MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by targeting a mRNA for degradation or translational repression. Not surprisingly, aberrant miRNA expression can cause disease, in particular cancers. Herein, we propose a series of investigations to explore the therapeutic potential of compounds that bind a miRNA precursor associated with breast cancer and inhibit its biogenesis in breast cancer cells. These compounds were developed as a direct result of our currently funded R01 grant (R01- GM097455) in which we proposed to develop computational methods to design small molecules that avidly and selectively bind miRNA precursors. These studies have the potential to advance new anti-cancer therapies to the clinic and to enable development of general strategies to target miRNAs that cause other diseases such as Hepatitis C infections, Alzheimer's disease, and heart disease, among others. Our parent R01 grant proposes to: (i) establish a computational approach to identify lead small molecules for a desired RNA target using a database of RNA motif-small molecule interactions and to identify novel RNA targets from transcriptomes; and (ii) use these computational tools to identify lead small molecules that bind human miRNAs and evaluate them for inhibiting biogenesis in mammalian cell lines. Indeed, this approach was employed to rationally design a small molecule that selectively binds an oncogenic miRNA precursor, miR- 96, and inhibits its biogenesis in breast cancer cells. Importantly, miRNA profiling studies reveal that our small molecule is more selective than an antagomir, the state-of-the-art miRNA targeting modality! Thus, this small molecule is an exemplary case to push forward into animal models of cancer and test the hypothesis that small molecules can indeed drug undruggable RNA targets. The Specific Aims are: Aim 1: Evaluate the therapeutic potential of our designed pri-miR-96 small molecule in models of triple negative and metastatic breast cancer. Aim 2: Validate in vivo targets, compound selectivity and mechanism of action of our designer compounds. Previously, our group has shown that small molecules can be designed to react with RNA targets in live cells. This covalent approach significantly improves bioactivity (>2500-fold) and can be used to validate the cellular targets of small molecules. Aim 3: Evaluate a dimeric small molecule that simultaneously binds the Drosha processing site and an adjacent internal loop in pri-miR-96. We have designed a potent dimeric small molecule that targets pri-miR-96 in vitro (~500-fold more potent than our lead monomeric small molecule). We will evaluate the therapeutic potential of this compound in MDA-MB-231 breast cancer cells and metastatic and drug-resistant variants, using orthotopic xenograft models.