The overall objective of the research described in this proposal is to bring a small molecule therapeutic agent into phase 1 clinical trails for treatment of cancers that depend upon c-Myc overexpression for growth. The specific aims are (1) structural characterization of the i-motif(s) in the NHE 1111 of the c-Myc promoter and its complex(es) with TMPyP4, (2) to determine the conformational state of the NHE 1111 element that exists in a short linear duplex DMA molecule, in a supercoiled plasmid state, and in vivo, and then to determine how binding of TMPyP4 affects the structure of these forms, (3) to establish in vitro biochemical and cell-based screens to identify small molecules that interact specifically with the i-motif structure in the silencer element of the c-Myc promoter, (4) To discover and optimize new i-motif-interactive compounds using structure-based approaches for virtual screening of compound libraries, de novo design, and follow-up optimization of active lead molecules, and (5) in vivo and in vitro evaluation and subsequent preclinical development. We have recently uncovered a novel mechanism for silencing of gene expression involving secondary DMA structures that is amenable to small molecule targeting to specifically modulate gene expression. Our recent results show that the i-motif in the NHE 1111 element is the druggable target for modulation of c-Myc gene expression. Proof of principle already exists in vitro and in vivo using a cationic porphyrin. High-field NMR and footprinting techniques will be used to determine the structure of the DNA element and its drug complexes in cell-free, in vitro biochemical, and in vivo systems. Structure-based design and high through-put screening methods will be used to identify small molecule drug leads. In vitro and in vivo evaluation will be carried out to evaluate these leads prior to preclinical development and phase 1 clinical trials carried out in collaboration with Cylene Pharmaceuticals.