PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US. Most CRCs either are inherently insensitive to therapeutic treatment or acquire resistance upon relapse. Intrinsic and acquired therapeutic resistance is one of the most significant obstacles in the treatment of CRCs. Mcl-1, a pro-survival Bcl-2 family protein, has recently emerged as a key mediator of therapeutic resistance of CRCs. However, none of the previously described Mcl-1 inhibitors are suitable for clinical development owing to insufficient in vivo efficacy or undesirable pharmacokinetic properties. Utilizing integrated screening approaches and structure-based design, we have recently developed a new class of Mcl-1 inhibitors with novel chemical structures. The lead compound, 483, is highly selective for Mcl-1 and has potent activity against Mcl-1-mediated drug resistance in CRC cells. It also exhibited promising pharmacological properties and completely overcame Mcl-1-mediated drug resistance in vivo. Based on these findings, we propose to test the hypothesis that the 483 class of Mcl-1 inhibitors can be further developed to improve CRC therapy by targeting drug-resistant CRC cells. Aim 1: Design and synthesis of novel Mcl-1 inhibitors with improved activity and properties; Aim 2: Biophysical, biochemical, structural and functional characterization of novel Mcl-1 inhibitors; Aim 3: Determine the efficacy and mechanism of action of novel Mcl-1 inhibitors in CRC cells; Aim 4. Analyze the pharmacological properties and in vivo efficacy of novel Mcl-1 inhibitors. These studies may provide a major advance towards our long-term goal of developing a novel class of therapy for human cancer by targeting Mcl-1.