Continuation funds are requested for a project that has been funded for three+ years and that supports collaborating laboratories in nucleic acid targeted drug design and synthesis and in the biophysical chemistry of nucleic acid complexes. Based on results obtained under initial funding, it is clear that stacked-dimers of heterocyclic cations represent a powerful new motif for specific, strong recognition of mixed base pair DNA sequences and inhibition of protein-DNA complexes. The compounds have low toxicity with excellent biological activity and a compound from this class is set to enter Phase III clinical trials. Since the two strands of the double helix contain different chemical information, compounds that recognize DNA can do so with the highest specificity if they read both strands. Based on these observations our hypothesis is that stacked dimer recognition of DNA by designed heterocyclic cations offers unique opportunities for development of new classes of drugs for broad-based selective molecular recognition of the duplex and inhibition of DNA-protein complexes. Such dimers have significant potential for development as new types of therapeutics, for reagents in biotechnology, as agents for control of cellular gene expression, and for inhibition of specific protein-DNA complexes. We have designed and are studying two dimer-stacking motifs that recognize the minor groove in new and different ways. One system is being developed to inhibit bZlP transcription factors such as the Fos/Jun oncoproteins, while the second system will target the minor groove HMGA oncoproteins. We will prepare and study compounds that (i) increase the sequence recognition capability of the dimer systems by using a variety of heterocycles; (ii) extend the length of the base sequence that is read by extending the length of the dimers; (iii) enhance the affinity and specificity of binding by preparation of covalent dimers. The compound-DNA complexes will be characterized with a powerful array of biophysical methods including NMR and x-ray; enzymatic and chemical footprinting; biosensor-SPR and ITC. At the most fundamental level, the heterocyclic dimer systems will provide significant new understanding of the molecular basis of DNA recognition and how it is affected by the local interplay of base pair chemistry, solvent and ligand properties. The heterocyclic system is the only motif for targeting mixed sequences of DNA with clinically useful and cell permeable agents. ? ?
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