Metabolism-Guided Optimization of Tryptophanol-Derived Isoindolinone p53 Activators.

For the first time, the pharmacokinetic (PK) profile of tryptophanol-derived isoindolinones, previously reported as p53 activators, was investigated. From the metabolites' identification, performed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS), followed by their preparation and structural elucidation, it was possible to identify that the indole C2 and C3 are the main target of the cytochrome P450 (CYP)-promoted oxidative metabolism in the tryptophanol-derived isoindolinone scaffold. Based on these findings, to search for novel p53 activators a series of 16 enantiopure tryptophanol-derived isoindolinones substituted with a bromine in indole C2 was prepared, in yields of 62-89%, and their antiproliferative activity evaluated in human colon adenocarcinoma HCT116 cell lines with and without p53. Structural optimization led to the identification of two (S)-tryptophanol-derived isoindolinones 3.9-fold and 1.9-fold more active than hit SLMP53-1, respectively. Compounds' metabolic stability evaluation revealed that this substitution led to a metabolic switch, with the impact of Phase I oxidative metabolism being minimized. Through differential scanning fluorimetry (DSF) experiments, the most active compound of the series in cell assays led to an increase in the protein melting temperature (Tm) of 10.39 °C, suggesting an effective binding to wild-type p53 core domain.

Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance.

Melanoma is the deadliest form of skin cancer, primarily due to its high metastatic propensity and therapeutic resistance in advanced stages. The frequent inactivation of the p53 tumour suppressor protein in melanomagenesis may predict promising outcomes for p53 activators in melanoma therapy. Herein, we aimed to investigate the antitumor potential of the p53-activating agent SLMP53-2 against melanoma. Two- and three-dimensional cell cultures and xenograft mouse models were used to unveil the antitumor activity and the underlying molecular mechanism of SLMP53-2 in melanoma. SLMP53-2 inhibited the growth of human melanoma cells in a p53-dependent manner through induction of cell cycle arrest and apoptosis. Notably, SLMP53-2 induced p53 stabilization by disrupting the p53-MDM2 interaction, enhancing p53 transcriptional activity. It also promoted the expression of p53-regulated microRNAs (miRNAs), including miR-145 and miR-23a. Moreover, it displayed anti-invasive and antimigratory properties in melanoma cells by inhibiting the epithelial-to-mesenchymal transition (EMT), angiogenesis and extracellular lactate production. Importantly, SLMP53-2 did not induce resistance in melanoma cells. Additionally, it synergized with vemurafenib, dacarbazine and cisplatin, and resensitized vemurafenib-resistant cells. SLMP53-2 also exhibited antitumor activity in human melanoma xenograft mouse models by repressing cell proliferation and EMT while stimulating apoptosis. This work discloses the p53-activating agent SLMP53-2 which has promising therapeutic potential in advanced melanoma, either as a single agent or in combination therapy. By targeting p53, SLMP53-2 may counteract major features of melanoma aggressiveness.

Tryptophanol-Derived Oxazolopyrrolidone Lactams as Potential Anticancer Agents against Gastric Adenocarcinoma.

Gastric cancer is one of the deadliest cancers in modern societies, so there is a high level of interest in discovering new drugs for this malignancy. Previously, we demonstrated the ability of tryptophanol-derived polycyclic compounds to activate the tumor suppressor protein p53, a relevant therapeutic target in cancer. In this work, we developed a novel series of enantiomerically pure tryptophanol-derived small molecules to target human gastric adenocarcinoma (AGS) cells. From an initial screening of fourteen compounds in AGS cell line, a hit compound was selected for optimization, leading to two derivatives selective for AGS gastric cells over other types of cancer cells (MDA-MB-231, A-549, DU-145, and MG-63). More importantly, the compounds were non-toxic in normal cells (HEK 293T). Additionally, we show that the growth inhibition of AGS cells induced by these compounds is mediated by apoptosis. Stability studies in human plasma and human liver microsomes indicate that the compounds are stable, and that the major metabolic transformations of these molecules are mono- and di-hydroxylation of the indole ring.

Potency and Selectivity Optimization of Tryptophanol-Derived Oxazoloisoindolinones: Novel p53 Activators in Human Colorectal Cancer.

To search for novel p53 activators, four series of novel (S)- and (R)-tryptophanol-derived oxazoloisoindolinones were synthesized in a straightforward manner and their antiproliferative activity was evaluated in the human colorectal cancer HCT116 cell line. Structural optimization of the hit compound SLMP53-1 led to the identification of a (R)-tryptophanol-derived isoindolinone that was found to be six-fold more active, with increased selectivity for HCT116 cells with p53 and with low toxicity in normal cells. Binding studies with MDM2 showed that the antiproliferative activity of tryptophanol-derived isoindolinones does not involve inhibition of the main negative regulator of the p53 protein. Molecular docking simulations showed that although these molecules establish hydrophobic interactions with MDM2, they do not possess the required features to bind MDM2.

SLMP53-1 Inhibits Tumor Cell Growth through Regulation of Glucose Metabolism and Angiogenesis in a P53-Dependent Manner.

The Warburg effect is an emerging hallmark of cancer, which has the tumor suppressor p53 as its major regulator. Herein, we unveiled that p53 activation by (S)-tryptophanol-derived oxazoloisoindolinone (SLMP53-1) mediated the reprograming of glucose metabolism in cancer cells and xenograft human tumor tissue, interfering with angiogenesis and migration. Particularly, we showed that SLMP53-1 regulated glycolysis by downregulating glucose transporter 1 (GLUT1), hexokinase-2 (HK2), and phosphofructokinase-2 isoform 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3) (key glycolytic enzymes), while upregulating the mitochondrial markers synthesis of cytochrome c oxidase 2 (SCO2), cytochrome c oxidase subunit 4 (COX4), and OXPHOS mitochondrial complexes. SLMP53-1 also downregulated the monocarboxylate transporter 4 (MCT4), causing the subsequent reduction of lactate export by cancer cells. Besides the acidification of the extracellular environment, SLMP53-1 further increased E-cadherin and reduced metalloproteinase-9 (MMP-9) expression levels in both cancer cells and xenograft human tumor tissue, which suggested the interference of SLMP53-1 in extracellular matrix remodeling and epithelial-to-mesenchymal transition. Consistently, SLMP53-1 depleted angiogenesis, decreasing endothelial cell tube formation and vascular endothelial growth factor (VEGF) expression levels. SLMP53-1 also exhibited synergistic growth inhibitory activity in combination with the metabolic modulator dichloroacetic acid. These data reinforce the promising application of the p53-activating agent SLMP53-1 in cancer therapy, by targeting p53-mediated pathways of growth and dissemination.

SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations.

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.

SLMP53-2 Restores Wild-Type-Like Function to Mutant p53 through Hsp70: Promising Activity in Hepatocellular Carcinoma.

Half of human cancers harbor TP53 mutations that render p53 inactive as a tumor suppressor. In these cancers, reactivation of mutant p53 (mutp53) through restoration of wild-type-like function constitutes a valuable anticancer therapeutic strategy. In order to search for mutp53 reactivators, a small library of tryptophanol-derived oxazoloisoindolinones was synthesized and the potential of these compounds as mutp53 reactivators and anticancer agents was investigated in human tumor cells and xenograft mouse models. By analysis of their anti-proliferative effect on a panel of p53-null NCI-H1299 tumor cells ectopically expressing highly prevalent mutp53, the compound SLMP53-2 was selected based on its potential reactivation of multiple structural mutp53. In mutp53-Y220C-expressing hepatocellular carcinoma (HCC) cells, SLMP53-2-induced growth inhibition was mediated by cell cycle arrest, apoptosis, and endoplasmic reticulum stress response. In these cells, SLMP53-2 restored wild-type-like conformation and DNA-binding ability of mutp53-Y220C by enhancing its interaction with the heat shock protein 70 (Hsp70), leading to the reestablishment of p53 transcriptional activity. Additionally, SLMP53-2 displayed synergistic effect with sorafenib, the only approved therapy for advanced HCC. Notably, it exhibited potent antitumor activity in human HCC xenograft mouse models with a favorable toxicological profile. Collectively, SLMP53-2 is a new mutp53-targeting agent with promising antitumor activity, particularly against HCC.

An Update on MDMX and Dual MDM2/X Inhibitors.

The tumor suppressor protein p53 is inactivated in all types of human cancers, either by negative regulation, by mutation or deletion of its gene. Specifically, in tumors that retain wild-type (wt) p53 status, p53 is inactivated by interaction with negative regulators, such as MDM2 and MDMX. These two proteins are found to be overexpressed in several different types of cancers, and the restoration of p53 activity by inhibition of these proteins is now considered an important approach for cancer treatment. The first studies using this strategy to reactivate wt p53 were focused on the development of small molecules that could inhibit MDM2. In this way, p53 could be liberated and act again as a tumor suppressor. From these studies, nine small molecules have reached clinical trials. More recently, MDMX was also identified as an important therapeutic target to efficiently reactivate wt p53, and it is now considered that, for full p53 reactivation, dual inhibition of MDM2 and MDMX is required. In this review we will focus on the most recent advances in the discovery of novel small molecules and stapled peptides that act as selective MDMX inhibitors or as dual MDM2/X inhibitors.