Our long-term goal is to characterize the thermodynamic nature of the molecular forces that dictate and control the affinity and specificity of drug binding to DNA structures. Such a characterization of drug-DNA interactions is a prerequisite for the development of a rational basis for drug design. Our approach is to determine complete thermodynamic binding profiles for the complexation of antitumor and antiviral drugs and their analogues to various DNA host duplexes. Specifically, spectroscopic and calorimetric techniques will be employed to characterize thermodynamically the binding event as a function of the structure of the drug and the sequence of both oligomeric and polymeric host DNA's. The resulting thermodynamic binding profiles will allow us to: define the nature of the forces that drive complexation and predict the temperature-dependent stability of the complex; determine the thermodynamic origin(s) of sequence binding preferences; define the thermodynamic basis for cooperative binding; evaluate the contribution that specific structural features of a drug make to its DNA binding affinity and specificity by comparing thermodynamic binding data for a series of drug analogues; correlate the thermodynamic data with the mode of binding and the molecular picture of the complex; define the thermodynamic basis for chiral selectivity; resolve drug-induced conformational changes from local, specific drug-DNA interactions by comparing binding data on corresponding oligomeric and polymeric DNA hosts; evaluate the thermodynamic basis for the drug synergism (such as occurs in combined chemotherapy regiments) by comparing DNA binding data for a drug in the presence and absence of other drugs. Differential scanning calorimetry will be used to detect, monitor, and thermodynamically characterize the influence of drug binding on the melting behavior of the host duplex. In particular, the size of the cooperative melting unit for each host duplex will be determined in the presence and absence of each drug. This parameter will provide a measure of the influence of drug binding and base sequence on the ability of a polymer chain to propagate molecular distortions required for melting cooperativity -- a property which could be of considerable importance in numerous biological processes. Calorimetry represents the only experimental method by which the relevant thermodynamic data can be obtained in a direct and model-independent manner. In conjunction with standard spectroscopic techniques, this proposal is designed to exploit the unique powers of isothermal mixing and differential scanning calorimetry to obtain complete thermodynamic and extra-thermodynamic profiles of the solution properties of drug binding and the resultant drug-DNA complexes. Most significantly, in conjunction with structural data, our thermodynamic binding data on families of drugs with systematically altered structures will allow us to define the contribution(s) that specific drug-DNA interactions make to the DNA binding affinities and specificities exhibited by each ligand. As noted above, such a dissection and characterization of the thermodynamic contributions made by specific drug-DNA interactions represent an essential step in the long journey required to develop a rational basis for drug design.
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