SHAPE-DEPENDENT MOLECULAR RECOGNITION OF SPECIFIC SEQUENCES OF DNA BY HETEROCYCLIC CATIONS by YI MIAO Under the Direction of Dr. W. David Wilson ABSTRACT DB921 and DB911 are biphenyl-benzimidazole-diamidine isomers with a central para- and meta-substituted phenyl group, respectively. Unexpectedly, linear DB921 has much stronger binding affinity with DNA than its curved isomer, DB911. This is quite surprising and intriguing since DB911 has the classical curved shape generally required for strong minor groove binding while DB921 clearly does not match the groove shape. Several biophysical techniques including thermal melting (Tm), circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) have been utilized to investigate the interactions between these compounds and DNA. The structure of the DB921-DNA complex reveals that DB921 binds to DNA with a reduced twist of the biphenyl for better fit of DB921 into the minor groove. A bound water molecule complements the curvature of DB921 and contributes for tight binding by forming H-bonds with both DNA and DB921. Structure-affinity relationship studies of a series of DB921 analogs show that the benzimidazole group is one of the key groups of DB921 for its strong binding to the minor groove. Thermodynamic studies show that the stronger binding of DB921 is due to a more favorable binding enthalpy compared to DB911 even though the complex formation with DNA for these compounds are all predominantly entropically driven. DB921 also has more negative heat capacity change than DB911. The initial studies of inhibition of the interaction between an AT hook peptide of HMGA proteins and its target DNA by a set of diamidine AT-minor groove binders using biosensor-SPR technique show that the inhibitory ranking order is consistent with that of binding affinity and linear-shaped DB921 still has excellent inhibitory effects. These heterocyclic cations rapidly inhibit the binding of DBD2 peptide to the DNA and may only block the specific AT binding of the peptide without hindering the non-specific binding interaction. The results of this project have shown that DB921 represents a new novel effective minor groove binder that does not fit the traditional model and is a potential inhibitor for DNA/protein complexes. INDEX WORDS: Molecular recognition, DNA binding, Minor groove binding, Linear shape, Compound curvature, Binding affinity, Binding kinetics, Thermodynamics, Surface plasmon resonance, Isothermal titration calorimetry, Inhibition

<p>Accurate calculations of free energies for molecular association and solvation are important for the understanding of biochemical processes, and are useful in many pharmaceutical applications. In this thesis, molecular dynamics (MD) simulations are used to calculate thermodynamic properties for solvation and ligand binding.</p><p>The thermodynamic integration technique is used to calculate p<i>K</i>_a values for three aspartic acid residues in two different proteins. MD simulations are carried out in explicit and Generalized-Born continuum solvent. The calculated p<i>K</i>_a values are in qualitative agreement with experiment in both cases. A combination of MD simulations and a continuum electrostatics method is applied to examine p<i>K</i>_a shifts in wild-type and mutant epoxide hydrolase. The calculated p<i>K</i>_a values support a model that can explain some of the pH dependent properties of this enzyme.</p><p> Development of the linear interaction energy (LIE) method for calculating solvation and binding free energies is presented. A new model for estimating the electrostatic term in the LIE method is derived and is shown to reproduce experimental free energies of hydration. An LIE method based on a continuum solvent representation is also developed and it is shown to reproduce binding free energies for inhibitors of a malaria enzyme. The possibility of using a combination of docking, MD and the LIE method to predict binding affinities for large datasets of ligands is also investigated. Good agreement with experiment is found for a set of non-nucleoside inhibitors of HIV-1 reverse transcriptase.</p><p>Approaches for decomposing solvation and binding free energies into enthalpic and entropic components are also examined. Methods for calculating the translational and rotational binding entropies for a ligand are presented. The possibility to calculate ion hydration free energies and entropies for alkali metal ions by using rigorous free energy techniques is also investigated and the results agree well with experimental data.</p>

Accurate calculations of free energies for molecular association and solvation are important for the understanding of biochemical processes, and are useful in many pharmaceutical applications. In this thesis, molecular dynamics (MD) simulations are used to calculate thermodynamic properties for solvation and ligand binding. The thermodynamic integration technique is used to calculate pKa values for three aspartic acid residues in two different proteins. MD simulations are carried out in explicit and Generalized-Born continuum solvent. The calculated pKa values are in qualitative agreement with experiment in both cases. A combination of MD simulations and a continuum electrostatics method is applied to examine pKa shifts in wild-type and mutant epoxide hydrolase. The calculated pKa values support a model that can explain some of the pH dependent properties of this enzyme. Development of the linear interaction energy (LIE) method for calculating solvation and binding free energies is presented. A new model for estimating the electrostatic term in the LIE method is derived and is shown to reproduce experimental free energies of hydration. An LIE method based on a continuum solvent representation is also developed and it is shown to reproduce binding free energies for inhibitors of a malaria enzyme. The possibility of using a combination of docking, MD and the LIE method to predict binding affinities for large datasets of ligands is also investigated. Good agreement with experiment is found for a set of non-nucleoside inhibitors of HIV-1 reverse transcriptase. Approaches for decomposing solvation and binding free energies into enthalpic and entropic components are also examined. Methods for calculating the translational and rotational binding entropies for a ligand are presented. The possibility to calculate ion hydration free energies and entropies for alkali metal ions by using rigorous free energy techniques is also investigated and the results agree well with experimental data.