Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53–MDM2 interaction

One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53–MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53–MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53+/+) and in its isogenic derivative without p53 (HCT116 p53−/−). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53+/+ but not in p53−/− HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53–MDM2 interaction in HCT116 p53+/+ cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of chemotypes activators of a wild-type p53-pathway with promising antitumor activity. Moreover, it may open the way to the development of a new class of p53–MDM2 interaction inhibitors.

Joana Soares a,b, Nuno A.L. Pereira d, Ângelo Monteiro d, Mariana Leão a,b, Cláudia Bessa a,b, Daniel J.V.A. dos Santos d,e, Liliana Raimundo a,b, Glória Queiroz a,c, Alessandra Bisio f, Alberto Inga f, Clara Pereira a,b, Maria M.M. Santos d,⇑, Lucília Saraiva a,b,⇑ a

a REQUIMTE, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto, Portugal

b Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto, Portugal

c Laboratório de Farmacologia, Departamento de Ciências do Medicamento, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313
Porto, Portugal

d Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal

e REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal

f CIBIO, Centre for Integrative Biology, Laboratory of Transcriptional Networks, University of Trento, Via delle Regole, 101, 38123 Trento, Italy



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