Targeting a distinct binding pocket in the pregnane X receptor with natural agonist TRLW-2 ameliorates murine...

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.

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.

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.

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.