Peroxisome proliferator-activated receptor gamma (PPARγ) is a key target for metabolic disorders that contribute to obesity and type 2 diabetes mellitus (T2DM). However, full agonists such as thiazolidinediones (TZDs) have limitations in terms of side effects. Selective PPARγ modulators (SPPARγMs) that target alternative binding pockets offer the potential for safer partial agonists. Here, we employed six computational algorithms (Fpocket, DeepSite, CavityPlus, DoGSiteScorer, CASTpFold, POCASA) to identify a novel allosteric pocket (pocket 6–5) in the PPARγ ligand-binding domain (LBD), localized at the helix 3 (H3), helix 2 (H2), helix 2'(H2′), and β-sheet interface. A virtual screening of 4,097 natural compounds from traditional Chinese medicine (TCM) libraries was conducted, which led to the identification of ginsenoside Rg5 (TWSZ-5) as a top hit. Molecular docking and molecular dynamics (MD) dynamics revealed TWSZ-5 stabilizes pocket 6–5 through hydrogen bonds with Ser342, Gln345, Lys261, and Lys263. TWSZ-5 promoted beige adipocyte differentiation in adipose-derived stem cells (ADSCs) in vitro, upregulating Ucp1, Prdm16, Cpt1α, and Pgc1α. The present study identifies TWSZ-5 as a novel SPPARγM that utilizes an allosteric binding pocket to enhance thermogenesis while mitigating adverse effects. These findings emphasize the potential of TCM derivatives and structure-based screening strategies to develop safer antidiabetic therapies with precision pharmacology.

Background: The therapeutic development of pregnane X receptor (PXR) agonists for ulcerative colitis (UC) is hindered by the poor selectivity of the canonical ligand-binding pocket. This study aimed to identify a novel, selectivity-enhancing binding site on PXR and a corresponding natural ligand for UC treatment. Methods: A distinct binding pocket (Pocket 1–5) within the PXR ligand-binding domain was identified using a multi-algorithm computational approach (SiteMap, Fpocket, Prank, CASTpFold). Structure-based virtual screening of 6,058 natural compounds from a traditional Chinese medicine library was performed via Glide docking (High-Throughput Virtual Screening/Standard Precision/Extra Precision modes, HTVS/SP/XP), with binding affinities refined by molecular mechanics/generalized Born surface area (MM/GBSA). The top candidate, TRLW-2 (catechin), was evaluated using luciferase reporter assays, quantitative real-time PCR (Qpcr), and functional assays (CCK-8, EdU, Annexin V-FITC/PI) in HEK293T and mouse colonic epithelial cells (MCECs). In vivo efficacy was assessed in a DSS-induced murine colitis model. Results: TRLW-2 exhibited high affinity for pocket 1-5, forming key hydrogen bonds with residues including Ser350, Asp352, Asp363, Thr398, and Arg401, which was validated by molecular dynamics simulations (MD) and site-directed mutagenesis. Functionally, TRLW-2 acted as a potent PXR agonist, significantly upregulating detoxifying enzymes (such as Cyp2b10, Cyp3a11 and Ugt1a1) and proliferation markers (PCNA, CDK1, Cyclin B1, and Ki67) in vitro . It promoted epithelial cell proliferation and inhibited apoptosis in MCECs. In DSS-induced colitis mice, TRLW-2 treatment significantly attenuated weight loss, reduced disease activity index DAI) and colonic mucosa damage index (CMDI) scores, ameliorated colon shortening, and improved histopathology. Conclusion: This study identified pocket 1–5 as a selectivity-enhancing site on PXR. The natural product TRLW-2, discovered via virtual screening, potently engages this pocket and demonstrates robust anti-inflammatory and mucosal healing effects, validating a promising strategy for developing precision therapeutics for UC.