Identification of a New Promising BAG3 Modulator Featuring the Imidazopyridine Scaffold

Abstract

The antiapoptotic BAG3 protein plays a crucial role in cellular proteostasis and it is involved in several signalling pathways governing cell proliferation and survival. Owing to its multimodular structure, it possesses an extensive interactome including the molecular chaperone HSP70 and other specific cellular partners, which make it an eminent factor in several pathologies, particularly in cancer. Despite its potential as a therapeutic target, very few BAG3 modulators have been disclosed so far. Here we describe the identification of a promising BAG3 modulator able to bind the BAG domain of the protein featuring an imidazopyridine scaffold and obtained through the application of the Groebke-Blackburn-Bienaymé chemical synthesis procedure. The disclosed compound 10 showed a relevant cytotoxic activity, and in line with the biological profile of BAG3 disruption, it induced the activation of caspase 3 and 9.

Keywords: BAG domain modulator; BAG3 protein; Groebke–Blackburn–Bienaymé reaction; Surface Plasmon Resonance assay; imidazopyridine scaffold.

Figure 1

Structures and synthesis of 1–14 by Groebke–Blackburn–Bienaymé reaction. Reagents and conditions: (i) MeOH, CH₃COOH, r.t., o.n., HCl, 30 min.

Figure 1

Structures and synthesis of 1–14 by Groebke–Blackburn–Bienaymé reaction. Reagents and conditions: (i) MeOH, CH₃COOH, r.t., o.n., HCl, 30 min.

Figure 2

Antiproliferative activity of compounds 10, 12 and 14 on human cervical adenocarcinoma cell line (HeLa) after 72 h of treatment at 25, 50 and 100 µM. The positive control (CTRL POS) consisted of 10% DMSO, while the negative control (CTRL NEG) consisted of 0.1% DMSO. The results are showed as mean ± standard deviation (SD) from three independent experiments. **, *** denote respectively p < 0.01 and p < 0.001 vs. Ctrl.

Figure 3

Pro-apoptotic activity on HeLa cells treated for 72 h with compound 10 at 50, 30 and 20 μM. (A) Representative flow cytometry plots using Annexin V-FITC/PI staining for apoptosis. (B) Related quantitative analysis is reported. Data are expressed as a percentage of live, apoptotic and necrotic cells. (C) Cell cycle analysis. (D) Hypodiploid nuclei determination. Results are shown as mean ± standard deviation (SD) from three independent experiments. *, **, *** denote, respectively, p < 0.05; p < 0.01 and p < 0.001 vs. Ctrl.

Figure 4

Activation of caspases on HeLa cells treated for 72 h with compound 10 at 50, 30 and 20 μM. (A) Caspase 3 and (B) caspase 9 determination via cytofluorimetric technique. (C) Western blot analyses. Levels of cleaved caspase were evaluated. Βactin was used to check the equal loading of protein extracts. (D) Densitometric analysis of western blots. Results are shown as mean ± standard deviation (SD) from three independent experiments. *, ** denote, respectively, p < 0.05 and p < 0.01 vs. Ctrl.

Figure 5

Binding mode of (A) compound 10 (colored by atom type: C green, N blue, Cl dark green, Br dark red, polar H white), (B) compound 12 (colored by atom type: C pink, N blue, Cl dark green, Br dark red, polar H white), (C) compound 14 (colored by atom type: C orange, N blue, O red, Br dark red, Cl dark green, polar H white). H bonds, halogen bonds, and π interactions are reported in yellow, purple and green dotted lines, respectively.