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

Abstract

Recently, our group identified that harmine is able to induce β-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 β-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 β-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 β-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.

Human Beta Cell Mass Expansion In Vivo With A Harmine and Exendin-4 Combination: Quantification and Visualization By iDISCO+ 3D Imaging

“…Chemicals. Harmine-HCl was synthesized in the Drug Discovery Institute at Mount Sinai (15,16). Exendin-4 was purchased from MedChemExpress (Monmouth Junction, NJ).…”

Structure–Activity Relationships and Biological Evaluation of 7-Substituted Harmine Analogs for Human β-Cell Proliferation

“…Interestingly, other analogs with longer carbon lengths did not provide any improvement for DYRK1A inhibition, a result that did not align with our original docking hypothesis, which indicated that extension into solvent would be possible. Subsequent docking studies of these novel 7-C harmine analogs based on the crystal structure of DYRK1A bound to harmine analog 2-2c [39] were performed and suggested that the loss of activity with longer substituents at the 7-position could be due to interplay of several factors that diminish interactions with the DYRK1A back bone H-bond at Leu 241. These include steric hindrance, the entropic effects of longer, more flexible side chains, combined with the possibility of partially compensating additional new contacts ( Figure 3A, subpanel A-F).…”

“…After assessing the effect of these modifications on DYRK1A inhibition, we next explored the structure-activity relationships for in vitro β-cell proliferation activity of the most potent 7-O-substituted DYRK1A inhibitor harmine analogs. Compounds 1-2b and 1-3b, with DYRK1A inhibitory activities (IC 50 ) of 89 and 91 nM, respectively, were studied as previously reported [5,39,[43][44][45], for their ability to induce human β-cell proliferation in vitro as assessed by Ki-67-insulin co-immunolabeling after 4 days, our standard assay protocol [49] ( Figure 4A). Both the harmine analogues, 1-2b and 1-3b exhibited human β-cell proliferation at 10 µM ( Figure 4A).…”

“…We first determined the effect of these modifications on DYRK1A inhibition, measured by FRET-based LanthaScreen ® Eu Kinase Binding Assay [42]. Since the overall goal of the project is to develop DYRK1A inhibitors that can induce β-cells to proliferate, we also assayed potent 7-substituted harmine analogs for human β-cell proliferation by quantifying Ki67 immunolabeling in beta cells [5,39,[43][44][45].…”

“…Previously, we reported the optimization of the 1-position of harmine which led to two compounds with robust human β-cell proliferation at doses of 3-30 µ M and one of those, compound I (R = CH2OH), showing improved kinase selectivity as compared to harmine ( Figure 1) [38]. Next, we recently reported the optimization of the N-9 position to identify 2-2c, a highly optimized in vivo active harmine based DYRK1A inhibitor (Figure 1) [39]. To extend this systematic medicinal chemistry strategy, we last focused our effort on the harmine 7-position to understand the impact of modifications on DYRK1A inhibition and β-cell proliferation (Figure 1).…”