Abstract
Small molecule inhibitors of dual specificity, tyrosine phosphorylation-regulated kinase 1A (DYRK1A), including harmine and others, are able to drive human β cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only or the most important target is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human β cell RNA sequencing, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B. Simultaneous silencing of both DYRK1A and DYRK1B yields greater β cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to 7 targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human β cell proliferation, and we provide clarity for future efforts in structure-based drug design for human β cell regenerative drugs.
Highlights
Diabetes results from inadequate numbers of normally functioning insulin-producing pancreatic β cells [1,2,3,4]
We explored the abundance of these kinases in human β cells via our RNA sequencing (RNA-seq) database of FACS-sorted human β cell (Table 1 and refs. 23–25) and found that, in human β cells, each of the DYRK, CLK, and GSK3 family members is present, as are CSNK1A, CSNK1D, and CSNK1E, all in comparable abundance with other important kinases such as PI3 kinase, MAP kinase, and Jun kinase
We developed a panel of small molecule inhibitors of the potential dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor targets in Table 1 and tested their ability to induce human β cells to replicate, as assessed by insulin and Ki67 coimmunolabeling of dispersed cadaveric human islets treated with these drugs
Summary
Diabetes results from inadequate numbers of normally functioning insulin-producing pancreatic β cells [1,2,3,4]. Kinome screens performed by multiple groups reveal that each of these DYRK1A inhibitors inhibit other kinases, members of the CMGC (cyclin-dependent kinase [CDK], mitogen-activated protein [MAP] kinase, glycogen synthase kinase-3 [GSK3], and CDC-like kinase [CLK]) classes — notably DYRK1B, DYRK2, DYRK3, DYRK4, CLK1, CLK2, CLK4, GSK3α, GSK3β, and casein kinases (CSNK) 1A, 1D, and E [7,8,9,10,11,12,13]. Each of these could, in theory, participate in human β cell proliferation.
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