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Discovery of spirooxadiazoline oxindoles with dual-stage antimalarial activity
Publication . Lopes, Elizabeth A.; Mestre, Raquel; Fontinha, Diana; Legac, Jenny; Pei, Jinxin V.; Sanches-Vaz, Margarida; Mori, Mattia; Lehane, Adele M.; Rosenthal, Philip J.; Prudêncio, Miguel; Santos, Maria M. M.
Malaria remains a prevalent infectious disease in developing countries. The first-line therapeutic options are based on combinations of fast-acting artemisinin derivatives and longer-acting synthetic drugs. However, the emergence of resistance to these first-line treatments represents a serious risk, and the discovery of new effective drugs is urgently required. For this reason, new antimalarial chemotypes with new mechanisms of action, and ideally with activity against multiple parasite stages, are needed. We report a new scaffold with dual-stage (blood and liver) antiplasmodial activity. Twenty-six spirooxadiazoline oxindoles were synthesized and screened against the erythrocytic stage of the human malaria parasite P. falciparum. The most active compounds were also tested against the liver-stage of the murine parasite P. berghei. Seven compounds emerged as dual-stage antimalarials, with IC50 values in the low micromolar range. Due to structural similarity with cipargamin, which is thought to inhibit blood-stage P. falciparum growth via inhibition of the Na + efflux pump PfATP4, we tested one of the most active compounds for anti-PfATP4 activity. Our results suggest that this target is not the primary target of spirooxadiazoline oxindoles and further studies are ongoing to identify the main mechanism of action of this scaffold.
Stereoselective Synthesis of Spirooxindole Derivatives Using One-Pot Multicomponent Cycloaddition Reaction and Evaluation of Their Antiproliferative Efficacy
Publication . Ghosh, Rajat; Vítor, Jorge M. B.; Mendes, Maria Eduarda; Paulo, Alexandra; Acharya, Pratap Chandra
A highly stereoselective, one-pot, multicomponent method has been developed to synthesize pyrrolizidine- and N-methyl pyrrolidine-substituted spirooxindole derivatives. The [3 + 2] cycloaddition reaction involves the reaction between the dipole azomethine ylides, generated in situ from the reaction between isatin and secondary amino acids such as L-proline or sarcosine, and α,β-unsaturated carbonyl compounds as the dipolarophile. The reaction condition was optimized to achieve excellent regio- and stereoselectivity. Products were obtained in good yield using ethanol as a solvent at the reflux temperature. The newly synthesized spirooxindole derivatives were evaluated for their antiproliferative efficacy against National Cancer Institute (NCI)-60 cancer cell lines and DNA G-quadruplex (G4) interaction capacity. Compound 14b produced selective cytotoxicity against leukemia, renal, colon, and prostate cancer cell lines at a 10 μM concentration. The G4 interaction studies further suggested that these spirooxindole derivatives were devoid of any activity as DNA G4 ligands.
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.
Discovering New Molecules for Diagnosis and Non-Toxic Differentiation Therapy in Colorectal Cancer
Publication . Paiva, Nuno André Silva; Solá, Susana; Rodrigues, Cecília Maria Pereira
Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide. Therefore, better diagnostic tools and anticancer therapies have been some of the most sought out topics in CRC research. MicroRNAs (miRNAs), a type of short non-coding single strand RNA molecules capable of repressing mRNA translation, are rising as potential and powerful diagnostic and prognostic biomarkers in cancer. In CRC, differences in the expression pattern of certain miRNAs in tissue have been already described, suggesting that miRNAs could represent an effective diagnostic option for this particular disease. On the other hand, cancer stem cells (CSCs), a subpopulation of cells within tumors with phenotypic resemblance to normal stem cells, have been implicated in tumorigenesis, metastization and recurrence processes. Indeed, these undifferentiated cells are not entirely eliminated with the classical anti-proliferative approaches, being responsible for tumorigenic potential and rapid relapse. Thus, efficient therapies targeting this specific type of cancer cells are being developed, with special emphasis on combinatorial drug therapies, to sensitize cells to conventional anticancer treatments and therefore reduce the toxicity associated with high doses of chemotherapeutic treatments.
Two major objectives were pursued in this project: 1) to optimize methodologies to assess the diagnostic and prognostic potential of a panel of miRNAs in patients with CRC, prior to and after being submitted to chemoradiotherapy; and 2) to evaluate the effectiveness of two chemical compounds, from an in-house library, as potential non-toxic differentiation drivers in CRC cells and characterize their specific signaling pathways.
The qPCR analysis of human colonic samples revealed differential expression profiles for several miRNAs derived from normal and tumor tissues, as well as before and after chemoradiotherapy. Nevertheless, these data are still preliminary and further studies are necessary to validate the prognostic and diagnostic value of these miRNAs. In addition, we identified the biological effects of two potential candidate molecules, a spiropyrazoline oxindole (DS6) and a 3-piperidinyl-indole (SAS9), capable of promoting differentiation in human colorectal carcinoma cells. Although both compounds had impact on CRC cells, DS6 was particularly successful, being capable of reducing the levels of stemness and pluripotency markers, increasing differentiation, inhibiting self-renewal and exhibiting a slight synergistic effect in sensitizing cells to a classical chemotherapeutic, 5-fluorouracil.
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Interestingly, by using isogenic p53+/+ and p53-/- HCT116 cell lines, we were able to observe a tendency for DS6 to develop its differentiation effect only in p53+/+ cells, suggesting a p53-dependent mechanism of action for this molecule. Further studies are still required to validate the DS6 mechanism of action, as well as consolidate the data on its therapeutic potential for differentiation in CRC.
Altogether, our results shed light on two aspects of CRC research, related with diagnosis and treatment- We open the way to optimize the discovery of novel miRNAs as biomarkers for diagnosis and prognosis and contribute to uncover new regulators of differentiation in CRC.
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/29664/2017