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- Insights into the in vitro antitumor mechanism of action of a new pyranoxanthonePublication . Palmeira, Andreia; Paiva, Ana; Sousa, Emília; Seca, Hugo; Almeida, Gabriela M.; Lima, Raquel T.; Fernandes, Miguel X.; Pinto, Madalena; Vasconcelos, M. HelenaNaturally occurring xanthones have been docu mented as having antitumor properties, with some of them presently undergoing clinical trials. In an attempt to improve the biological activities of dihydroxyxanthones, prenylation and other mole cular modifications were performed. All the com pounds reduced viable cell number in a leukemia cell line K-562, with the fused xanthone 3, 4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano[3, 2-b]xanthen-6-one (5) being the most potent. The pyranoxanthone 5 was particularly effective in additional leukemia cell lines (HL-60 and BV-173). Furthermore, the pyranoxanthone 5 decreased cel lular proliferation and induced an S-phase cell cycle arrest. In vitro, the pyranoxanthone 5 increased the percentage of apoptotic cells which was confirmed by an appropriate response at the protein level (e.g., PARP cleavage). Using a com puter screening strategy based on the structure of several anti- and pro-apoptotic proteins, it was verified that the pyranoxanthone 5 may block the binding of anti-apoptotic Bcl-xL to pro-apoptotic Bad and Bim. The structure-based screening revealed the pyranoxanthone 5 as a new scaffold that may guide the design of small molecules with better affinity profile for Bcl-xL.
- New uses for old drugs: pharmacophore‐based screening for the discovery of P‐glycoprotein inhibitorsPublication . Palmeira, Andreia; Rodrigues, Freddy; Sousa, Emília; Pinto, Madalena; Vasconcelos, M. Helena; Fernandes, Miguel X.P-glycoprotein (P-gp) is one of the best character ized transporters responsible for the multidrug resistance phenotype exhibited by cancer cells. Therefore, there is widespread interest in eluci dating whether existing drugs are candidate P-gp substrates or inhibitors. With this aim, a pharma cophore model was created based on known P-gp inhibitors and it was used to screen a database of existing drugs. The P-gp modulatory activity of the best hits was evaluated by several methods such as the rhodamine-123 accumulation assay using K562Dox cell line, and a P-gp ATPase activ ity assay. The ability of these compounds to enhance the cytotoxicity of doxorubicin was assessed with the sulphorhodamine-B assay. Of the 21 hit compounds selected in silico, 12 were found to significantly increase the intracellular accumulation of Rhodamine-123, a P-gp substrate. In addition, amoxapine and loxapine, two tetracy clic antidepressant drugs, were discovered to be potent non-competitive inhibitors of P-gp, causing a 3.5-fold decrease in the doxorubicin GI50 in K562Dox cell line. The overall results provide important clues for the non-label use of known drugs as inhibitors of P-gp. Potent inhibitors with a dibenzoxazepine scaffold emerged from this study and they will be further investigated in order to develop new P-gp inhibitors.
- Discovery of a new small-molecule inhibitor of p53–MDM2 interaction using a yeast-based approachPublication . Leão, Mariana; Pereira, Clara; Bisio, Alessandra; Ciribilli, Yari; Paiva, Ana M.; Machado, Neuza; Palmeira, Andreia; Fernandes, Miguel X.; Sousa, Emília; Pinto, Madalena; Inga, Alberto; Saraiva, LucíliaThe virtual screening of a library of xanthone derivatives led us to the identification of potential novel MDM2 ligands. The activity of these compounds as inhibitors of p53–MDM2 interaction was investigated using a yeast phenotypic assay, herein developed for the initial screening. Using this approach, in association with a yeast p53 transactivation assay, the pyranoxanthone (3,4-dihydro-12- hydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one) (1) was identified as a putative small molecule inhibitor of p53–MDM2 interaction. The activity of the pyranoxanthone 1 as inhibitor of p53–MDM2 interaction was further investigated in human tumor cells with wild-type p53 and overexpressed MDM2. Notably, the pyranoxanthone 1 mimicked the activity of known p53 activators, leading to p53 stabilization and activation of p53- dependent transcriptional activity. Additionally, it led to increased protein levels of p21 and Bax, and to caspase-7 cleavage. By computational docking studies, it was predicted that, like nutlin-3a, a known small-molecule inhibitor of p53–MDM2 interaction, pyranoxanthone 1 binds to the p53-binding site of MDM2. Overall, in this work, a novel small-molecule inhibitor of p53–MDM2 interaction with a xanthone scaffold was identified for the first time. Besides its potential use as molecular probe and possible lead to develop anticancer agents, the pyranoxanthone 1 will pave the way for the structure-based design of a new class of p53–MDM2 inhibitors.
- Dual inhibitors of P-glycoprotein and tumor cell growth: (re)discovering thioxanthonesPublication . Palmeira, Andreia; Vasconcelos, M. Helena; Paiva, Ana; Fernandes, Miguel X.; Pinto, Madalena; Sousa, EmíliaFor many pathologies, there is a crescent effort to design multiple ligands that interact with a wide variety of targets. 1-Aminated thioxanthone derivatives were synthesized and assayed for their in vitro dual activity as antitumor agents and P-glycoprotein (P-gp) inhibitors. The approach was based on molecular hybridization of a thioxanthone scaffold, present in known antitumor drugs, and an amine, described as an important pharmacophoric feature for P-gp inhibition. A rational approach using homology modeling and docking was used, to select the molecules to be synthesized by conventional or microwave-assisted Ullmann C–N cross-coupling reaction. The obtained aminated thioxanthones were highly effective at inhibiting P-gp and/or causing growth inhibition in a chronic myelogenous leukemia cell line, K562. Six of the aminated thioxanthones had GI50 values in the K562 cell line below 10 mM and 1-{[2-(diethylamino)ethyl]amino}-4-propoxy-9H-thioxanthen-9-one (37) had a GI50 concentration (1.90 mM) 6-fold lower than doxorubicin (11.89 mM) in the K562Dox cell line. The best P-gp inhibitor found was 1-[2-(1H-benzimidazol-2-yl)ethanamine]-4-propoxy-9H-thioxanthen-9-one (45), which caused an accumulation rate of rhodamine-123 similar to that caused by verapamil in the K562Dox resistant cell line, and a decrease in ATP consumption by P-gp. At a concentration of 10 mM, compound 45 caused a decrease of 12.5-fold in the GI50 value of doxorubicin in the K562Dox cell line, being 2-fold more potent than verapamil. From the overall results, the aminated thioxanthones represent a new class of P-gp inhibitors with improved efficacy in sensitizing a resistant P-gp overexpressing cell line (K562Dox) to doxorubicin.
- Enantioseparation and chiral recognition mechanism of new chiral derivatives of xanthones on macrocyclic antibiotic stationary phasesPublication . Fernandes, Carla; Tiritan, Maria Elizabeth; Cass, Quezia; Kairys, Visvaldas; Fernandes, Miguel Xavier; Pinto, MadalenaA chiral HPLC method using four macrocyclic antibiotic chiral stationary phases (CSPs) has been inves tigated for determination of the enantiomeric purity of fourteen new chiral derivatives of xanthones (CDXs). The separations were performed with the CSPs Chirobiotic T, Chirobiotic TAG, Chirobiotic V and Chirobiotic R under multimodal elution conditions (normal-phase, reversed-phase and polar ionic mode). The analyses were performed at room temperature in isocratic mode and UV and CD detection at a wavelength of 254 nm. The best enantioselectivity and resolution were achieved on Chirobiotic R and Chirobiotic T CSPs, under normal elution conditions, with RS ranging from 1.25 to 2.50 and from 0.78 to 2.06, respectively. The optimized chromatographic conditions allowed the determination of the enan tiomeric ratio of eight CDXs, always higher than 99%. In order to better understand the chromatographic behavior at a molecular level, and the structural features associated with the chiral recognition mech anism, computational studies by molecular docking were carried out using VDock. These studies shed light on the mechanisms involved in the enantioseparation for this important class of chiral compounds.
- Structure and ligand-based design of P-glycoprotein inhibitors: a historical perspectivePublication . Palmeira, Andreia; Sousa, Emilia; Vasconcelos, M. Helena; Pinto, Madalena; Fernandes, Miguel X.Computer-assisted drug design (CADD) is a valuable approach for the discovery of new chemical entities in the field of cancer therapy. There is a pressing need to design and develop new, selective, and safe drugs for the treatment of multidrug resistance (MDR) cancer forms, specifically active against P-glycoprotein (P-gp). Recently, a crystallographic structure for mouse P-gp was obtained. However, for decades the design of new P-gp inhibitors employed mainly ligand-based approaches (SAR, QSAR, 3D-QSAR and phar macophore studies), and structure-based studies used P-gp homology models. However, some of those results are still the pillars used as a starting point for the design of potential P-gp inhibitors. Here, pharmacophore mapping, (Q)SAR, 3D-QSAR and homology modeling, for the discovery of P-gp inhibitors are reviewed. The importance of these methods for understanding mechanisms of drug resistance at a molecular level, and design P-gp inhibitors drug candidates are discussed. The examples mentioned in the review could provide insights into the wide range of possibilities of using CADD methodologies for the discovery of efficient P-gp inhibitors.
- Sulfated small molecules targeting EBV in Burkitt lymphoma: from in silico screening to the evidence of in vitro effect on viral episomal DNAPublication . Lima, Raquel T.; Seca, Hugo; Palmeira, Andreia; Fernandes, Miguel X.; Castro, Felipe; Correia-da-Silva, Marta; Nascimento, Maria S. J.; Sousa, Emília; Pinto, Madalena; Vasconcelos, M. HelenaEpstein–Barr virus (EBV) infects more than 90% of the world population. Following primary infection, Epstein– Barr virus persists in an asymptomatic latent state. Occasionally, it may switch to lytic infection. Latent EBV infection has been associated with several diseases, such as Burkitt lymphoma (BL). To date, there are no available drugs to target latent EBV, and the existing broad-spec trum antiviral drugs are mainly active against lytic viral infection. Thus, using computational molecular docking, a virtual screen of a library of small molecules, including xanthones and flavonoids (described with potential for antiviral activity against EBV), was carried out targeting EBV proteins. The more interesting molecules were selected for further computational analysis, and sub sequently, the compounds were tested in the Raji (BL) cell line, to evaluate their activity against latent EBV. This work identified three novel sulfated small molecules capable of decreasing EBV levels in a BL. Therefore, the in silico screening presents a good approach for the development of new anti-EBV agents.