Fig 1.
The flowchart of high-throughput screening by AlphaFold 3.
There are four steps for the high-throughput in the present work, including the environment setup, database preparation, input file design and the screening.
Fig 2.
Results of AI virtual screening.
A. Chemical structures of the top six candidate compounds identified from high-throughput virtual screening using AlphaFold 3 with CB2R receptor structures. Among these, COCA (N-(3-chloro-2-methylphenyl)-5-(4-hydroxyphenyl)-1,2-oxazole-3-carboxamide) exhibited the most favorable predicted binding affinity and was selected for further analysis. B. The three-dimensional structure of the CB2 receptor (CB2R), highlighting the orthosteric ligand-binding pocket as the primary target site for docking. C. Predicted binding pose of COCA within the orthosteric site of CB2R, as determined through molecular docking. COCA is shown embedded in the binding cavity, forming multiple stabilizing interactions with surrounding amino acid residues. Key contacts include π–π stacking interactions with PHE94 and TRP194, and hydrophobic interactions involving PHE87, PHE91, ILE110, VAL113, THR111, PHE106, PHE183, PRO184, ILE186, and TYR190. The docking score of −6.772 kcal/mol indicates a high predicted affinity, supporting COCA’s potential as a lead compound for further pharmacological evaluation.
Fig 3.
Weight change curve (left) and Lung Coefficient (right) of mice in each group (NSP > 0.05, *P < 0.05, **P < 0.01).
Fig 4.
HE staining and Masson staining of lung tissues of mice in each group (200× & 1250×).
A. HE staining and Masson staining of lung tissues of mice in each group (200× & 1250×).The Control group had normal alveolar structure with no inflammation on day 7. The BLM group showed severe damage, including disorganized alveoli, thickened septa, and inflammation. The treatment groups had milder changes, with less inflammation and more uniform tissue structure.Masson’s staining showed high collagen levels, structural damage, and fibroblast growth in the BLM group—signs of advanced fibrosis. COCA and pirfenidone reduced collagen buildup and structural damage. The COCA HD group showed the best improvement, with clearer lung structure and less collagen. B. fibrosis scores of rats lung tissues.
Fig 5.
Cell count map of mouse BALF (left) and Expression of TNF-α (middle), IL-6 (right) in lung tissues (NSP > 0.05, **P < 0.01).
Fig 6.
Expression of Col-Ⅰ and Col-Ⅲ proteins in lung tissues of mice in each group (×40).
A. Immunofluorescence analysis showed that green fluorescence corresponds to Collagen Type I (Col-I) and red fluorescence to Collagen Type III (Col-III). The BLM group had higher Col-I and Col-III fluorescence intensity in lung tissues than the Control group, indicating increased expression. In contrast, the COCA LD and COCA HD groups showed lower fluorescence and reduced collagen expression compared to the BLM group. B. Quantitative results confirmed that Col-I and Col-III levels were significantly higher in the BLM group (P < 0.001). COCA and pirfenidone treatment significantly reduced these levels (P < 0.01), with no significant difference between the COCA and PF groups (P > 0.05).
Fig 7.
Representative Western blots and corresponding densitometric analyses of CB2R, Nrf2 and Smad7 in lungs from control and COCA-treated IPF mice (NSP > 0.05, *P < 0.05, *P < 0.01).
The entire assay was independently repeated three times; the individual data points on the bar graph represent the mean densitometric value from each independent experimental repeat.