Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (‘fuzzy’ binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Pharmacologic expansion of endogenous β cells is a promising therapeutic strategy for diabetes. To elucidate the molecular pathways that control β-cell growth we screened ∼2400 bioactive compounds for rat β-cell replication-modulating activity. Numerous hit compounds impaired or promoted rat β-cell replication, including CC-401, an advanced clinical candidate previously characterized as a c-Jun N-terminal kinase inhibitor. Surprisingly, CC-401 induced rodent (in vitro and in vivo) and human (in vitro) β-cell replication via dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B inhibition. In contrast to rat β cells, which were broadly growth responsive to compound treatment, human β-cell replication was only consistently induced by DYRK1A/B inhibitors. This effect was enhanced by simultaneous glycogen synthase kinase-3β (GSK-3β) or activin A receptor type II-like kinase/transforming growth factor-β (ALK5/TGF-β) inhibition. Prior work emphasized DYRK1A/B inhibition-dependent activation of nuclear factor of activated T cells (NFAT) as the primary mechanism of human β-cell-replication induction. However, inhibition of NFAT activity had limited effect on CC-401-induced β-cell replication. Consequently, we investigated additional effects of CC-401-dependent DYRK1A/B inhibition. Indeed, CC-401 inhibited DYRK1A-dependent phosphorylation/stabilization of the β-cell-replication inhibitor p27Kip1. Additionally, CC-401 increased expression of numerous replication-promoting genes normally suppressed by the dimerization partner, RB-like, E2F and multivulval class B (DREAM) complex, which depends upon DYRK1A/B activity for integrity, including MYBL2 and FOXM1. In summary, we present a compendium of compounds as a valuable resource for manipulating the signaling pathways that control β-cell replication and leverage a DYRK1A/B inhibitor (CC-401) to expand our understanding of the molecular pathways that control β-cell growth.

Small molecule stimulation of β-cell regeneration has emerged as a promising therapeutic strategy for diabetes. Although chemical inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is sufficient to enhance β-cell replication, current lead compounds have inadequate cellular potency for in vivo application. Herein, we report the clinical stage anti-cancer kinase inhibitor OTS167 as a structurally novel, remarkably potent DYRK1A inhibitor and inducer of human β-cell replication. Unfortunately, OTS167's target promiscuity and cytotoxicity curtails utility. To tailor kinase selectivity towards DYRK1A and reduce cytotoxicity we designed a library of fifty-one OTS 167 derivatives based upon a modeled structure of the DYRK1A-OTS167 complex. Indeed, derivative characterization yielded several leads with exceptional DYRK1A inhibition and human β-cell replication promoting potencies but substantially reduced cytotoxicity. These compounds are the most potent human β-cell replication-promoting compounds yet *

Gene activator proteins comprise distinct DNA‐binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD‐Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (“fuzzy” binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

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