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The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment. ; We thank Gilberto Fronza (from Mutagenesi e Prevenzione Oncologica, Ospedale Policlinico San Martino, Genova, Italy), for providing us with the pLS76 vector. We acknowledge the European Synchrotron Radiation Facility for the provision of synchrotron radiation facilities and access to beamline ID30B. This work received financial support from the European Union (FEDER, Fundo Europeu de Desenvolvimento Regional, funds POCI/01/0145/FEDER/007728 through Programa Operacional Factores de Competitividade–COMPETE) and National Funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) under the Partnership Agreement PT2020 UID/MULTI/04378/2013, and projects (3599-PPCDT) PTDC/DTP-FTO/1981/2014–POCI-01-0145-FEDER-016581 and RECI/BBB-BEP/0124/2012. FCT fellowships: PD/BD/114046/2015 (Ana Sara Gomes) and SFRH/BD/96189/2013 (Sara Gomes) (thanks FCT PhD ...

PD/BD/114046/2015 SFRH/BD/96189/2013 SFRH/BPD/110640/2015 We thank Gilberto Fronza (from Mutagenesi e Prevenzione Oncologica, Ospedale Policlinico San Martino, Genova, Italy), for providing us with the pLS76 vector. We acknowledge the European Synchrotron Radiation Facility for the provision of synchrotron radiation facilities and access to beamline ID30B. This work received financial support from the European Union (FEDER, Fundo Europeu de Desenvolvimento Regional, funds POCI/01/0145/FEDER/007728 through Programa Operacional Factores de Competitividade–COMPETE) and National Funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) under the Partnership Agreement PT2020 UID/MULTI/04378/2013, and projects (3599-PPCDT) PTDC/DTP-FTO/1981/2014–POCI-01-0145-FEDER-016581 and RECI/BBB-BEP/0124/2012. FCT fellowships: PD/BD/114046/2015 (Ana Sara Gomes) and SFRH/BD/96189/2013 (Sara Gomes) (thanks FCT PhD Doctoral Programme BiotechHealth), and SFRH/BPD/110640/2015 (Carla Oliveira). ; The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment. ; publishersversion ; published

Background Half of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1. Methods and results By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced. Conclusions SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53. General Significance This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status. ; European Union (FEDER funds through Programa Operacional Factores de Competitividade – COMPETE) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) through the projects UID/QUI/50006/2019, COMPETE 2020 (POCI-01-0145-FEDER-006684/POCI-01-0145-FEDER-007440) and the BioTecNorte operation (NORTE-01-0145-FEDER-000004), (3599-PPCDT) PTDC/DTP-FTO/1981/2014 – POCI-01-0145-FEDER-016581 and UID/QUI/0081/2013; the Italian Association for Cancer Research, AIRC (IG#5506 to G.F.), Compagnia S. Paolo, Turin, Italy (Project 2017.0526 to G.F.) and Ministry of Health, (Project 5 × 1000, 2013 and 2015; ...