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Development of protein p53 activators to tackle colon cancer
Publication . Barcherini, Valentina; Santos, Maria Manuel Duque Vieira Marques dos; Antunes, Alexandra Maria Moita; Wang, Shaomeng
Colorectal cancer (CRC) figures currently as the third most diagnosed cancer and ranks second as leading cause of cancer death globally. Due to unmet screening programs, limited therapeutic strategies, and increasing incidence rates, CRC accounts for 10% of global cancer incidence and 9.4% of cancer deaths, just lower than lung cancer. Despite the latest progresses made in understanding CRC pathophysiology, poor therapeutic options are currently available. Targeted therapy represents a fundamental therapeutic option for the cure of CRC. In this respect, the tumor suppressor p53 figures as key therapeutic target. p53 is a multifunctional protein that regulates cell cycle, DNA repair, apoptosis and metabolic pathways. In CRC, mutations of the TP53 gene occur in 60% of patients and are associated with a more aggressive tumor phenotype and multi drug resistance. Unfortunately, there are still very few examples of mutant p53 reactivators, that restore wild-type p53 function, with low adverse effects on normal cells.
Following our research on the design and synthesis of novel wild-type p53 activators, tryptophanol-derived isoindolinones SLMP53-1 (43a), DIMP53-1 (43d) and SLMP53-2 (44a) were identified, with promising p53-dependent in vitro and in vivo biological activity in wild-type and mutant p53 expressing human cancer cells. In this PhD project, the optimization of the tryptophanol-derived isoindolinone family was addressed. Towards this goal, an initial screening of the pharmacokinetic profile of hit compounds SLMP53-1 (43a) and DIMP53-1 (43d) was conducted. Investigations through in vitro procedures on the stability profile of these new chemical entities were performed in physiological conditions, in human plasma and in human liver microsomes. Determination of the Phase I and Phase II metabolites and identification of the possible reactive metabolites allowed to identify the metabolic liabilities of the tryptophanol-derived isoindolinone scaffold. For the first time, non-heme containing iron(II) complexes were employed to prepare the major Phase I metabolites of tryptophanol derivatives, and their biological potential was subsequently evaluated. This allowed to understand that the parent compounds SLMP53-1 (43a) and DIMP53-1 (43d) are responsible for the observed antiproliferative activity. Compounds SLMP53-1 (43a) and SLMP53-2 (44a) were selected for hit-to-lead optimization to improve the efficacy, selectivity and metabolic stability of the scaffold. The first series of compounds was prepared through stereoselective cyclocondensation of enantiopure forms of amino alcohol tryptophanol and selected oxoacids, with yields of 41-86%. Chemical derivatization of para and meta positions of the C-9b phenyl ring and the impact of the stereochemistry were considered. From series 1, compound 66f’ resulted to exhibit 6-fold increase of the antiproliferative activity and 3.3-fold increase selectivity for the p53 pathway in human colorectal carcinoma HCT116 cell line, when compared to hit compound SLMP53-1 (43a). Importantly, the compound showed low toxicity in normal colon cells. Subsequently, a small series of compound 66f’ analogues were prepared exploring further derivatization of the para and meta positions of the C-9b phenyl ring and derivatizing the N-indole moiety, with yields of 66-95%. Based on the structure-metabolism relationships acquired for hit compound SLMP53-1 (43a), a series of halogen-enriched tryptophanol-derived isoindolinones was prepared by pyridinium bromide perbromide-promoted bromination with yields 75-92%. Two compounds, 73k and 73d, showed 1.9- and 3.9-fold higher antiproliferative activity in HCT116 cell line, once compared to hit compound SLMP53-1 (43a), and exhibited 3.8- and 1.9-fold selectivity towards p53 pathway, respectively. Through differential scanning fluorimetry experiments most active tryptophanol-derived isoindolinone compounds were screened against wild-type p53 core domain. Compound 66f’ enhances the thermostability of wt p53 core domain by melting temperature (Tm) increment of 1.64°C and compound 73d increases wt p53 core domain by Tm value of 10.39°C. This result may indicate that the compounds promote p53 stability. Additionally, screening of the metabolic stability of compounds 66f’, 73k and 73d revealed that the optimized compounds display a more adequate metabolic profile once compared with hit SLMP53-1 (43a). Derivatization of the indole nitrogen and introduction of a bromine atom promotes higher metabolic stability of the tryptophanol-derived isoindolinone scaffold and induces a decrement of the oxidative metabolism in positions 2 and 3 of the indole core. A metabolic switch is promoted under these conditions and metabolization of the 6-membered ring of the indole is observed.
In general, the results collected in this PhD, give relevant contributions in the development of p53 modulators with adequate metabolic profile and optimized efficacy and selectivity.
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.
Design, synthesis and biological evaluation of novel p53 activators by targeting p53 protein-protein interactions
Publication . Espadinha, Margarida; Santos, Maria Manuel Duque Vieira Marques dos; Conway, Stuart John; Rodrigues, Cecília Maria Pereira
The p53 protein, also known as the “guardian of the genome”, has an important role in the tumor suppression and regulation of cell processes. The majority of human cancers show inactivation of the p53 pathway. This perturbation can occur either by negative regulation, either by mutation or deletion of its gene. In tumors harboring wt p53, the MDM2 and MDMX homologous proteins are the main contributors for suppressing the p53 functions. In the last years, the development of p53-MDM2 PPI small molecule inhibitors has been one of the most popular approaches to reactivate wt p53, with eight clinical candidates under evaluation. However, it is now considered that, to achieve a full p53 reactivation, a dual inhibition of MDM2 and MDMX is required. Until today, there is no dual small molecule inhibitors of p53-MDM2/X PPIs in clinical trials.
In the last years, our group has been working on the design of five-membered spirooxindoles to develop novel anticancer agents. This work explores the design of the spiropyrazoline oxindole family to act as MDM2/X dual inhibitors. Here, we report an in silico-guided design, synthetic optimization, and biological evaluation of two libraries of spiropyrazoline oxindoles.
p53 also interacts with CREBBP. The inhibition of the p53-CREBBP PPI in certain biological circumstances can result in the p53 stabilization. For this purpose, PROTAC technology that allows to degrade the CREBBP protein was implemented, based on a lead CREBBP ligand developed in the Conway group.
Also, a yeast target-based screening of enantiopure tryptophanol derivatives led to the identification of dual p53-MDM2/X inhibitors, which were further optimized to compounds DIMP53-1, SYNAP and SLMP53-1, the last being also a mut p53 reactivator. To better understand the mechanism of action of this chemical family, in particular of SLMP53-1, two types of chemical probes were prepared, and preliminary in vitro cell assays were performed to evaluate their potential in future applications. Also, the preliminary biological and photocrosslinking results for SLMP53-1 photoaffinity-based probe showed its potential for the biological target profile of this compound.
Overall, this PhD thesis has provided valuable insights in the development of p53 activators.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/QUI-QOR/1304/2020