Abstract/Summary Facioscapulohumeral dystrophy (FSHD) is the third most common form of muscular dystrophy affecting over 30,000 Americans. FSHD is a progressive disease where patients initially lose muscle cells in the face, shoulders and upper arms before degeneration expands to include nearly all skeletal muscles and 20% become wheelchair bound. 95% of FSHD patients display a contraction of the highly polymorphic D4Z4 repeat (FSHD1) containing an open reading frame for the transcription factor (TF) Double Homeobox 4 (DUX4). DUX4 misexpression is associated with myoblast toxicity and is thought to be the driver of FSHD. It is believed that DUX4 induces myoblast death by upregulating target genes including MBD3L2, TRIM43, ZSCAN4, and LEUTX that are not normally expressed in muscle. Although there are no FDA approved therapies for FSHD, losmapimod, a small molecule p38 inhibitor, was shown to inhibit DUX4 transcription in FSHD patient cells and demonstrated clinical benefit in several outcome measures. However, losmapimod is not specific to DUX4 and FSHD, therefore developing multiple targeted therapies with different modes of inhibition would increase the success rate in treating FSHD with minimum long term side effects. We hypothesize that targeting DUX4 will block multiple pathways and reduce muscle cell death. Although TFs like DUX4 are attractive therapeutic targets, they are challenging to target with small molecules because they lack clear binding pockets, have large surface areas important for protein-protein interactions and contain large intrinsically disordered domains. At Altay Therapeutics, we developed a platform that enables identification of small binding pockets within intrinsically disordered domains in previously undruggable TFs, allowing a novel druggable approach for targeting DUX4 and development of potent and highly specific DUX4 inhibitors (DUX4i). We completed in-silico screening and identified inhibitors that reduced DUX4 DNA binding by targeting the disordered linker domain. Importantly, these DUX4i had minimal cytotoxicity, reduced DUX4 target genes and rescued DUX4 driven cell viability, important to treat FSHD. We propose three aims to identify and characterize the most promising lead and continue our efforts to develop a viable treatment for FSHD based on inhibiting DUX4. The successful completion of our proposal is intended to nominate a lead DUX4 drug candidate with the following aims, 1) Determine DUX4 target gene inhibition with our DUX4is and measure cytotoxicity in a broader panel of normal cells 2) Determine specificity of our DUX4is against other homeobox-containing genes such as PAX3/PAX7 and measure potential non-specific inhibition of normal myoblast differentiation 3) Measure in vivo efficacy of DUX4is in mice implanted with human FSHD myoblasts in the tibialis anterior muscles (xenograft model of FSHD). We will then pursue an SBIR phase 2 grant that will include medicinal chemistry efforts and additional...