具有肿瘤多药耐药逆转作用的金雀异黄素衍生物的合成及生物活性研究

细胞膜P-糖蛋白(P-gp)介导的药物外流是肿瘤多药耐药(MDR)产生的重要机制,异黄酮类化合物可以通过抑制P-gp活性发挥MDR逆转作用.通过对P-gp抑制剂进行结构分析,以金雀异黄素为母体,在其7位、8位及4'位分别引进碱性边链,设计、合成了20个金雀异黄素衍生物(其中16个未见文献报道),并检测了其多药耐药逆转活性.结果表明,大多数目标化合物对人白血病耐药细胞株K562/A02具有不同程度的耐药逆转作用.其中目标化合物8a,8b,8d,8e逆转作用较强,逆转倍数分别为8.97,6.36,5.19和5.82.     

Electrochemical Method to Characterize Multidrug Resistance

Multidrug resistance（MDR） is a main factor to make the failure of chemotherapy. It is closely related to the over-expression of P-glycoprotein（P-gp）, multidrug resistance protein（MRP） and breast cancer resistance protein（BCRP）. Herein we reported a novel method to characterize MDR, taking advantage of the electrochemical properry of chemotherapeutic drugs. Meanwhile, the definition of accumulation phase and retention phase has been improved. Furthermore, with specific modulators introduced to inhibit the relevant efflux pumps, the exact protein that mainly works in the cells employed in this study can be identified.     

Theoretical analysis of bacterial efflux pumps inhibitors: Strategies in-search of competent molecules and develop next

Multi-drug resistance (MDR) bacteria pose a significant threat to our ability to effectively treat infections due to the development of several antibiotic resistant mechanisms. A major component in the development of the MDR phenotype in MDR bacteria is over expression of different-type of efflux pumps, which actively pump out antibacterial agents and biocides from the periplasm to the outside of the cell. Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments. Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. Several potent inhibitors of efflux pumps have been reported which has been summarized here. All the natural and synthetic EPIs were optimized with Gaussian and Avogadro software. The optimized structures were docked with each class of efflux pumps and their bonding parameters were computed. The theoretical analyses were performed with density functional theory (DFT). Overall, computational study revealed a good trend of electrophilicity and ionization potential of the EPIs, the obtained average values are within in the range of 0.001414 AU ± 0.00032 and 0.208821 AU ± 0.015545, respectively. Interestingly, cathinone interacts with most of the efflux pumps among the tested inhibitors. The electrophilicity and ionization potential of cathinone are 0.00198 and 0.2388 AU, respectively. The study opens a new road for designing future-generation target-specific efflux pump inhibitors, as well as one molecule with multiple inhibition abilities.     

新型紫杉烷类衍生物的制备及生物活性评价

多药耐药性问题是导致第一代紫杉烷药物在临床化疗失败的主要原因。本文对紫杉醇C7、C10、C14、C3′多个位点的取代基进行改造,针对合成的6个新型的紫杉烷化合物,在体外考察其对多药耐药肿瘤细胞株以及人结肠癌HCT-116干细胞的增殖抑制活性,实验结果表明6个化合物的抗多药耐药活性均优于紫杉醇。采用P-gp高表达的犬肾细胞MDCK-MDR1进一步研究高活性候选化合物JT-3与P-gp的相互作用。以此研发抗多药耐药型的新一代紫杉烷类药物,对开发扩大抗癌新适应症的新一代紫杉烷类抗癌药意义重大。     

Effects of Two Natural Bisbenzylisoquinolines,Curine and Guattegaumerine,Extracted from Isolona hexaloba on Rhodamine Efflux by Abcb1b from Rat Glycocholic-Acid-Resistant Hepatocarcinoma Cells

To develop new therapeutic molecules, it is essential to understand the biological effects and targets of clinically relevant compounds. In this article, we describe the extraction and characterization of two alkaloids from the roots of Isolona hexaloba—curine and guattegaumerine. The effect of these alkaloids on the multidrug efflux pump ABCB1 (MDR1/P-Glycoprotein) and their antiproliferative properties were studied. Compared to verapamil, a widely used inhibitor of P-gp, curine and guattegaumerine were found to be weak inhibitors of MDR1/P-Glycoprotein. The highest inhibition of efflux produced by verapamil disappeared in the presence of curine or guattegaumerine as competitors, and the most pronounced effect was achieved with curine. Altogether, this work has provided new insights into the biological effects of these alkaloids on the rat Mdr1b P-gp efflux mechanism and would be beneficial in the design of potent P-gp inhibitors.     

Differences in Sensitivity to UVC, UVB and UVA Radiation of a Multidrug-Resistant Cell Line Overexpressing P-Glycoprotein

Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells, selected for resistance to one chemotherapeutic agent, simultaneously acquire resistance to several apparently unrelated drugs. The MDR phenotype is multifactorial. The best-studied mechanism involves the expression of a membrane protein that acts as an energy-dependent efflux pump, known as P-glycoprotein (Pgp), capable of extruding toxic materials from the cell. In this work, resistance to UVA radiation, but not to UVC nor UVB, was observed in an MDR leukemia cell line. This cell line overexpresses Pgp. To study the role of Pgp in the resistance to UVA radiation, two MDR modulators or reversing agents (verapamil and cyclosporin A) capable of blocking Pgp activity were used. Cell viability was assessed and the techniques of flow cytometry and fluorescence microscopy were employed to measure the extrusion of rhodamine 123 by the efflux pump. The results show that MDR modulators did not modify the resistance to UVA radiation. Furthermore, although cell viability was not significantly altered, Pgp function was impaired after UVA treatment, suggesting that this glycoprotein may be a physical target for oxidative damage, and that other factors may be responsible for the UVA resistance. In agreement with this, it was found that the resistant cell line presented a higher catalase activity than the parental (non-MDR) cell line.     

Machine learning-, rule- and pharmacophore-based classification on the inhibition of P-glycoprotein and NorA

The efflux pumps P-glycoprotein (P-gp) in humans and NorA in Staphylococcus aureus are of great interest for medicinal chemists because of their important roles in multidrug resistance (MDR). The high polyspecificity as well as the unavailability of high-resolution X-ray crystal structures of these transmembrane proteins lead us to combining ligand-based approaches, which in the case of this study were machine learning, perceptual mapping and pharmacophore modelling. For P-gp inhibitory activity, individual models were developed using different machine learning algorithms and subsequently combined into an ensemble model which showed a good discrimination between inhibitors and noninhibitors (acc_{train-diverse} = 84%; acc_{internal-test} = 92% and acc_{external-test} = 100%). For ligand promiscuity between P-gp and NorA, perceptual maps and pharmacophore models were generated for the detection of rules and features. Based on these in silico tools, hit compounds for reversing MDR were discovered from the in-house and DrugBank databases through virtual screening in an attempt to restore drug sensitivity in cancer cells and bacteria.     

FG020326 sensitized multidrug resistant cancer cells to docetaxel-mediated apoptosis via enhancement of caspases activation

Apoptotic resistance is the main obstacle for treating cancer patients with chemotherapeutic drugs. Multidrug resistance (MDR) is often characterized by the expression of P-glycoprotein (P-gp), a 170-KD ATP-dependent drug efflux protein. Functional P-gp can confer resistance to activate caspase-8 and -3 dependent apoptosis induced by a range of different stimuli, including tumor necrosis and chemotherapeutic drugs such as docetaxel and vincristine. We demonstrated here that comparison of sensitive KB cells, P-gp positive (P-gp(+ve)) KBv200 cells were extremely resistant to apoptosis induced by docetaxel. FG020326, a pharmacological inhibitor of P-gp function, could enhance concentration-dependently the effect of docetaxel on cell apoptosis and sensitize caspase-8, -9 and -3 activation in P-gp overexpressing KBv200 cells, but not in KB cells. Therefore, the enhancement of caspase-8, -9 and -3 activation induced by docetaxel may be one of the key mechanisms of the reversal of P-gp mediated docetaxel resistance by FG020326.     

Doxorubicin Encapsulated in TPGS-Modified 2D-Nanodisks Overcomes Multidrug Resistance

Multidrug resistance (MDR) is regarded as a main obstacle for effective chemotherapy, and P-glycoprotein (P-gp)-mediated drug efflux has been demonstrated to be the key factor responsible for MDR. In this study, a novel pH-responsive hybrid drug delivery system was developed by conjugating d -α-tocopheryl polyethylene glycol 1000 succinate (TPGS), a kind of P-gp inhibitor, on the surface of laponite nanodisks to overcome MDR. The prepared LM-TPGS display excellent colloidal stability, a high encapsulation efficiency of doxorubicin (DOX), and a pH-responsive drug release profile. In vitro experiments verified that LM-TPGS/DOX could exhibit significantly enhanced therapeutic efficacy in treating DOX-resistant breast cancer cells (MCF-7/ADR) through inhibiting the activity of P-gp-mediated drug efflux and effectively accumulating DOX within cancer cells. In vivo results revealed that LM-TPGS/DOX outstandingly suppressed MCF-7/ADR tumors with low side effects. Therefore, the high drug payload, enhanced inhibition efficacy to drug-resistant cells, and low side effects make the LM-TPGS/DOX a promising nanoplatform to reverse MDR for effective chemotherapy.     

In silico model for P-glycoprotein substrate prediction: insights from molecular dynamics and in vitro studies

P-glycoprotein (P-gp) is a plasma membrane efflux transporter belonging to ATP-binding cassette superfamily, responsible for multidrug resistance in tumor cells. Over-expression of P-gp in cancer cells limits the efficacy of many anticancer drugs. A clear understanding of P-gp substrate binding will be advantageous in early drug discovery process. However, substrate poly-specificity of P-gp is a limiting factor in rational drug design. In this investigation, we report a dynamic trans-membrane model of P-gp that accurately identified the substrate binding residues of known anticancer agents. The study included homology modeling of human P-gp based on the crystal structure of C. elegans P-gp, molecular docking, molecular dynamics analyses and binding free energy calculations. The model was further utilized to speculate substrate propensity of in-house anticancer compounds. The model demonstrated promising results with one anticancer compound (NSC745689). As per our observations, the molecule could be a potential lead for anticancer agents devoid of P-gp mediated multiple drug resistance. The in silico results were further validated experimentally using Caco-2 cell lines studies, where NSC745689 exhibited poor permeability (P _app 1.03 ± 0.16 × 10^?6 cm/s) and low efflux ratio of 0.26.     

Exploring Ovarian Cancer Cell Resistance to Rhenium Anticancer Complexes

Rhenium tricarbonyl complexes have been recently investigated as novel anticancer agents. However, little is understood about their mechanisms of action, as well as the means by which cancer cells respond to chronic exposure to these compounds. To gain a deeper mechanistic insight into these rhenium anticancer agents, we developed and characterized an ovarian cancer cell line that is resistant to a previously studied compound [Re(CO)₃(dmphen)(ptolICN)]⁺, where dmphen=2,9‐dimethyl‐1,10‐phenanthroline and ptolICN=para‐tolyl isonitrile, called TRIP. This TRIP‐resistant ovarian cancer cell line, A2780TR, was found to be 9 times less sensitive to TRIP compared to the wild‐type A2780 ovarian cancer cell line. Furthermore, the cytotoxicities of established drugs and other rhenium anticancer agents in the TRIP‐resistant cell line were determined. Notably, the drug taxol was found to exhibit a 184‐fold decrease in activity in the A2780TR cell line, suggesting that mechanisms of resistance towards TRIP and this drug are similar. Accordingly, expression levels of the ATP‐binding cassette transporter P‐glycoprotein, an efflux transporter known to detoxify taxol, were found to be elevated in the A2780TR cell line. Additionally, a gene expression analysis using the National Cancer Institute 60 cell line panel identified the MT1E gene to be overexpressed in cells that are less sensitive to TRIP. Because this gene encodes for metallothioneins, this result suggests that detoxification by this class of proteins is another mechanism for resistance to TRIP. The importance of this gene in the A2780TR cell line was assessed, confirming that its expression is elevated in this cell line as well. As the first study to investigate and identify the cancer cell resistance pathways in response to a rhenium complex, this report highlights important similarities and differences in the resistance responses of ovarian cancer cells to TRIP and conventional drugs.     

In silico analysis of a few dietary phytochemicals as potential tumor chemo-sensitizers

P-glycoprotein (P-gp) is a membrane ATP-binding cassette (ABC) transporter that extrudes different xenobiotics out of cells. Besides its tissue protection role, overexpression of P-gp on the surface of many neoplastic cells restricts the cell entry of many anti-cancer drugs, the phenomenon which is known as multidrug resistance (MDR). It has been demonstrated that MDR cells can be sensitized toward anti-cancer agents when treated with P-gp inhibitors/modulators known as chemo-sensitizers. Due to the clinical significance and also considering the fact that many P-gp inhibitors are transported by P-gp, the search for more potent and low toxic non-transported chemo-sensitizers is an active area of research. Regarding this, several naturally occurring compounds were reported as MDR reversal agents, a category which is generally referred to as “fourth-generation P-gp inhibitors.” Dietary supplements containing natural products are widely used, and it is possible that they interact with co-administered pharmaceutical substances that are P-gp substrates, leading to altered pharmacokinetic profile. In silico approaches for quantitative and quantitative prediction of binding mechanism of dietary natural products to P-gp may be regarded as appropriate strategy in the early phase of drug discovery projects since they describe structural features of various phytochemicals for interaction with P-gp and pave the way toward alternative and novel anti-MDR scaffolds. In the present contribution, some phytochemicals of turmeric, black pepper, and green tea as commonly consumed dietary sources were subjected to systematic combined in silico analysis including molecular docking and amino acid decomposition analysis through B3LYP functional in association with 6-31G basis set. On the basis of major identified drug binding sites within P-gp internal pocket, modeled natural compounds were categorized as substrate, inhibitor, or modulator while structure binding relationship of each category was developed and elucidated.     

Exploring Natural Product Activity and Species Source Candidates for Hunting ABCB1 Transporter Inhibitors: An In Silico Drug Discovery Study

The P-glycoprotein (P-gp/ABCB1) is responsible for a xenobiotic efflux pump that shackles intracellular drug accumulation. Additionally, it is included in the dud of considerable antiviral and anticancer chemotherapies because of the multidrug resistance (MDR) phenomenon. In the search for prospective anticancer drugs that inhibit the ABCB1 transporter, the Natural Product Activity and Species Source (NPASS) database, containing >35,000 molecules, was explored for identifying ABCB1 inhibitors. The performance of AutoDock4.2.6 software to anticipate ABCB1 docking score and pose was first assessed according to available experimental data. The docking scores of the NPASS molecules were predicted against the ABCB1 transporter. Molecular dynamics (MD) simulations were conducted for molecules with docking scores lower than taxol, a reference inhibitor, pursued by molecular mechanics-generalized Born surface area (MM-GBSA) binding energy estimations. On the basis of MM-GBSA calculations, five compounds revealed promising binding affinities as ABCB1 inhibitors with ΔG_binding < −105.0 kcal/mol. The binding affinity and stability of the identified inhibitors were compared to the chemotherapeutic agent. Structural and energetical analyses unveiled great steadiness of the investigated inhibitors within the ABCB1 active site throughout 100 ns MD simulations. Conclusively, these findings point out that NPC104372, NPC475164, NPC2313, NPC197736, and NPC477344 hold guarantees as potential ABCB1 drug candidates and warrant further in vitro/in vivo tests.     

Synthesis of Phenoxazinone Derivatives and Antiproliferative Activities on Wild-type and Drug-resistant Tumor Cells

Some natural occurring phenoxazinones, such as xanthommatin, actinomycin D (AMD), and questiomycin A (APO), all contain a coplanar tricyclic iminoquinone system (Figure 1). Polycyclic iminoquinone is an important chemical structural moiety of action- mycins and other antitumor drugs. One of the cytostatic mechanisms of coplanar polycyclic compounds, that they can intercalate into human DNA, to cause enzymatic blocking and reading errors during the replication process. AMD is a well known…     

Traceless solid-phase synthesis and biological evaluation of purine analogs as inhibitors of multidrug resistance protein 4

The traceless solid-phase syntheses of 6-oxopurines and pyrazolo[3,4-d]pyrimidines are presented. The effects of these compounds on multidrug resistance protein 4 (MRP4/ABCC4) facilitated efflux was examined. Four of the compounds, 7b, 7c, 15a, and 17e, were active in inhibiting MRP4-mediated efflux of the bimane-glutathione conjugate. In addition, all four compounds were also able to reverse MRP4-mediated resistance to the anticancer drug 6-thioguanine. In the presence of 25 microM 15a or 17e, there was complete reversal. The reversal of resistance was achieved without any effects on the uptake and metabolism of 6-thioguanine.     

Development of a double-layer microfluidic chip with flow medium for chemotherapy resistance analysis of lung cancer

Integration and miniaturization are main advantages of microchip-based systems. Vertical integration of the multiple operations within a multiple-layer chip is expected to satisfy the urgent demand for high-throughput and large-scale applications. This study aimed at establishing a double-layer chip to integrate the operations including the cell culture, the identification of the protein and the detection of the cell viability onto a platform systematically and supplied with flow fresh medium continuously via a syringe pump to mimic the microenvironment in vivo. With this device, human non-small cell lung cancer cell line (SPCA-1) was cultured well; the expression and the activity of multidrug resistance-associated protein (MRP1) were detected by immunofluorescence assay for the cells pretreated with or without MK-571, a known inhibitor of MRP1; apoptosis percentages were assayed for the cells after being treated by the anticancer drug etoposide (VP-16). The results demonstrated that the function of the MRP1 was inhibited by MK-571, and the percentage of apoptotic for the cells pretreated with MK-571 was higher than that of the control (38.2±2.5% versus 12.3±0.85%, p<0.005). All these indicated that the new device could provide a suitable condition for cell culture and functional analysis in biomedical research, and MK-571 is an effective inhibitor of MRP1 associated with the viability of SPCA-1 cell line treated by VP-16.     

多芳基取代咪唑的合成及其逆转多药耐药性研究

合成了一系列新的多芳基取代咪唑类化合物,其结构经元素分析、IR、¹HNMR和MS等确定,并采用MTT法测定了它们对由P-糖蛋白(P-gp)介导的肿瘤多药耐药性(MDR)的逆转效果.结果表明,化合物和具有很好的体外逆转MDR活性     

Investigation of the Uptake and Transport of Two Novel Camptothecin Derivatives in Caco-2 Cell Monolayers

Irinotecan and Topotecan are two Camptothecin derivatives (CPTs) whose resistance is associated with the high expression of breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). To reverse this resistance, two novel CPTs, FL77-28 (7-(3-Fluoro-4-methylphenyl)-10,11-methylenedioxy-20(S)-CPT) and FL77-29 (7-(4-Fluoro-3-methylphenyl)-10,11-methylenedioxy-20(S)-CPT), were synthesized by our group. In this study, the anti-tumor activities of FL77-28, FL77-29, and their parent, FL118 (10,11-methylenedioxy-20(S)-CPT), were evaluated and the results showed that FL77-28 and FL77-29 had stronger anti-tumor activities than FL118. The transport and uptake of FL118, FL77-28, and FL77-29 were investigated in Caco-2 cells for the preliminary prediction of intestinal absorption. The apparent permeability coefficient from apical to basolateral (P_{app AP-BL}) values of FL77-28 and FL77-29 were (2.32 ± 0.04) × 10⁻⁶ cm/s and (2.48 ± 0.18) × 10⁻⁶ cm/s, respectively, suggesting that the compounds had moderate absorption. Since the transport property of FL77-28 was passive diffusion and the efflux ratio (ER) was less than 2, two chemical inhibitors were added to further confirm the involvement of efflux proteins. The results showed that FL77-28 was not a substrate of P-gp or BCRP, but FL77-29 was mediated by P-gp. In conclusion, FL77-28 might be a promising candidate to overcome drug resistance induced by multiple efflux proteins.     

Lubeluzole Repositioning as Chemosensitizing Agent on Multidrug-Resistant Human Ovarian A2780/DX3 Cancer Cells

In a previous paper, we demonstrated the synergistic action of the anti-ischemic lubeluzole (Lube S) on the cytotoxic activity of doxorubicin (Dox) and paclitaxel in human ovarian cancer A2780 and lung cancer A549 cells. In the present paper, we extended in vitro the study to the multi-drug-resistant A2780/DX3 cell line to verify the hypothesis that the Dox and Lube S drug association may potentiate the antitumor activity of this anticancer compound also in the context of drug resistance. We also evaluated some possible mechanisms underlying this activity. We analyzed the antiproliferative activity in different cancer cell lines. Furthermore, apoptosis, Dox accumulation, MDR1 downregulation, ROS, and NO production in A2780/DX3 cells were also evaluated. Our results confirm that Lube S improves Dox antiproliferative and apoptotic activities through different mechanisms of action, all of which may contribute to the final antitumor effect. Moderate stereoselectivity was found, with Lube S significantly more effective than its enantiomer (Lube R) and the corresponding racemate (Lube S/R). Docking simulation studies on the ABCB1 Cryo-EM structure supported the hypothesis that Lube S forms a stable MDR1-Dox-Lube S complex, which hampers the protein transmembrane domain flipping and blocks the efflux of Dox from resistant A2780/DX3 cells. In conclusion, our in vitro studies reinforce our previous hypothesis for repositioning the anti-ischemic Lube S as a potentiating agent in anticancer chemotherapy.