Bcl-2-associated athanogene 3 (BAG3) is a multifunctional co-chaperone protein that regulates apoptosis, autophagy, and proteostasis through interactions with HSP70 and other partners. Overexpression of BAG3 contributes to tumor cell survival, metastasis, and chemotherapy resistance, making it an appealing but challenging anticancer target due to its intrinsic disorder and lack of structural data. Here, we report a fragment-based drug discovery (FBDD) approach to identify novel small molecules targeting human BAG3. A fragment library of 783 compounds was screened using a thermal shift assay (TSA) against recombinant BAG3 expressed in mammalian cells, followed by hit validation through ligand-observed NMR (WaterLOGSY). Eleven fragments stabilized the protein, and seven were confirmed as binders. Among them, a 6-chloro-2-oxindole fragment ( Fr1 ) exhibited the strongest interaction, with a dissociation constant (K D ) of 97.8 ± 11.1 μM. Structure–activity relationship (SAR) studies focused on maintaining the 6-chloro-2-oxindole core and optimizing substitutions at position 3, identified derivative 7 as a promising lead. Derivative 7 bound BAG3 with improved affinity (K D ≈ 22 μM), as confirmed by grating-coupled interferometry, and displaced Fr1 in competition NMR assays. This work demonstrates the feasibility of applying FBDD to intrinsically disordered and structurally unresolved proteins such as BAG3, providing a validated chemical starting point for the development of selective BAG3 inhibitors. These findings expand the druggability landscape of BAG3 and highlight fragment-based methodologies as powerful tools to explore protein–protein interaction targets previously considered intractable.
Favaro et al. (Fri,) studied this question.