Structure-based identification of natural small molecules as potential CTLA4 inhibitors: a computational approach

CTLA4 is a critical immune checkpoint receptor that downregulates T-cell activation by binding with CD80 and CD86, facilitating tumour immune evasion. Although monoclonal antibodies targeting CTLA4 have shown clinical success, their application is limited by immune-related adverse events, cost, and tissue penetration challenges. On the other hand, due to structural challenges, small-molecule inhibitor development is mostly focused on targeting the CD80 ligand rather than directly engaging CTLA4. Hence, in this study, we performed structure-based virtual screening of approximately 5 lakh natural compounds, followed by in silico ADMET analysis, molecular dynamics simulations, and MM-PBSA binding energy calculations, which led to the identification of two promising natural compounds predicted to bind CTLA4 directly. This integrated workflow identified two natural compounds predicted to bind within the CC′ and FG loop regions of CTLA4, with docking scores better than those of previously reported small molecules A9 and D11, and better than the computed CTLA4–CD80/B7-1 reference. Both ligands were predicted to have favourable pharmacokinetic properties, including good drug‑likeness and low toxicity risk, as well as less potential off-target effects. Both candidates exhibited stable interactions with CTLA4 over 200 ns MD simulations, maintained consistent centre‑of‑mass distances, and showed strong MM‑PBSA binding free energies. Furthermore, protein–protein docking and MD simulations of CTLA4 with CD80/CD86 in the absence and presence of the selected ligands indicated that ligand binding may reduce CTLA4–B7 binding free energies by possibly restricting the flexibility of the CC′ and FG loops, potentially disrupting the CTLA4-B7 pathway. These findings present natural compounds as promising new small molecules with the potential to modulate the CTLA4-CD80 pathway, paving the way for further in vitro and preclinical validation to enable a safer, more accessible cancer immunotherapy.