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 IC 50 values of 1.26, 1.30, and 1.25 µM, respectively, in comparison with nutlin‐3 (IC 50 = 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.