Development of p53-MDMs Protein-Protein Interaction Inhibitors to Tackle Cancer

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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.

2022-12Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

Programa de financiamento

OE

Número da atribuição

PD/BD/143126/2019