Excavating Podocyte-Protective Targets of phloroglucinol-terpene hybrids Utilizing AI Models and Omics Analysis

Lupus nephritis (LN) is a multifactorial autoimmune inflammatory disorder, with podocyte dysfunction or structural abnormalities playing a pivotal role in its pathogenesis. Eucalyptus leaves, traditionally employed in folk medicine, have demonstrated notable efficacy in managing non-infectious inflammations. Eucarbwenstols D (R14) is a novel phloroglucinol-terpene that has been isolated from Eucalyptus robusta. To further refine potential targets, we utilized an AI scoring system and successfully identified the epidermal growth factor receptor (EGFR) as the novel target of R14. This discovery was built upon the foundation of transcriptome and connectivity mapping (CMap) analysis. Molecular docking and molecular dynamics simulations were employed to analyze the interaction between R14 and EGFR, surface plasmon resonance (SPR) and homogeneous time-resolved fluorescence (HTRF) assays were utilized to accurately quantify the binding affinity between R14 and EGFR. Western blot (WB) and immunofluorescence assays were then conducted to explore the downstream signaling pathways initiated by EGFR, specifically the PI3K/AKT/mTOR pathway. These assays demonstrated that R14 modulates the expression of apoptotic proteins Bax and Bcl-2 through this pathway, thereby exerting a protective effect on podocytes from undergoing apoptosis. The modulation of key podocyte proteins, nephrin and podocin, further substantiates this protective effect. Overall, we have applied a novel AI algorithm model that transcends bioinformatics analysis to precise identification of the novel podocyte target of R14. In this study, R14 was identified for the first time as a novel scaffold compound for EGFR inhibition. Moreover, R14 has demonstrated its capacity to inhibit podocyte apoptosis, thereby protecting these critical cells. This discovery establishes a robust theoretical foundation for the use of phloroglucinol-terpene hybrids (PTHs) in the treatment of LN.