Synthesis, Biological Evaluation, and In Silico Studies of Novel Aminated Xanthones as Potential p53-Activating Agents.

Xanthone scaffold has been regarded as an attractive chemical tool in the search for bioactive molecules with antitumor activity, and in particular two xanthone derivatives, 12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano [3,2-b]xanthen-6-one (4) and 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (5), were described as a murine double minute 2 (MDM2)-p53 inhibitor and a TAp73 activator, respectively. The xanthone 5 was used as a starting point for the construction of a library of 3,4-dioxygenated xanthones bearing chemical moieties of described MDM2-p53 inhibitors. Eleven aminated xanthones were successfully synthesized and initially screened for their ability to disrupt the MDM2-p53 interaction using a yeast cell-based assay. With this approach, xanthone 37 was identified as a putative p53-activating agent through inhibition of interaction with MDM2. Xanthone 37 inhibited the growth of human colon adenocarcinoma HCT116 cell lines in a p53-dependent manner. The growth inhibitory effect of xanthone 37 was associated with the induction of G1-phase cell cycle arrest and increased protein expression levels of p53 transcriptional targets. These results demonstrated the potential usefulness of coupling amine-containing structural motifs of known MDM2-p53 disruptors into a 3,4-dioxygenated xanthone scaffold in the design of novel and potent p53 activators with antitumor activity and favorable drug-like properties. Moreover, in silico docking studies were performed in order to predict the binding poses and residues involved in the potential MDM2-p53 interaction.

p53 and glucose metabolism: an orchestra to be directed in cancer therapy.

Metabolic reprogramming is a hallmark of cancer with a strong impact on tumor cell survival, proliferation, dissemination, and resistance to therapy. As such, it has represented a promising therapeutic target for cancer. Although cancer cells may exhibit a wide range of metabolic profiles, the enhancement of aerobic glycolysis to generate lactate and ATP (Warburg effect) is a cancer-associated trait, which is under regulation of both oncogenes and tumor suppressor genes. Particularly, the tumor suppressor protein p53 was shown to revert the Warburg effect, and to negatively influence the oncogenic metabolic adaption of cancer cells. This review provides a systematization of the p53 influence on glycolysis and oxidative phosphorylation (OXPHOS), giving attention to the interplay of p53 with key signaling pathways, including c-Myc, HIF-1, LKB1/AMPK, and PI3K/Akt, as well as to mutant p53 gain-of-function. It also contributes to a better understanding of distinct metabolic profiles in heterogeneous tumor cell populations, and of its impact on cancer therapeutic resistance. Additionally, a reflection on current strategies adopted in clinical trials to overcome therapeutic resistance is presented, highlighting the main limitations and future therapeutic perspectives based on metabolic reprogramming. In particular, this review emphasizes the p53 activation as a promising therapeutic strategy to reprogram tumor glucose metabolism, conducting to cell death. Moreover, potential synergisms between p53-activating agents and metabolic inhibitors are discussed, fostering the improvement of cancer therapy.

The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding.

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.

The Antitumor Activity of a Lead Thioxanthone is Associated with Alterations in Cholesterol Localization.

The search for novel anticancer small molecules and strategies remains a challenge. Our previous studies have identified TXA1 (1-{[2-(diethylamino)ethyl]amino}-4-propoxy-9H- thioxanthen-9-one) as a hit compound, with in vitro antitumor potential by modulating autophagy and apoptosis in human tumor cell lines. In the present study, the mechanism of action and antitumor potential of the soluble salt of this molecule (TXA1.HCl) was further investigated using in vitro and mouse xenograft tumor models of NSCLC. Our results showed that TXA1.HCl affected steroid biosynthesis, increased RagD expression, and caused abnormal cellular cholesterol localization. In addition, TXA1.HCl treatment presented no toxicity to nude mice and significantly reduced the growth of human NSCLC cells xenografts in mice. Overall, this work provides new insights into the mechanism of action of TXA1, which may be relevant for the development of anticancer therapeutic strategies, which target cholesterol transport.

Screening a Small Library of Xanthones for Antitumor Activity and Identification of a Hit Compound which Induces Apoptosis.

Our previous work has described a library of thioxanthones designed to have dual activity as P-glycoprotein modulators and antitumor agents. Some of these compounds had shown a significant cell growth inhibitory activity towards leukemia cell lines, without affecting the growth of non-tumor human fibroblasts. However, their effect in cell lines derived from solid tumors has not been previously studied. The present work aimed at: (i) screening this small series of compounds from an in-house library, for their in vitro cell growth inhibitory activity in human tumor cell lines derived from solid tumors; and (ii) initiate a study of the effect of the most potent compound on apoptosis. The tumor cell growth inhibitory effect of 27 compounds was first analysed in different human tumor cell lines, allowing the identification of a hit compound, TXA1. Its hydrochloride salt TXA1·HCl was then synthesized, to improve solubility and bioavailability. Both TXA1 and TXA1·HCl inhibited the growth of MCF-7, NCI-H460, A375-C5, HeLa, 786-O, Caki-2 and AGS cell lines. The effect of TXA1·HCl in MCF-7 cells was found to be irreversible and was associated, at least in part, with an increase in cellular apoptosis.

Folic acid-mesoporous silicon nanoparticles enhance the anticancer activity of the p73-activating small molecule LEM2.

Many drugs with anticancer potential fail in their translation to the clinics due to problems related to pharmacokinetics. LEM2 is a new dual inhibitor of MDM2/mutp53-TAp73 interactions with interesting in vitro anticancer activity, which opens new hopes as an unconventional anticancer therapeutic strategy against cancers lacking p53 or with impaired p53 pathways. As others xanthone derivatives, LEM2 has limited aqueous solubility, posing problems to pursue in vivo assays, and therefore limiting its potential clinical translation. In this work, a mesoporous silicon (PSi)-based nanodelivery system was developed with folate functionalization (APTES-TCPSi-PEG-FA) for targeted delivery, which successfully increased LEM2 solubility when compared to bulk LEM2, evidenced in payload release study. Such effect was reflected on the increase of LEM2 cytotoxicity in HCT116 and MDA-MB-231 cancer cells when treated with LEM2-loaded APTES-TCPSi-PEG-FA, by reducing cell viability lower than 50% in comparison with bulk LEM2. Despite the reduced LEM2 loading degree, which still limits its application in further in vivo assays, the results obtained herein recognize PSi-based nanodelivery systems as a promising strategy to improve LEM2 anticancer activity and bioavailability, which will be relevant for the potential use of this potent TAp73 activator in anticancer therapy.

Structural and Drug Targeting Insights on Mutant p53.

p53 is a transcription factor with a pivotal role in cell homeostasis and fate. Its impairment is a major event in tumor onset and development. In fact, about half of human cancers bear TP53 mutations that not only halt the normal function of p53, but also may acquire oncogenic gain of functions that favor tumorigenesis. Although considered undruggable for a long time, evidence has proven the capability of many compounds to restore a wild-type (wt)-like function to mutant p53 (mutp53). However, they have not reached the clinic to date. Structural studies have strongly contributed to the knowledge about p53 structure, stability, dynamics, function, and regulation. Importantly, they have afforded relevant insights into wt and mutp53 pharmacology at molecular levels, fostering the design and development of p53-targeted anticancer therapies. Herein, we provide an integrated view of mutp53 regulation, particularly focusing on mutp53 structural traits and on targeting agents capable of its reactivation, including their biological, biochemical and biophysical features. With this, we expect to pave the way for the development of improved small molecules that may advance precision cancer therapy by targeting p53.

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.

DIMP53-1: a novel small-molecule dual inhibitor of p53-MDM2/X interactions with multifunctional p53-dependent anticancer properties.

The transcription factor p53 plays a crucial role in cancer development and dissemination, and thus, p53-targeted therapies are among the most encouraging anticancer strategies. In human cancers with wild-type (wt) p53, its inactivation by interaction with murine double minute (MDM)2 and MDMX is a common event. Simultaneous inhibition of the p53 interaction with both MDMs is crucial to restore the tumor suppressor activity of p53. Here, we describe the synthesis of the new tryptophanol-derived oxazoloisoindolinone DIMP53-1 and identify its activity as a dual inhibitor of the p53-MDM2/X interactions using a yeast-based assay. DIMP53-1 caused growth inhibition, mediated by p53 stabilization and upregulation of p53 transcriptional targets involved in cell cycle arrest and apoptosis, in wt p53-expressing tumor cells, including MDM2- or MDMX-overexpressing cells. Importantly, DIMP53-1 inhibits the p53-MDM2/X interactions by potentially binding to p53, in human colon adenocarcinoma HCT116 cells. DIMP53-1 also inhibited the migration and invasion of HCT116 cells, and the migration and tube formation of HMVEC-D endothelial cells. Notably, in human tumor xenograft mice models, DIMP53-1 showed a p53-dependent antitumor activity through induction of apoptosis and inhibition of proliferation and angiogenesis. Finally, no genotoxicity or undesirable toxic effects were observed with DIMP53-1. In conclusion, DIMP53-1 is a novel p53 activator, which potentially binds to p53 inhibiting its interaction with MDM2 and MDMX. Although target-directed, DIMP53-1 has a multifunctional activity, targeting major hallmarks of cancer through its antiproliferative, proapoptotic, antiangiogenic, anti-invasive, and antimigratory properties. DIMP53-1 is a promising anticancer drug candidate and an encouraging starting point to develop improved derivatives for clinical application.

Drug-like Properties and ADME of Xanthone Derivatives: The Antechamber of Clinical Trials.

Xanthone derivatives have been described as compounds with a privileged scaffold exhibiting diverse biological/pharmacological activities, what directed the interest to pursue the development of these derivatives into drug candidates. Nevertheless, to achieve this purpose it is crucial to study their pharmacokinetics and toxicity (PK/tox) properties as decision endpoints to continue or interrupt the development investment. This review aims to expose the most relevant analytical methods used in the physicochemical and PK/tox studies in order to detect, quantify, and identify different bioactive xanthones. Analyzing the main results from in vitro and in vivo systems towards ADME properties such as solubility, lipophilicity, pKa, chemical and metabolic stability, permeability, transporters modulation, and plasma protein binding, it is possible to uncover some threats governing the PK properties and to understand the bioavailability and drugability of xanthone derivatives. The last section of this review focuses on a case-study of the development of the drug candidate DMXAA, which has reached clinical trials, to provide the paths and the importance of PK/tox parameters of this scaffold. The data assembled in this review intends to guide for tackling issues in the design of potential lead compounds and drug candidates with a xanthone scaffold.