Exploring the binding dynamics of triazolyl-thio-oxazines for MDM2-p53 disruption: Design, synthesis, and p53 upregulation.

The mouse double minute 2 homolog (MDM2) - p53 interaction inactivates p53, a key tumor suppressor, and has been validated as a target for cancer therapy. Four triazolyl-thio-oxazines 4(a-d) were rationally designed based on a three-finger pharmacophore model, which parallels the binding conformation of p53 to suppress this oncogenic interaction. All compounds were characterized by 1H/13C NMR, IR, and mass spectrometry, whereas 4a was characterized by X-ray crystallography. All compounds bound to the MDM2 binding site at key residues (Leu54, Leu57, Ile61, Met62, Phe86, and Tyr100) with similar binding affinities (-7.88 to -8.75 kcal/mol). Further 200 ns molecular dynamics simulations and MM-GBSA analyses were performed to determine the precise differences. Complex 4a demonstrated better binding stability (RMSD <2.0 Å) and MM-GBSA Binding Score ΔGbind (-172.718 kcal/mol) than either Nutlin-3a or the p53 peptide. Its fluorobenzene dives further into the MDM2 cleft than the chlorobenzene ring in Nutlin-3a, resulting in stronger π-π stacking and hydrophobic interactions. SwissADME profiling also predicted that 4a has a good balance of pharmacokinetic properties. This was evident from the cytotoxicity studies (MCF-7 breast cancer cells), which revealed 4a to be the most potent (IC₅₀ = 4.379 μM). Further Western blot analysis showed significant upregulation of p53 and p21 in MCF-7 cells upon treatment with 4(a-d). In contrast, no significant induction of p53 or p21 was observed in MDA-MB-468 cells, confirming the upregulation of wild-type p53. These results suggest that 4a is a promising candidate for further biological evaluation as an MDM2-p53 interaction inhibitor.