Facilitating access to health research through a participatory research register: a feasibility study in outpatient clinics.

BACKGROUND: A research register (Reach West) has been established to facilitate recruitment of people and patients to health-related research. We conducted a prospective feasibility study to investigate the practicality of recruiting through outpatient clinics. METHODS: Patients over 18 years of age attending dental, eye or oncology outpatient clinics in an acute hospital in the West of England were provided with the opportunity to participate in Reach West. In Phase I, recruitment packs were handed to clinic attendees who could place completed consent forms in secure drop-box or return them later on-line or by post. In Phase II, recruitment packs were posted directly to patients with consent forms to be returned by post or on-line. Response rates by age, sex, postcode (for level of deprivation), and clinic type were recorded for those agreeing to participate on paper or on-line. RESULTS: In Phase I, 2,314 of 4,500 (51.4%) of recruitment packs were handed out to clinic attendees, and 114 (5%) consented to join Reach West. In Phase II, 7,173 of 9000 packs were posted (79.7%), and 387 (5.4%) consented to participate. The overall consent rate was 6% (580), with the majority doing so on paper (87%) rather than on-line. The sample was balanced by sex, but mostly comprised people over 50 years located in less deprived postcodes. Non-staff costs for postal recruitment were lower than hand-outs in clinic (£6.84 compared with £8.05 per participant). CONCLUSIONS: Recruiting participants to the Reach West register was feasible among those with oncology, dental or eye outpatient appointments by post or with packs given out in the clinic. Response rates were similar to those achieved for other registers. Recruitment of participants can be achieved through outpatient clinics but other strategies will also be required to attract large numbers of participants and more diverse populations.

Structure and Function in Homodimeric Enzymes: Simulations of Cooperative and Independent Functional Motions.

Large-scale conformational change is a common feature in the catalytic cycles of enzymes. Many enzymes function as homodimers with active sites that contain elements from both chains. Symmetric and anti-symmetric cooperative motions in homodimers can potentially lead to correlated active site opening and/or closure, likely to be important for ligand binding and release. Here, we examine such motions in two different domain-swapped homodimeric enzymes: the DcpS scavenger decapping enzyme and citrate synthase. We use and compare two types of all-atom simulations: conventional molecular dynamics simulations to identify physically meaningful conformational ensembles, and rapid geometric simulations of flexible motion, biased along normal mode directions, to identify relevant motions encoded in the protein structure. The results indicate that the opening/closure motions are intrinsic features of both unliganded enzymes. In DcpS, conformational change is dominated by an anti-symmetric cooperative motion, causing one active site to close as the other opens; however a symmetric motion is also significant. In CS, we identify that both symmetric (suggested by crystallography) and asymmetric motions are features of the protein structure, and as a result the behaviour in solution is largely non-cooperative. The agreement between two modelling approaches using very different levels of theory indicates that the behaviours are indeed intrinsic to the protein structures. Geometric simulations correctly identify and explore large amplitudes of motion, while molecular dynamics simulations indicate the ranges of motion that are energetically feasible. Together, the simulation approaches are able to reveal unexpected functionally relevant motions, and highlight differences between enzymes.

Unique DC-SIGN clustering activity of a small glycomimetic: A lesson for ligand design.

DC-SIGN is a dendritic cell-specific C-type lectin receptor that recognizes highly glycosylated ligands expressed on the surface of various pathogens. This receptor plays an important role in the early stages of many viral infections, including HIV, which makes it an interesting therapeutic target. Glycomimetic compounds are good drug candidates for DC-SIGN inhibition due to their high solubility, resistance to glycosidases, and nontoxicity. We studied the structural properties of the interaction of the tetrameric DC-SIGN extracellular domain (ECD), with two glycomimetic antagonists, a pseudomannobioside (1) and a linear pseudomannotrioside (2). Though the inhibitory potency of 2, as measured by SPR competition experiments, was 1 order of magnitude higher than that of 1, crystal structures of the complexes within the DC-SIGN carbohydrate recognition domain showed the same binding mode for both compounds. Moreover, when conjugated to multivalent scaffolds, the inhibitory potencies of these compounds became uniform. Combining isothermal titration microcalorimetry, analytical ultracentrifugation, and dynamic light scattering techniques to study DC-SIGN ECD interaction with these glycomimetics revealed that 2 is able, without any multivalent presentation, to cluster DC-SIGN tetramers leading to an artificially overestimated inhibitory potency. The use of multivalent scaffolds presenting 1 or 2 in HIV trans-infection inhibition assay confirms the loss of potency of 2 upon conjugation and the equal efficacy of chemically simpler compound 1. This study documents a unique case where, among two active compounds chemically derived, the compound with the lower apparent activity is the optimal lead for further drug development.

Selective targeting of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) with mannose-based glycomimetics: synthesis and interaction studies of bis(benzylamide) derivatives of a pseudomannobioside.

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and Langerin are C-type lectins of dendritic cells (DCs) that share a specificity for mannose and are involved in pathogen recognition. HIV is known to use DC-SIGN on DCs to facilitate transinfection of T-cells. Langerin, on the contrary, contributes to virus elimination; therefore, the inhibition of this latter receptor is undesired. Glycomimetic molecules targeting DC-SIGN have been reported as promising agents for the inhibition of viral infections and for the modulation of immune responses mediated by DC-SIGN. We show here for the first time that glycomimetics based on a mannose anchor can be tuned to selectively inhibit DC-SIGN over Langerin. Based on structural and binding studies of a mannobioside mimic previously described by us (2), a focused library of derivatives was designed. The optimized synthesis gave fast and efficient access to a group of bis(amides), decorated with an azide-terminated tether allowing further conjugation. SPR inhibition tests showed improvements over the parent pseudomannobioside by a factor of 3-4. A dimeric, macrocyclic structure (11) was also serendipitously obtained, which afforded a 30-fold gain over the starting compound (2). The same ligands were tested against Langerin and found to exhibit high selectivity towards DC-SIGN. Structural studies using saturation transfer difference NMR spectroscopy (STD-NMR) were performed to analyze the binding mode of one representative library member with DC-SIGN. Despite the overlap of some signals, it was established that the new ligand interacts with the protein in the same fashion as the parent pseudodisaccharide. The two aromatic amide moieties showed relatively high saturation in the STD spectrum, which suggests that the improved potency of the bis(amides) over the parent dimethyl ester can be attributed to lipophilic interactions between the aromatic groups of the ligand and the binding site of DC-SIGN.

Protein dynamics and enzyme catalysis: insights from simulations.

The role of protein dynamics in enzyme catalysis is one of the most active and controversial areas in enzymology today. Some researchers claim that protein dynamics are at the heart of enzyme catalytic efficiency, while others state that dynamics make no significant contribution to catalysis. What is the biochemist - or student - to make of the ferocious arguments in this area? Protein dynamics are complex and fascinating, as molecular dynamics simulations and experiments have shown. The essential question is: do these complex motions have functional significance? In particular, how do they affect or relate to chemical reactions within enzymes, and how are chemical and conformational changes coupled together? Biomolecular simulations can analyse enzyme reactions and dynamics in atomic detail, beyond that achievable in experiments: accurate atomistic modelling has an essential part to play in clarifying these issues. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.