Acute kidney injury (AKI) is a severe clinical condition with high morbidity and mortality. Apoptosis of renal tubular epithelial cells is a key pathological driver, and upregulation of the anti-apoptotic protein BCL-2 is a validated protective mechanism. The BCL-2 promoter harbors a C-rich sequence capable of forming an i-motif, a non-canonical DNA secondary structure with regulatory functions. Previous studies have reported steroid-like molecules as potential ligands for the BCL-2 i-motif, but their interactions were generally weak and lacked systematic pharmacophore insight. Here, we identify Sanguinarine ( SG ), a benzophenanthridine alkaloid, as a more potent ligand from a natural product library. Surface plasmon resonance (SPR) revealed broad-spectrum binding of SG to multiple promoter i-motifs, yet circular dichroism (CD) and melting assays suggested a relatively stronger stabilizing effect on the BCL-2 C-rich structure under our assay conditions. This apparent conformational preference, rather than strict binding selectivity, led to transcriptional activation of BCL-2 and protection of HK-2 cells from cisplatin-induced apoptosis. While i-motifs have only rarely been explored in AKI, our study provides new mechanistic insight by defining an aromatic–cationic pharmacophore and suggesting that structure-dependent stabilization may serve as a plausible mechanism for promoter-specific modulation. These findings support the biological relevance of the BCL-2 i-motif and highlight natural products as valuable scaffolds for nucleic-acid–interacting agents in renal injury models. • Sanguinarine is identified as a natural ligand of the BCL-2 promoter i-motif. • Sanguinarine selectively stabilizes the formation of BCL-2 i-motif. • Multi-site recognition might contribute to the i-motif stabilization mechanism. • BCL-2 activation protects renal tubular cells from cisplatin-induced apoptosis. • Findings suggest a structure-guided approach for nucleic acid–targeted therapy.

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.