Bardoxolone conjugation enables targeted protein degradation of BRD4

Abstract

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3's has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.

Figure 1

(A) Targeted protein degradation using bifunctional small molecules. (B) Selected examples of E3 ligase recruiters of varying degrees of covalent engagement.

Figure 2

Bardoxolone-based protein degradation. (A) Synthesis of CDDO–JQ1. (B) Effect of CDDO–JQ1 on BRD4 and KEAP1 levels in 231MFP cells treated with DMSO vehicle or CDDO–JQ1 for 12 h, assessed by Western blotting. (C) BRD4 levels in 231MFP cells pre-treated with vehicle, proteasome inhibitor bortezomib (BTZ) (1 μM), or E1 activating enzyme inhibitor MLN7243 (1 μM) for 30 min prior to treatment with vehicle or CDDO–JQ1 (200 nM) for 12 h. (D) BRD4 levels in 231MFP cells pre-treated with DMSO vehicle or NEDD8 inhibitor MLN4924 (1 μM) for 30 min prior to treatment with DMSO vehicle or CDDO–JQ1 (200 nM) for 12 h. Blots are representative of n = 3 biological replicates/group. Data in bar graphs is expressed as individual replicate values and average ± sem. Significance shown as *p < 0.05 compared to vehicle-treated control groups and ^#p < 0.05 compared to CDDO-JQ1-treated groups.

Figure 3

Degraders derived from unreactive variants of CDDO do not support the degradation of BRD4. (A) Synthesis of H₂-CDDO–JQ1 and 3-oxo-oleanoic acid–JQ1 (3-OOA–JQ1). (B) Effect of CDDO–JQ1, H₂-CDDO–JQ1, and 3-OOA–JQ1degraders on BRD4 and KEAP1 levels in 231MFP cells assessed by Western blotting. DMSO vehicle or compounds were treated at 1 μM for 12 h. Blots are representative of n = 3 biological replicates/group. Data in bar graphs is expressed as individual replicate values and average ± sem. Significance shown as *p < 0.05 compared to vehicle-treated control groups and ^#p < 0.05 compared to CDDO–JQ1-treated groups.

Figure 4

Functional significance of the α-cyanoenone moiety. (A) Synthesis of de-CN-CDDO–JQ1. (B) Effect of CDDO–JQ1 and de-CN-CDDO–JQ1 on BRD4 and GAPDH levels in 231MFP cells assessed by Western blotting. DMSO vehicle or compounds were treated at 1 μM for 12 h. Blots are representative of n = 3 biological replicates/group. Data in bar graphs is expressed as individual replicate values and average ± sem. Significance shown as *p < 0.05 compared to vehicle-treated control groups and ^#p < 0.05 compared to CDDO–JQ1-treated groups.