Zinc-Chelating BET Bromodomain Inhibitors Selectively Accumulate and Affect Gene Expression in Pancreatic beta-Cells

The islets of Langerhans are a heterogeneous mixture of endocrine (hormone-secreting) cells in the mammalian pancreas. Of particular interest are the β-cells responsible for maintaining glucose homeostasis by secreting insulin in response to increased blood glucose. Type 1 diabetes (T1D) is characterized by autoimmune-mediated destruction of β-cells, resulting in an inability to produce and release insulin in response to changing glucose levels and subsequent reliance on daily insulin injections. There is currently no cure for T1D. The bromodomain and extra-terminal domain (BET) family of proteins, comprised of BRD2, BRD3, BRD4 and BRDT, are a druggable family of proteins that can be targeted by pan-BET bromodomain inhibitors (BETi), including the small molecule (+)-JQ1. Recent studies have shown that targeting the BET family of proteins with BETi may prevent and treat T1D. However, the systemic use of current generation BETi is associated with off-target, adverse effects including fatigue, gastrointestinal toxicity and memory loss. Since any treatment for T1D would be widely used in a pediatric patient population, avoiding off-target effects is imperative. Here, we propose to ameliorate the off-target effects of BET bromodomain inhibition by targeting BETi directly to pancreatic β-cells. To achieve this, we exploited the high concentration of Zn ions (~20 mM) present in β-cells relative to other cells in the body. We report the synthesis of a novel, zinc-chelating analogue of (+)-JQ1, [(+)-JQ1-DPA], in which the BETi (+)-JQ1 was conjugated, via an ethylenediamine linker, to dipicolyl amine (DPA). DPA chelates Zn and thus enables (+)-JQ1 to accumulate in β-cells. As control compounds we also synthesized (+)-JQ1-DBA, a non-zinc-chelating analogue of (+)-JQ1-DPA, in which (+)-JQ1 was conjugated to dibenzyl amine (DBA) and (-)-JQ1-DPA, a zinc-chelating compound that is inactive as a BETi. Molecular modeling and biophysical assays showed that (+)-JQ1-DPA and (+)-JQ1-DBA, but not (-)-JQ1-DPA, retain binding to both bromodomains of BRD4 in vitro. Cellular assays demonstrated that (+)-JQ1-DPA retains efficacy; in particular, (+)-JQ1-DPA, attenuated the expression of NF-ĸB target genes in β-cells stimulated with the pro-inflammatory cytokine, IL-1β, a model for β-cell destruction. Finally, using islets isolated from the INS1 ; ROSA26 mouse, an established mouse model that expresses enhanced green fluorescent protein in insulin-positive cells and mTomato in insulin-negative cells, we demonstrated that (+)-JQ1-DPA affects gene transcription in pancreatic β-cells but not in other pancreatic cell types. In conclusion, we have synthesized a zinc-chelating analogue of (+)-JQ1 that is selectively active in pancreatic β-cells over other islet cell types, including macrophages and α-cells.