In depth analysis of various aspects of thrombin-ligand binding energetics
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Molecular recognition is the process of selective, weak reversible binding between two molecular entities to form a complex. It is one of the most important processes in molecular biology. Understanding molecular recognition is centrally important to elucidate and control the set of molecular processes that make up the cell and life. The problem of understanding molecular recognition translates into the challenge of being able to design ligands/drugs/small molecules that bind to specific sites on the macromolecules, usually proteins. This idea is enormously important since solving it would provide ways to modulate the activity of proteins which is the central idea of "Rational Drug Design". There have been substantial advances in the determination of the structures of proteins and their complexes by X-ray crystallography and NMR methods. However the ability to predict binding affinity from structure using computational methods is still a significant challenge. Predicting binding affinity has posed to be so challenging since the observed binding affinity between the protein-ligand complexes is not only due to collective weak non-covalent interactions like hydrophobic effect, hydrogen bonding, van der waals interactions etc but also include the solvation and entropy of the protein, the ligand and the complex formed. Furthermore these noncovalent interactions in biological systems are poorly understood. The computational methods used for predicting binding affinity make the assumption that the binding affinity contributed by each of these interactions is the sum of the binding energy contributed by each of the interactions and doesn't depend on the surrounding interactions. This assumption ignores one of the fundamental properties of non-covalent interactions', namely that of cooperativity and enthalpyxix entropy compensation. The focus of the research is to understand these fundamental properties of non-covalent interactions. To explore the phenomenon of cooperativity two separate experiments were designed, one directed towards unraveling cooperativity between hydrophobic interactions and hydrogen bonding and the other directed towards understanding cooperativity between hydrogen bonds. The best way to answer this complex problem was by designing a series of carefully designed inhibitors with small modifications so that the data obtained can be correlated to the changes made. The serine protease thrombin was used as the model system. In order to demonstrate, cooperativity between hydrophobic interactions and hydrogen bonds, two separate series of 28 thrombin inhibitors were designed, synthesized and tested using biochemical assay. The data obtained demonstrated cooperativity for both series. The binding affinity improved by 75% for every sq. Å of hydrophobic contact surface area added in the presence of an additional hydrogen bond in one series and 57% for the other series. The underlying cause of this observation was further studied by X-ray crystallography, Isothermal Calorimetry and Molecular dynamics simulations. Another series of 8 inhibitors was designed to reveal cooperativity between hydrogen bonds. The data obtained on this series suggest that hydrogen bonds also act cooperatively. However this data is preliminary and definite conclusions cannot be made for this series. Along with these series of inhibitors, a follow up study was designed for a previously observed classic case of enthalpy-entropy compensation, wherein two ligands have very similar binding affinities however one was enthalpy driven and the other was entropy driven. Also to investigate the surprising high activity of the thrombin inhibitors having the bis-phenyl methane moiety in the S-3 hydrophobic pocket of thrombin, two thrombin inhibitors having the bisphenyl methane moiety in the S-3 pocket were synthesized, tested and analyzed by X-ray crystallography and Isothermal calorimetry. From the data it was concluded that the high affinity was contributed to the rotation of a glutamate residue adjacent to the binding pocket forming a salt-bridge to a lysine moiety giving rise to an enhanced enthalpic contribution. Overall, the results obtained from this project would improve our understanding of the underlying interactions involved in the biological molecular recognition. The data obtained would be valuable in rational drug design and more accurate prediction of binding affinities.