This is a competing renewal for R-01 DK 105015, which ran for four years from 3/01/16 to 2/29/20. The project focuses on developing drugs that induce human beta cells to regenerate for people with Type 1 and Type 2 diabetes (T1D and T2D), both of which ultimately result from inadequate numbers of normally functioning beta cells. Small molecule drugs that inhibit the kinase, DYRK1A, such as harmine and others, are very reproducibly able to induce adult human beta cells to replicate, but at low rates (~2%/day). More recently, we have shown that adding any small molecule DYRK1A inhibitor to either a TGF-beta superfamily inhibitor (TGFβI’s) or to a GLP1 receptor agonist (GLP1RA), such as GLP1 or exendin-4, markedly enhances beta cell proliferation to rates averaging 5- 8%/day, and that this translates to marked increases in actual numbers and mass of human beta cells in vivo, when transplanted into NOD-SCID-gamma (NSG) mice. Thus, during the period of this grant, through the work of ourselves and others, the field of human beta cell regeneration for T1D and T2D has transitioned from impossible to clearly possible. In this proposed renewal, we seek to learn why some DYRK1A inhibitors - but not others - are able to enhance human beta cell differentiation and function, while also driving beta cell proliferation. Maintaining beta cell differentiation will be an essential feature of any beta cell regenerative drug. We expect to identify the underlying signaling pathway(s) that explain this surprising and very positive effect. We also will explore the efficacy of DYRK1A inhibitors, in combination with GLP1RA’s, in essential but previously unexplored animal models of T1D and T2D, strengthening pre-clinical relevance. Finally, using sophisticated medicinal chemistry approaches, we have identified, synthesized and patented entirely novel and extremely potent human beta cell regenerative DYRK1A inhibitors. We now need to optimize, using computational predictions and advanced medicinal chemical syntheses, cleavable linkers designed to attach our next-generation potent and selective human beta cell DYRK1A inhibitors to potential beta cell-targeting molecules. Thus, the Specific Aims of this application are to: 1. Decipher Mechanisms Underlying the Beneficial Pro-Differentiation Effects of Select DYRK1A Inhibitors. 2. Define in vivo Efficacy of DYRK1A inhibition in Type 1 and Type 2 Diabetes Models. 3. Develop Linkers to Novel Classes of DYRK1A Inhibitors for Targeted Delivery to Beta Cells. These goals are both achievable and directly responsive to the goals of aims of the NIDDK. They provide a realistic basis for generating a new and urgently needed class of therapeutics for T1D and T2D.