DRUG DISCOVERY FOR p53 PPI-TARGETS

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Development of índole based scaffolds to tackle cancer and malaria

Publication . Lopes, Elizabeth A.; Santos, Maria Manuel Duque Vieira Marques dos; Mori, Mattia

Genetic and epigenetic alterations are key drivers of tumorigenesis, disrupting fundamental cellular processes like growth and proliferation. Central to maintaining genetic stability is the “guardian of the genome” p53 protein, which regulates deoxyribonucleic acid (DNA) repair, cell division, and cell death, thereby preventing the accumulation of genetic mutations. In normal cells, p53 levels are tightly regulated by endogenous negative regulators, mouse double minute (MDM) 2 and MDM4. However, in cancer, dysregulation of these regulators or mutations in the TP53 gene lead to the inactivation of p53 function. Consequently, p53 is a significant target for anticancer therapies. Small molecule targeted therapies aim to restore p53 function by inhibiting or degrading MDM2 and MDM4 in cancers with wild-type p53. Conversely, strategies for tumors with mutant p53 involve restoring its wild-type conformation, improving DNA interactions, inhibiting mutant p53 functions, and preventing protein aggregation. Interestingly, p53 exhibits multifaceted roles beyond cancer, presenting opportunities for p53-based therapies in diseases such as malaria, highlighting its versatility in human health. Despite significant progress, the clinical treatment with p53 activators remains challenging, with a current dearth of such activators available for clinical use. Addressing this gap, our research team is developing novel five-membered spirooxindole and tryptophanol-based oxazoloisoindolinone compounds with enhanced pharmacological properties. These compounds are designed to disrupt p53–MDM2/4 interactions and restore DNA contacts and proper folding, thereby effectively reactivating p53 function with minimal collateral effects. This thesis focuses on developing new small molecule reactivators of p53 function to treat cancer and malaria. It includes developing dual p53–MDM2/4 protein-protein interaction inhibitors through different approaches and designing novel small molecules to restore mutant p53’s DNA-binding ability. The work within developed is organized into four chapters: the first three chapters cover dual inhibitors of p53–MDM2/4 protein-protein interactions, and the fourth addresses reactivating two of the most prevalent mutant p53 associated with difficult-to-treat cancers. For the first time, the antiplasmodial activity of spirooxadiazoline oxindoles containing the 1,3,4- oxadiazole core is being reported. A series of spirooxadiazoline oxindoles was synthesized to activate the p53 pathway. While they showed no significant antiproliferative activity in cancer cell lines with wild-type p53, seven derivatives demonstrated dual-stage antiplasmodial properties against Plasmodium berghei and Plasmodium falciparum malaria parasites without cytotoxicity in mammalian cells. The most promising compound inhibited both sodium-dependent and independent membrane ATPase activity, suggesting its potential to impair the parasite's metabolic processes, ion homeostasis, and energy production. Alternative strategies were developed to inhibit the interaction between p53 and MDM2/4. The spiropyrazoline oxindole scaffold, known for its ability to reactivate the p53 pathway, was further investigated. Molecular docking studies uncovered that by mimicking the interactions of p53 residues and establishing additional hydrogen bonds within the solvent-exposed pocket of the MDM2 binding site, the inhibition of the p53–MDM2 protein-protein interaction could be enhanced. This approach also held great promise for targeting the p53–MDM4 protein-protein interaction. The two most active compounds in cancer cell lines overexpressing MDM2 and/or MDM4, induced apoptosis and controlled cell growth. Moreover, three derivatives showed dual p53–MDM2/4 protein-protein interaction inhibition with nanomolar IC50 values, while one derivative selectively dissociated p53–MDM2 protein-protein interaction. Alternatively, a structure-based virtual screening of nearly a million compounds identified four potential chemical scaffolds capable of acting as dual inhibitors of MDM2/4. Notably, etrasimod, a recently approved drug, demonstrated binding to both MDM2 and MDM4 proteins in fluorescence polarization binding assays. Delving deeper into the MDM4 binding site prompted the design of new 1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl derivatives, heightening their inhibitory effectiveness. The rationale behind this study involved modifying specific groups within the MDM4 binding pocket to mimic p53 residues’ interactions. Given the inadequacy of the current synthetic pathway for etrasimod in synthesizing the new optimized derivatives, a new synthetic route was devised to obtain novel trifluoromethyl-containing compounds. Particularly, synthesizing the trifluoromethyl-containing building block involved three highly efficient steps, followed by a C(sp2) selective Suzuki-Miyaura coupling, representing the pivotal synthetic step for introducing chemical diversity. Introducing chemical diversity at this late stage of synthesis is particularly advantageous, as it allows for the generation of multiple derivatives with fewer synthetic steps required. Currently, new derivatives are being synthesized through the developed methodology. Exploring mutant p53 reactivation, the tryptophanol-derived oxazoloisoindolinone SLMP53-1's mechanism in restoring wild-type p53 function in R273H and R280K mutants was investigated. Molecular dynamics highlighted SLMP53-1's putative binding site, a crevice in the interface of mutant p53 and DNA's minor groove. Acting as a molecular glue, it facilitated p53 residues interaction network and re-establishment of DNA contacts. Screening of in-house tryptophanol-derived oxazoloisoindolinones revealed hydrogen bonds with both p53 and DNA while maintaining SLMP53-1’s analogous docking pose feature crucial for interaction. These findings guide the structure-based optimization of this scaffold for potent p53 reactivation in cancers with R273H and R280K mutations. Collectively, this thesis represents a significant step forward in advancing both antimalarial therapeutics and p53 reactivation strategies in cancers with MDM2/4 overexpression and R273H/R280K mutations. Specifically, the groundbreaking discovery of dual-stage antiplasmodial activity of spirooxadiazoline oxindoles opens up exciting possibilities for the development of next-generation antimalarials able to overcome drug resistance. Moreover, the optimization of spiropyrazoline oxindoles represents a remarkable achievement, with dissociation potency increasing up to 19-fold compared to the initial compound. Spiropyrazoline oxindoles are reported to dually inhibit p53–MDM2/4 protein protein interactions at the nanomolar level for the first time. In addition, the discovery of etrasimod binding to both MDM2 and MDM4 suggests its 1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl core as a promising and distinct scaffold for the development of dual MDM2/4 inhibitors. Finally, the identification of SLMP53-1 as a molecular glue in the interface of p53 and DNA lays a solid foundation for the structure-guided optimization of tryptophanol-derived oxazoloisoindolinones, paving the way for innovative therapeutics for R273H and R280K mutant p53-expressing cancer.

2024-07Tese de doutoramento

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

Concurso para Financiamento de Projetos de Investigação Científica e Desenvolvimento Tecnológico em Todos os Domínios Científicos - 2017

Número da atribuição

PTDC/QUI-QOR/29664/2017