Andreia Palmeira

Three Decades of P-gp Inhibitors: Skimming Through Several Generations and Scaffolds

Many tumor cells become resistant to commonly used cytotoxic drugs due to the overexpression of ATP-binding cassette (ABC) transporters, namely P-glycoprotein (P-gp). The discovery of the reversal of multidrug resistance (MDR) by verapamil occured in 1981, and in 1968 MDR Chinese hamster cell lines were isolated for the first time. Since then, P-gp inhibitors have been intensively studied as potential MDR reversers. Initially, drugs to reverse MDR were not specifically developed for inhibiting P-gp; in fact, they had other pharmacological properties, as well as a relatively low affinity for MDR transporters. An example of this first generation P-gp inhibitors is verapamil. The second generation included more specific with less side-effect inhibitors, such as dexverapamil or dexniguldipine. A third generation of P-gp inhibitors comprised compounds such as tariquidar, with high affinity to P-gp at nanomolar concentrations. These generations of inhibitors of P-gp have been examined in preclinical and clinical studies; however, these trials have largely failed to demonstrate an improvement in therapeutic efficacy. Therefore, new and innovative strategies, such as the fallback to natural products, the design of peptidomimetics and dual activity ligands emerged as a fourth generation of P-gp inhibitors. The chemistry of P-gp inhibitors, as well as their in vitro, in vivo and clinical trials are discussed, and the most recent advances concerning Pgp modulators are reviewed.

Dual inhibitors of P-glycoprotein and tumor cell growth: (Re)discovering thioxanthones

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 GI(50) values in the K562 cell line below 10 μM and 1-{[2-(diethylamino)ethyl]amino}-4-propoxy-9H-thioxanthen-9-one (37) had a GI(50) concentration (1.90 μM) 6-fold lower than doxorubicin (11.89 μM) 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 μM, compound 45 caused a decrease of 12.5-fold in the GI(50) 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.

New uses for old drugs: pharmacophore-based screening for the discovery of P-glycoprotein inhibitors.

P‐glycoprotein (P‐gp) is one of the best characterized transporters responsible for the multidrug resistance phenotype exhibited by cancer cells. Therefore, there is widespread interest in elucidating whether existing drugs are candidate P‐gp substrates or inhibitors. With this aim, a pharmacophore 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 activity 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 tetracyclic 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.

Insights into the In Vitro Antitumor Mechanism of Action of a New Pyranoxanthone

Naturally occurring xanthones have been documented 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 molecular modifications were performed. All the compounds 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 cellular 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 computer 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.

Discovery of a new small-molecule inhibitor of p53-MDM2 interaction using a yeast-based approach.

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.

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 pharmacophore 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.

Medicinal Chemistry Strategies to Disrupt the p53-MDM2/MDMX Interaction.

The growth inhibitory activity of p53 tumor suppressor is tightly regulated by interaction with two negative regulatory proteins, murine double minute 2 (MDM2) and X (MDMX), which are overexpressed in about half of all human tumors. The elucidation of crystallographic structures of MDM2/MDMX complexes with p53 has been pivotal for the identification of several classes of inhibitors of the p53–MDM2/MDMX interaction. The present review provides in silico strategies and screening approaches used in drug discovery as well as an overview of the most relevant classes of small‐molecule inhibitors of the p53–MDM2/MDMX interaction, their progress in pipeline, and highlights particularities of each class of inhibitors. Most of the progress made with high‐throughput screening has led to the development of inhibitors belonging to the cis‐imidazoline, piperidinone, and spiro‐oxindole series. However, novel potent and selective classes of inhibitors of the p53–MDM2 interaction with promising antitumor activity are emerging. Even with the discovery of the 3D structure of complex p53–MDMX, only two small molecules were reported as selective p53–MDMX antagonists, WK298 and SJ‐172550. Dual inhibition of the p53–MDM2/MDMX interaction has shown to be an alternative approach since it results in full activation of the p53‐dependent pathway. The knowledge of structural requirements crucial to the development of small‐molecule inhibitors of the p53–MDMs interactions has enabled the identification of novel antitumor agents with improved in vivo efficacy.

Colchicine effect on P-glycoprotein expression and activity: In silico and in vitro studies

Colchicine is a P-glycoprotein (P-gp) substrate that induces its expression, thus increasing the risk for unexpected pharmacokinetic interactions with this drug. Because increased P-gp expression does not always correlate with increased activity of this efflux pump, we evaluated the changes in both P-gp expression and activity induced by colchicine using an in vitro model. Caco-2 cells were incubated with 0.1-100 μM colchicine up to 96 h. Cytotoxicity was evaluated by the MTT and LDH leakage assays, P-gp expression and activity were evaluated by flow cytometry and P-gp ATPase activity was measured in MDR1-Sf9 membrane vesicles. Furthermore, colchicine fitting in P-gp induction and competitive inhibition pharmacophore hypothesis, and docking studies evaluating the interaction between colchicine and P-gp drug binding pocket were tested in silico. Significant cytotoxicity was noted after 48 h. At 24 h a significant increase in P-gp expression was observed, which was not accompanied by an increase in transport activity. Moreover, colchicine significantly increased P-gp ATPase activity, demonstrating to be actively transported by the pump. New pharmacophores were constructed to predict P-gp modulatory activity. Colchicine fitted both the P-gp induction and competitive inhibition models. In silico, colchicine was predicted to bind to the P-gp drug-binding pocket suggesting a competitive mechanism of transport. These results show that colchicine induced P-gp expression in Caco-2 cells but the activity of the protein remained unchanged, highlighting the need to simultaneously evaluate P-gp expression and activity. With the newly constructed pharmacophores, new drugs can be initially screened in silico to predict such potential pharmacokinetic interactions.

Development of novel rifampicin-derived P-glycoprotein activators/inducers. synthesis, in silico analysis and application in the RBE4 cell model, using paraquat as substrate.

P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat’s blood–brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre- or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp.

Enantioresolution of chiral derivatives of xanthones on (S,S)-Whelk-O1 and L-phenylglycine stationary phases and chiral recognition mechanism by docking approach for (S,S)-Whelk-O1.

The resolution of seven enantiomeric pairs of chiral derivatives of xanthones (CDXs) on (S,S)-Whelk-O1 and L-phenylglycine chiral stationary phases (CSPs) was systematically investigated using multimodal elution conditions (normal-phase, polar-organic, and reversed-phase). The (S,S)-Whelk-O1 CSP, under polar-organic conditions, demonstrated a very good power of resolution for the CDXs possessing an aromatic moiety linked to the stereogenic center with separation factor and resolution factor ranging from 1.91 to 7.55 and from 6.71 to 24.16, respectively. The chiral recognition mechanisms were also investigated for (S,S)-Whelk-O1 CSP by molecular docking technique. Data regarding the CSP-CDX molecular conformations and interactions were retrieved. These results were in accordance with the experimental chromatographic parameters regarding enantioselectivity and enantiomer elution order. The results of the present study fulfilled the initial objectives of enantioselective studies of CDXs and elucidation of intermolecular CSP-CDX interactions.