ABSTRACT
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.
Subject(s)
Harmine/analogs & derivatives , Harmine/pharmacology , Insulin-Secreting Cells/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Cell Proliferation/drug effects , Cells, Cultured , Harmine/chemical synthesis , Humans , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Male , Mice, Inbred C57BL , Molecular Docking Simulation , Nervous System/drug effects , Nervous System/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Rats, Wistar , Dyrk KinasesABSTRACT
Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) induce human beta cells to proliferate, generating a labeling index of 1.5%-3%. Here, we demonstrate that combined pharmacologic inhibition of DYRK1A and transforming growth factor beta superfamily (TGFßSF)/SMAD signaling generates remarkable further synergistic increases in human beta cell proliferation (average labeling index, 5%-8%, and as high as 15%-18%), and increases in both mouse and human beta cell numbers. This synergy reflects activation of cyclins and cdks by DYRK1A inhibition, accompanied by simultaneous reductions in key cell-cycle inhibitors (CDKN1C and CDKN1A). The latter results from interference with the basal Trithorax- and SMAD-mediated transactivation of CDKN1C and CDKN1A. Notably, combined DYRK1A and TGFß inhibition allows preservation of beta cell differentiated function. These beneficial effects extend from normal human beta cells and stem cell-derived human beta cells to those from people with type 2 diabetes, and occur both in vitro and in vivo.
Subject(s)
Diabetes Mellitus, Type 2 , Harmine/pharmacology , Insulin-Secreting Cells , Monoamine Oxidase Inhibitors/pharmacology , Transforming Growth Factor beta/antagonists & inhibitors , Adolescent , Adult , Aged , Animals , Cell Line , Cell Proliferation , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Female , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Humans , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/pathology , Male , Mice , Mice, Inbred C57BL , Middle Aged , Myeloid-Lymphoid Leukemia Protein/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Smad Proteins/antagonists & inhibitors , Stem Cells , Young Adult , Dyrk KinasesABSTRACT
Acetylcholinesterase (AChE) that has been covalently inhibited by organophosphate compounds (OPCs), such as nerve agents and pesticides, has traditionally been reactivated by using nucleophilic oximes. There is, however, a clearly recognized need for new classes of compounds with the ability to reactivate inhibited AChE with improved in vivo efficacy. Here we describe our discovery of new functional groups--Mannich phenols and general bases--that are capable of reactivating OPC--inhibited AChE more efficiently than standard oximes and we describe the cooperative mechanism by which these functionalities are delivered to the active site. These discoveries, supported by preliminary in vivo results and crystallographic data, significantly broaden the available approaches for reactivation of AChE.
