Abstract
The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra-BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent with selective suppression of BRD4. Our discovery opens up new opportunities to elucidate the cellular phenotypes and therapeutic implications associated with selective targeting of BRD4.
Highlights
The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1
The Bromo- and Extra-terminal (BET) family of proteins, including the ubiquitously expressed BRD2, BRD3, and BRD4 and the testis-specific BRDT, recruit transcriptional regulatory complexes to acetylated chromatin thereby controlling specific networks of genes involved in cellular proliferation and cell cycle progression.[1]
RNAi screens have identified BRD4 as a therapeutic target in acute myeloid leukemia,[3] ovarian carcinoma,[4] and siRNA knock down of BRD4, but not of BRD2 or BRD3, induced upregulation of apolipoprotein A1 (ApoA1), which protects from atherosclerosis progression and other inflammatory processes.[5]
Summary
Bromodomain inhibitors JQ1 and I-BET762 and binders of von Hippel-Lindau protein VHL-1 and VHL-2. (b) Scheme of the synthesis of PROTAC compounds MZ1−3 and cisMZ1; for detailed synthetic procedures see the Supporting Information. (c) Isothermal titration calorimetry data for titration of MZ1 into the individual members of the BET-bromodomain subfamily. To study the activities of our PROTACs over time, HeLa cells were treated with 1 μM or 0.1 μM MZ1, and cellular BET protein levels were monitored in a time course experiment (Figure 2b for representative data with MZ1, see Figure S2 for additional data with other compounds and concentrations). The washed cells showed detectable recovery of intracellular BRD4 only by 20 h after washout, while in the absence of the wash step, no protein could be detected even after 48 h (Figure 3e, see Figure S7 for the same experiment monitoring time-dependent levels of BRD2 and BRD3) Taken together, these results demonstrate that PROTAC-induced protein degradation is strictly dependent on binding to VHL, on proteasome activity, and does not interfere with the normal endogenous levels of both VHL and HIF-1α. Potent chemical probes that bind to human bromodomains outside the BET subfamily are beginning to emerge,[37] which could be conjugated to a VHL ligand to induce selective intracellular degradation of their respective target bromodomain-containing proteins
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