Biochemical approaches to study protein-ligand interaction for pharmacological applications

This work is focused on the application of simple biochemical techniques to study protein-ligand interaction involved in some diseases of frequent incidence, such as Alzheimer's disease (AD) and cancer. The current therapies for AD, still symptomatic and palliative, from one side act to inhibit AChE, in order to restore the natural level of ACh, and, from the other side act as inhibitors of ?-secretase 1 (BACE-1), useful to prevent the A? aggregation. Under this regard, efforts have been recently devoted to the development of dual inhibitors of AChE and BACE-1. In this frame, donepezil-like compounds were synthesised, in order to identify novel effective drugs for the treatment of AD. For all these new synthesized analogues, more rigid and diversely substituted compared to donepezil structure, the inhibitor activity on AChE, the selectivity vs BuChE, the side-activity on BACE-1 and the effect on SHSY-5Y neuroblastoma cell viability were tested. Two potential new lead compounds for a dual therapeutic strategy against Alzheimer disease were envisaged. Activation of p53 tumor suppressor by antagonizing its negative regulators MDM2/X has been considered an attractive strategy for cancer therapy. Great effort has been given in the development of drugs able to dissociate the p53†¢MDM2/X complex. Under this regard, a simple and rapid technique to study the p53†"MDM2/X interaction has been developed. This method is based on the different mobility between the interacting domains of the oncosuppressor p53 and its protein ligands MDM2/X on polyacrylamide gels under native conditions. While the two proteins MDM2/X alone were able to enter the gel, the formation of a binary complex between p53 and MDM2/X prevented the gel entry. The novel technique is reliable for determining the different affinity elicited by MDM2 or MDMX toward p53, and for analyzing the dissociation power exerted by small molecules on the complex taking advantage of the appearance of migrating MDM2 or MDMX, when inhibitors are added to the complex mixture. Despite the fact that some other different methods have been employed to study this kind of interaction, including NMR technology, surface plasmon resonance or fluorescence polarization, the relevance of the method here described resides in the fact that it is much more simple and it does not require any tagging/derivatization procedure of the protein fragments employed. This simple and rapid technique can be useful to easily discriminate, among a library of compounds endowed with a potential pharmacological activity against cancer development, the molecules with the highest dissociative potency, preventing the use of expensive and more sophisticated technologies.