Ricardo L. Mancera

The Structure of Glycosaminoglycans and their Interactions with Proteins

Glycosaminoglycans (GAGs) are important complex carbohydrates that participate in many biological processes through the regulation of their various protein partners. Biochemical, structural biology and molecular modelling approaches have assisted in understanding the molecular basis of such interactions, creating an opportunity to capitalize on the large structural diversity of GAGs in the discovery of new drugs. The complexity of GAG–protein interactions is in part due to the conformational flexibility and underlying sulphation patterns of GAGs, the role of metal ions and the effect of pH on the affinity of binding. Current understanding of the structure of GAGs and their interactions with proteins is here reviewed: the basic structures and functions of GAGs and their proteoglycans, their clinical significance, the three‐dimensional features of GAGs, their interactions with proteins and the molecular modelling of heparin binding sites and GAG–protein interactions. This review focuses on some key aspects of GAG structure–function relationships using classical examples that illustrate the specificity of GAG–protein interactions, such as growth factors, anti‐thrombin, cytokines and cell adhesion molecules. New approaches to the development of GAG mimetics as possible new glycotherapeutics are also briefly covered.

New anti-tuberculosis drugs in clinical trials with novel mechanisms of action.

Tuberculosis is a major health problem worldwide, with approximately 1.7 million people dying annually from the disease. The long current drug regimen, the emergence of drug resistant strains and HIV co-infection have resulted in a resurgence in research efforts to address the urgent need for new anti-tuberculosis drugs. A number of new potential anti-tuberculosis drug candidates with novel modes of action have entered clinical trials in recent years. These agents are most likely to be effective against resistant strains. We provide a concise review of their structure-activity relationships, in vitro and in vivo activity, pharmacokinetics, mechanism of action and combination regimens.

Ligand−Protein Docking with Water Molecules

The presence of water molecules plays an important role in the accuracy of ligand-protein docking predictions. Comprehensive docking simulations have been performed on a large set of ligand-protein complexes whose crystal structures contain water molecules in their binding sites. Only those water molecules found in the immediate vicinity of both the ligand and the protein were considered. We have investigated whether prior optimization of the orientation of water molecules in either the presence or absence of the bound ligand has any effect on the accuracy of docking predictions. We have observed a statistically significant overall increase in accuracy when water molecules are included during docking simulations and have found this to be independent of the method of optimization of the orientation of water molecules. These results confirm the importance of including water molecules whenever possible in a ligand-protein docking simulation. Our findings also reveal that prior optimization of the orientation of water molecules, in the absence of any bound ligand, does not have a detrimental effect on the improved accuracy of ligand-protein docking. This is important, given the use of docking simulations to predict the binding modes of new ligands or drug molecules.

Ligand-protein cross-docking with water molecules

The accuracy of ligand-protein docking may be affected by the presence of water molecules on the surface of the protein. Cross-docking simulations have been performed on a number of ligand-protein complexes for various proteins whose crystal structures contain water molecules in their binding sites. Only common sets of water molecules found in the binding site of the proteins were considered. A statistically significant overall increase in accuracy was observed when water molecules were included in cross-docking simulations. These results confirm the importance of including water molecules whenever possible in ligand-protein docking simulations.

Expanded Interaction Fingerprint Method for Analyzing Ligand Binding Modes in Docking and Structure-Based Drug Design

An expanded interaction fingerprint method has been developed for analyzing the binding modes of ligands in docking and structure-based design methods. Taking the basic premise of representing a ligand in terms of a binary string that denotes its interactions with a target protein, we have expanded the method to include additional interaction-specific information. By considering the hydrogen-bonding strength and/or accessibility of the hydrogen bonding groups within a binding site as well as their geometric arrangement we aim to provide a better representation of a ligand-protein interaction. These expanded methods have been applied to the postprocessing of binding poses generated in a docking study for 220 different proteins and to the analysis of ligands generated by an automated ligand-generation algorithm for the anthrax oedema factor. In the docking study, the application of the interaction fingerprint method as a postprocessing tool resulted in an increased success rate in identifying the crystallographic binding mode. In the analysis of the ligands generated for the anthrax oedema factor, the incorporation of additional interaction-specific information resulted in a more intuitive and comprehensive analysis of automated ligand-generation output.

A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

Molecular dynamics simulation of dilute aqueous DMSO solutions. A temperature-dependence study of the hydrophobic and hydrophilic behaviour around DMSO

We have studied the temperature dependence of the hydrophobic and hydrophilic hydration properties of dimethyl sulfoxide (DMSO) in water by performing a series of molecular dynamics simulations of such aqueous solutions at a concentration of 0.055 mole fraction at 298, 318 and 338 K. Our results corroborate the existence of an enhancement in the structure of water. There is a well-defined hydration structure around the oxygen atom of DMSO, which establishes strong linear hydrogen-bonds with water molecules. Such interactions increase the lifetime of water–water hydrogen bonds in the vicinity of the OS group. Hydrophobic hydration around the Me groups of DMSO is observed, shown by the formation of an ordered hydration shell around these groups, with strong water–water hydrogen bonds that have longer lifetimes than in the bulk of the solution. We could find no evidence for a temperature-dependent hydrophobic interaction between these groups. All these combined effects reveal that the hydrophilic hydration of DMSO dominates over any hydrophobic effects as the temperature is increased.

Prediction of heparin binding sites in bone morphogenetic proteins (BMPs)

Heparin is a glycosaminoglycan known to bind bone morphogenetic proteins (BMPs) and the growth and differentiation factors (GDFs) and has strong and variable effects on BMP osteogenic activity. In this paper we report our predictions of the likely heparin binding sites for BMP-2 and 14. The N-terminal sequences upstream of TGF-β-type cysteine-knot domains in BMP-2, 7 and 14 contain the basic residues arginine and lysine, which are key components of the heparin/HS-binding sites, with these residues being highly non-conserved. Importantly, evolutionary conserved surfaces on the beta sheets are required for interactions with receptors and antagonists. Furthermore, BMP-2 has electropositive surfaces on two sides compared to BMP-7 and BMP-14. Molecular docking simulations suggest the presence of high and low affinity binding sites in dimeric BMP-2. Histidines were found to play a role in the interactions of BMP-2 with heparin; however, a pK(a) analysis suggests that histidines are likely not protonated. This is indicative that interactions of BMP-2 with heparin do not require acidic pH. Taken together, non-conserved amino acid residues in the N-terminus and residues protruding from the beta sheet (not overlapping with the receptor binding sites and the dimeric interface) and not C-terminal are found to be important for heparin-BMP interactions.

Including Tightly-Bound Water Molecules in de Novo Drug Design. Exemplification through the in Silico Generation of Poly(ADP-ribose)polymerase Ligands

Different strategies for the in silico generation of ligand molecules in the binding site of poly(ADP-ribose)polymerase (PARP) were studied in order to observe the effect of the targeting and displacement of tightly bound water molecules. Several molecular scaffolds were identified as having better interactions in the binding site when targeting one or two tightly bound water molecules in the NAD binding site. Energy calculations were conducted in order to assess the ligand-protein and ligand-water-protein interactions of different functional groups of the generated ligands. These calculations were used to evaluate the energetic consequences of the presence of tightly bound water molecules and to identify those that contribute favorably to the binding of ligands.

A molecular mechanism of solvent cryoprotection in aqueous DMSO solutions

Dimethyl sulfoxide (DMSO) in aqueous solution is widely used for the preservation of biological tissues under freezing conditions. DMSO and other agents are believed to act colligatively to depress the freezing point of water and, importantly, to promote the vitrification of water to prevent its crystallisation and the ensuing damage arising from the formation of intracellular ice. However, there has been no direct evidence of the precise effect of these agents on the vitrification properties of water. Here we report direct computational evidence, using molecular dynamics annealing simulations carried out within the experimentally inaccessible region in supercooled water, of a broadening of the glass transition of water, corresponding to the formation of a stronger glass in aqueous DMSO solutions. These findings provide insight at the molecular level into the mechanism of solvent cryoprotection by suggesting that the resulting thermodynamic stability of the glassy state of water reduces the probability of its nucleation and the subsequent formation of ice as the temperature is decreased.