Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics

Abstract

We have developed a heterobifunctional all-small molecule PROTAC (PROteolysis TArgeting Chimera) capable of inducing proteasomal degradation of the androgen receptor. This cell permeable PROTAC consists of a non-steroidal androgen receptor ligand (SARM) and the MDM2 ligand known as nutlin, connected by a PEG-based linker. The SARM-nutlin PROTAC recruits the androgen receptor to MDM2, which functions as an E3 ubiquitin ligase. This leads to the ubiquitination of the androgen receptor, and its subsequent degradation by the proteasome. Upon treatment of HeLa cells with 10microM PROTAC for 7h, we were able to observe a decrease in androgen receptor levels. This degradation is proteasome dependent, as it is mitigated in cells pre-treated with 10microM epoxomicin, a specific proteasome inhibitor. These results have implications for the potential study and treatment of various cancers with increased androgen receptor levels.

Figure 1

PROTAC mechanism of action: The heterobifunctional PROTAC molecule contains two binding motifs: one recruits an E3 ligase, and another recruits the target protein. Following binding to the PROTAC, the E3 ligase catalyzes the synthesis of a polyubiquitin chain on the target, leading to its recognition by the 26S proteasome, and subsequent target protein degradation.

Figure 2

The SARM-nutlin PROTAC effectively degrades the androgen receptor (AR) in vivo. HeLa cells transiently expressing AR were treated with either vehicle, 10 μM PROTAC 14, or pre-treated with 10 μM epoxomicin and then 10 μM PROTAC, for 7h. Cells were then lysed and separated by SDS-PAGE. The total level of AR was detected by Western blotting using an anti-AR antibody (top). As a loading control, the same membrane was probed using an anti-α-tubulin antibody (bottom).

Scheme 1

Synthesis of nutlin derivative 4 Reagents and conditions: (a) 2, CH₂Cl₂, 0°C, 2h, then NBS, 0°C-rt, 16h, 88%; (b) Triphosgene, Et₃N, THF, 0°C, 2.5h; (c) 8, CH₂Cl₂, 0°C, 1.5h, 96%; (d) TFA, CH₂Cl₂, 96%; (e) 5, NaH, DMF, 0°C, 30min, then 6, rt, 4h; (f) H₂, 10% Pd/C, MeOH, rt, 16h

Scheme 2

Synthesis of derivatized PEG linker 11 Reagents and conditions: (a) NaH, DMF, 0°C, 30 min., then NaIAc, rt, 32h; (b) CHCl₂, 4-aminophenol hydrochloride, HOBt, DIPEA, rt, then 0°C, EDCI, then rt, 18h

Scheme 3

Synthesis of SARM-nutlin PROTAC 14 Reagents and conditions: (a) (i) K₂CO₃, 2-PrOH, 85°C; (ii) PPh₃, H₂O, THF, rt; (b) 4, HATU, DIPEA, DMF, rt