Kir channels serve diverse and important roles throughout the human body and malfunctions of these channels are implicated in various channelopathies. Specific inhibitors for different subtypes of Kir channels are not available. However, Tertiapin-Q (TPNQ), a polypeptide isolated from honey bee venom, differentially inhibits certain subtypes of Kir channels with nanomolar affinity: ROMK1 (Kir1.1) and GIRK1/GIRK4 (Kir3.1/Kir3.4). Modification of TPNQ to increase selectivity for target channels bears great therapeutic potential. The in silico studies based on TPNQ-docked channel models, ROMK1_IRK2 (Kir1.1_Kir2.2) and GIRK2 (Kir3.2), predicted specific paired residue interactions and were experimentally validated here. In ROMK1 E123A mutant, the TPNQ sensitivity was decreased by ~2-fold while GIRK2 E127A mutant reduced the TPNQ sensitivity by greater than 10-fold. Also, we could observe the additional effect, ~ 18 fold, of GIRK1 subunits, ~1.7 fold, and E127A mutation, ~10 fold, on the TPNQ sensitivity in the heteromeric mutant channel, GIRK1/GIRK2 E152D_E127A as compared with the homomeric GIRK2 E152D. Finally, we introduced the Kir3.2 E152D mutant as a good representative of wild-type behavior particularly for the TPNQ study. Overall, this type of structure-function studies suggests an efficient and cost effective way toward design and development of specific Kir channel blockers by targeting on specific paired interactions between TPNQ and the Kir channels.

In this thesis, molecular dynamics simulations, molecular docking, and homology modeling methods have been used in combination to design possible inhibitors as well as to study the structural changes and function of target proteins related to diseases that today are in the spotlight of drug discovery. The inwardly rectifying potassium (Kir) channels constitute the first target in this study; they are involved in cardiac problems. On the other hand, tensin, a promising target in cancer research, is the second target studied here. The first chapter includes a brief update on computational methods and the current proposal of the combination of MD simulations and docking techniques, a procedure that is applied for the engineering of a new blocker for Kir2.1 ion channels and for the design of possible inhibitors for Tensin. Chapter two focuses in Kir ion channels that belong to the family of potassium-selective ion channels which have a wide range of physiological activity. The resolved crystal structure of a eukaryotic Kir channel was used as a secondary structure template to build the Kir-channels whose crystallographic structures are unavailable. Tertiapin (TPN), a 21 a.a. peptide toxin found in honey bee venom that blocks a type of Kir channels with high affinity was also used to design new Kir channel blockers. The computational methods homology modeling and protein-protein docking were employed to yield Kir channel-TPN complexes that showed good binding affinity scores for TPN-sensitive Kir channels, and less favorable for Kir channels insensitive to TPN block. The binding pocket of the insensitive Kir-channels was studied to engineer novel TPN-based peptides that show favorable binding scores via thermodynamic mutant-cycle analysis. Chapter three is focused on the building of homology models for Tensin 1, 2 and 3 domains C2 and PTP using the PTEN X-ray crystallographic structure as a secondary structure template. Molecular docking was employed for the screening of druggable small molecules and molecular dynamics simulations were also used to study the tensin structure and function in order to give some new insights of structural data for experimental binding and enzymatic assays. Chapter four describes the conformational changes of FixL, a protein of bradyrhizobia japonicum. FixL is a dimer known as oxygen sensor that is involved in the nitrogen fixation process of root plants regulating the expression of genes. Ligand behavior has been investigated after the dissociation event, also the structural changes that are involved in the relaxation to the deoxy state. Molecular dynamics simulations of the CO-bound and CO-unbound bjFixL heme domain were performed during 10 ns in crystal and solution environments then analyzed using Principal Component Analysis (PCA). Our results show that the diffusion of the ligand is influenced by internal motions of the bound structure of the protein before CO dissociation, implying an important role for Arg220. In turn, the location of the ligand after dissociation affects the conformational changes within the protein. The study suggests the presence of a cavity close to the methine bridge C of the heme group in agreement with spectroscopic probes and that Arg220 acts as a gate of the heme cavity.