In-Silico Transcriptomic Characterization Analysis Identifies SMAD5, USP2, and CUL4A as Key Biomarkers and Therapeutic Targets in Colorectal Cancer

Colorectal cancer (CRC) is a molecularly heterogeneous malignancy requiring reproducible biomarkers and structurally tractable therapeutic targets. We applied an integrated transcriptomic, network, virtual-screening, docking, ADMET, and molecular-dynamics (MD) workflow using GSE8671, GSE14333, and GSE89076. Differential expression analysis (adjusted p < 0.05, |log2FC| ≥ 1) identified 2,092 upregulated/9130 downregulated genes in GSE8671, 926/945 in GSE14333, and 2,317/1,456 in GSE89076. Cross-dataset concordance was highest between GSE8671 and GSE89076 ( r = 0.70), with 1,040 shared upregulated and 500 shared downregulated genes, whereas GSE14333 showed weak concordance with GSE8671 ( r = −0.11) and GSE89076 ( r = −0.01). Integrated network topology, enrichment, and expression profiling prioritized SMAD5, USP2, and CUL4A. Structure-based virtual screening of approximately 25,000 approved/bioactive compounds identified Metergotamine–SMAD5 (−10.0 kcal/mol), Ravidasvir–USP2 (−12.5 kcal/mol), and Hesperidin–CUL4A (−10.8 kcal/mol) as leading screening hits. Refined molecular docking subsequently prioritized MK3207–SMAD5 (−10.6 kcal/mol), R428–USP2 (−12.1 kcal/mol), and Hesperidin–CUL4A (−8.7 kcal/mol). ADMET profiling indicated the most balanced drug-likeness for MK3207, poor predicted solubility for R428, and multiple rule-based violations for Hesperidin. Each complex underwent a 100 ns all-atom MD simulation in explicit solvent. CUL4A–Hesperidin retained hydrogen bonds with GLU680, THR537, GLN673, and THR535 in >83% of frames and a LYS459 π-cation interaction in 73.3%. USP2–R428 maintained key PHE364, SER190, and ARG363 contacts with a protein-referenced ligand RMSD of 4.84 ± 0.58 Å. Conversely, SMAD5–MK3207 showed substantial displacement (14.09 ± 0.62 Å) and <4% docking hydrogen-bond occupancy. Single-frame 100 ns MM-GBSA endpoint energies were −107.62, −95.99, and −88.00 kcal/mol, respectively. Collectively, the results support CUL4A–Hesperidin and USP2–R428 as the most dynamically persistent complexes, while the initial SMAD5–MK3207 binding mode requires re-evaluation and experimental validation.