Probing Torpedo californica acetylcholinesterase catalytic gorge with two novel bis-functional galanthamine derivatives.

N-Piperidinopropyl-galanthamine (2) and N-saccharinohexyl-galanthamine (3) were used to investigate interaction sites along the active site gorge of Torpedo californica actylcholinesterase (TcAChE). The crystal structure of TcAChE-2 solved at 2.3 A showed that the N-piperidinopropyl group in 2 is not stretched along the gorge but is folded over the galanthamine moiety. This result was unexpected because the three carbon alkyl chain is just long enough for the bulky piperidine group to be placed above the bottleneck (Tyr121, Phe330) midway down the gorge. The crystal structure of TcAChE-3 at 2.2 A confirmed that a dual interaction with the sites at the bottom, and at the entrance of the gorge, enhances inhibitory activity: a chain of six carbon atoms has, in this class of derivatives, the correct length for optimal interactions with the peripheral anionic site (PAS).

Time resolved structure analysis of growing beta-amyloid fibers.

Formation of beta-amyloid plaques is a crucial feature of Alzheimer's disease. In the present work time resolved static light scattering was applied to investigate the size and shape of growing beta-amyloid aggregates preceding plaque formation. The beta-amyloid protein with 40 amino acid residues was used. Salt free buffer solutions and solutions with 0.15M NaCl at 37 degrees C served as the aggregation medium. The focus lay on the first 2h following initiation of the aggregation process which corresponds to the protofibril phase. Addition of the NaCl accelerated the aggregation process considerably. Scattering data from aggregation in saline solutions indicated formation of long fibers which suggest interpretation of data with the worm-like chain model. Two important results were revealed: (i) At the end of the time resolved recordings, the worm-like chain model provided a fully adequate picture for the growing aggregates. Chain stiffness is characterised in terms of the persistence length, which is close to 50 nm. The linear mass density of the growing fibers approached a value of two monomers per nm corresponding to single stranded fibers, which is in accordance with presently existing models for the aggregation of beta-amyloid. The fibers finally reached contour lengths of several thousand nanometers. (ii) The plateau values for the persistence length and linear mass density observed in the final regime are gradually approached from higher values. This observation is inconsistent with simple worm-like chains. Rather does it indicate existence of another species during the initial phase of the aggregation, in addition to monomers and fibers. Aside from further insight into fundamental aspects of beta-amyloid aggregation, time resolved static light scattering provides an appropriate tool for assay tests with drugs designed to interfere with the aggregation process.

A QXP-based multistep docking procedure for accurate prediction of protein-ligand complexes.

The two great challenges of the docking process are the prediction of ligand poses in a protein binding site and the scoring of the docked poses. Ligands that are composed of extended chains in their molecular structure display the most difficulties, predominantly because of the torsional flexibility. On the basis of the molecular docking program QXP-Flo+0802, we have developed a procedure particularly for ligands with a high degree of rotational freedom that allows the accurate prediction of the orientation and conformation of ligands in protein binding sites. Starting from an initial full Monte Carlo docking experiment, this was achieved by performing a series of successive multistep docking runs using a local Monte Carlo search with a restricted rotational angle, by which the conformational search space is limited. The method was established by using a highly flexible acetylcholinesterase inhibitor and has been applied to a number of challenging protein-ligand complexes known from the literature.