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Research Project
Strategic Project - UI 4040 - 2011-2012
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Publications
Sulfated small molecules targeting EBV in Burkitt lymphoma: from in silico screening to the evidence of in vitro effect on viral episomal DNA
Publication . 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. Helena
Epstein–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.
Dual inhibitors of P-glycoprotein and tumor cell growth: (re)discovering thioxanthones
Publication . Palmeira, Andreia; Vasconcelos, M. Helena; Paiva, Ana; Fernandes, Miguel X.; Pinto, Madalena; Sousa, Emília
For 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 phases
Publication . Fernandes, Carla; Tiritan, Maria Elizabeth; Cass, Quezia; Kairys, Visvaldas; Fernandes, Miguel Xavier; Pinto, Madalena
A 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.
Discovery of a new small-molecule inhibitor of p53–MDM2 interaction using a yeast-based approach
Publication . 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, Lucília
The 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.
Structure and ligand-based design of P-glycoprotein inhibitors: a historical perspective
Publication . 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.
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Funders
Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
6817 - DCRRNI ID
Funding Award Number
PEst-OE/SAU/UI4040/2011