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Reversing multidrug resistance (MDR) in cancer cells by targeting p-glycoprotein (P-gp) : insights into the mechanism of MDR reversal from in silico P-gp modeling
Publication . Ferreira, Ricardo J; Ferreira, Maria José Umbelino, 1956-; Santos, Daniel J. V. A. dos
Multidrug resistance (MDR) in cancer is one of the major impairments in the success of chemotherapy. The main objective of this work was the identification and optimization of MDR reversers, derived from Euphorbia species, and to gain insights on the drug efflux mechanism by P-gp. The phytochemical study of Euphorbia pedroi yielded four new diterpenes, two macrocyclic lathyranes (9, 12), one jatrophane (10) and an unprecedented rearranged tigliane (13). While 9 is characterized by a rare double α,β-unsaturated ketone system, 13 has a new skeleton that may result from a pinacol rearrangement as proposed in a possible biogenetic pathway. Furthermore, a new spiroterpenoid (6) was also isolated, together with several known terpenoids (1-5, 7, 8, 14-16) and flavonoids (17-18). Molecular derivatization of compounds 15 and 17 yielded two set of new derivatives (19-24 and 25-71, respectively). In this way, reaction of 15 with hydroxylamine hydrochloride gave compound 19 that was further acylated with acyl anhydrides (20) and chlorides (21-24). Flavanone derivatives were obtained through three main approaches. Firstly, the methylation of naringenin (17) yielded compounds 46 and 47. Following, while hydrazones (25-28, 48-53) and carbohydrazides (37, 38, 40-42, 54-63) were obtained from compounds 17, 46 and 47, azines (29-36) were prepared by the reaction of 28 with aldehydes. A thiosemicarbazone derivative (39) was also prepared from 17. Other flavanone derivatives were additionally synthesized through a Mannich-type reaction (43-45) or by alkylation of compound 47 with epichlorohydrin (64, 65) followed by the reaction with amines, indole or thiophenol to yield 66-71. The chemical structures of all compounds were deduced from physical and spectroscopic data (IR, MS, 1D- and 2D-NMR experiments). The P-gp-mediated MDR reversal activity of compounds was evaluated by combining transport and chemosensitivity assays, in mouse lymphoma L5178Y-MDR (1-71) and Colo320 (1-18) cell models. While 6 showed high modulation activity even at 0.2 μM, compound 9 combined a good P-gp modulatory activity with a strong cytotoxic effect in both cell lines. When compared to the parent compound (15), the derivatives 20 and 22 and 23 were stronger efflux modulators towards the L5178Y-MDR cells. Most of the flavanone derivatives (25-71) were also more active than the parent compound (17) in L5178Y-MDR cells, being the most significant results observed for propanolamines 66-69, where compound 69 was found to be a strong P-gp modulator even at 2.0 μM. When in combination with doxorubicin, the natural compounds 6, 9, 10, 12 and 13 synergistically enhanced the cytotoxic effects of the drug. Strong synergistic effects were also observed for the derivatives 22 and 69. The ability of compounds 25-45 to modulate drug efflux by MRP1 and BCRP was also assessed, using human MRP1- and BCRP-transfected cell models. For this set of compounds, a second P-gp-transfected cell model was used. Azines (29-36) displayed significant activity towards BCRP while hydrazides (38-42) showed a good selectivity profile for MRP1. Oppositely, derivatives 35 and 36 displayed a good activity profile in both efflux pumps, when tested at 20 μM. Based on these results, new structure-activity relationships (SAR) for the selective BCRP and MRP1 inhibitors were obtained, unveiling which structural features could be directly correlated with the observed biological activity. The efflux mechanism of P-gp was studied by means of molecular dynamics and docking studies. The ‘linker’ polypeptide sequence was found to be important to absorb stronger motions and acting as a ‘damper’ between both NBDs, stabilizing the cytosolic portion of the transporter. Following, based on a previously refined P-gp structure, three distinct drugbinding sites could be identified and characterized, in a good agreement with published experimental data. Together with a new classification scheme, cross interactions between the substrate/modulator and each halve of P-gp were identified as an important mechanism in efflux modulation. Drug transit from bulk water into the DBP was also characterized as an overall free-energy downhill process, with no activation energy required for crossing the gate found between transmembrane helices 10 and 12. Furthermore, from the analysis on drug adsorption to the cytoplasmic domains in P-gp substrates and modulators were show to have different free energies of adsorption in both lipid/water and protein/ water interfaces and important differences in drug–protein interactions, protein dynamics and membrane biophysical characteristics were observed between non-substrates, substrates and modulators.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BD/84285/2012