Hybrid Enzalutamide Derivatives with Histone Deacetylase Inhibitor Activity Decrease Heat Shock Protein 90 and Androgen Receptor Levels and Inhibit Viability in Enzalutamide-Resistant C4-2 Prostate Cancer Cells

Abstract

Histone deacetylase inhibitors (HDACIs) can disrupt the viability of prostate cancer (PCa) cells through modulation of the cytosolic androgen receptor (AR) chaperone protein heat shock protein 90 (HSP90). However, toxicities associated with their pleiotropic effects could contribute to the ineffectiveness of HDACIs in PCa treatment. We designed hybrid molecules containing partial chemical scaffolds of enzalutamide and suberoylanilide hydroxamic acid (SAHA), with weakened intrinsic pan-HDACI activities, to target HSP90 and AR in enzalutamideresistant PCa cells. The potency of the new molecules, compounds 2-75 [4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide] and 1005 [(E)-3-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluorophenyl)-N-hydroxyacrylamide], as inhibitors of nuclear and cytosolic histone deacetylases was substantially lower than that of SAHA in cell-free and in situ assays. Compounds 2-75 and 1005 antagonized gene activation by androgen without inducing chromatin association of AR. Enzalutamide had no effect on the levels of AR or HSP90, whereas the hybrid compounds induced degradation of both AR and HSP90, similar to (compound 1005) or more potently than (compound 2-75) SAHA. Similar to SAHA, compounds 2-75 and 1005 decreased the level of HSP90 and induced acetylation in a predicted approximately 55 kDa HSP90 fragment. Compared with SAHA, compound 2-75 induced greater hyperacetylation of the HDAC6 substrate a-tubulin. In contrast with SAHA, neither hybrid molecule caused substantial hyperacetylation of histones H3 and H4. Compounds 2-75 and 1005 induced p21 and caused loss of viability in the enzalutamide-resistant C4-2 cells, with efficacies that were comparable to or better than SAHA. The results suggest the potential of the new compounds as prototype antitumor drugs that would downregulate HSP90 and AR in enzalutamide-resistant PCa cells with weakened effects on nuclear HDACI targets.

Targeting prostate cancer cells with enzalutamide‐HDAC inhibitor hybrid drug 2‐75

“…There, AR binds to the androgen response element (ARE) to initiate transcription of target genes . As an Enz analog, 2‐75 was shown to reduce the expression of AR‐modulated genes and the recruitment of AR to ARE . In this study, we conducted ARE‐luciferase assay to compare the inhibitory functions of 2‐75 and its analogs 2 and 3 on AR transcriptional activity, and used a receptor translocation assay to define 2‐75 ’s effects on AR localization.…”

“…Converting cyano group to amide abrogated AR antagonist activities of 2 and 3 (Figure A), supporting the ineffective binding of 2 and 3 to AR. Compound 10 (Figure S1), the methyl ester analog of 2‐75 , also inhibited AR‐driven luciferase activity, however, with lower potency reflected by the higher IC 50 at 1.89 µM (graph not is shown). This finding suggests that the hydroxamic acid moiety of 2‐75 contributed to AR antagonist activity.…”

“…The primary antibodies against acetyl‐histone H3 (06‐599) and acetyl‐histone H4 (06‐598) were purchased from Millipore Sigma‐Aldrich (Burlington, MA); acetyl‐α‐Tubulin (5335), horseradish peroxidase‐conjugated anti‐rabbit (7074) or anti‐mouse (7076) antibodies were from Cell Signaling Technology (Danvers, MA); AR (sc‐816), Hsp70 (sc‐59569), Hsp90 (sc‐7947), glyceraldehyde 3‐phosphate dehydrogenase (sc‐25778), HER2 (sc‐23684), and c‐Myc (sc‐40) antibodies were from Santa Cruz Biotechnology (Dallas, TX); AR‐V7 antibody (AG10008) was from Precision Antibody (Columbia, MD); 5α‐dihydrotestosterone (DHT) was from Sigma‐Aldrich; luciferase assay kit (E2520) was from Promega (Madison, WI); JC‐1 mitochondria membrane potential assay kit (10009172) was from Cayman (Ann Arbor, MI); and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining kit was from Roche (Basel, Switzerland). 2‐75 was prepared as described previously . The synthesis of 2 and 3 was described in detail in Figure S1.…”

“…To address this need, we recently designed a class of multifunctional anti‐PCa agents by attaching HDACi pharmacophore to the Enz scaffold, forming Enz‐HDACi hybrid drugs, exemplified by compounds 2‐75 (Figure ) and 1005 , and demonstrated their AR antagonistic and FL AR downregulating properties in the androgen‐independent PCa C4‐2 cell line . In the present study, we further investigated molecular and cellular actions of 2‐75 and tested its anti‐PCa effects in vivo.…”

Chimeric HDAC inhibitors: Comprehensive review on the HDAC‐based strategies developed to combat cancer

“…On another aspect, it was found that the inhibition of HDAC‐6 leads to hyperacetylation of HSP‐90 that leads to the disturbance in the stability, nuclear localization, and activation of ARs. These findings suggested the synergistic effect between the antiandrogens and HDAC inhibitors . This directed Gryder et al .…”