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- Escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides BP100 and pepRPublication . Alves, Carla S.; Melo, Manuel N.; Franquelim, Henri G.; Ferre, Rafael; Planas, Marta; Feliu, Lidia; Bardají, Eduard; Kowalczyk, Wioleta; Andreu, David; Santos, Nuno C.; Fernandes, Miguel X.; Castanho, Miguel A.R.B.The potential of antimicrobial peptides (AMPs) as an alter native to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mech anisms adopted by two AMPs in disrupting the Gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic Gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neu tralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-depen dent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.
- 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.
- Effects of hydroxycinnamic acids on the glycolysis pathwayPublication . Serina, J.; Fernandes, M. X.; Castilho, P. C.Glycolysis is a metabolic pathway vital to the production of energy and some organisms rely on it solely to meet their energy requirements. It is also a central pathway in the metabolism of carbohydrates and a source of therapeutic targets against diabetes and cancer. Caffeoylquinic acids (CQAs) have been extensively studied for their role in the treatment and prevention of diabetes (and cancer) but their mechanisms of action remain mostly unknown. As such, molecular docking was used to find possible targets of CQAs in the glycolysis pathway. The molecular docking assays showed that CQAs were docked preferably to the Rossman fold (nicotinamide adenine dinucleotide — NAD(H) binding site) of oxidoreductases, that use NAD(H) as a cofactor, than to any other site. In-vitro assays were then performed using two NAD(H) dependent oxidoreductases from glycolysis (alcohol dehydrogenase and L-lactate dehydrogenase) in order confirm if CQAs would compete with the cofactor to inhibit the reaction. The results from these assays indicate that CQAs can act as both inhibitors and activators of NAD(H) dependent oxidoreductases of the glycolysis pathway.
- Caffeoylquinic acids as inhibitors for HIV-I protease and HIV-I integrase: a molecular docking studyPublication . Serina, João C.; Castilho, Paula C.; Fernandes, Miguel X.Caffeoylquinic acids are ubiquitous phenolic compounds with several health benefits to humans and they have been shown to be promisinganti-HIV compounds. In this work, molecular docking was used to study the inhibition of HIV-I integrase and protease using caffeoylquinic acids. It was possible to establish that the naturally occurring caffeoylquinic acids are not suitable as inhibitors for protease but are very good inhibitors for integrase. A new binding site was found for 3, 4-O-di-Caffeoylquinic acid between the chains of HIV-I integrase that could possibly lead to a disruption of the catalytic process of HIV-I integrase.
- Generation of artificial neural networks models in anticancer studyPublication . Sousa, Inês J.; Padrón, José M.; Fernandes, Miguel X.Artificial neural networks (ANNs) have several applications; one of them is the prediction of biological activity. Here, ANNs were applied to a set of 32 compounds with anticancer activity assayed experimentally against two cancer cell lines (A2780 and T-47D). Using training and test sets, the obtained correlation coefficients between experimental and calculated values of activity, for A2780, were 0.804 and 0.829, respectively, and for T-47D, we got 0.820 for the training set and 0.927 for the test set. Com paring multiple linear regression and ANN models, the latter were better suited in establishing relationships between compounds’ structure and their anticancer activity.
- QSAR models for prediction of PPARδ agonistic activity of indanylacetic acid derivativesPublication . Lather, Viney; Fernandes, Miguel X.Peroxisome Proliferator Activated Receptor b/d (PPAR b/d), one of three PPAR isoforms is a member of nuclear receptor superfamily and ubiquitously expressed in several metabolically active tissues such as liver, muscle, and fat. Tissue specific expression and knock-out studies suggest a role of PPARd in obesity and metabolic syndrome. Specific and selective PPARd ligands may play an important role in the treatment of metabolic disorders. Indanylacetic acid derivatives reported as potent and specific ligands against PPARd have been studied for the Quantitative Structure – Activity Relationships (QSAR). Molecules were represented by chemical descriptors that encode constitutional, topological, geometrical, and electronic structure features. Four different approaches, i.e., random selection, hierarchical clustering, k-means clustering, and sphere exclusion method were used to classify the dataset into training and test subsets. Forward stepwise Multiple Linear Regression (MLR) approach was used to linearly select the subset of descriptors and establish the linear relationship with PPARd agonistic activity of the molecules. The models were validated internally by Leave One Out (LOO) and externally for the prediction of test sets. The best subset of descriptors was then fed to the Artificial Neural Networks (ANN) to develop non-linear models. Statistically significant MLR models; with R2 varying from 0.80 to 0.87 were generated based on the different training and test set selection methods. Training of ANNs with different architectures for the different training and test selection methods resulted in models with R2 values varying from 0.83 to 0.94, which indicates the high predictive ability of the models.
- 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.
- A small molecule tubulin depolymerizing agent identified by a phenotypic drug discovery approachPublication . Ríos-Luci, Carla; Díaz-Rodríguez, Elena; Buey, Ruben M.; Sousa, Inês J.; Fernandes, Miguel X.; Pandiella, Atanasio; Padrón, José M.In the scenario of drug discovery, numerous in vitro testing initiatives had been established. Thus far, no general methodology is reputable and literature on this hot topic is scarce. In this respect, we propose a strategy based on a Phenotypic Drug Discovery approach. Within our program directed at the discovery of new antitumor agents, we have focused our attention on compounds that disturb the cell cycle. Our strategy relies on the use of a set of biological assays organized in a modular fashion. Herein, we exemplified this strategy with a family of propargylic enol ether derivatives. Using different assays in sequential stages and in a stepwise manner, our studies allowed us to understand the bioactivity of this family of compounds and led us to identify tubulin as the main molecular target.
- Erratum: Design of mutation-resistant HIV protease inhibitors with the substrate envelope hypothesis (Chemical Biology and Drug Design (2007) 69, (298-313)Publication . Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X.; Schiffer, Celia; Gilson, Michael K.There is a clinical need for HIV protease inhibitors that can evade resistance mutations. One possible approach to designing such inhibitors relies upon the crystallographic observation that the sub strates of HIV protease occupy a rather constant region within the binding site. In particular, it has been hypothesized that inhibitors which lie within this region will tend to resist clinically relevant mutations. The present study offers the first pros pective evaluation of this hypothesis, via compu tational design of inhibitors predicted to conform to the substrate envelope, followed by synthesis and evaluation against wild-type and mutant pro teases, as well as structural studies of complexes of the designed inhibitors with HIV protease. The results support the utility of the substrate envel ope hypothesis as a guide to the design of robust protease 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.