Abstract The dysfunction and death of pancreatic β cells are key features in all types of diabetes. It was recently shown that increased proinsulin misfolding occurs well before the onset of diabetes and is responsible for events, including endoplasmic reticulum (ER) stress, leading to β-cell dysfunction and death in diabetes. There is currently no interventional means that suppresses proinsulin misfolding. In our preliminary studies, we identified a small molecule (named as PTTD) that protects β-cells from ER stress-induced death in a high- throughput screen. We discovered that PTTD suppressed the ER stress-induced activation of all three branches (IRE1, PERK, and ATF6) of unfolded protein response (UPR) pathways in β-cells under ER stress. We then observed that PTTD eliminated the accumulation of misfolded proinsulin while increasing mature insulin production in β-cells and that PTTD suppressed purified insulin protein misfolding/aggregation in cell- free biochemical assays. Importantly, in in vivo animal studies, PTTD significantly ameliorated hyperglycemia in multiple mouse diabetes models of β-cell failure. These exciting results demonstrate that suppression of proinsulin misfolding by PTTD protects β-cells and ameliorates diabetes. In this application, we will use PTTD as the starting molecule to develop potent analogs as first-in-class proinsulin misfolding inhibitors. To achieve this, we will use an approach that integrates iterative and parallel medicinal chemistry with in vitro and in vivo efficacy and DMPK studies with specific aims. In Aim 1, we will improve and optimize our lead compound, PTTD, through medicinal chemistry-based structure-activity relationship studies. In Aim 2, compounds with improved potency will be characterized physicochemically and pharmacologically using standardized ADMET and in vivo PK assays. In Aim 3, we will evaluate the in vivo efficacy of lead PTTD analogs in two well- established diabetes models of proinsulin misfolding and progressive β cell loss. Completion of this work will not only identify PTTD and its analogs as first-in-class chemical suppressors of proinsulin misfolding for β-cell protection, but also establish the suppression of proinsulin misfolding as a new therapeutic direction for diabetes, which will serve as a foundation and provide a lead compound that may guide further development of proinsulin folding therapeutics.