Background Non-small cell lung cancer (NSCLC) patients have a 5-year survival rate of < 18%, which causes severe therapeutic, social and economic impacts. Significant challenges are the toxicity and resistance induced by existing treatments, i.e. platinum salts or immune checkpoint and EGF receptor kinase inhibitors. It is thus necessary to improve NSCLC treatment with complementary approaches that target other vulnerable cellular pathways. Splicing is a key step in RNA maturation and splicing defects are often oncogenic. With respect to healthy cells, cancer cells display specific splicing patterns and susceptibility to splicing inhibition. Thus, splicing is a vulnerable pathway potentially exploitable for NSCLC treatment. New therapies could be established if we better understood the impact of splicing inhibitors on cell physiology, but it is necessary to develop more potent and selective compounds. Towards this goal, the recently-determined high-resolution structures of splicing complexes provide unique new opportunities for rational drug design. Hypothesis By integrating cell/structural biology with computational/medicinal chemistry, we have discovered a novel class of splicing inhibitors active against bacterial and eukaryotic splicing complexes with single-digit micromolar potency, and we have elucidated their molecular mechanism by crystallography. We hypothesize that by targeting RNA splicing in NSCLC cells our inhibitors would reduce their proliferation, and might thus represent a powerful approach to overcome toxicity and resistance in NSCLC patients. Interestingly, some of our compounds establish sequence-specific contacts with splice junctions. Thus, we further hypothesize that these compounds could modulate splicing in a gene-specific, hence less toxic, manner. To test our hypotheses, we will pursue two aims. Aims Aim 1: Improve the potency of our inhibitors through structure-based, mechanistic studies, and characterize their phenotypic effects in healthy and NSCLC lung cells. Aim 2: Develop and characterize new compounds that establish gene-specific contacts and selectively induce the overexpression of potent tumor-suppressing genes. Experimental Design We will expand our current SAR study by synthesizing second-generation compounds and determining their mechanism of inhibition using in vitro and cell-based splicing assays, X-ray crystallography, and cryo-EM. Our goal is to identify new compounds active with nanomolar potency on the spliceosome, study their cellular phenotypes in healthy and NSCLC lung cells, and test their pharmacological properties through toxicity, stability, solubility, and ADME assays. Expected Results We expect to develop at least 1-2 lead compounds and characterize them from atomic resolution to the preclinical stage to understand their molecular and physio-pathological mechanisms of splicing modulation in NSCLC. Our lead compounds will constitute new therapeutic options for addressing the urgent unmet needs of NSCLC patients, particularly those experiencing resistance. Impact On Cancer Our project will reinforce the emerging view that targeting splicing provides clinical benefits to cancer patients. Considering that splicing is a fundamental and ubiquitous biological process, our work will also more broadly set novel paradigms in cancer research, serving as a reference study for targeting other cancer types, or for targeting healthy cells indirectly involved in NSCLC, such as endothelial cells, which promote angiogenesis within the tumor mass.