Zinc-chelating BET bromodomain inhibitors equally target islet endocrine cell types.

Inhibition of the bromodomain and extraterminal domain (BET) protein family is a potential strategy to prevent and treat diabetes; however, the clinical use of BET bromodomain inhibitors (BETis) is associated with adverse effects. Here, we explore a strategy for targeting BETis to ß cells by exploiting the high-zinc (Zn2+) concentration in ß cells relative to other cell types. We report the synthesis of a novel, Zn2+-chelating derivative of the pan-BETi (+)-JQ1, (+)-JQ1-DPA, in which (+)-JQ1 was conjugated to dipicolyl amine (DPA). As controls, we synthesized (+)-JQ1-DBA, a non-Zn2+-chelating derivative, and (-)-JQ1-DPA, an inactive enantiomer that chelates Zn2+. Molecular modeling and biophysical assays showed that (+)-JQ1-DPA and (+)-JQ1-DBA retain potent binding to BET bromodomains in vitro. Cellular assays demonstrated (+)-JQ1-DPA attenuated NF-ĸB target gene expression in ß cells stimulated with the proinflammatory cytokine interleukin 1ß. To assess ß-cell selectivity, we isolated islets from a mouse model that expresses green fluorescent protein in insulin-positive ß cells and mTomato in insulin-negative cells (non-ß cells). Surprisingly, Zn2+ chelation did not confer ß-cell selectivity as (+)-JQ1-DPA was equally effective in both ß and α cells; however, (+)-JQ1-DPA was less effective in macrophages, a nonendocrine islet cell type. Intriguingly, the non-Zn2+-chelating derivative (+)-JQ1-DBA displayed the opposite selectivity, with greater effect in macrophages compared with (+)-JQ1-DPA, suggesting potential as a macrophage-targeting molecule. These findings suggest that Zn2+-chelating small molecules confer endocrine cell selectivity rather than ß-cell selectivity in pancreatic islets and provide valuable insights and techniques to assess Zn2+ chelation as an approach to selectively target small molecules to pancreatic ß cells.NEW & NOTEWORTHY Inhibition of BET bromodomains is a novel potential strategy to prevent and treat diabetes mellitus. However, BET inhibitors have negative side effects. We synthesized a BET inhibitor expected to exploit the high zinc concentration in ß cells to accumulate in ß cells. We show our inhibitor targeted pancreatic endocrine cells; however, it was less effective in immune cells. A control inhibitor showed the opposite effect. These findings help us understand how to target specific cells in diabetes treatment.

N-terminal BET bromodomain inhibitors disrupt a BRD4-p65 interaction and reduce inducible nitric oxide synthase transcription in pancreatic ß-cells.

Chronic inflammation of pancreatic islets is a key driver of ß-cell damage that can lead to autoreactivity and the eventual onset of autoimmune diabetes (T1D). In the islet, elevated levels of proinflammatory cytokines induce the transcription of the inducible nitric oxide synthase (iNOS) gene, NOS2, ultimately resulting in increased nitric oxide (NO). Excessive or prolonged exposure to NO causes ß-cell dysfunction and failure associated with defects in mitochondrial respiration. Recent studies showed that inhibition of the bromodomain and extraterminal domain (BET) family of proteins, a druggable class of epigenetic reader proteins, prevents the onset and progression of T1D in the non-obese diabetic mouse model. We hypothesized that BET proteins co-activate transcription of cytokine-induced inflammatory gene targets in ß-cells and that selective, chemotherapeutic inhibition of BET bromodomains could reduce such transcription. Here, we investigated the ability of BET bromodomain small molecule inhibitors to reduce the ß-cell response to the proinflammatory cytokine interleukin 1 beta (IL-1ß). BET bromodomain inhibition attenuated IL-1ß-induced transcription of the inflammatory mediator NOS2 and consequent iNOS protein and NO production. Reduced NOS2 transcription is consistent with inhibition of NF-κB facilitated by disrupting the interaction of a single BET family member, BRD4, with the NF-κB subunit, p65. Using recently reported selective inhibitors of the first and second BET bromodomains, inhibition of only the first bromodomain was necessary to reduce the interaction of BRD4 with p65 in ß-cells. Moreover, inhibition of the first bromodomain was sufficient to mitigate IL-1ß-driven decreases in mitochondrial oxygen consumption rates and ß-cell viability. By identifying a role for the interaction between BRD4 and p65 in controlling the response of ß-cells to proinflammatory cytokines, we provide mechanistic information on how BET bromodomain inhibition can decrease inflammation. These studies also support the potential therapeutic application of more selective BET bromodomain inhibitors in attenuating ß-cell inflammation.

Human induced pluripotent stem cell derived hepatocytes provide insights on parenteral nutrition associated cholestasis in the immature liver.

Parenteral nutrition-associated cholestasis (PNAC) significantly limits the safety of intravenous parenteral nutrition (PN). Critically ill infants are highly vulnerable to PNAC-related morbidity and mortality, however the impact of hepatic immaturity on PNAC is poorly understood. We examined developmental differences between fetal/infant and adult livers, and used human induced pluripotent stem cell-derived hepatocyte-like cells (iHLC) to gain insights into the contribution of development to altered sterol metabolism and PNAC. We used RNA-sequencing and computational techniques to compare gene expression patterns in human fetal/infant livers, adult liver, and iHLC. We identified distinct gene expression profiles between the human feta/infant livers compared to adult liver, and close resemblance of iHLC to human developing livers. Compared to adult, both developing livers and iHLC had significant downregulation of xenobiotic, bile acid, and fatty acid metabolism; and lower expression of the sterol metabolizing gene ABCG8. When challenged with stigmasterol, a plant sterol found in intravenous soy lipids, lipid accumulation was significantly higher in iHLC compared to adult-derived HepG2 cells. Our findings provide insights into altered bile acid and lipid metabolizing processes in the immature human liver, and support the use of iHLC as a relevant model system of developing liver to study lipid metabolism and PNAC.

FoxO1 and FoxA1/2 form a complex on DNA and cooperate to open chromatin at insulin-regulated genes.

We have previously shown that cooperative, interdependent binding by the pioneer factors FoxO1 and FoxA1/2 is required for recruitment of RNA polymerase II and H3K27 acetylation to the promoters of insulin-regulated genes. However, the underlying mechanisms are unknown. In this study, we demonstrate that, in HepG2 cells, FoxO1 and FoxA2 form a complex on DNA that is disrupted by insulin treatment. Insulin-mediated phosphorylation of FoxO1 and FoxA2 does not impair their cooperative binding to mononucleosome particles assembled from the IGFBP1 promoter, indicating that direct disruption of complex formation by phosphorylation is not responsible for the loss of interdependent FoxO1:FoxA1/2 binding following insulin treatment. Since FoxO1 and FoxA1/2 binding is required for the establishment and maintenance of transcriptionally active chromatin at insulin-regulated genes, we hypothesized that cooperative FoxO1 and FoxA1/2 binding dictates the chromatin remodeling events required for the initial activation of these genes. In support of this idea, we demonstrate that FoxO1 and FoxA2 cooperatively open linker histone compacted chromatin templates containing the IGFBP1 promoter. Taken together, these results provide a mechanism for how interdependent FoxO1:FoxA1/2 binding is negatively impacted by insulin and provide a developmental context for cooperative gene activation by these factors.

Spinal muscular atrophy astrocytes exhibit abnormal calcium regulation and reduced growth factor production.

Spinal muscular atrophy (SMA) is a genetic disorder caused by the deletion of the survival motor neuron 1 (SMN1) gene that leads to loss of motor neurons in the spinal cord. Although motor neurons are selectively lost during SMA pathology, selective replacement of SMN in motor neurons does not lead to full rescue in mouse models. Due to the ubiquitous expression of SMN, it is likely that other cell types besides motor neurons are affected by its disruption and therefore may contribute to disease pathology. Here we show that astrocytes in SMAΔ7 mouse spinal cord and from SMA-induced pluripotent stem cells exhibit morphological and cellular changes indicative of activation before overt motor neuron loss. Furthermore, our in vitro studies show mis-regulation of basal calcium and decreased response to adenosine triphosphate stimulation indicating abnormal astrocyte function. Together, for the first time, these data show early disruptions in astrocytes that may contribute to SMA disease pathology.