MOLECULAR MODELLING STUDIES OF THE NF-κB BIOLOGICAL SYSTEM AS RELEVANT DRUG TARGET

Background Virtual screening (VS) is now a well-established method for finding small molecular modulators of a biological relevant macromolecule function by using much older computational techniques, such as docking simulations. The NF-kB family consists of a group of eukaryotic inducible transcription factors that have evoked widespread interest until now. As a matter of fact, the dysfunction of NF-kB is associated with many serious disease states. In spite of the well-defined solved NF-kB 3D structures, to the best of our knowledge, nowadays VS applications against this interesting target for the discovery of new NF-kB/DNA inhibitors have not been published yet. Aims The project described in this thesis is aimed to the identification of low molecular weight compounds interacting with NF-kB and able to inhibit DNA/NF-kB complex formation and biological dependent functions. Methods As a first example towards the development of a docking protocol fit to the target under study (NF-kB p50 monomer and homodimer), a data set of 27 bioactive natural compounds were prepared. In the second example a chemical library was purpose-made for VS applying a filtering procedure on 3D ZINC commercial database containing more than 2 million compounds. Successively a focus furocoumarin library of 1700 molecules was prepared for subsequent docking runs. The three dimensional structure of the complex NF-kB-DNA was retrieved from the RCSB Protein Data Bank. The cocrystallized DNA macromolecule was removed from the structure. p50-p50 homodimer, p50-p65 heteodimer and p50 monomers were selected for the docking simulations and prepared using the graphical interface Maestro. All molecules under study were docked in to the binding site of the target using Glide software from Schrodinger. Grids were prepared for each proteins. The coordinates of the enclosing box were defined starting from the set of active site residues involved in hydrogen bonds with the NF-kB recognition site of DNA and optimised including the double strands DNA helices volume by visual inspection. Docking studies were based on rigid X-ray crystal transcription factor structures (1LE9, 1IKN) and no information is reported in literature on dynamic features of this protein. Thus, 14ns of canonical MD simulations were carried out in explicit solvent on structural models of NF-kB systems as free and bound both to DNA and IkB. CHARMM27 force field, TIP3P model for water and P.M.E. for electrostatic force calculation were used. Main Results We found that the adopted docking sampling method and the scoring function were successful in predict the ranking of active and inactive natural compounds into DNA binding site of the ensemble protein (NF-kB p50). Further EMSA assays and biological evaluation were in agreement with computational findings. A successful identification of a micromolar active compound from docking-based VS of the prepared compound library is described. Subsequent in silico screening of the developed focus furocoumarin library carried forward the identifications of five micromolar p50 binders. The binding modes of the best-scored compounds showed the involvement of an essential residue for p50 in vivo activity in DNA interaction. In depth investigations of these interesting hits are also reported, including docking analysis into p50-p65 heterodimer. The ligands showed a different occupation of the DNA binding region of the heterodimer. The structural and dynamics properties of NF-kB p50-p65 in free form and in complex with DNA and IkB have been analyzed showing quite pertinent differences. The most relevant configurations of the transcription factor have been clustered and selected for further docking studies and drug design. Conclusions Discovered micromolar bioactive compounds can be considered as “leads” for developing stronger NF-kB/DNA inhibitors. The encouraging computational and biological results here reported could open new perspectives in discovery of NF-kB/DNA interaction inhibitors by taking advantages from recent VS strategies