Hijacking the MDM2 E3 Ligase with Novel BRD4-Targeting Proteolysis-Targeting Chimeras in Pancreatic Cancer Cells

Abstract

The phenotypic effect induced by a Proteolysis-Targeting Chimera (PROTAC) can depend on several factors, including the E3 ligase recruited. For the discovery of a first-in-class PROTAC for a target of interest, the E3 ligases commonly hijacked remain the Von Hippel-Lindau (VHL) and Cereblon (CRBN) since potent and accessible ligands are readily available to recruit them. Mouse double minute 2 (MDM2) E3 ligase stands out because it regulates p53 levels to maintain cellular homeostasis. However, the synthesis of the most potent MDM2 ligands remains very complex. Herein, the discovery of novel MDM2-recruiting PROTACs incorporating rac-Nutlin-3 as a ligand with an easier synthetic tractability is reported, further demonstrating its potential in this technology. The most promising degrader, PROTAC 3, showed preferential degradation of the BRD4 short isoform (BRD4 S) and c-Myc compared with MZ1, a validated VHL-based PROTAC.

Keywords: BRD4; HDM2; MDM2; MIA PaCa‐2; PROTACs; pancreatic cancer; targeted protein degradation.

Figure 1

Chemical structures of: A) representative MDM2 inhibitors,^{[ 26} , ^{37 ]} B) previously reported MDM2‐recruiting PROTACs,^{[ 27} , ²⁸ , ^{29 ]} and C) the library of MDM2‐recruiting PROTACs described in this work.

Scheme 1

Synthesis of intermediates 28–30 and 31–33. Reagents and conditions: a) K₂CO₃, ⁱPrBr, Bu₄NBr, THF, 70°C, 24 h, 69%; b) NBS, DCM, 0°C–rt, 16 h, 44–82%; c) NaH, methyl bromoacetate, THF, 0°C–rt, 18 h, 80–96%; d) 4 M HCl/1,4‐dioxane, rt, 18 h, 65–99%; e) triphosgene, Et₃N, DCM, 0°C–rt, 18 h; f) 13, DCM, rt, 2–18 h, 71–88% (over 2 steps); g) LiOH•H₂O, THF, MeOH, H₂O, rt, 18 h, 44%–quant.; h) corresponding amine 16–21, HATU, DIPEA, DCM, DMF, rt, 18–60 h, 30–58%; i) 4 M HCl/1,4‐dioxane, 0°C, 1–7 h, 67%–quant.

Scheme 2

Synthesis of MDM2‐based PROTACs 1–6. Reagents and conditions: a) HCOOH, rt, 48 h, 96%; b) HATU, DIPEA, DMF, rt, 1–16 h, 14–43%.

Figure 2

Novel MDM2‐BRD4 PROTACs gave a more pronounced effect on BRD4 S and c‐Myc than MZ1. A) MIA PaCa‐2 cells were treated for 24 h with selected PROTAC 3, MZ1, and cisMZ1 at the indicated concentrations. The increase in BRD4 levels at lower concentrations of PROTAC 3 (0.5–1 μM) could potentially indicate that PROTAC 3 behaves initially as an inhibitor. This feedback is precedented for (+)‐JQ1 and other inhibitors.^{[ 38} , ³⁹ , ^{40 ]} B) Quantified data are shown as mean ± SD from three independent biological replicates (quantified with ImageJ and analyzed in GraphPad Prism 8.2.0, n = 3, except PROTAC 3 at 0.5 μM, n = 2). The value for each condition was divided by the loading control, then the result was normalized against the left DMSO value to obtain the % remaining of the corresponding protein. *The third band on the BRD4 membrane could indicate proteolytic cleavage of BRD4.