Unlocking the Anticancer Potential of New Spirooxindoles via p53‐MDM2/MDMX Dual Inhibition: In Vitro and In Silico Assessments

Abstract

Blocking the p53-MDM2 and/or p53-MDMX protein-protein interaction (PPI) by small-molecule inhibitors has been tracked as a potentially effective cancer treatment approach. Herein, we present the discovery of a new series of spirooxindole-tethered pyrazolopyridine derivatives. The development of the new congeners was based on the analysis of the co-crystal structures of the inhibitors bound to both MDM2 and MDMX and studying the binding interactions between the substituents of small molecules and the three subpockets of the p53-MDM2/MDMX. The spirooxindoles 6b, 6k, and 6n attained the most pronounced activity in the MULTI-ARRAY MDM2-p53 complex assay with IC₅₀ values of 1.26, 1.30, and 1.25 µM, respectively, in comparison with nutlin-3 (IC₅₀ = 2.03 µM). The counterparts 6b, 6k, and 6n also revealed notable inhibitory potential against p53-MDMX. The antiproliferative efficacy of the most active target compounds was assessed in HCT-116 colon cancer cell line that overexpressed MDM2 and harbored wild-type p53. The derivative 6k accomplished the highest antiproliferative activity against HCT-116 compared to nutlin-3. Moreover, 6k displayed minimal toxicity compared to the reference nutlin-3 when examined on a normal cell line. Flow cytometric analysis revealed that 6k controlled cell growth via cell cycle arrest at the G1 phase and induced cell death via apoptosis. Additionally, compound 6k revealed a prominent effect in raising p53 levels with a 6.464-fold increase compared to the control. Molecular docking and molecular dynamics simulations justified the observed efficacy. Collectively, this study showcased a new class of potent p53-MDM2/MDMX dual inhibitors possessing a spirooxindole scaffold which can be subjected to future development.