Browsing by Author "Gorai, Sukhamoy"

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    Membrane Binding Mechanism of Phosphoinositide Interacting Domains and Development of Their Inhibitors
    (2015) Gorai, Sukhamoy
    The content of this thesis entitled “Membrane Binding Mechanism of Phosphoinositide Interacting Domains and Development of Their Inhibitors” have been divided into five chapters based on the results of experimental work carried out during the research period. The introductory chapter (Chapter1) describes the role of phosphoinositides (PIP) in cell signaling, several PIP binding domains including PX, PH domains and small molecule based non-lipid antagonists those are used as inhibitors in PIP-PH interactions. Chapter 2 determines the roles of basic and hydrophobic residues of Tks5-Phox homology domain in membrane binding. In Chapter 3, we have determined the mechanistic insight into PIP binding properties of Lpd-PH domain. Chapter 4 demonstrates the inhibition of phosphatidylinositol-3,4,5-trisphosphate binding to AKT pleckstrin homology domain by 4-Amino-1,2,5-oxadiazole derivatives. In Chapter 5, we have demonstrated the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain. To elucidate the role of basic and hydrophobic residues of Tks5-PX and Lpd-PH domain in membrane binding, we quantitatively determined the binding parameters using a number of biophysical studies including surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET) and monolayer penetration analyses. Mutational studies revealed that presence of basic residues within the PIP-binding pocket and hydrophobic residues at the putative membrane binding surface of the PX and PH domain are significant for its PIP-dependent membrane binding properties. Under normal growth conditions, cellular localization patterns of the PX and PH domain and its mutants in A549 cells are consistent with their in vitro membrane binding properties. We also used a series of small molecule antagonists for PI(3,4,5)P3/PH-domain and PI(4,5)P2/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance (SPR) analysis. To elucidate their binding selectivity, we also used PI(3,4,5)P3, PI(3,4)P2, PI(4,5)P2 specific PH-domains. For further understanding of their PH-domain inhibition mechanism, we also performed various physicochemical analyses. The results showed that these water-soluble compounds do not significantly interact with the model membranes. The oxadiazole and N′-hydroxy moieties of the compounds are essential for their exothermic interaction with the PH-domains and their bindings do not alter the secondary structure of the PH-domain. Further, this idea can be extended for PIP/PH-domain interactions based drug development..