Structure-based tailoring of compound libraries for high-throughput screening: discovery of novel EphB4 kinase inhibitors

High-throughput docking is a computational tool frequently used to discover small-molecule inhibitors of enzymes or receptors of known three-dimensional structure. Because of the large number of molecules in chemical libraries, automatic procedures to prune multimillion compound collections are useful for high-throughput docking and necessary for in vitro screening. Here, we propose an anchor-based library tailoring approach (termed ALTA) to focus a chemical library by docking and prioritizing molecular fragments according to their binding energy which includes continuum electrostatics solvation. In principle, ALTA does not require prior knowledge of known inhibitors, but receptor-based pharmacophore information (hydrogen bonds with the hinge region) is additionally used here to identify molecules with optimal anchor fragments for the ATP-binding site of the EphB4 receptor tyrosine kinase. The 21,418 molecules of the focused library (from an initial collection of about 730,000) are docked into EphB4 and ranked by force-field-based energy including electrostatic solvation. Among the 43 compounds tested in vitro, eight molecules originating from two different anchors show low-micromolar activity in a fluorescence-based enzymatic assay. Four of them are active in a cell-based assay and are potential anti-angiogenic compounds.     

Protein dynamics: From the native to the unfolded state and back again

Simulations to study protein unfolding and folding were performed. The unfolding simulations make use of molecular dynamics and treat an atomic model of barnase in aqueous solvent. The cooperative nature of the unfolding transition and the important role of water are described. The folding simulations are based on a bead model of the protein on a cubic lattice. It is shown for the 27-mer model that a large energy gap between the lowest energy (native) state and the excited states is a necessary and sufficient condition for fast folding.     

Reduced oxidative activity of circulating neutrophils in patients after myocardial infarction

Circulating neutrophils isolated from patients 3–4 h after a myocardial infarction produced less $ {\rm O}\frac{ \cdot }{{\rm 2}} $ compared with controls, when stimulated with phorbol myrystate acetate or formyl-methionine-leucine-phenylalanine. Three days after the infraction the $ {\rm O}\frac{ \cdot }{{\rm 2}} $ generation elicited by both stimuli further decreased markedly. Seven and 15 days after infarction the $ {\rm O}\frac{ \cdot }{{\rm 2}} $ stimulated production was only slightly lower than or similar to the control values. The neutrophils of infarcted patients showed an augmented latency period before $ {\rm O}\frac{ \cdot }{{\rm 2}} $ production compared with controls in response to exogenous stimuli, particularly three days after infarction. Electron microscopy revealed that the neutrophils isolated from the infarcted patients displayed signs of cell exhaustion with few alterations of the plasma membranes when stimulated with phorbol ester. In contrast, control neutrophils displayed alterations of the plasma membranes characteristic of active neutrophils. The results of this study indicate that the circulating neutrophils appear exhausted and functionally inhibited immediately after myocardial infarction.     

Wordom: a program for efficient analysis of molecular dynamics simulations

Wordom is a versatile program for manipulation of molecular dynamics trajectories and efficient analysis of simulations. Original tools in Wordom include a procedure to evaluate significance of sampling for principal component analysis as well as modules for clustering multiple conformations and evaluation of order parameters for folding and aggregation. The program was developed with special emphasis on user-friendliness, effortless addition of new modules and efficient handling of large sets of trajectories.     

Growth and nutrient absorption of two submerged aquatic macrophytes in mesocosms, for reinsertion in a eutrophicated shallow lake

Aquatic macrophytes play a central role in preserving the ecological equilibrium of shallow lakes and in the restoration of eutrophic lakes that have switched to phytoplankton-dominated turbid water. Massaciuccoli Lake, a shallow lake located along the Tuscan coast in Italy, has shown a constant and progressive simplification of the submerged plant community, for anthropogenic reasons, leading, in recent years, to turbid water. The growth and nutrient absorption capability of two macrophyte species, Myriophyllum verticillatum L. and Elodea canadensis Michaux, in the lake was investigated, with the prospect of a future lake restoration programme centred on their replacement. Mesocosm experiments were conducted to monitor the plant growth and nutrient (NO₂⁻, NO₃⁻, NH₄⁺, Ntot, PO₄³⁻, Ptot) content in the plant dry matter and water at the beginning and at the end of the trial. Bacterial activity was analysed in the water in order to verify the possible nutrient absorption contribution by organisms other than plants. Both M. verticillatum and E. canadensis showed satisfactory growth and nutrient reduction in the water body. Moreover, their different growth patterns suggested that optimal replacement can be performed with their introduction in two steps, starting with M. verticillatum, which shows the best capacity to colonise the aquatic environment, due to its tendency towards lengthening.     

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure–activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC₅₀ values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC₅₀ values with steep dose–response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay’s utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein–ligand interactions.     

Amyloid Aggregation on Lipid Bilayers and Its Impact on Membrane Permeability

Fibrillar protein aggregates (amyloids) are involved in several common pathologies, e.g., Alzheimer's disease and type II diabetes. Accumulating evidence suggests that toxicity in amyloid-related diseases originates from the deposition of protein aggregates on the cell membrane, which results in bilayer disruption and cell leakage. The molecular mechanism of damage to the membrane, however, is still obscure. To shed light on it we have performed coarse-grained molecular dynamics simulations of fibril-forming amphipathic peptides in the presence of lipid vesicles. The simulation results show that highly amyloidogenic peptides fibrillate on the surface of the vesicle, damaging the bilayer and promoting leakage. In contrast, the ordered aggregation of peptides with low amyloidogenicity is hindered by the vesicles. Remarkably, leakage from the vesicle is caused by growing aggregates, but not mature fibrils. The simulation results provide a basis for understanding the range of aggregation behavior that is observed in experiments with fibril-forming (poly)peptides.