Modeling Smad domains and their interaction with Smurf-1, c-Ski and DNA promoter motif to design inhibitory compounds.

摘要

Transforming Growth Factor-beta (TGF-beta) superfamily members are known for regulating wide array of cellular processes such as growth, differentiation, proliferation, and apoptosis. In the downstream of TGF-beta signaling there are important growth and differentiation factors known as Smad proteins, which carry out the TGF-beta responsive signaling and elicit various responses once inside the nucleus. The goal of this dissertation is texplore the available structural data of some of the molecules involved in TGF-beta signaling process and to apply state of the art molecular modeling, docking and virtual screening tools and techniques to gain insight into the TGF-beta signaling pathway. This study mainly concentrates on the interaction of Smad proteins with the DNA promoter motif, and other proteins c-Ski and Smurf-1 with which they interact in the signaling process. Initially MH1 domain of mammalian Smad proteins were modeled based on known crystal structure of Smad3 MH1-DNA complex (PDB ID: 1OZJ) followed by modeling of interaction pose of MH1 domain of BMP regulated Smads (Smad1/5/8) with their corresponding DNA sequence motif 5'-GCCG-3'. In this work the key residues of MH1 domain of Smad1/5/8 interacting with 'GCCG' motif were identified. To investigate further the solvent accessibile contact area of key residues and binding energy calculations of modeled Smad1/5/8 MH1 with the GCCG DNA motif and GTCT DNA motif were computed. Higher free energy of binding for Smad1/5/8-MH1 complexed with nonspecific 'GTCT' DNA motif compared to the GCCG motif confirmed high specificity of Smad1/5/8 with 'GCCG' motif indicating that these Smads may not bind with 'GTCT' DNA. Further, homology modeling approach was followed to build Smad binding domain of c-Ski, a proto-oncoprotein, which acts as co-repressor in Smad mediated TGF-beta signaling. Various protein-protein docking methods were applied to study the interactions between the model c-Ski domain and Smad3-MH2 domain. Knowledge of biochemical data, contacts observed between key residues and solvent accessibility calculations of residues of both proteins in our top models were applied to finalize four best favored complexes of Smad3-Ski that can be used to design small molecule inhibitors antagonizing the c-Ski binding which may lead to anti-cancer drug design by appropriately regulating Smad3-Ski interaction.;Besides homology modeling and docking, this thesis work also include virtual screening of small molecular databases to identify high scoring lead molecules against Smad4 binding site of c-Ski and also against Smad binding site of Smurf1 (a key regulator of BMP regulated Smad proteins). Widely used structure-based high throughput virtual screening methods, the GLIDE and the GOLD, were applied for molecular docking studies. Previously identified active site of Smurf1-WW2 domain was targeted, which is known to interact with PPXY motif in Smad1/5 for inhibiting the ubiquitination of the bone inducing Smads by Smurf1. BMP signaling is inhibited due to strong Smad4-Ski binding so in the subsequent studies we focused on designing small molecule inhibitors of c-Ski at Smad4 interacting sites. Both these virtual screening experiments aim at developing a simple, safe and cost effective bone inducing drug that inhibit c-Ski binding to Smad4 and Smurf1 binding to Smad1/5 to increase the BMP responsiveness.