3-Aminophthalic acid, a new cereblon ligand for targeted protein degradation by O'PROTAC

Abstract

In this study, we identified 3-aminophthalic acid as a new ligand of cereblon (CRBN) E3 ubiquitin ligase and developed a phthalic acid-based O'PROTAC for degradation of the ERG transcription factor. This phthalic acid-based O'PROTAC presented an efficacy in degrading ERG comparable to those displayed by pomalidomide-based ERG O'PROTACs. Moreover, phthalic acid-being more chemically stable and economical than classical immunomodulatory drugs (IMiDs)-represents, as a ligand, a new alternative for the development of PROTACs, especially O'PROTACs.

Fig.1

Phthalic acid-based ERG O’PROTAC degrades ERG oncoprotein. (A) The linear structure of ERG OP-C-P1. (B) The VCaP cells were transfected with control or four indicated ERG O’PROTACs at a final concentration of 100 nM and harvested for western blot analysis. (C and D) The VCaP cells were transfected with increasing concentrations of ERG OP-C-P1 for 36 h and treated with 20 μg/ml of cycloheximide (CHX) for another 12 h. Cells were harvested for western blot analysis of ERG protein expression (C). The remaining ERG protein (%) was calculated by normalizing the value of each group to that of the group without ERG OP-C-P1 treatment. The concentration of ERG OP-C-P1 degrading 50% of ERG protein (DC50) was calculated with Prism software in (D).

Fig.2

Phthalic acid-based ERG OP degrades ERG via CRBN and the proteasome pathway. (A) The VCaP cells were transfected with a final concentration of 100 nM of control OP or ERG OP-C-P1 for 36 h and treated with or without MG132 (20 μM) for another 12 h before harvested for western blot analysis. (B) The VCaP cells were transfected with the indicated plasmids and ERG OP-C-P1 at a final concentration of 100 nM for 36 h and treated with the proteasome inhibitor MG132 (20 μM) for 12 h before harvested for protein extraction. ERG protein was immunoprecipitated with ERG antibody by protein A/G beads to detect its ubiquitination level by western blot analysis. (C) The VCaP cells were transfected with a final concentration of 100 nM of control OP or ERG OP-C-P1 and siRNA control (siNS) or siCRBN for 48 h before harvested for western blot analysis. (D) The VCaP cells were transfected with control OP or ERG OP-C-P1 at a final concentration of 100 nm and incubated with 1-, 10-, or 50-fold of CRBN ligand pomalidomide for 36 h, followed by western blot analysis of ERG protein level.

Fig.3

Docking of CRBN binding of thalidomide (PDB:4CI1) (A) and 3-N-substituted- aminophthalic acid (B). Dotted black lines represent hydrogen bond and dotted cyan lines represent pi-pi interaction.

Fig.4

Phthalic acid-based ERG OP inhibits ERG target gene expression and prostate cancer cell growth and invasion. (A and B) The VCaP cells were embedded in matrigel and cultured for 5 days, followed by the treatment of 200 nM of control OP or ERG OP-C-P1 for another five days. The representative images with three-dimension (3D) spheres are shown in (A) and the quantified diameters of 3D spheres are shown in (B). Data are demonstrated with box and whiskers; whiskers represent min to max, and each point is one value of an individual 3D sphere (n = 50). The P value was determined using the unpaired two-tailed Student’s t-test; *** P < 0.001. (C and D) The 22Rv1 cells were transfected with pCMV-HA-ERG and 100 nM of control OP or ERG OP-C-P1, followed by plating on matrigel-coated chambers and incubating for 48 h in 37 °C incubator. The invaded cells were stained with 0.5% of crystal violet. The representative fields are shown in (C) and the quantification data are shown in (D). Data represents means ± SD (n = 4). The P value was determined using the unpaired two-tailed Student’s t-test. ** P < 0.01.