The CETSA-Luminex integrated platform for drug targets screening identifies IL-4-IN-1 as an anti-pulmonary fibrosis therapeutic candidate.

This study established an innovative platform integrating Cellular Thermal Shift Assay (CETSA) with Luminex technology. It overcomes traditional single-target limitations and the complexity of thermal proteome profiling (TPP), enabling rapid, high-throughput multi-target drug screening. The platform detects drug-induced protein stabilisation via CETSA while utilising Luminex for multiplex cytokine quantification. Validation using 21 cytokines and IL-4 inhibitor IL-4-IN-1 revealed distinct modulation: IL-4-IN-1 produced dose-dependent up-regulation of IL-4 and unexpectedly TNF-α (P < 0.01). Binding studies (DSF/BLI) confirmed specific IL-4-IN-1 interactions with IL-4 (ΔTm = 5.83 °C) and TNF-α (Kd = 1.30 × 10-6 M; ΔTm = 5.03 °C). Given their association with pulmonary fibrosis, anti-fibrotic investigations showed IL-4-IN-1 significantly suppressed α-SMA and COL1A1 expression (P < 0.01) in A549 cells. It also dose-dependently downregulated key fibrosis markers (MMP2, FN1, TGFB1; P < 0.001), substantially reducing FN1, Vimentin, and α-SMA (P < 0.001). The CETSA-Luminex platform provides efficient multi-target screening, while IL-4-IN-1 demonstrates therapeutic potential against fibrosis by targeting IL-4/TNF-α pathways and inhibiting profibrotic mediators, offering a novel efficient approach for drug target discovery.