Understanding pathogenic single-nucleotide polymorphisms in multidomain proteins--studies of isolated domains are not enough.

Studying the effects of pathogenic mutations is more complex in multidomain proteins when compared with single domains: mutations occurring at domain boundaries may have a large effect on a neighbouring domain that will not be detected in a single-domain system. To demonstrate this, we present a study that utilizes well-characterized model protein domains from human spectrin to investigate the effect of disease- and non-disease-causing single point mutations occurring at the boundaries of human spectrin repeats. Our results show that mutations in the single domains have no clear correlation with stability and disease; however, when studied in a tandem model system, the disease-causing mutations are shown to disrupt stabilizing interactions that exist between domains. This results in a much larger decrease in stability than would otherwise have been predicted, and demonstrates the importance of studying such mutations in the correct protein context.

Drug release from PLGA microspheres attached to solids using supercritical CO2.

Functionalization of a porous orthopedic implant with dexamethasone, a widely used anti-inflammatory drug, encapsulated within a biodegradable polymer for controlled release could help reduce or eliminate the inflammation response by the local tissue. In this research, we investigated the possibility of using supercritical carbon dioxide (CO2) for attaching dexamethasone-loaded PLGA (polylactic-co-glycolic acid) microspheres to porous CoCrMo alloy for continuous delivery of dexamethasone. Supercritical CO2 has been shown to be effective for attachment of PLGA microspheres to glass plates and porous CoCrMo alloy. Attached microspheres showed similar dexamethasone release profiles but different magnitude of burst release. Microspheres attached to the porous alloy samples using supercritical CO2 at 10 bar and 40 °C for 30 min showed a release profile similar to that of the nonattached microspheres. The microsphere morphology and the release profiles of microspheres attached to the glass plates and to the porous alloy samples suggest that dexamethasone burst release is enhanced by PLGA swelling at higher CO2 pressures and better dispersion of microspheres. This study shows that microspheres can be incorporated into porous solids using supercritical CO2, allowing for a wide variety of drug-biodegradable polymer formulations prepared using the proven emulsion/solvent evaporation method to be tested.

A fragment-based approach to probing adenosine recognition sites by using dynamic combinatorial chemistry.

A new strategy that combines the concepts of fragment-based drug design and dynamic combinatorial chemistry (DCC) for targeting adenosine recognition sites on enzymes is reported. We demonstrate the use of 5'-deoxy-5'-thioadenosine as a noncovalent anchor fragment in dynamic combinatorial libraries templated by Mycobacterium tuberculosis pantothenate synthetase. A benzyl disulfide derivative was identified upon library analysis by HPLC. Structural and binding studies of protein-ligand complexes by X-ray crystallography and isothermal titration calorimetry informed the subsequent optimisation of the DCC hit into a disulfide containing the novel meta-nitrobenzyl fragment that targets the pantoate binding site of pantothenate synthetase. Given the prevalence of adenosine-recognition motifs in enzymes, our results provide a proof-of-concept for using this strategy to probe adjacent pockets for a range of adenosine binding enzymes, including other related adenylate-forming ligases, kinases, and ATPases, as well as NAD(P)(H), CoA and FAD(H2) binding proteins.