Molecular docking, molecular dynamics simulations and in vitro screening reveal cefixime and ceftriaxone as GSK3β covalent inhibitors

GSK3β is a serine/threonine kinase that has been suggested as a putative drug target for several diseases. Recent studies have reported the beneficial effects of cephalosporin antibiotics in cancer and Alzheimer's disease, implying potential inhibition of GSK3β. To investigate this mechanism, four cephalosporins, namely, cefixime, ceftriaxone, cephalexin and cefadroxil were docked into the GSK3β binding pocket. The third-generation cephalosporins, cefixime and ceftriaxone, exhibited the best docking scores due to the exclusive hydrogen bonding between their aminothiazole group and hinge residues of GSK3β. The stability of top-ranked poses and the possibility of covalent bond formation between the carbonyl carbon of the β-lactam ring and the nucleophilic thiol of Cys-199 were evaluated by molecular dynamics simulations and covalent docking. Finally, the in vitro inhibitory activities of the four cephalosporins were measured against GSK3β with and without preincubation. In agreement with the results of molecular docking, cefixime and ceftriaxone exhibited the best inhibitory activities with IC(50) values of 2.55 μM and 7.35 μM, respectively. After 60 minutes preincubation with GSK3β, the IC(50) values decreased to 0.55 μM for cefixime and 0.78 μM for ceftriaxone, supporting a covalent bond formation as suggested by molecular dynamics simulations and covalent docking. In conclusion, the third-generation cephalosporins are reported herein as GSK3β covalent inhibitors, offering insight into the mechanism behind their benefits in cancer and Alzheimer's disease.