Structural Refinement of 2,4-Thiazolidinedione Derivatives as New Anticancer Agents Able to Modulate the BAG3 Protein

Abstract

The multidomain BAG3 protein is a member of the BAG (Bcl-2-associated athanogene) family of co-chaperones, involved in a wide range of protein-protein interactions crucial for many key cellular pathways, including autophagy, cytoskeletal dynamics, and apoptosis. Basal expression of BAG3 is elevated in several tumor cell lines, where it promotes cell survival signaling and apoptosis resistance through the interaction with many protein partners. In addition, its role as a key player of several hallmarks of cancer, such as metastasis, angiogenesis, autophagy activation, and apoptosis inhibition, has been established. Due to its involvement in malignant transformation, BAG3 has emerged as a potential and effective biological target to control multiple cancer-related signaling pathways. Recently, by using a multidisciplinary approach we reported the first synthetic BAG3 modulator interfering with its BAG domain (BD), based on a 2,4-thiazolidinedione scaffold and endowed with significant anti-proliferative activity. Here, a further in silico-driven selection of a 2,4-thiazolidinedione-based compound was performed. Thanks to a straightforward synthesis, relevant binding affinity for the BAG3BD domain, and attractive biological activities, this novel generation of compounds is of great interest for the development of further BAG3 binders, as well as for the elucidation of the biological roles of this protein in tumors. Specifically, we found compound 6 as a new BAG3 modulator with a relevant antiproliferative effect on two different cancer cell lines (IC₅₀: A375 = 19.36 μM; HeLa = 18.67 μM).

Keywords: 2,4-thiazolidinedione scaffold; BAG domain modulators; BAG3 protein; cancer; chaperones; surface plasmon resonance (SPR) assay; virtual screening.

Figure 1

Virtual screening workflow leading to the selection of compounds 2–9.

Figure 2

Chemical structures of new 2,4-thiazolidinedione derivatives 2–9 and lead compound 1.

Scheme 1

Synthetic strategy for compound 2–9. Reagents and conditions: (i) piperidine, EtOH, reflux 16–24 h; (ii) NaH, DMF dry, 80 °C 16–20 h; (iii) HOBt, DIC, DMF, r.t. overnight.

Figure 3

Cell cycle analysis: (a) DNA content and (b) hypodiploid nuclei, with propidium iodide staining, were evaluated by flow cytometric assay. A375 cells were treated respectively with 1 or 6 (both 5-10-25 μM) for 48 h. Results are expressed as mean ± S.E.M. of three independent experiments, each performed in duplicate. Data were analyzed by Student’s t-test. * p < 0.05 and ** p < 0.005 vs. non-treated.

Figure 4

Cell cycle analysis: (a) DNA content and (b) hypodiploid nuclei, with propidium iodide staining, were evaluated by flow cytometric assay. HeLa cells were treated with 6 (5-10-25 μM) for 48 h. Results are expressed as mean ± S.E.M. of three independent experiments each performed in duplicate. Data were analyzed by Student’s t-test. * p < 0.05 and ** p < 0.005 vs. non-treated.

Figure 5

(a) Caspase 3 and (b) caspase 9 expressions were detected by flow cytometric analysis. HeLa cells were treated with 6 (5-10-25 μM) for 48 h. Results are expressed as mean ± S.E.M. from at least three independent experiments each performed in duplicate. Data were analyzed by Student’s t-test. *** p < 0.0001 vs. non-treated.

Figure 6

HeLa cells were treated with 6 (5-10-25 μM) for 48 h. BAG3 expression was detected by Western blotting. Tubulin protein expression was used as a loading control. Results are expressed as mean ± S.E.M. from at least three independent experiments each performed in duplicate. Data were analyzed by Student’s t test vs. non-treated.