Discovery of a Covalent Small-Molecule eEF1A1 Inhibitor via Structure-Based Virtual Screening

Pancreatic cancer remains a formidable health challenge due to its late-stage diagnosis and limited therapeutic options, underscoring the need for novel targets and modalities. Our previous work revealed that the natural product, BE-43547A₂, could effectively inhibit the progression of pancreatic cancer by the covalent binding to eukaryotic translation elongation factor 1 α 1 (eEF1A1) at Cys234 (C234). Considering the critical role in protein synthesis and the association with pancreatic cancer progression, eEF1A1 is a novel promising target for pancreatic cancer. However, the rational drug design methods for eEF1A1 are extremely lacking. Herein, using microsecond-scale molecular dynamics (MD) simulations, we identify a suitable eEF1A1 conformation for structure-based virtual screening (SBVS) by targeting the residue of C234. Through a tailored SBVS pipeline, we identified AKOS-04 as a novel small-molecule covalent inhibitor with nanomolar-level potency (IC₅₀ = 28.5 ± 2.86 nM in the PATU8988T cell line). Notably, cellular thermal shift assays (CETSA), with the treatments of dithiothreitol (DTT) and iodoacetamide (IAM), confirmed the covalent Cys-involved interaction of AKOS-04 and eEF1A1. Further structural modification validated the critical contribution of a double bond in the acrylamide group of AKOS-04 for its covalent binding with eEF1A1, manifested by the abolished inhibitory activity of compound <b>9</b> with the changed single bond in the acrylamide group. MST experiments confirmed direct binding of the compounds to eEF1A1 protein. AKOS-04 exhibited the strongest binding among the tested compounds, consistent with effective covalent target. Finally, MD simulations and pair-interaction energy analyses highlighted Lys84, Arg218, and Glu230 of eEF1A1 as key residues for driving its binding interactions to AKOS-04. These results reveal that AKOS-04, screened by SBVS against C234 of eEF1A1, represents a promising lead for eEF1A1-targeted pancreatic cancer therapy, highlighting the power of computational approaches in covalent drug discovery.