Daniel Santos

Molecular docking characterizes substrate-binding sites and efflux modulation mechanisms within P-glycoprotein.

P-Glycoprotein (Pgp) is one of the best characterized ABC transporters, often involved in the multidrug-resistance phenotype overexpressed by several cancer cell lines. Experimental studies contributed to important knowledge concerning substrate polyspecificity, efflux mechanism, and drug-binding sites. This information is, however, scattered through different perspectives, not existing a unifying model for the knowledge available for this transporter. Using a previously refined structure of murine Pgp, three putative drug-binding sites were hereby characterized by means of molecular docking. The modulator site (M-site) is characterized by cross interactions between both Pgp halves herein defined for the first time, having an important role in impairing conformational changes leading to substrate efflux. Two other binding sites, located next to the inner leaflet of the lipid bilayer, were identified as the substrate-binding H and R sites by matching docking and experimental results. A new classification model with the ability to discriminate substrates from modulators is also proposed, integrating a vast number of theoretical and experimental data.

Insights on P-Glycoprotein's Efflux Mechanism Obtained by Molecular Dynamics Simulations.

P-Glycoprotein (P-gp) is often involved in multidrug resistance (MDR) to the pharmacological action of a wide number of anticancer agents. In this article, a series of molecular dynamics simulations of murines P-gp were developed, elucidating the importance of the lipid membrane and linker sequence in the protein structure stability. The behavior of several molecules inside the drug-binding pocket revealed a striking difference between substrates or modulators, and motion patterns were identified that could be correlated with conformational alterations due to substrate binding, corresponding to the initial step in the efflux mechanism. Only one entrance gate to the drug-binding pocket was found and, in the presence of a substrate, leads to changes in the motion patterns of the transporter into an efflux-like movement.

Toward a Better Pharmacophore Description of P-Glycoprotein Modulators, Based on Macrocyclic Diterpenes from Euphorbia Species

Multidrug resistance related to the increased expression of P-glycoprotein (P-gp) by cancer cells is the major contributor for the failure of chemotherapeutic treatments. Starting from pharmacophores and data already published and in macrocyclic diterpenes isolated from Euphorbia species, a comprehensive study of pharmacophore definitions of features was performed in order to obtain a new improved four-point pharmacophore able to detect literature and in-house modulators and simultaneously specific enough to avoid the detection of most nonactive molecules in a universe of 152 (literature), 74 (in-house), and 46 (inactive) molecules. This pharmacophore detects 84.2% of the molecules described in the literature, along with 100% detection of in-house isolated compounds and 19.5% of false positives. The importance of the hydrophobic and electron acceptor moieties as essential features for recognition of different molecules by the P-gp drug-binding site is clarified. The best combination of acceptor, donor, hydrophobic, and aromatic characteristics that contribute for the increased selectivity shown by the described pharmacophore is evaluated, and the protonation state of the molecules is also addressed.

Interfacial Excess Free Energies of Solid-Liquid Interfaces by Molecular Dynamics Simulation and Thermodynamic Integration

A method to compute the interfacial excess free energy of systems where a liquid phase is interacting with a solid phase is presented. The calculations are carried out by means of molecular dynamics simulations. The algorithm is based on a thermodynamic integration scheme that reversibly turns a flexible atomistically detailed solid surface that interacts with a liquid phase into a flat surface and allows the calculation of the variation in Gibbs free energy. The approach is probed by applying it to a model system of Lennard-Jones particles and comparing to previous calculations on similar systems.

Enhancing macrocyclic diterpenes as multidrug-resistance reversers: structure-activity studies on jolkinol D derivatives.

The phytochemical study of Euphorbia piscatoria yielded jolkinol D (1) in a large amount, whose derivatization gave rise to 12 ester derivatives (2-13) and hydrolysis to compound 14. The in vitro modulation of P-gp of compounds 1-14 was evaluated through a combination of transport and chemosensitivity assays, using the L5178 mouse T lymphoma cell line transfected with the human MDR1 gene. Apart from jolkinol D, all derivatives (2-14) showed potential as MDR reversal agents. In this small library of novel bioactive macrocyclic lathyrane diterpene derivatives, designed to evaluate structure-activity relationships essential in overcoming multidrug resistance (MDR), some correlations between MDR reversal and molecular weight, accessible solvent areas, and octanol/water partition coefficient were identified that can contribute to the development of new selective P-gp reversal agents.

Synthesis and evaluation of vinyl sulfones as caspase-3 inhibitors. A structure-activity study

The first structure-activity relationship study of vinyl sulfones as caspase-3 inhibitors is reported. A series of 12 vinyl sulfones was synthesized and evaluated for two downstream caspases (caspases-3 and -7). Dipeptidyl derivatives were significantly superior to their counterparts containing only Asp at P(1), as caspase-3 inhibitors. Fmoc-Val-Asp-trans-CH=CH-SO(2)Me was the most potent inhibitor of caspase-3 in the series, with a IC(50) of 29 microM and a second-order rate constant of inactivation, k(inact)/K(i), of 1.5 M(-1) s(-1). Computational studies suggest that the second amino acid occupies position S(3) of the enzyme. In addition, Fmoc-Val-Asp-trans-CH=CH-SO(2)Ph was inactive for caspase-7 for the tested concentrations.

Properties and Permeability of Hypericin and Brominated Hypericin in Lipid Membranes.

The promising photosensitizing properties of hypericin, a substituted phenanthroperylene quinone naturally found in Saint Johns wort, has led to the proposal that it can be utilized in photodynamic therapy. Structurally modified derivatives are at the present time being investigated to generate a more effective hypericin photosensitizer. Neither the detailed mechanism behind the powerful action of hypericin, arising as a result of light excitation, nor the intracellular localization and transportation is still fully understood. In the present work, molecular dynamics simulations have been performed to study the properties and the permeability of hypericin and modifications thereof, substituted with one or four bromine atoms, in a dipalmitoylphosphatidylcholine lipid membrane. The molecules were found to accumulate in the most dense region of the lipids due to competing interactions with the hydrophobic lipid interior and the polar aqueous environment. This was confirmed by analyzing the radial distribution functions and by the density profiles of the system components. Calculated free energy profiles display large negative changes in free energy for the transport process of the molecules into the lipids, which also support this finding. Permeability coefficients show overall fastest diffusion in the membrane system for hypericin containing one bromine.

Probing the aurone scaffold against Plasmodium falciparum: design, synthesis and antimalarial activity.

A library comprising 44 diversely substituted aurones derivatives was synthesized by straightforward aldol condensation reactions of benzofuranones and the appropriately substituted benzaldehydes. Microwave enhanced synthesis using palladium catalyzed protocols was introduced as a powerful strategy for extending the chemical space around the aurone scaffold. Additionally, Mannich-base derivatives, containing a 7-aminomethyl-6-hydroxy substitution pattern at ring A, were also prepared. Screening against the chloroquine resistant Plasmodium falciparum W2 strain identified novel aurones with IC50 values in the low micromolar range. The most potent compounds contained a basic moiety, with the ability to accumulate in acidic digestive vacuole of the malaria parasite. However, none of those aurones revealed significant activity against hemozoin formation and falcipain-2, two validated targets expressed during the blood stage of P. falciparum infection and functional in digestive vacuole of the parasite. Overall, this study highlight (i) the usefulness of aurones as platforms for synthetic procedures using palladium catalyzed protocols to rapidly deliver lead compounds for further optimization and (ii) the potential of novel aurone derivatives as promising antimalarial compounds.

Reversing cancer multidrug resistance: insights into the efflux by ABC transports from in silico studies

One of the greatest threats to cancer treatment is the development, by some tumors, of resistance to the pharmacological action of several structurally unrelated cytotoxic agents—multidrug resistance (MDR). As P‐glycoprotein (P‐gp) is one of the most studied ATP‐dependent efflux pumps that are frequently involved in drug efflux from cancer cells, the development of MDR modulators with the ability to inhibit P‐gp efflux is considered a promising approach for overcoming MDR. However, the development of P‐gp modulators have been hampered due to the absence of knowledge on the intrinsic molecular aspects by which efflux occurs, namely the specific steps that correlates drug recognition, ATP binding and efflux‐related conformational changes. Experimental evidences for these processes are also difficult to obtain and only provide small pieces of information that need to be assembled for better comprehension of a wider and complex process that is drug efflux. A promising alternative relies on cutting‐edge computational techniques to provide new insights on key aspects that are determinant to understand how P‐gp efflux can be effectively reversed. With the contribution of ligand‐based or structure‐based computational methods, P‐gp drug efflux is slowly becoming a dynamic and reactive process rather than a simple response to drug binding, with the complex architecture of ABC transporters playing a determinant role not only in drug recognition but in the coordination of ATP‐driven conformational changes that ultimately drives drug efflux. The major enlightenments that computational studies provided toward a better comprehension of MDR and P‐gp efflux phenomena are hereby described. WIREs Comput Mol Sci 2015, 5:27–55. doi: 10.1002/wcms.1196

Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53-MDM2 interaction

One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53-MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53-MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53(+/+)) and in its isogenic derivative without p53 (HCT116 p53(-/-)). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53(+/+) but not in p53(-/-) HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53-MDM2 interaction in HCT116 p53(+/+) cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of chemotypes activators of a wild-type p53-pathway with promising antitumor activity. Moreover, it may open the way to the development of a new class of p53-MDM2 interaction inhibitors.