Synthesis and Biological Validation of a Harmine-Based, Central Nervous System (CNS)-Avoidant, Selective, Human ß-Cell Regenerative Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase A (DYRK1A) Inhibitor.

Recently, our group identified that harmine is able to induce ß-cell proliferation both in vitro and in vivo, mediated via the DYRK1A-NFAT pathway. Since, harmine suffers from a lack of selectivity, both against other kinases and CNS off-targets, we therefore sought to expand structure-activity relationships for harmine's DYRK1A activity, to enhance selectivity for off-targets while retaining human ß-cell proliferation activity. We carried out optimization of the 9-N-position of harmine to synthesize 29 harmine-based analogs. Several novel inhibitors showed excellent DYRK1A inhibition and human ß-cell proliferation capability. An optimized DYRK1A inhibitor, 2-2c, was identified as a novel, efficacious in vivo lead candidate. 2-2c also demonstrates improved selectivity for kinases and CNS off-targets, as well as in vivo efficacy for ß-cell proliferation and regeneration at lower doses than harmine. Collectively, these findings demonstrate that 2-2c is a much improved in vivo lead candidate as compared to harmine for the treatment of diabetes.

Cell-Based Methods to Identify Inducers of Human Pancreatic Beta-Cell Proliferation.

Diabetes is the result of the insufficiency or dysfunction of pancreatic beta cells alone or in combination with insulin resistance. The replacement or regeneration of beta cells can effectively reverse diabetes in humans and rodents. Therefore, the identification of novel small molecules that promote pancreatic beta-cell proliferation is an attractive approach for diabetic therapy. While numerous hormones, small molecules, and growth factors are able to drive rodent beta cells to replicate, only a few small molecules have demonstrated the ability to stimulate human beta-cell proliferation. Hence, there is an urgent need for therapeutic agents that induce regeneration and expansion of adult human beta cells. Here, we describe a detailed protocol for coating chamber slides, culturing primary islets, performing islet cell disassociation, seeding cells on chamber slides, treating islet cells with compounds or infecting them with adenovirus, immunostaining of proliferation markers and imaging, and data analysis.

Development of Kinase-Selective, Harmine-Based DYRK1A Inhibitors that Induce Pancreatic Human ß-Cell Proliferation.

DYRK1A has been implicated as an important drug target in various therapeutic areas, including neurological disorders and oncology. DYRK1A has more recently been shown to be involved in pathways regulating human ß-cell proliferation, thus making it a potential therapeutic target for both Type 1 and Type 2 diabetes. Our group, using a high-throughput phenotypic screen, identified harmine that is able to induce ß-cell proliferation both in vitro and in vivo. Since harmine has suboptimal kinase selectivity, we sought to expand structure-activity relationships for harmine's DYRK1A activity, to enhance selectivity, while retaining human ß-cell proliferation capability. We carried out the optimization of the 1-position of harmine and synthesized 15 harmine analogues. Six compounds showed excellent DYRK1A inhibition with IC50 in the range of 49.5-264 nM. Two compounds, 2-2 and 2-8, exhibited excellent human ß-cell proliferation at doses of 3-30 µM, and compound 2-2 showed improved kinase selectivity as compared to harmine.