Mediated Multidrug Resistance Research Articles

Reversal of P-glycoprotein Mediated Multidrug Resistance in MCF-7/R Cancer Cells by Esculetin Derivatives: Experimental and MD Simulation Studies

Coumarins of natural origin have been explored as potential inhibitors of P-glycoprotein (P-gp). Esculetin which belongs to the class of coumarin has been derivatized with known hydrazine pharmacophores viz; benzoyl hydrazine (BH), isonicotinyl hydrazine (INH), and hydrazino benzoic acid. The homology modeling approach was used to predict the three-dimensional structure of human P-gp. An <i>in-silico</i> study has been performed for the structural insight into the molecular mechanism of P-gp inhibition of the esculetin derivatives by molecular docking (MD) and simulation studies. The cell cytotoxic activities of the synthesized compounds were evaluated using in-vitro studies. The sublines resistant doxorubicin (MCF-7/R) were generated and the activities of P-gp proteins were estimated using fluorescent dye accumulation assays. The E-BH showed promising P-gp inhibitory activity and cell cytotoxicity against MCF7 and MCF7/R (resistant) breast cancer cell lines. In line with experimental observations, the E-BH (Esculetin benzoyl hydrazine) has yielded the lowest energy stable complex with P-gp and is stabilized by intermolecular hydrogen bonding and more hydrophobic interactions during 100 ns of simulation. This suggested that the activity of P-gp is probably controlled by hydrophobic interactions. Performed experimental and computational studies has helped to elucidate the mechanism of P-gp inhibition by E-BH. Thus, amongst the three derivatives; E-BH exhibits greater efficacy in blocking the efflux mechanism.

Discovery of novel pyrazolo[1,5-a]pyrimidine derivatives as potent reversal agents against ABCB1-mediated multidrug resistance

The P-glycoprotein (ABCB1)-mediated multidrug resistance (MDR) has emerged as a significant impediment to the efficacy of cancer chemotherapy in clinical therapy, which could promote the development of effective agents for MDR reversal. In this work, we reported the exploration of novel pyrazolo [1,5-a]pyrimidine derivatives as potent reversal agents capable of enhancing the sensitivity of ABCB1-mediated MDR MCF-7/ADR cells to paclitaxel (PTX). Among them, compound 16q remarkably increased the sensitivity of MCF-7/ADR cells to PTX at 5 μM (IC50 = 27.00 nM, RF = 247.40) and 10 μM (IC50 = 10.07 nM, RF = 663.44). Compound 16q could effectively bind and stabilize ABCB1, and does not affect the expression and subcellular localization of ABCB1 in MCF-7/ADR cells. Compound 16q inhibited the function of ABCB1, thereby increasing PTX accumulation, and interrupting the accumulation and efflux of the ABCB1-mediated Rh123, thus resulting in exhibiting good reversal effects. In addition, due to the potent reversal effects of compound 16q, the abilities of PTX to inhibit tubulin depolymerization, and induce cell cycle arrest and apoptosis in MCF-7/ADR cells under low-dose conditions were restored. These results indicate that compound 16q might be a promising potent reversal agent capable of revising ABCB1-mediated MDR, and pyrazolo [1,5-a]pyrimidine might represent a novel scaffold for the discovery of new ABCB1-mediated MDR reversal agents.

Discovery and mechanistic insights into thieno[3,2-d]pyrimidine and heterocyclic fused pyrimidines inhibitors targeting tubulin for cancer therapy

Guided by the X-ray cocrystal structure of the lead compound 4a, we developed a series of thieno[3,2-d]pyrimidine and heterocyclic fused pyrimidines demonstrating potent antiproliferative activity against four tumor cell lines. Two analogs, 13 and 25d, exhibited IC50 values around 1 nM and overcame P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). At low concentrations, 13 and 25d inhibited both the colony formation of SKOV3 cells in vitro and tubulin polymerization. Furthermore, mechanistic studies showed that 13 and 25d induced G2/M phase arrest and apoptosis in SKOV3 cells, as well as dose-dependent inhibition of tumor cell migration and invasion at low concentrations. Most notably, the X-ray cocrystal structures of compounds 4a, 25a, and the optimal molecule 13 in complex with tubulin were elucidated. This study identifies thieno[3,2-d]pyrimidine and heterocyclic fused pyrimidines as representatives of colchicine-binding site inhibitors (CBSIs) with potent antiproliferative activity.

RN486, a Bruton's Tyrosine Kinase inhibitor, antagonizes multidrug resistance in ABCG2-overexpressing cancer cells.

Overcoming ATP-binding cassette subfamily G member 2 (ABCG2)-mediated multidrug resistance (MDR) has attracted the attention of scientists because one of the critical factors resulting in MDR in cancer is the overexpression of ABCG2. RN486, a Bruton's Tyrosine Kinase (BTK) inhibitor, was discovered to potentially reverse ABCB1-mediated MDR. However, there is still uncertainty about whether RN486 has a reversal off-target impact on ABCG2-mediated MDR. MTT assay was used to detect the reversal effect of RN486 on ABCG2-overexpressing cancer cells. The ABCG2 expression level and subcellular localization were examined by Western blotting and immunofluorescence. Drug accumulation and eflux assay and ATPase assay were performed to analyze the ABCG2 transporter function and ATPase activity. Molecular modeling predicted the binding between RN486 and ABCG2 protein. Non-toxic concentrations of RN486 remarkably increased the sensitivity of ABCG2-overexpressing cancer cells to conventional anticancer drugs mitoxantrone and topotecan. The reversal mechanistic studies showed that RN486 elevated the drug accumulation because of reducing the eflux of ABCG2 substrate drug in ABCG2-overexpressing cancer cells. In addition, the inhibitory efect of RN486 on ABCG2-associated ATPase activity was also verified. Molecular docking study implied a strong binding afinity between RN486 and ABCG2 transporter. Meanwhile, the ABCG2 subcellular localization was not altered by the treatment of RN486, but the expression level of ABCG2 was down-regulated. Our studies propose that RN486 can antagonize ABCG2-mediated MDR in cancer cells via down-regulating the expression level of ABCG2 protein, reducing ATPase activity of ABCG2 transporter, and inhibiting the transporting function. RN486 could be potentially used in conjunction with chemotherapy to alleviate MDR mediated by ABCG2 in cancer.

Abstract 3102: A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University

Abstract Background: Drug development is important for cancer therapy to improve patient outcomes. In most cases, drug development starts in discovering candidate compounds from a chemical library in high-throughput screening (HTS). Tohoku University Graduate School of Pharmaceutical Science owns the original chemical library consisting of thousands of original chemical compounds. P-glycoprotein (P-gp), a major drug efflux transporter mediates multi-drug resistance (MDR) in cancer cells. Therefore, P-gp modulators could revere MDR and increase chemosensitivity. The aim of this study is to reveal an adequate method of HTS for discovering potent P-gp modulators from the chemical library in Tohoku University. Methods: The chemical library in Tohoku University had 5861 compounds in the present study, and the collection contains over 7 thousand compounds in 2023. P-gp overexpressing KB-V1, human epidermal carcinoma cell lines were obtained, and calcein AM, P-gp substrate was employed for fluorescent substrate efflux assays. A plate reader-based calcein AM efflux assay for HTS was established to select candidate compounds as P-gp modulators. After the primary HTS, structures of the candidate compounds were analyzed, then several analogous compounds were extracted from the chemical library. For the validation of the hit compounds in HTS and the analogs, a flow cytometry-based calcein AM efflux assay was conducted. In addition, reversal effects of P-gp modulators on the cytotoxicity of paclitaxel were measured in a cell proliferation assay. Results: Of 5861 compounds in the chemical library, 53 compounds showed 2-fold or higher fluorescence intensity, and then 13 compounds showed 3-fold or higher fluorescence intensity in a plate reader-based assay for the HTS. Especially 2 candidate compounds showed over 5-fold fluorescence intensity. In the analysis for structures of the candidate compounds, 20 analogous compounds were extracted from the chemical library. To verify the result of the plate reader-based assay, a flow cytometry-based assay was conducted for 53 hits and 20 analogs. As a result, the 2 candidate compounds that selected in the plate reader-based assay showed 60-fold, the highest fluorescence intensity in a flow cytometry-based assay. The other compounds showed less fluorescence intensity than these 2 candidates. In addition, both candidates enhanced the paclitaxel-induced cytotoxicity in a cell proliferation assay. Conclusions: A plate reader-based calcein AM efflux assay in HTS was an adequate method for discovering potent P-gp modulators from the chemical library in Tohoku University. Citation Format: Norihiko Sugisawa, Shinobu Ohnuma, Takayuki Doi, Michiaki Unno. A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3102.

Design and Synthesis of Cyclolipopeptide Mimics of Dysoxylactam A and Evaluation of the Reversing Potencies against P-Glycoprotein-Mediated Multidrug Resistance.

Inspired by the structure of dysoxylactam A (DLA) that has been demonstrated to reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) effectively, 61 structurally simplified cyclolipopeptides were thus designed and synthesized via an effective method, and their reversing P-gp-mediated MDR potentials were evaluated, which provided a series of more potent analogues and allowed us to explore their structure-activity relationship (SAR). Among them, a well-simplified compound, 56, with only two chiral centers that all derived from amino acids dramatically reversed drug resistance in KBV200 cells at 10 μM in combination with vinorelbine (VNR), paclitaxel (PTX), and adriamycin (ADR), respectively, which is more promising than DLA. The mechanism study showed that 56 reversed the MDR of tumor cells by inhibiting the transport function of P-gp rather than reducing its expression. Notably, compound 56 effectively restored the sensitivity of MDR tumors to VNR in vivo at a dosage without obvious toxicity.

Reversal of Chemoresistance via Staged Liberation of Chemodrug and siRNA in Hierarchical Response to ROS Gradient.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) often leads to the failure of antitumor chemotherapy, and codelivery of chemodrug with P-gp siRNA (siP-gp) represents a promising approach for treating chemoresistant tumors. To maximize the antitumor efficacy, it is desired that the chemodrug be latently released upon completion of siP-gp-mediated gene silencing, which however, largely remains an unmet demand. Herein, core-shell nanocomplexes (NCs) are developed to overcome MDR via staged liberation of siP-gp and chemodrug (doxorubicin, Dox) in hierarchical response to reactive oxygen species (ROS) concentration gradients. The NCs are constructed from mesoporous silica nanoparticles (MSNs) surface-decorated with cRGD-modified, PEGylated, ditellurium-crosslinked polyethylenimine (RPPT), wherein thioketal-linked dimeric doxorubicin (TK-Dox2) and photosensitizer are coencapsulated inside MSNs while siP-gp is embedded in the RPPT polymeric layer. RPPT with ultrahigh ROS-sensitivity can be efficiently degraded by the low-concentration ROS inside cancer cells to trigger siP-gp release. Upon siP-gp-mediated gene silencing and MDR reversal, light irradiation is performed to generate high-concentration, lethal amount of ROS, which cleaves thioketal with low ROS-sensitivity to liberate the monomeric Dox. Such a latent release profile greatly enhances Dox accumulation in Dox-resistant cancercells (MCF-7/ADR) in vitro and in vivo, which cooperates with the generated ROS to efficiently eradicate MCF-7/ADR xenograft tumors.

Outer Membrane Proteins and Efflux Pumps Mediated Multi-Drug Resistance in Salmonella: Rising Threat to Antimicrobial Therapy.

Despite colossal achievements in antibiotic therapy in recent decades, drug-resistant pathogens have remained a leading cause of death and economic loss globally. One such WHO-critical group pathogen is Salmonella. The extensive and inappropriate treatments for Salmonella infections have led from multi-drug resistance (MDR) to extensive drug resistance (XDR). The synergy between efflux-mediated systems and outer membrane proteins (OMPs) may favor MDR in Salmonella. Differential expression of the efflux system and OMPs (influx) and positional mutations are the factors that can be correlated to the development of drug resistance. Insights into the mechanism of influx and efflux of antibiotics can aid in developing a structurally stable molecule that can be proficient at escaping from the resistance loops in Salmonella. Understanding the strategic responsibilities and developing policies to address the surge of drug resistance at the national, regional, and global levels are the needs of the hour. In this Review, we attempt to aggregate all the available research findings and delineate the resistance mechanisms by dissecting the involvement of OMPs and efflux systems. Integrating major OMPs and the efflux system's differential expression and positional mutation in Salmonella may provide insight into developing strategic therapies for one health application.

Repositioning of HMG-CoA Reductase Inhibitors as Adjuvants in the Modulation of Efflux Pump-Mediated Bacterial and Tumor Resistance.

Efflux pump (EP)-mediated multidrug resistance (MDR) seems ubiquitous in bacterial infections and neoplastic diseases. The diversity and lack of specificity of these efflux mechanisms raise a great obstacle in developing drugs that modulate efflux pumps. Since developing novel chemotherapeutic drugs requires large investments, drug repurposing offers a new approach that can provide alternatives as adjuvants in treating resistant microbial infections and progressive cancerous diseases. Hydroxy-methyl-glutaryl coenzyme-A (HMG-CoA) reductase inhibitors, also known as statins, are promising agents in this respect. Originally, statins were used in the therapy of dyslipidemia and for the prevention of cardiovascular diseases; however, extensive research has recently been performed to elucidate the functions of statins in bacterial infections and cancers. The mevalonate pathway is essential in the posttranslational modification of proteins related to vital eukaryotic cell functions. In this article, a comparative review is given about the possible role of HMG-CoA reductase inhibitors in managing diseases of bacterial and neoplastic origin. Molecular research and clinical studies have proven the justification of statins in this field. Further well-designed clinical trials are urged to clarify the significance of the contribution of statins to the lower risk of disease progression in bacterial infections and cancerous diseases.

Pregnane steroid glycosides with multidrug resistance reversal activity from the stems of Marsdenia tenacissima

Eighteen previously unreported pregnane glycosides, namely marsdenosides S1–S18, along with 15 known analogues, have been isolated from the stems of Marsdenia tenacissima. The structures of the undescribed compounds were elucidated by spectroscopic means, and their absolute configurations were established on the basis of time-dependent density functional theory (TD-DFT) based electronic circular dichroism (ECD) calculation, X-ray crystallography and acid hydrolysis. All the isolates were evaluated for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and nine ones displayed moderate MDR reversal activity with reversal folds in the range of 2.45–9.01. The most active 12-O-acetyl-20-O-benzoyl-(14,17,18-orthoacetate)-dihydrosarcostin-3-O-β-d-thevetopyranosyl-(1 → 4)-O-β-d-oleandropyranosyl-(1 → 4)-O-β-d-cymaropyranoside increased the sensitivity of MCF-7/ADR cell to adriamycin comparably to the reference drug verapamil (RF = 8.93).

Pregnane steroids and steroid glycosides with multidrug resistance reversal activity from Marsdenia tenacissima

Twenty compounds comprising four pregnane steroids (2-4 & 20) and 16 pregnane glycosides (1 & 5-19) have been obtained from the ethanol extract of the roots of a Dai ethnological herb, Marsdenia tenacissima. Their structures were characterized on the basis of comprehensive spectroscopic analyses with 17 ones (1-17) being reported for the first time, including the rare cases (2 & 3) of free C21 steroids with 17α-acetyl substitution, compounds 4-7 bearing an unusual 14α-OH, and the first examples with simultaneous 14α-OH/17α-acetyl substitution (7) and glycosylation at C-12 position (10 & 11). An empirical rule for the identification of C-17 configuration, in C21 steroids incorporating the marsdenin constitution structure, was also proposed. All the isolates, along with an array of previously reported analogues in our compound library, were screened for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and six compounds exhibited moderate MDR reversal activity with reversal folds ranging from 1.92 to 4.44.

A multi-functional nano-system combining PI3K-110α/β inhibitor overcomes P-glycoprotein mediated MDR and improves anti-cancer efficiency

P-glycoprotein (P-gp/ABCB1)-mediated multidrug resistance (MDR) in cancers severely limit chemotherapeutic efficacy. We recently reported that phosphatidylinositol-3-kinase (PI3K) 110α and 110β subunits can be novel targets for reversal of P-gp mediated MDR in cancers, and BAY-1082439 as an inhibitor specific for PI3K 110α and 110β subunits could reverse P-gp-mediated MDR by downregulating P-gp expression in cancer cells. However, BAY-1082439 has very low solubility, short half-life and high in-vivo clearance rate. Till now, nano-system with the functions to target PI3K P110α and P110β and reverse P-gp mediated MDR in cancers has not been reported. In our study, a tumor targeting drug delivery nano-system PBDF was established, which comprised doxorubicin (DOX) and BAY-1082439 respectively encapsulated by biodegradable PLGA-SH nanoparticles (NPs) that were grafted to gold nanorods (Au NRs) modified with FA-PEG-SH, to enhance the efficacy to reverse P-gp mediated MDR and to target tumor cells, further, to enhance the efficiency to inhibit MDR tumors overexpressing P-gp. In-vitro experiments indicated that PBDF NPs greatly enhanced uptake of DOX, improved the activity to reverse MDR, inhibited the cell proliferation, and induced S-phase arrest and apoptosis in KB-C2 cells, as compared with free DOX combining free BAY-1082439. In-vivo experiments further demonstrated that PBDF NPs improved the anti-tumor ability of DOX and inhibited development of KB-C2 tumors. Notably, the metastasis of KB-C2 cells in livers and lungs of nude mice were inhibited by treatment with PBDF NPs, which showed no obvious in-vitro or in-vivo toxicity.

Abstract 2843: RN486, a Bruton’s tyrosine kinase inhibitor, reverses multidrug resistance in ABCG2-overexpressing cancer cells

Abstract Overcoming ATP-binding cassette subfamily G member 2 (ABCG2)-mediated multidrug resistance (MDR) has attracted the attention of scientists because one of the critical factors leading to MDR is the overexpression of ABCG2. Identification of novel chemotherapeutic reagents as inhibitors of ABCG2 has been considered an effective strategy. RN486, a Bruton’s Tyrosine Kinase (BTK) inhibitor, was discovered to potentially reverse ABCB1-mediated MDR. In this study, we reported that RN486 effectively antagonizes ABCG2-mediated MDR in cancer cells. Non-toxic concentrations of RN486 remarkably increased the sensitivity of ABCG2-overexpressing cancer cells to conventional anticancer drugs mitoxantrone and topotecan. The reversal mechanistic studies showed that RN486 increased the accumulation and decreased the efflux of ABCG2 substrate drug in ABCG2-overexpressing cancer cells. In addition, the inhibitory effect of RN486 on ABCG2-associated ATPase activity was also verified. Docking analysis indicated a strong binding between RN486 and ABCG2 transporter. Meanwhile, the AGCG2 subcellular localization and expression level were not altered by the treatment of RN486. Taken together, our studies suggest that RN486 can antagonize ABCG2-mediated MDR in cancer cells via interacting with ABCG2 and inhibiting the transporting function. RN486 could be potentially used in combination with chemotherapy against ABCG2-mediated MDR in cancers. Citation Format: Xing-Duo Dong, Qisi Lu, Yi-Dong Li, Qiu-Xu Teng, Zi-Ning Lei, Zhe-Sheng Chen. RN486, a Bruton’s tyrosine kinase inhibitor, reverses multidrug resistance in ABCG2-overexpressing cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2843.

Exploration of the inhibition action of TPGS on tumor cells and its combined use with chemotherapy drugs

D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a commonly used nonionic surfactant used as a pharmaceutical carrier in different drug delivery systems. TPGS can reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) and also has anticancer activities. It suggests that when designing antitumor drug preparation, it’s necessary to take into account the antitumor activity of TPGS. Our in vivo studies showed that TPGS exerted the strongest cytotoxicity in MCF-7-ADR cells when compared with seven other tumor cell lines. The further study revealed TPGS caused apoptosis and blocked MCF-7 cell growth in G2/M phase. Mechanistic insights suggested that TPGS increased intracellular calcium ion concentrations, leading to apoptosis via the mitochondrial pathway. Furthermore, two in vivo experiments were performed. One was TPGS, and DOX solution was administered by tail vein injection on MCF-7-ADR tumor bearing nude mice. The other was temperature sensitive TPGS gel (TPGS-TG) was administered by intratumoral injection on MCF-7-ADR tumor bearing nude mice combined with paclitaxel albumin nanoparticles (Abraxane®) administered by tail vein injection. The findings confirmed that TPGS could play its role in anti-tumor to reduce the toxicity of chemotherapeutic drugs and improve the efficiency of drug-resistant tumors, thereby enhancing the development of safe oncology therapeutics.

Enhanced delivery of quercetin and doxorubicin using β-cyclodextrin polymer to overcome P-glycoprotein mediated multidrug resistance

In this study, we prepared a β-cyclodextrin polymer (β-CDP) co-loaded quercetin (QCT) and doxorubicin (DOX) nanocarrier (β-CDP/QD NCs) by freeze-dried method to combat P-glycoprotein (P-gp) mediated multidrug resistance (MDR) in KB-ChR 8–5 cancer cells. Various microscopic and spectroscopic techniques were employed to characterize the prepared nanocarrier. The molecular docking studies confirm the effective binding interactions of QCT and DOX with the synthesized β-CD polymer. The in vitro drug release study illustrates the sustainable release of DOX and QCT from the β-CDP nanocarrier. Further, we noticed that the QCT released from the β-CDP nanocarrier improved the intracellular availability of DOX via modulating P-gp drug efflux function in KB-ChR 8–5 cells and MCF-7/DOX cancer cells. Cell uptake results confirmed the successful internalization of DOX in KB-ChR 8–5 cells compared with free DOX. Cell-based assays such as nuclear condensation, alteration in the mitochondrial membrane potential (MMP), and apoptosis morphological changes confirmed the enhanced anticancer effect of β-CDP/QD NCs in the resistant cancer cells. Hence, QCT and DOX co-loaded β-CDP may be considered effective in achieving maximum cell death in the P-gp overexpressing MDR cancer cells.

Modular Biomimetic Strategy Enabled Discovery of Simplified Pseudo-Natural Macrocyclic P-Glycoprotein Inhibitors Capable of Overcoming Multidrug Resistance.

Natural macrocycles have shown impressive activity to overcome P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). However, the total synthesis and structural modification of natural macrocycles are challenging, which would hamper the deeper investigations of their structure-activity relationship (SAR) and drug likeness. Herein, we describe a modular biomimetic strategy to expeditiously achieve a new class of macrocycles featuring polysubstituted 1,3-diene, which efficiently inhibited P-gp and reversed MDR in cancer cells. The SAR analysis revealed that the size and linker of the macrocycles are important structural characteristics to restore activity. Particularly, 32 containing a naphthyl group and (d)-Phe moiety has higher potency with an excellent reversal fold than verapamil at a concentration of 5 μM, which induces conformational change of P-gp and inhibits its function instead of altering P-gp expression. Furthermore, 23 and 32 were identified to be attractive leads, which possess a good pharmacokinetic profile and antitumor activity in a KBV200 xenograft mouse model.

Concise Total Synthesis of Dysoxylactam A and a Simplified Analog

Comprehensive SummaryA total synthesis of 17‐membered macrocyclolipopeptide dysoxylactam A, a potent agent reversing P‐glycoprotein (P‐gp)‐mediated multidrug resistance (MDR) in cancer cells, was developed from the starting material (S)‐2‐methylbutanal in a linear sequence of 12 steps with 23.2% overall yield. The key steps include proline‐catalyzed asymmetric aldol reaction, Evans aldol reaction and Krische allylation to construct the multiple stereocenters containing fragment, and ring‐closing metathesis to furnish the macrocycle. This total synthesis facilitates the preparation of large amount samples of dysoxylactam A and the side chain simplified derivatives for further biological tests and preliminary structure‐activity relationship exploration.

A New Twist in ABC Transporter Mediated Multidrug Resistance – Pdr5 is a Drug/proton Co-transporter

The two major efflux pump systems that are involved in multidrug resistance (MDR) are (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ATP binding cassette (ABC) transporters that are involved in multidrug resistance (MDR) and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of drug transport and substrate specificity remain elusive. Here, we provide electrophysiological data on the reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but at the same time generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

Interaction of crown ethers with the ABCG2 transporter and their implication for multidrug resistance reversal.

Overexpression of ABC transporters, such as ABCB1 and ABCG2, plays an important role in mediating multidrug resistance (MDR) in cancer. This feature is also attributed to a subpopulation of cancer stem cells (CSCs), having enhanced tumourigenic potential. ABCG2 is specifically associated with the CSC phenotype, making it a valuable target for eliminating aggressive and resistant cells. Several natural and synthetic ionophores have been discovered as CSC-selective drugs that may also have MDR-reversing ability, whereas their interaction with ABCG2 has not yet been explored. We previously reported the biological activities, including ABCB1 inhibition, of a group of adamantane-substituted diaza-18-crown-6 (DAC) compounds that possess ionophore capabilities. In this study, we investigated the mechanism of ABCG2-inhibitory activity of DAC compounds and the natural ionophores salinomycin, monensin and nigericin. We used a series of functional assays, including real-time microscopic analysis of ABCG2-mediated fluorescent substrate transport in cells, and docking studies to provide comparative aspects for the transporter-compound interactions and their role in restoring chemosensitivity. We found that natural ionophores did not inhibit ABCG2, suggesting that their CSC selectivity is likely mediated by other mechanisms. In contrast, DACs with amide linkage in the side arms demonstrated noteworthy ABCG2-inhibitory activity, with DAC-3Amide proving to be the most potent. This compound induced conformational changes of the transporter and likely binds to both Cavity 1 and the NBD-TMD interface. DAC-3Amide reversed ABCG2-mediated MDR in model cells, without affecting ABCG2 expression or localization. These results pave the way for the development of new crown ether compounds with improved ABCG2-inhibitory properties.

Cytotoxicity of cinchona alkaloid organocatalysts against MES-SA and MES-SA/Dx5 multidrug-resistant uterine sarcoma cell lines

Since the first application of natural quinine as an anti-malarial drug, cinchona alkaloids and their derivatives have been exhaustively studied for their biological activity. In our work, we tested 13 cinchona alkaloid organocatalysts, synthesised from quinine. These derivatives were screened against MES-SA and Dx5 uterine sarcoma cell lines for in vitro anticancer activity and to investigate their potential to overcome P-glycoprotein (P-gp) mediated multidrug resistance (MDR). Decorating quinine with hydrogen-bond donor units, such as thiourea and (thio)squaramide, resulted in decreased half-maximal growth inhibition values on both cell lines (1.3–21 µM) compared to quinine and other cinchona alcohols (47–111 µM). Further cytotoxicity studies conducted in the presence of the P-gp inhibitor tariquidar indicated that several analogues, especially cinchona amines and squaramides, but not thiosquaramide, were expelled from MDR cells by P-gp. Similarly to the established P-gp inhibitor quinine, 6 cinchona analogues were shown to inhibit calcein-AM efflux. Interestingly, quinine and didehydroquinine exhibited a marginally increased toxicity against the multidrug resistant Dx5 cells. Collateral sensitivity of the MDR cell line was more pronounced when the cinchona thiosquaramide was complexed with Cu(II) acetate. Based on the results, cinchona derivatives are good anticancer candidates for further drug development.