombinant human adenosine A2A receptor functional assay. In order to further explore these molecules, we investigated the biological effects of assorted linkers attached to different positions on selected adenosine A2A receptor antagonists, and assessed their potential binding modes using molecular docking studies. The results suggest that linking from the phenolic oxygen of selected adenosine A2A receptor antagonists is relatively well tolerated due to the extension towards extracellular space, and leads to the potential of attaching further functionality from this position.

AutoMap: a tool for analyzing protein-ligand recognition using multiple ligand binding modes.

Prediction of the protein residues most likely to be involved in ligand recognition is of substantial value in structure-based drug design. Considering multiple ligand binding modes is of potential relevance to studying ligand recognition, but is generally ignored by currently available techniques. We have previously presented the site mapping technique, which considers multiple ligand binding modes in its analysis of protein-ligand recognition. AutoMap is a partially automated implementation of our previously developed site mapping procedure. It consists of a series of Perl scripts that utilize the output of molecular docking to generate "site maps" of a protein binding site. AutoMap determines the hydrogen bonding and van der Waals interactions taking place between a target protein and each pose of a ligand ensemble. It tallies these interactions according to the protein residues with which they occur, then normalizes the tallies and maps these to the surface of the protein. The residues involved in interactions are selected according to specific cutoffs. The procedure has been demonstrated to perform well in studying carbohydrate-protein and peptide-antibody recognition. An automated procedure to optimize cutoff selection is demonstrated to rapidly identify the appropriate cutoffs for these previously studied systems. The prediction of key ligand binding residues is compared between AutoMap using automatically optimized cutoffs, AutoMap using a previously selected cutoff, the top ranked pose from docking and the predictions supplied by FTMap. AutoMap using automatically optimized cutoffs is demonstrated to provide improved predictions, compared to other methods, in a set of immunologically relevant test cases. The automated implementation of the site mapping technique provides the opportunity for rapid optimization and deployment of the technique for investigating a broad range of protein-ligand systems.

Antibody recognition of cancer-related gangliosides and their mimics investigated using in silico site mapping.

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.

Characterization of the N-methyltransferase activities of the multifunctional polypeptide cyclosporin synthetase.

This study demonstrates a critical role for N-methylation in cyclosporin biosynthesis and maintenance of the biologically active cyclosporin conformation. The structural requirements for the AdoMet binding to CySyn were defined. N-methylation of specific amide positions in the cyclosporin backbone is critical for the complete assembly and cyclization of the cyclosporin peptide. A maximum of two desmethyl positions is tolerated before peptide assembly stalls. Subinhibitory concentrations of AdoMet analogs directed peptide assembly towards cyclosporins with less than seven N-methylated amide bonds. Molecular modeling and nuclear magnetic resonance analyses indicate that N-methylation of specific amide bond positions in the cyclosporin backbone is mandatory for the formation of a product-like conformation and recognition by the acceptor site of the downstream peptide bond forming C-domain.

Carbohydrate-mimetic peptides: structural aspects of mimicry and therapeutic implications.

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.

Peptide inhibitors of xenoreactive antibodies mimic the interaction profile of the native carbohydrate antigens.

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 blood transfusion 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, Galalpha (1,3)Gal (alphaGal), 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-alphaGal 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-alphaGal antibodies. The method involves molecular docking of carbohydrates to antibodies and uses the docked carbohydrate poses to generate maps of th 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-alphaGal 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.

Free Ig light chains interact with sphingomyelin and are found on the surface of myeloma plasma cells in an aggregated form.

Free κ L chains (FκLCs) 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 FκLCs interact with membranes remains unresolved. In this study, we show that FκLCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound FκLCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of FκLCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated FκLCs 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.

Molecular docking of carbohydrate ligands to antibodies: structural validation against crystal structures.

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.

Gas phase supramolecular cluster ions of deoxyguanosine induced by binding to (2,2':6'2''-terpyridine)-platinum(II) and (diethylenetriamine)-platinum(II).

Electrospray ionization (ESI) of solutions containing deoxyguanosine (dG) and tridentate platinum chloride complexes yields clusters of general formula [Pt(II)(L)(dG)(n)](2+), where L = 2,2':6',2'' terpyridine (terpy), or diethyltriamine (dien). When these clusters were mass selected and subjected to collision induced dissociation (CID), the primary fragmentation channels arise from loss of dG and protonated dG to yield the fragment ions [Pt(II)(L)(dG)(n-x)](2+) and [Pt(II)(L)(dG)(n-x)-H](+) respectively. The relative abundances of the [Pt(II)(L)(dG)(n-x)](2+) fragments depend on the nature of the ligand, L. The most abundant peaks observed were: n-x = 5 for terpy and n-x = 4 for dien. In order to further understand these gas phase reactions, molecular dynamics calculations were carried out. These simulated collision calculations not only predict with fidelity the experimental profiles for the relative abundances for the [Pt(II)(L)(dG)(n-x)](2+) fragments, for both terpy and dien ligand systems, but they also provide structural insights into the complex interplay of hydrogen bonding and stacking interactions within these clusters.

Three-dimensional structures of carbohydrate determinants of Lewis system antigens: implications for effective antibody targeting of cancer.

Lewis system carbohydrate antigens have been shown to be expressed at high levels in many cancers of epithelial cell origin, including those of colon, breast, lung, prostate and ovary. The type 1 (Le(a) and Le(b)) antigens are important histo-blood groups, while type 2 (Le(x) and Le(y)) antigens in healthy individuals are only expressed, at relatively low levels, by a few tissues, including some epithelial cells. Thus, the type 2 antigens are considered to be tumour-associated antigens and are promising targets for cancer treatment, including antibody-based immunotherapy. In this review, we discuss the conformational characteristics of the free and bound forms of Lewis oligosaccharides and the 3D structures of antibodies in complex with Le(y) and Le(x) antigens. Collectively, the structural studies have demonstrated that the Lewis determinants are rigid structures, which generally maintain the same conformation in the free and bound states. The rigid nature and similarities in shape of type 1 and 2 Lewis oligosaccharides appear to make them perfectly suited to driving a structurally convergent immune response (at least in the case of Le(y) specific antibodies) toward a highly specific recognition of individual carbohydrate determinants, which is a goal in the development of effective antibody-based cancer treatments.

A glycopeptide in complex with MHC class I uses the GalNAc residue as an anchor.

Peptides bind MHC class I molecules by anchoring hydrophobic side chains into pockets in the peptide binding groove. Here, we report an immunogenic (in vitro and in vivo) MUC1 glycopeptide (MUC1-8-5GalNAc) bound to H-2Kb, fully crossreactive with the nonglycosylated variant. Molecular modeling showed that the central P5-Thr-GalNAc residue points into the C pocket and forms van der Waals and hydrogen bond interactions with the MHC class I. As predicted, GalNAc, a modified peptide carrying an additional anchor in the central C anchor pocket, increased the affinity by approximately 100-fold compared with the native low-affinity peptide (MUC1-8). The findings demonstrate that glycopeptides associated with MHC class I molecules can use GalNAc to anchor the peptide in the groove and enable high-affinity binding.

Mcg light chain dimer as a model system for ligand design: a docking study.

Mcg light chain dimer has been extensively studied by crystallography and peptide binding studies to investigate its peptide cross-reactivity as well as to use it as a model system for designing space filling peptide ligands. Here we extend these investigations by utilizing automated docking. Mcg light chain dimer is an ideal model system for such study due to the availability of experimental data for both the native structure and the 14 complexes with various peptide ligands. We show the ability of the docking approach to reproduce the experimental structures and discuss the limitations associated with such outcomes. We demonstrate the usefulness of the docking approach in generating structural information otherwise not available from the experiment.