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The development of multidrug resistance (MDR) is a major cause of failure in cancer chemotherapy. Several abietane diterpenes with antitumoral activities have been isolated from Plectranthus spp. such as 6,7-dehydroroyleanone (DHR, 1) and 7α-acetoxy-6β-hydroxyroyleanone (AHR, 2). Several royleanone derivatives were prepared through hemisynthesis from natural compounds 1 and 2 to achieve a small library of products with enhanced anti-P-glycoprotein activity. Nonetheless, some derivatives tend to be unstable. Therefore, to reason such lack of stability, the electron density based local reactivity descriptors condensed Fukui functions and dual descriptor were calculated for several derivatives of DHR. Additionally, molecular docking and molecular dynamics studies were performed on several other derivatives to clarify the molecular mechanisms by which they may exert their inhibitory effect in P-gp activity. The analysis on local reactivity descriptors was important to understand possible degradation pathways and to guide further synthetic approaches toward new royleanone derivatives. A molecular docking study suggested that the presence of aromatic moieties increases the binding affinity of royleanone derivatives toward P-gp. It further suggests that one royleanone benzoylated derivative may act as a noncompetitive efflux modulator when bound to the M-site. The future generation of novel royleanone derivatives will involve (i) a selective modification of position C-12 with chemical moieties smaller than unsubstituted benzoyl rings and (ii) the modification of the substitution pattern of the benzoyloxy moiety at position C-6. This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in ACS Medicinal Chemistry Letters, copyright © American Chemical Society after peer review. To access the final edited and published work see [https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00642]. Related to: [http://ibiss-r.rcub.bg.ac.rs/handle/123456789/3817].

Aim: Naringenin (1), isolated in large amount from the aerial parts of Euphorbia pedroi, was chemically derivatized to yield 18 imine derivatives (2–19) and three alkylated derivatives through a Mannich-type reaction (20–22) that were tested as multidrug resistance (MDR) reversers in cancer cells. Results/methodology: While hydrazone (2–4) and azine (5–13) derivatives showed an improvement in their MDR reversal activities against the breast cancer resistance protein, carbohydrazides 14–19 revealed an enhancement in MDR reversal activity toward the multidrug resistance protein 1. Conclusion: The observed activities, together with pharmacophoric analysis and molecular docking studies, identified the spatial orientation of the substituents as a key structural feature toward a possible mechanism by which naringenin derivatives may reverse MDR in cancer.

Background: From a dataset obtained by chemical derivatization of a macrocyclic diterpenic scaffold, in silico approaches identified which structural features correlate with experimental modulation of P-gp activity. Results/methodology: Ninety-two percent of the strongest MDR modulators were positively identified within the dataset by virtual screening. Quantitative structure–activity relationships models with high robustness and predictability were obtained for both MDR1-transfected L5178Y mouse lymphoma T-cells (q2 0.875, R2 pred 0.921) and human colon adenocarcinoma (q2 0.820, R2 pred 0.951) cell lines. A new pharmacophoric model suggests that charge distribution within the molecule is important for biological activity. Conclusion: For the studied diterpenes, the conformation of the macrocyclic scaffold and its substitution pattern are the main determinants for the biological activity, being related with steric and electrostatic factors.

Naringenin (1), isolated from Euphorbia pedroi, was previously derivatized yielding compounds 2-13. In this study, aiming at expanding the pool of analogues of the flavanone core towards better multidrug resistance (MDR) reversal agents, alkylation reactions and chemical modification of the carbonyl moiety was performed (15-39). Compounds structures were assigned mainly by 1D and 2D NMR experiments. Compounds 1-39 were assessed as MDR reversers, in human ABCB1-transfected mouse T-lymphoma cells, overexpressing P-glycoprotein (P-gp). The results revealed that O-methylation at C-7, together with the introduction of nitrogen atoms and aromatic moieties at C-4 or C-4', significantly improved the activity, being compounds 27 and 37 the strongest P-gp modulators and much more active than verapamil. In combination assays, synergistic interactions of selected compounds with doxorubicin substantiated the results. While molecular docking suggested that flavanone derivatives act as competitive modulators, molecular dynamics showed that dimethylation promotes binding to a modulator-binding site. Moreover, flavanones may also interact with a vicinal ATP-binding site in both nucleotide-binding domains, hypothesizing an allosteric mode of action.

The phytochemical study of Euphorbia pedroi led to the isolation of a new tetracyclic triterpenoid with an unusual spiro scaffold, spiropedroxodiol (1), along with seven known terpenoids (2-8). Aiming at obtaining compounds with improved multidrug-resistance (MDR) reversal activity, compound 8, an ent-abietane diterpene, was derivatized by introducing nitrogen-containing and aromatic moieties, yielding compounds 9-14. The structures of compounds were characterized by detailed spectroscopic analysis, including 2D NMR experiments (COSY, HMQC/HSQC, HMBC, and NOESY). Compounds 1-14 were evaluated for their MDR-reversing activity on human ABCB1 gene transfected mouse lymphoma cells (L5178Y-MDR) through a combination of functional and chemosensitivity assays. The natural compounds 1-8 were further evaluated on resistant human colon adenocarcinoma cells (Colo320), and, additionally, their cytotoxicity was assessed on noncancerous mouse (NIH/3T3) and human (MRC-5) embryonic fibroblast cell lines. While spiropedroxodiol (1) was found to be a very strong MDR reversal agent in both L5178Y-MDR and Colo320 cells, the chemical modifications of helioscopinolide E (8) at C-3 positively contributed to increase the MDR reversal activity of compounds 10, 12, and 13. Furthermore, in combination assays, compounds 1 and 7-14 enhanced synergistically the cytotoxicity of doxorubicin. Finally, by means of molecular docking, the key residues and binding modes by which compounds 1-14 may interact with a murine P-glycoprotein model were identified, allowing additional insights on the efflux modulation mechanism of these compounds.

Multidrug-resistance (MDR) phenomena are a worldwide health concern. ATP-binding cassette efflux pumps as P-glycoprotein have been thoroughly studied in a frantic run to develop new efflux modulators capable to reverse MDR phenotypes. The study of efflux pumps has provided some key aspects on drug extrusion, however the answers could not be found solely on ATP-binding cassette transporters. Its counterpart – the plasma membrane – is now emerging as a critical structure able to modify drug behavior and efflux pump activity. Alterations in the membrane surrounding P-glycoprotein are now known to modulate drug efflux, with membrane-related biophysical, biochemical and mechanical aspects further increasing the complexity of an already multifaceted phenomena. This review summarizes the main knowledge comprising the plasma membrane role in MDR.

Herein, collateral sensitivity effect was exploited as a strategy to select effective compounds to overcome multidrug resistance in cancer. Thus, eleven macrocyclic diterpenes, namely jolkinol D (1), isolated from Euphorbia piscatoria, and its derivatives (2-11) were evaluated for their activity on three different Human cancer entities: gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29) each with a variant selected for resistance to mitoxantrone (EPG85-257RN; EPP85-181RN; HT-29RN) and one to daunorubicin (EPG85-257RD; EPP85-181RD; HT-29RD). Jolkinol D (1) and most of its derivatives (2-11) exhibited significant collateral sensitivity effect towards the cell lines EPG85-257RN (associated with P-glycoprotein overexpression) and HT-29RD (altered topoisomerase II expression). The benzoyl derivative, jolkinoate L (8) demonstrated ability to target different cellular contexts with concomitant high antiproliferative activity. These compounds were previously assessed as P-glycoprotein modulators, at non-cytotoxic doses, on MDR1-mouse lymphoma cells. A regression analysis between the antiproliferative activity presented herein and the previously assessed P-glycoprotein modulatory effect showed a strong relation between the compounds that presented both high P-glycoprotein modulation and cytotoxicity.

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.

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., This study was supported by FCT (Fundação para a Ciência e a ̂Tecnologia, Portugal) by Research Projects PTDC/QUI-QUI/099815/2008 and PEst-OE/SAU/UI4013/2011. M.R. acknowledges FCT for her Ph.D. Grant SFRH/BD/72915/2010. The authors thank Dr. Teresa Vasconcelos, Instituto Superior de Agronomia, Universidade de Lisboa, Portugal, for the taxonomic work on the plant material.

This paper focuses on the importance of the intermediate linker sequence for the stabilization of the cytoplasmic portion of murine P-glycoprotein, an ABC transporter involved in Multidrug Resistance (MDR) in cancer. Three putative protein-protein interaction areas were predicted to exist, two of them next to the C-terminal nucleotide-binding domain (NBD2) and the third one next to the inner leaflet interface of the lipid bilayer. These contact spots were confirmed by detailed contact maps from structures obtained before and after a 100 ns molecular dynamics production run, allowing a more thorough characterization of the type and number of residues involved in protein-protein contacts. It was found that these contact surfaces are located next to several highly conserved motifs of ABC transporters, serving as anchor points and assisting the linker's 'damper' function.

The failure of chemotherapeutics in cancer is mainly imputed to multidrug resistance (MDR). Among several proposed approaches to overcome MDR, the modulation of P-glycoprotein (Pgp) is the most highlighted [1]. Another valuable strategy is to take advantage of the collateral sensitivity effect, where some compounds may selectively exert a more pronounced cytotoxicity on cells with MDR phenotype than in the parental ones [1]. Macrocyclic diterpenes isolated from Euphorbia species, have been recognized as potent Pgp modulators and as having a collateral sensitivity effect on MDR cell variants [2]. Our main goal is to explore these two approaches, since tackling more than one MDR mechanism simultaneously might be a solution to control MDR in its complexity. The fractionation of the methanol extract of the aerial parts of E. piscatoria led to the isolation of a macrocyclic diterpene that allowed to build a small library of derivatives [3]. In this work, eleven compounds were evaluated for their potential collateral sensitivity effect on nine cell lines, gastric, pancreatic and colon cancer cell models (drug sensitive and drug resistant sublines), using the SRB assay. Most of the derivatives exhibited significant collateral sensitivity effect towards the MDR gastric cell line (with Pgp overexpression) and the MDR colon cells. The most significant results were obtained with a benzoyl derivative that demonstrated ability to target different cellular contexts with concomitant high antiproliferative activity. These compounds were previously assessed as Pgp modulators, at non-cytotoxic doses, on MDR1-mouse lymphoma cells [3]. A regression analysis between the antiproliferative activity presented herein and the previously assessed Pgp modulatory effect showed a strong relation between the compounds that presented both high Pgp modulation and cytotoxicity. Acknowledgements: FCT, Portugal (PTDC/QUI-QUI/099815/2008; PTDC/QEQ-MED/0905/2012; SFRH/BD/72915/2010). References: [1] Hall MD, Handley MD, Gottesman MM. Is resistance useless? Multidrug resistance and collateral sensitivity. Trends Pharmacol Sci 2009; 30: 546 – 556. [2] Ferreira M-JU, Duarte N, Reis M, Madureira AM, Molnar J. Euphorbia and Momordica metabolites for overcoming multidrug resistance. Phytochem Rev 2014. [3] Reis M, Ferreira RJ, Santos MMM, dos Santos DJV a, Molnar J, Ferreira M-JU. Enhancing macrocyclic diterpenes as multidrug-resistance reversers: structure-activity studies on jolkinol D derivatives. J Med Chem 2013; 56: 748 – 760.