Second-site suppressor mutations reveal connection between the drug-binding pocket and nucleotide-binding domain 1 of human P-glycoprotein (ABCB1).

Human P-glycoprotein (P-gp) or ABCB1 is overexpressed in many cancers and has been implicated in altering the bioavailability of chemotherapeutic drugs due to their efflux, resulting in the development of chemoresistance. To elucidate the mechanistic aspects and structure-function relationships of P-gp, we previously utilized a tyrosine (Y)-enriched P-gp mutant (15Y) and demonstrated that at least 15 conserved residues in the drug-binding pocket of P-gp are responsible for optimal substrate interaction and transport. To further understand the role of these 15 residues, two new mutants were generated, namely 6Y with the substitution of six residues (F72, F303, I306, F314, F336 and L339) with Y in transmembrane domain (TMD) 1 and 9Y with nine substitutions (F732, F759, F770, F938, F942, M949, L975, F983 and F994) in TMD2. Although both the mutants were expressed at normal levels at the cell surface, the 6Y mutant failed to transport all the tested substrates except Bodipy-verapamil, whereas the 9Y mutant effluxed all tested substrates in a manner very similar to that of the wild-type protein. Further mutational analysis revealed that two second-site mutations, one in intracellular helix (ICH) 4 (F916Y) and one in the Q loop of nucleotide-binding domain (NBD) 1 (F480Y) restored the transport function of 6Y. Additional biochemical data and comparative molecular dynamics simulations of the 6Y and 6Y+F916Y mutant indicate that the Q-loop of NBD1 of P-gp communicates with the substrate-binding sites in the transmembrane region through ICH4. This is the first evidence for the existence of second-site suppressors in human P-gp that allow recovery of the loss of transport function caused by primary mutations. Further study of such mutations could facilitate mapping of the communication pathway between the substrate-binding pocket and the NBDs of P-gp and possibly other ABC drug transporters.

ABCG2 Mediates Resistance to the Dual EGFR and PI3K Inhibitor MTX-211 in Cancer Cells.

MTX-211 is a first-in-class dual inhibitor of epidermal growth factor receptor (EGFR) and phosphoinositide-3 kinase (PI3K) signaling pathways with a compelling pharmaceutical profile and could enhance the effectiveness of mitogen-activated protein kinase kinase (MEK) inhibitor therapy in colorectal tumors with KRAS mutations. However, the specific mechanisms contributing to the acquired resistance to MTX-211 in human cancers remain elusive. Here, we discovered that the overexpression of the ATP-binding cassette (ABC) drug transporter ABCG2, a prevalent mechanism associated with multidrug resistance (MDR), could diminish the effectiveness of MTX-211 in human cancer cells. We showed that the drug efflux activity of ABCG2 substantially decreased the intracellular accumulation of MTX-211 in cancer cells. As a result, the cytotoxicity and effectiveness of MTX-211 in suppressing the activation of the EGFR and PI3K pathways were significantly attenuated in cancer cells overexpressing ABCG2. Moreover, the enhancement of the MTX-211-stimulated ATPase activity of ABCG2 and the computational molecular docking analysis illustrating the binding of MTX-211 to the substrate-binding sites of ABCG2 offered a further indication for the interaction between MTX-211 and ABCG2. In summary, our findings indicate that MTX-211 acts as a substrate for ABCG2, underscoring the involvement of ABCG2 in the emergence of resistance to MTX-211. This finding carries clinical implications and merits further exploration.

Infliximab Inhibits Colitis Associated Cancer in Model Mice by Downregulating Genes Associated with Mast Cells and Decreasing Their Accumulation.

Inflammatory bowel diseases (IBDs), such as Crohn's disease or ulcerative colitis, can be treated with anti TNF-alpha (TNF-α) antibodies (Abs), but they also put patients with IBDs at risk of cancer. We aimed to determine whether the anti TNF-α Ab induces colon cancer development in vitro and in vivo, and to identify the genes involved in colitis-associated cancer. We found that TNF-α (50 ng/mL) inhibited the proliferation, migration, and invasion of HCT8 and COLO205 colon cancer cell lines and that anti TNF-α Ab neutralized TNF-α inhibition in vitro. The effects of anti TNF-α Ab, infliximab (10 mg/kg) were investigated in mouse models of colitis-associated cancer induced by intraperitoneally injected azoxymethane (AOM: 10 mg/kg)/orally administered dextran sodium sulfate (DSS: 2.5%) (AOM/DSS) in vivo. Infliximab significantly attenuated the development of colon cancer in these mice. Microarray analyses and RT-qPCR revealed that mast cell protease 1, mast cell protease 2, and chymase 1 were up-regulated in cancer tissue of AOM/DSS mice; however, those mast cell related genes were downregulated in cancer tissue of AOM/DSS mice with infliximab. These results suggested that mast cells play a pivotal role in the development of cancer associated with colitis in AOM/DSS mice.

Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein.

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

Furmonertinib, a Third-Generation EGFR Tyrosine Kinase Inhibitor, Overcomes Multidrug Resistance through Inhibiting ABCB1 and ABCG2 in Cancer Cells.

ATP-binding cassette transporters, including ABCB1 (P-glycoprotein) and ABCG2 (BCRP/MXR/ABCP), are pivotal in multidrug resistance (MDR) development in cancer patients undergoing conventional chemotherapy. The absence of approved therapeutic agents for multidrug-resistant cancers presents a significant challenge in effectively treating cancer. Researchers propose repurposing existing drugs to sensitize multidrug-resistant cancer cells, which overexpress ABCB1 or ABCG2, to conventional anticancer drugs. The goal of this study is to assess whether furmonertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor overcomes drug resistance mediated by ABCB1 and ABCG2 transporters. Furmonertinib stands out due to its ability to inhibit drug transport without affecting protein expression. The discovery of this characteristic was validated through ATPase assays, which revealed interactions between furmonertinib and ABCB1/ABCG2. Additionally, in silico docking of furmonertinib offered insights into potential interaction sites within the drug-binding pockets of ABCB1 and ABCG2, providing a better understanding of the underlying mechanisms responsible for the reversal of MDR by this repurposed drug. Given the encouraging results, we propose that furmonertinib should be explored as a potential candidate for combination therapy in patients with tumors that have high levels of ABCB1 and/or ABCG2. This combination therapy holds the potential to enhance the effectiveness of conventional anticancer drugs and presents a promising strategy for overcoming MDR in cancer treatment.

Hydroxygenkwanin Improves the Efficacy of Cytotoxic Drugs in ABCG2-Overexpressing Multidrug-Resistant Cancer Cells.

Hydroxygenkwanin, a flavonoid isolated from the leaves of the Daphne genkwa plant, is known to have pharmacological properties; however, its modulatory effect on multidrug resistance, which is (MDR) mediated by ATP-binding cassette (ABC) drug transporters, has not been investigated. In this study, we examine the interaction between hydroxygenkwanin, ABCB1, and ABCG2, which are two of the most well-characterized ABC transporters known to contribute to clinical MDR in cancer patients. Hydroxygenkwanin is not an efflux substrate of either ABCB1 or ABCG2. We discovered that, in a concentration-dependent manner, hydroxygenkwanin significantly reverses ABCG2-mediated resistance to multiple cytotoxic anticancer drugs in ABCG2-overexpressing multidrug-resistant cancer cells. Although it inhibited the drug transport function of ABCG2, it had no significant effect on the protein expression of this transporter in cancer cells. Experimental data showing that hydroxygenkwanin stimulates the ATPase activity of ABCG2, and in silico docking analysis of hydroxygenkwanin binding to the inward-open conformation of human ABCG2, further indicate that hydroxygenkwanin sensitizes ABCG2-overexpressing cancer cells by binding to the substrate-binding pocket of ABCG2 and attenuating the transport function of ABCG2. This study demonstrates the potential use of hydroxygenkwanin as an effective inhibitor of ABCG2 in drug combination therapy trials for patients with tumors expressing higher levels of ABCG2.

A Phenylfurocoumarin Derivative Reverses ABCG2-Mediated Multidrug Resistance In Vitro and In Vivo.

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.

Evidence for the Interaction of A3 Adenosine Receptor Agonists at the Drug-Binding Site(s) of Human P-glycoprotein (ABCB1).

P-glycoprotein (P-gp) is a multidrug transporter that is expressed on the luminal surface of epithelial cells in the kidney, intestine, bile-canalicular membrane in the liver, blood-brain barrier, and adrenal gland. This transporter uses energy of ATP hydrolysis to efflux from cells a variety of structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs. In this regard, understanding the interaction with P-gp of drug entities in development is important and highly recommended in current US Food and Drug Administration guidelines. Here we tested the P-gp interaction of some A3 adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain, and hepatocellular carcinoma. Biochemical assays of the ATPase activity of P-gp and by photolabeling P-gp with its transport substrate [125I]-iodoarylazidoprazosin led to the identification of rigidified (N)-methanocarba nucleosides (i.e., compound 3 as a stimulator and compound 8 as a partial inhibitor of P-gp ATPase activity). Compound 8 significantly inhibited boron-dipyrromethene (BODIPY)-verapamil transport mediated by human P-gp (IC50 2.4 ± 0.6 µM); however, the BODIPY-conjugated derivative of 8 (compound 24) was not transported by P-gp. In silico docking of compounds 3 and 8 was performed using the recently solved atomic structure of paclitaxel (Taxol)-bound human P-gp. Molecular modeling studies revealed that both compounds 3 and 8 bind in the same region of the drug-binding pocket as Taxol. Thus, this study indicates that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. SIGNIFICANCE STATEMENT: Certain A3 adenosine receptor agonists are being developed for the treatment of chronic diseases. The goal of this study was to test the interaction of these agonists with the human multidrug resistance-linked transporter P-glycoprotein (P-gp). ATPase and photolabeling assays demonstrated that compounds with rigidified (N)-methanocarba nucleosides inhibit the activity of P-gp; however, a fluorescent derivative of one of the compounds was not transported by P-gp. Furthermore, molecular docking studies revealed that the binding site for these compounds overlaps with the site for paclitaxel in the drug-binding pocket. These results suggest that nucleoside derivatives, depending on structural functionalization, can modulate the function of P-gp.

Novel Potent ABCB1 Modulator, Phenethylisoquinoline Alkaloid, Reverses Multidrug Resistance in Cancer Cell.

ATP-binding cassette (ABC) transporters, which are concerned with the efflux of anticancer drugs from cancer cells, have a pivotal role in multidrug resistance (MDR). In particular, ABCB1 is a well-known ABC transporter that develops MDR in many cancer cells. Some ABCB1 modulators can reverse ABCB1-mediated MDR; however, no modulators with clinical efficacy have been approved. The aim of this study was to identify novel ABCB1 modulators by using high-throughput screening. Of the 5861 compounds stored at Tohoku University, 13 compounds were selected after the primary screening via a fluorescent plate reader-based calcein acetoxymethylester (AM) efflux assay. These 13 compounds were validated in a flow cytometry-based calcein AM efflux assay. Two isoquinoline derivatives were identified as novel ABCB1 inhibitors, one of which was a phenethylisoquinoline alkaloid, (±)-7-benzyloxy-1-(3-benzyloxy-4-methoxyphenethyl)-1,2,3,4-tetrahydro-6-methoxy-2-methylisoquinoline oxalate. The compound, a phenethylisoquinoline alkaloid, was subsequently evaluated in the cytotoxicity assay and shown to significantly enhance the reversal of ABCB1-mediated MDR. In addition, the compound activated the ABCB1-mediated ATP hydrolysis and inhibited the photolabeling of ABCB1 with [125I]-iodoarylazidoprazosin. Furthermore, the compound also reversed the resistance to paclitaxel without increasing the toxicity in the ABCB1-overexpressing KB-V1 cell xenograft model. Overall, we concluded that the newly identified phenethylisoquinoline alkaloid reversed ABCB1-mediated MDR through direct interaction with the substrate-binding site of ABCB1. These findings may contribute to the development of more potent and less toxic ABCB1 modulators, which could overcome ABCB1-mediated MDR.

Lymph nodes around the posterior gastric artery: their existence, frequency, and clinical implications.

PURPOSE: The lymphatic flow along the posterior gastric artery (PGA) is considered of possible clinical importance in terms of lymphatic metastasis; however, little is known about the lymph nodes (LNs) around this artery. The purpose of this study was to establish if LNs exist around the PGA and to evaluate their clinical implications. METHODS: We examined the tissues surrounding the PGA from 21 cadavers to search for LNs. We also investigated the patterns of lymphatic metastases in patients who underwent surgery for gastric neoplasms at our institute to detect their presence along the PGA. RESULTS: The PGA was identified in 11 cadavers, and LNs around the PGA were detected microscopically in 2 of these. Lymphatic metastasis directly to the LNs at the splenic artery without any metastases was regarded as skip metastasis along the PGA. Skip metastasis was found in two of ten patients who underwent surgery for remnant gastric cancer. CONCLUSIONS: The existence of LNs around the PGA was confirmed, and based on our findings, lymphatic metastasis through the PGA is possible in patients with remnant gastric cancer.

Synthetic Analogs of Curcumin Modulate the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCG2.

Multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) transporters in cancer cells is a major obstacle in cancer chemotherapy. Previous studies have shown that curcumin, a natural product and a dietary constituent of turmeric, inhibits the function of MDR-related ABC transporters, including ABCB1, ABCC1, and especially ABCG2. However, the limited bioavailability of curcumin prevents its use for modulation of the function of these transporters in the clinical setting. In this study, we investigated the effects of 24 synthetic curcumin analogs with increased bioavailability on the transport function of ABCG2. The screening of the 24 synthetic analogs by means of flow cytometry revealed that four of the curcumin analogs (GO-Y030, GO-Y078, GO-Y168, and GO-Y172) significantly inhibited the efflux of the ABCG2 substrates, mitoxantrone and pheophorbide A, from ABCG2-overexpressing K562/breast cancer resistance protein (BCRP) cells. Biochemical analyses showed that GO-Y030, GO-Y078, and GO-Y172 stimulated the ATPase activity of ABCG2 at nanomolar concentrations and inhibited the photolabeling of ABCG2 with iodoarylazidoprazosin, suggesting that these analogs interact with the substrate-binding sites of ABCG2. In addition, when used in cytotoxicity assays, GO-Y030 and GO-Y078 were found to improve the sensitivity of the anticancer drug, SN-38, in K562/BCRP cells. Taken together, these results suggest that nontoxic synthetic curcumin analogs with increased bioavailability, especially GO-Y030 and GO-Y078, inhibit the function of ABCG2 by directly interacting at the substrate-binding site. These synthetic curcumin analogs could therefore be developed as potent modulators to overcome ABCG2-mediated MDR in cancer cells.

Alpha-Mangostin Reverses Multidrug Resistance by Attenuating the Function of the Multidrug Resistance-Linked ABCG2 Transporter.

The ATP-binding cassette (ABC) drug transporter ABCG2 can actively efflux a wide variety of chemotherapeutic agents out of cancer cells and subsequently reduce the intracellular accumulation of these drugs. Therefore, the overexpression of ABCG2 often contributes to the development of multidrug resistance (MDR) in cancer cells, which is one of the major obstacles to successful cancer chemotherapy. Moreover, ABCG2 is highly expressed in various tissues including the intestine and blood-brain barrier (BBB), limiting the absorption and bioavailability of many therapeutic agents. For decades, the task of developing a highly effective synthetic inhibitor of ABCG2 has been hindered mostly by the intrinsic toxicity, the lack of specificity, and complex pharmacokinetics. Alternatively, considering the wide range of diversity and relatively nontoxic nature of natural products, developing potential modulators of ABCG2 from natural sources is particularly valuable. α-Mangostin is a natural xanthone derived from the pericarps of mangosteen (Garcinia mangostana L.) with various pharmacological purposes, including suppressing angiogenesis and inducing cancer cell growth arrest. In this study, we demonstrated that at nontoxic concentrations, α-mangostin effectively and selectively inhibits ABCG2-mediated drug transport and reverses MDR in ABCG2-overexpressing MDR cancer cells. Direct interactions between α-mangostin and the ABCG2 drug-binding site(s) were confirmed by stimulation of ATPase activity and by inhibition of photolabeling of the substrate-binding site(s) of ABCG2 with [125I]iodoarylazidoprazosin. In summary, our findings show that α-mangostin has great potential to be further developed into a promising modulator of ABCG2 for reversing MDR and for its use in combination therapy for patients with MDR tumors.

Overexpression of ATP-Binding Cassette Subfamily G Member 2 Confers Resistance to Phosphatidylinositol 3-Kinase Inhibitor PF-4989216 in Cancer Cells.

Deregulated activation of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently found in human cancers, which plays a key role in promoting cancer proliferation and resistance to anticancer therapies. Therefore, developing inhibitors targeting key components of the PI3K/Akt/mTOR signaling pathway has great clinical significance. PF-4989216 is a novel, orally available small-molecule drug that was developed to selectively inhibit the PI3K/Akt/mTOR signaling pathway and subsequent cancer cell proliferation. PF-4989216 exhibited potent and selective inhibition against PI3K kinase activity in preclinical small-cell lung cancer (SCLC) models, and was especially effective against the proliferation of SCLCs harboring PIK3CA mutation. Unfortunately, in addition to innate resistance mechanisms, drug extrusion by the efflux pumps may also contribute to the development of acquired resistance to PI3K inhibitors in cancer cells. The overexpression of ATP-binding cassette (ABC) drug transporters ABCB1 and ABCG2 is one of the most common mechanisms for reducing intracellular drug concentration and developing multidrug resistance, which remains a substantial challenge to the effective treatment of cancer. In this study, we report the discovery of ABCG2 overexpression as a mechanism of resistance to PI3K inhibitor PF-4989216 in human cancer cells. We demonstrated that the inhibition of Akt and downstream S6RP phosphorylation by PF-4989216 were significantly reduced in ABCG2-overexpressing human cancer cells. Moreover, overexpression of ABCG2 in various cancer cell lines confers significant resistance to PF-4989216, which can be reversed by an inhibitor or competitive substrate of ABCG2, indicating that ABCG2-mediated transport alone can sufficiently reduce the intracellular concentration of PF-4989216.

Epidermal Growth Factor Receptor Inhibitor Mobocertinib Resensitizes Multidrug-Resistant Cancer Cells by Attenuating the Human ATP-Binding Cassette Subfamily B Member 1 and Subfamily G Member 2.

ATP-binding cassette (ABC) transporters, notably ABCB1 (P-glycoprotein) and ABCG2, play a crucial role in the development of multidrug resistance (MDR) during the administration of chemotherapy for cancer patients. With a lack of approved treatments for addressing multidrug-resistant cancers, MDR remains a substantial challenge to the effective management of cancer. Rather than focusing on developing novel synthetic inhibitors, a promising approach to combat MDR involves repurposing approved therapeutic agents to enhance the sensitivity to cytotoxic antiproliferative drugs of multidrug-resistant cancer cells with high expression of ABCB1 or ABCG2. In this investigation, we observed a substantial reversal of MDR conferred by ABCB1 and ABCG2 in multidrug-resistant cancer cells through the use of mobocertinib, an approved third-generation inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. Mobocertinib demonstrated the ability to hinder drug transport function without causing changes in protein expression. The interactions between mobocertinib and ABCB1, as well as ABCG2, were validated through ATPase assays. Furthermore, in silico docking simulations were utilized to substantiate the binding of mobocertinib within the drug-binding pockets of both ABCB1 and ABCG2. We conclude that further testing of mobocertinib in combination therapy is warranted for patients with tumors expressing elevated levels of the ABC drug transporters ABCB1 and ABCG2.

Design, Synthesis and Biological Evaluation of Quinazolinamine Derivatives as Breast Cancer Resistance Protein and P-Glycoprotein Inhibitors with Improved Metabolic Stability.

A series of twenty-two quinazolinamine derivatives showing potent inhibitory activities on breast cancer resistance protein (BCRP) and p-glycoprotein (P-gp) were synthesized. A cyclopropyl-containing quinazolinamine 22 was identified as a dual BCRP and P-gp inhibitor, while azide-containing quinazolinamine 33 showed BCRP inhibitory activity. These lead compounds were further investigated in a battery of mechanistic experiments. Compound 22 changed the localization of BCRP and P-gp in cells, thus inhibiting the efflux of anticancer drugs by the two ATP-binding cassette (ABC) transporters. In addition, both 22 and 33 significantly stimulated the ATP hydrolysis of the BCRP transporter, indicating that they can be competitive substrates of the BCRP transporter, and thereby increase the accumulation of mitoxantrone in BCRP-overexpressing H460/MX20 cells. Azide derivative 33, exhibited a greater inhibitory effect on BCRP after UV activation and can serve as a valuable probe for investigating the interactions of quinazolinamine derivatives with BCRP. Notably, the dual BCRP and P-gp inhibitors 4-5, 22-24, 27, and BCRP inhibitor 33 showed improved metabolic stability compared to Ko143.

Imperatorin Restores Chemosensitivity of Multidrug-Resistant Cancer Cells by Antagonizing ABCG2-Mediated Drug Transport.

The high expression of the ATP-binding cassette (ABC) drug transporter ABCG2 in cancer cells contributes to the emergence of multidrug resistance (MDR) in individuals afflicted with either solid tumors or blood cancers. MDR poses a major impediment in the realm of clinical cancer chemotherapy. Recently, substantial endeavors have been dedicated to identifying bioactive compounds isolated from nature capable of counteracting ABCG2-mediated MDR in cancer cells. Imperatorin, a natural coumarin derivative renowned for its diverse pharmacological properties, has not previously been explored for its impact on cancer drug resistance. This study investigates the chemosensitizing potential of imperatorin in ABCG2-overexpressing cancer cells. Experimental results reveal that at sub-toxic concentrations, imperatorin significantly antagonizes the activity of ABCG2 and reverses ABCG2-mediated MDR in a concentration-dependent manner. Furthermore, biochemical data and in silico analysis of imperatorin docking to the inward-open conformation of human ABCG2 indicate that imperatorin directly interacts with multiple residues situated within the transmembrane substrate-binding pocket of ABCG2. Taken together, these results furnish substantiation that imperatorin holds promise for further evaluation as a potent inhibitor of ABCG2, warranting exploration in combination drug therapy to enhance the effectiveness of therapeutic agents for patients afflicted with tumors that exhibit high levels of ABCG2.

ABCB1 and ABCG2 Overexpression Mediates Resistance to the Phosphatidylinositol 3-Kinase Inhibitor HS-173 in Cancer Cell Lines.

Constitutive activation of the phosphoinositide-3-kinase (PI3K)/Akt signaling pathway is crucial for tumor growth and progression. As such, this pathway has been an enticing target for drug discovery. Although HS-173 is a potent PI3K inhibitor that halts cancer cell proliferation via G2/M cell cycle arrest, the resistance mechanisms to HS-173 have not been investigated. In this study, we investigated the susceptibility of HS-173 to efflux mediated by the multidrug efflux transporters ABCB1 and ABCG2, which are two of the most well-known ATP-binding cassette (ABC) transporters associated with the development of cancer multidrug resistance (MDR). We found that the overexpression of ABCB1 or ABCG2 significantly reduced the efficacy of HS-173 in human cancer cells. Our data show that the intracellular accumulation of HS-173 was substantially reduced by ABCB1 and ABCG2, affecting G2/M arrest and apoptosis induced by HS-173. More importantly, the efficacy of HS-173 in multidrug-resistant cancer cells could be recovered by inhibiting the drug-efflux function of ABCB1 and ABCG2. Taken together, our study has demonstrated that HS-173 is a substrate for both ABCB1 and ABCG2, resulting in decreased intracellular concentration of this drug, which may have implications for its clinical use.

Interaction of A3 adenosine receptor ligands with the human multidrug transporter ABCG2.

Various adenosine receptor nucleoside-like ligands were found to modulate ATP hydrolysis by the multidrug transporter ABCG2. Both ribose-containing and rigidified (N)-methanocarba nucleosides (C2-, N6- and 5'-modified), as well as adenines (C2-, N6-, and deaza modified), were included. 57 compounds out of 63 tested either stimulated (50) or inhibited (7) basal ATPase activity. Structure-activity analysis showed a separation of adenosine receptor and ABCG2 activities. The 7-deaza modification had favorable effects in both (N)-methanocarba nucleosides and adenines. Adenine 37c (MRS7608) and (N)-methanocarba 7-deaza-5'-ethyl ester 60 (MRS7343) were found to be potent stimulators of ABCG2 ATPase activity with EC50 values of 13.2 ± 1.7 and 13.2 ± 2.2 nM, respectively. Both had affinity in the micromolar range for A3 adenosine receptor and lacked the 5'-amide agonist-enabling group (37c was reported as a weak A3 antagonist, Ki 6.82 µM). Compound 60 significantly inhibited ABCG2 substrate transport (IC50 0.44 µM). Docking simulations predicted the interaction of 60 with 21 residues in the drug-binding pocket of ABCG2.

ABCB1 limits the cytotoxic activity of TAK-243, an inhibitor of the ubiquitin-activating enzyme UBA1.

BACKGROUND: One of the major concerns of cancer therapy is the emergence of multidrug resistance (MDR). The MDR-associated ATP-binding cassette sub-family B member 1 (ABCB1) transporter is established to mediate resistance against numerous anticancer drugs. In this study, we demonstrated that the Ubiquitin-like modifier activating enzyme 1 (UBA1) inhibitor TAK-243 is transported by the ABCB1. METHODS: MTT assay was performed to evaluate the cytotoxicity of TAK-243. Western blot was carried out to investigate if TAK-243 affect to ABCB1 protein expression in cancer cells. High Performance Liquid Chromatography (HPLC) and ATPase assay were carried out to confirm TAK-243 as an ABCB1 substrate. [3H]-paclitaxel accumulation assay was used to determine the MDR reversal effect of TAK-243. Computational docking analysis was performed to investigate the drug-transporter binding position. RESULTS: The cytotoxicity profile showed that TAK-243 was less effective in ABCB1-overexpressing cells than in the parental cells, but pharmacological inhibition or knockout the gene of ABCB1 was able to reverse TAK-243 resistance. Furthermore, TAK-243 potently stimulated ABCB1 ATPase activity and the HPLC analysis revealed that TAK-243 accumulation was significantly reduced in ABCB1-overexpressing cells. Finally, the computational docking analysis indicates a high binding affinity between TAK-243 and human ABCB1 transporter. CONCLUSIONS: Our in vitro data characterized TAK-243 as a substrate of ABCB1, which may predict limited anticancer effect of this compound in drug resistant tumors.

The multi-targeted tyrosine kinase inhibitor SKLB610 resensitizes ABCG2-overexpressing multidrug-resistant cancer cells to chemotherapeutic drugs.

The overexpression of ATP-binding cassette (ABC) transporter ABCB1 (P-glycoprotein) or ABCG2 (BCRP/MXR/ABCP) in cancer cells is frequently associated with the development of multidrug resistance (MDR) in cancer patients, which remains a major obstacle to effective cancer treatment. By utilizing energy derived from ATP hydrolysis, both transporters have been shown to reduce the chemosensitivity of cancer cells by actively effluxing cytotoxic anticancer drugs out of cancer cells. Knowing that there are presently no approved drugs or other therapeutics for the treatment of multidrug-resistant cancers, in recent years, studies have investigated the repurposing of tyrosine kinase inhibitors (TKIs) to act as agents against MDR mediated by ABCB1 and/or ABCG2. SKLB610 is a multi-targeted TKI with potent activity against vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor 2 (FGFR2). In this study, we investigate the interaction of SKLB610 with ABCB1 and ABCG2. We discovered that neither ABCB1 nor ABCG2 confers resistance to SKLB610, but SKLB610 selectively sensitizes ABCG2-overexpressing multidrug-resistant cancer cells to cytotoxic anticancer agents in a concentration-dependent manner. Our data indicate that SKLB610 reverses ABCG2-mediated MDR by attenuating the drug-efflux function of ABCG2 without affecting its total cell expression. These findings are further supported by results of SKLB610-stimulated ABCG2 ATPase activity and in silico docking of SKLB610 in the drug-binding pocket of ABCG2. In summary, we reveal the potential of SKLB610 to overcome resistance to cytotoxic anticancer drugs, which offers an additional treatment option for patients with multidrug-resistant cancers and warrants further investigation.