Human Estrogen Receptor Alpha Antagonists, Part 3: 3-D Pharmacophore and 3-D QSAR Guided Brefeldin A Hit-to-Lead Optimization toward New Breast Cancer Suppressants

Abstract

The estrogen receptor α (ERα) is an important biological target mediating 17β-estradiol driven breast cancer (BC) development. Aiming to develop innovative drugs against BC, either wild-type or mutated ligand-ERα complexes were used as source data to build structure-based 3-D pharmacophore and 3-D QSAR models, afterward used as tools for the virtual screening of National Cancer Institute datasets and hit-to-lead optimization. The procedure identified Brefeldin A (BFA) as hit, then structurally optimized toward twelve new derivatives whose anticancer activity was confirmed both in vitro and in vivo. Compounds as SERMs showed picomolar to low nanomolar potencies against ERα and were then investigated as antiproliferative agents against BC cell lines, as stimulators of p53 expression, as well as BC cell cycle arrest agents. Most active leads were finally profiled upon administration to female Wistar rats with pre-induced BC, after which 3DPQ-12, 3DPQ-3, 3DPQ-9, 3DPQ-4, 3DPQ-2, and 3DPQ-1 represent potential candidates for BC therapy.

Keywords: anticancer activity in vitro and in vivo; breast cancer; brefeldin a derivatives synthesis; estrogen receptor α; structure-based 3-D QSAR; structure-based 3-D pharmacophores.

Figure 3

The overall procedure workflow used for the definition of the 3-D pharmacophore/3-D QSAR models and their analysis is depicted as a “black” pathway. The application of generated 3-D pharmacophore/3-D QSAR models in structure-based and ligand-based virtual screening is depicted as a “red” pathway.

Figure 1

The active site of ERα in the apo form (PDB ID: 4Q13 [21]) (A); in complex with 17β-estradiol (PDB ID: 1ERE [13], i.e., agonist/partial agonist) (B); in complex with Raloxifene (PDB ID: 1ERR [13], i.e., SERM antagonist) (C); in complex with GW568 (PDB ID: 1R5K [21], i.e., SERD antagonist) (D). The residues depicted as white sticks and ribbons belong to the helices H3 (residues 332–354), H6 (residues 383–394), H7 (residues 429–438), H11 (residues 517–528), H12 (residues 531–547), loop (residues 418–428), and S1 and S2 antiparallel β-sheets (residues 402–410). H12 helix is depicted as a blue ribbon, as a crucial delimiter for partial agonists, SERMs, and SERDs.

Figure 2

SERMs and SERDs as FDA-approved drugs and compounds in clinical trials for BC treatment.

Figure 4

Experimental vs. recalculated (“actives”: green squares; “inactives”: purple squares) and predicted (“actives”: blue squares; “inactives”: orange squares) pIC₅₀s for ADDHHHP.13 hypothesis and LOO cross-validation (A); ADDRRRP.11 hypothesis and LOO cross-validation (B); ADDHHHP.13 hypothesis and LSO cross-validation (C); ADDRRRP.11 hypothesis and LSO cross-validation (D).

Figure 5

The 3-D PhypI features (D: hydrogen-bond donators, A: hydrogen-bond acceptors, H: hydrophobic features, P: positive ionizable features) and 3-D QSAR PLS-coefficients contour maps (GREEN_{PLS-coefficients}: positive steric interactions, YELLOW_{PLS-coefficients}; negative steric interactions, BLUE_{PLS-coefficients}: areas where positively charged functional groups and H-bond donators are favored whereas the negatively charged functional groups and H-bond acceptors are disfavored, RED_{PLS-coefficients}: areas negatively charged functional groups and H-bond acceptors are favored, whereas the positively charged functional groups and H-bond donators are disfavored) for 1ERR (A); 3ERD (B); 1XP1 (C); 1ERE (D); 2IOK (E); 2BJ4 (F). Amino acid residues are depicted in white. For the clarity of presentation, only the H12 helix is presented in a cornflower blue ribbon, as a crucial delimiter for partial agonists, SERMs, and SERDs.

Figure 6

The NCI89671 (viz., BFA) structure and nomenclature (A); the SB/LB virtually screened conformations of NCI89671, SB conformation blue, LB conformation pink (B).

Scheme 1

Synthesis of Brefeldin A derivatives 3DPQ-1 to 3DPQ-12. Reagents and conditions: (a) Me₂Zn, (−)-DBNE, toluene, 0 °C, 24 h, 87% ee; (b) HCl, THF, rt, 25 min; (c) (i) TBS-Cl, imidazole, DMAP, CH₂Cl₂, 0 °C, 3 h, (ii) PPh3, DEAD, 1-phenyl-1H-tetrazole-5-thiol, THF, 0 °C, 16h; (d) (NH₄)₆Mo₇O₂₄, H₂O₂, EtOH, rt, 16 h; (e) compound R6, KHDMS, 1,2-dimetoxyethane; -78 °C, 18h; (f) HCl, THF, rt, 1.5 h; (g) (i) LiOH, THF/H₂O, rt, 2h, (ii) 2,4,6-trichlobenzoylchloride, NEt₃, THF, rt, 1.5 h, (iii) DMAP, toluene, reflux, 5h; (h) (i) cc HBR, THF, rt, 1.5 h (ii) recrystallization; (i) TBSOTf, 2,6-lutidine, CH₂Cl₂, rt; (j) 3-acetyl-4-hydroxybenzoic acid, ECD, DMAP, CH₂Cl₂, reflux; (k) K₂CO₃, EtOH, reflux; (l) (i) TBAF, THF, rt, (ii) BBr₃, CH₂Cl₂, 0 °C, 3h, reflux.

Figure 7

The bioactive conformations of 3DPQ-12 (A); 3DPQ-3 (B); 3DPQ-9 (C); 3DPQ-4 (D); 3DPQ-2 (E); 3DPQ-1 (F) within the ERα active site. Amino acid residues are depicted in white, H12 helix is presented in cornflower blue ribbon.

Figure 8

ERα recruits transcriptional corepressors to repress p53-mediated transcriptional activation. (A) ChIP and sequential ChIP assays were performed on MCF-7 cells saturated with 3DPQ-1 to 3DPQ-12 in concentrations of 0.1 and 1 nM (for 3DPQ-5, 3DPQ-6, and 3DPQ-8 the concentrations were 1 and 10 nM) with primers specific to the p53-binding site of the p21 promoter. The primary ChIP was performed with anti-p53 antibody, and the immunoprecipitate was subjected to a second ChIP with anti-ERα antibody; (B) The immunoprecipitate from the ERα ChIP was then subjected to the third ChIP with antibodies against NCoR, SMRT, and HDAC1 antibodies; (C) qChIP was performed to analyze the ERα–p53 interaction on the p21 promoter in MCF-7 cells saturated with 3DPQ-1 to 3DPQ-12. Cells were grown in media with dextran-coated charcoal-treated FBS for 4 d and treated with E₂ (1 and 10 nM) with or without 3DPQ-1 to 3DPQ-12 for 3 h. * p < 0.05 when compared with control group; ^† p < 0.05 when compared with E₂; ^‡ p < 0.05 when compared with 4-OTH; ^§ p < 0.05 when compared with Ral.

Figure 9

Photomicrograph of breast section of a normal control rat showing lobuloalveolar unit (LaU) and cuboidal epithelial cells (CE) (A); photomicrograph of breast section treated with MNU showing mammary gland carcinoma alongside with massive proliferation of neoplastic epithelial cells (EC) (B); photomicrograph of breast section treated with 3DPQ-12 in a concentration of 5 mg/kg of bwt showing lobuloalveolar unit (LaU) and cuboidal epithelial cells (CE) (C); photomicrograph of breast section treated with 3DPQ-12 in concentration of 50 mg/kg of bwt showing lobuloalveolar unit (LaU) and cuboidal epithelial cells (CE) (D); photomicrograph of breast section treated with 4-OHT in a concentration of 5 mg/kg of bwt showing necrosis (NEC) (E); photomicrograph of breast section treated with 4-OHT in concentration of 50 mg/kg of bwt showing necrosis (NEC) (F); photomicrograph of breast section treated with Ral in a concentration of 5 mg/kg of bwt showing differentiated extralobular ducts (ED) (G); photomicrograph of breast section treated with Ral in a concentration of 50 mg/kg of bwt showing differentiated extralobular ducts (ED) (H), shown in ×200 magnification and stained with hematoxylin and eosin.