Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5ß/B6 and ABCB5ß/B9.

ABCB5 is a member of the ABC transporter superfamily composed of 48 transporters, which have been extensively studied for their role in cancer multidrug resistance and, more recently, in tumorigenesis. ABCB5 has been identified as a marker of skin progenitor cells, melanoma, and limbal stem cells. It has also been associated with multidrug resistance in several cancers. The unique feature of ABCB5 is that it exists as both a full transporter (ABCB5FL) and a half transporter (ABCB5ß). Several studies have shown that the ABCB5ß homodimer does not confer multidrug resistance, in contrast to ABCB5FL. In this study, using three complementary techniques, (1) nanoluciferase-based bioluminescence resonance energy transfer, (2) coimmunoprecipitation, and (3) proximity ligation assay, we identified two novel heterodimers in melanoma: ABCB5ß/B6 and ABCB5ß/B9. Both heterodimers could be expressed in High-Five insect cells and ATPase assays revealed that both functional nucleotide-binding domains of homodimers and heterodimers are required for their basal ATPase activity. These results are an important step toward elucidating the functional role of ABCB5ß in melanocytes and melanoma.

ABCB1-dependent collateral sensitivity of multidrug-resistant colorectal cancer cells to the survivin inhibitor MX106-4C.

AIMS: To investigate the collateral sensitivity (CS) of ABCB1-positive multidrug resistant (MDR) colorectal cancer cells to the survivin inhibitor MX106-4C and the mechanism. METHODS: Biochemical assays (MTT, ATPase, drug accumulation/efflux, Western blot, RT-qPCR, immunofluorescence, flow cytometry) and bioinformatic analyses (mRNA-sequencing, reversed-phase protein array) were performed to investigate the hypersensitivity of ABCB1 overexpressing colorectal cancer cells to MX106-4C and the mechanisms. Synergism assay, long-term selection, and 3D tumor spheroid test were used to evaluate the anti-cancer efficacy of MX106-4C. RESULTS: MX106-4C selectively killed ABCB1-positive colorectal cancer cells, which could be reversed by an ABCB1 inhibitor, knockout of ABCB1, or loss-of-function ABCB1 mutation, indicating an ABCB1 expression and function-dependent mechanism. MX106-4C's selective toxicity was associated with cell cycle arrest and apoptosis through ABCB1-dependent survivin inhibition and activation on caspases-3/7 as well as modulation on p21-CDK4/6-pRb pathway. MX106-4C had good selectivity against ABCB1-positive colorectal cancer cells and retained this in multicellular tumor spheroids. In addition, MX106-4C could exert a synergistic anti-cancer effect with doxorubicin or re-sensitize ABCB1-positive cancer cells to doxorubicin by reducing ABCB1 expression in the cell population via long-term exposure. CONCLUSIONS: MX106-4C selectively kills ABCB1-positive MDR colorectal cancer cells via a novel ABCB1-dependent survivin inhibition mechanism, providing a clue for designing CS compound as an alternative strategy to overcome ABCB1-mediated colorectal cancer MDR.

Magnolol derivatives as specific and noncytotoxic inhibitors of breast cancer resistance protein (BCRP/ABCG2).

The breast cancer resistance protein (BCRP/ABCG2) transporter mediates the efflux of numerous antineoplastic drugs, playing a central role in multidrug resistance related to cancer. The absence of successful clinical trials using specific ABCG2 inhibitors reveals the urge to identify new compounds to attend this critical demand. In this work, a series of 13 magnolol derivatives was tested as ABCG2 inhibitors. Only two compounds, derivatives 10 and 11, showed partial and complete ABCG2 inhibitory effect, respectively. This inhibition was selective toward ABCG2, since none of the 13 compounds inhibited neither P-glycoprotein nor MRP1. Both inhibitors (10 and 11) were not transported by ABCG2 and demonstrated a low cytotoxic profile even at high concentrations (up to 100 µM). 11 emerged as the most promising compound of the series, considering the ratio between cytotoxicity (IG50) and ABCG2 inhibition potency (IC50), showing a therapeutic ratio (TR) higher than observed for 10 (10.5 versus 1.6, respectively). This derivative showed a substrate-independent and a mixed type of inhibition. The effect of compound 11 on the ABCG2 ATPase activity and thermostability revealed allosteric protein changes. This compound did not affect the expression levels of ABCG2 and increased the binding of the conformational-sensitive antibody 5D3. A docking study showed that 11 did not share the same binding site with ABCG2 substrate mitoxantrone. Finally, 11 could revert the chemoresistance to SN-38 mediated by ABCG2.

Natural medicinal compounds target signal transduction pathways to overcome ABC drug efflux transporter-mediated multidrug resistance in cancer.

ATP-binding cassette (ABC) transporters such as ABCB1, ABCG2, and ABCC1 are the major players in drug efflux-mediated multidrug resistance (MDR), which severely affects the efficacy of chemotherapy. Several synthetic compounds block the drug transport by ABC transporters; however, they exhibit a narrow therapeutic window, and produce side effects in non-target normal tissues. Conversely, the downregulation of the expression of ABC drug transporters seems to be a promising strategy to reverse MDR in cancer cells. Several signaling pathways, such as NF-κB, STAT3, Gli, NICD, YAP/TAZ, and Nrf2 upregulate the expression of ABC drug transporters in drug-resistant cancers. Recently, natural medicinal compounds have gained importance to overcome the ABC drug-efflux pump-mediated MDR in cancer. These compounds target transcription factors and the associated signal transduction pathways, thereby downregulating the expression of ABC transporters in drug-resistant cancer cells. Several potent natural compounds have been identified as lead candidates to synergistically enhance chemotherapeutic efficacy, and a few of them are already in clinical trials. Therefore, modulation of signal transduction pathways using natural medicinal compounds for the reversal of ABC drug transporter-mediated MDR in cancer is a novel approach for improving the efficiency of the existing chemotherapeutics. In this review, we discuss the modulatory role of natural medicinal compounds on cellular signaling pathways that regulate the expression of ABC transporters in drug-resistant cancer cells.

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.

Residues forming the gating regions of asymmetric multidrug transporter Pdr5 also play roles in conformational switching and protein folding.

ATP-binding cassette (ABC) multidrug transporters are large, polytopic membrane proteins that exhibit astonishing promiscuity for their transport substrates. These transporters unidirectionally efflux thousands of structurally and functionally distinct compounds. To preclude the reentry of xenobiotic molecules via the drug-binding pocket, these proteins contain a highly conserved molecular gate, essentially allowing the transporters to function as molecular diodes. However, the structure-function relationship of these conserved gates and gating regions are not well characterized. In this study, we combine recent single-molecule, cryo-EM data with genetic and biochemical analyses of residues in the gating region of the yeast multidrug transporter Pdr5, the founding member of a large group of clinically relevant asymmetric ABC efflux pumps. Unlike the symmetric ABCG2 efflux gate, the Pdr5 counterpart is highly asymmetric, with only four (instead of six) residues comprising the gate proper. However, other residues in the near vicinity are essential for the gating activity. Furthermore, we demonstrate that residues in the gate and in the gating regions have multiple functions. For example, we show that Ile-685 and Val-1372 are required not only for successful efflux but also for allosteric inhibition of Pdr5 ATPase activity. Our investigations reveal that the gating region residues of Pdr5, and possibly other ABCG transporters, play a role not only in molecular gating but also in allosteric regulation, conformational switching, and protein folding.

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.

Emerging nanotechnology-based therapeutics to combat multidrug-resistant cancer.

Cancer often develops multidrug resistance (MDR) when cancer cells become resistant to numerous structurally and functionally different chemotherapeutic agents. MDR is considered one of the principal reasons for the failure of many forms of clinical chemotherapy. Several factors are involved in the development of MDR including increased expression of efflux transporters, the tumor microenvironment, changes in molecular targets and the activity of cancer stem cells. Recently, researchers have designed and developed a number of small molecule inhibitors and derivatives of natural compounds to overcome various mechanisms of clinical MDR. Unfortunately, most of the chemosensitizing approaches have failed in clinical trials due to non-specific interactions and adverse side effects at pharmacologically effective concentrations. Nanomedicine approaches provide an efficient drug delivery platform to overcome the limitations of conventional chemotherapy and improve therapeutic effectiveness. Multifunctional nanomaterials have been found to facilitate drug delivery by improving bioavailability and pharmacokinetics, enhancing the therapeutic efficacy of chemotherapeutic drugs to overcome MDR. In this review article, we discuss the major factors contributing to MDR and the limitations of existing chemotherapy- and nanocarrier-based drug delivery systems to overcome clinical MDR mechanisms. We critically review recent nanotechnology-based approaches to combat tumor heterogeneity, drug efflux mechanisms, DNA repair and apoptotic machineries to overcome clinical MDR. Recent successful therapies of this nature include liposomal nanoformulations, cRGDY-PEG-Cy5.5-Carbon dots and Cds/ZnS core-shell quantum dots that have been employed for the effective treatment of various cancer sub-types including small cell lung, head and neck and breast cancers.

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.

Overexpression of Human ABCB1 and ABCG2 Reduces the Susceptibility of Cancer Cells to the Histone Deacetylase 6-Specific Inhibitor Citarinostat.

Citarinostat (ACY-241) is a promising oral histone deacetylase 6 (HDAC6)-selective inhibitor currently in clinical trials for the treatment of multiple myeloma (MM) and non-small-cell lung cancer (NSCLC). However, the inevitable emergence of resistance to citarinostat may reduce its clinical effectiveness in cancer patients and limit its clinical usefulness in the future. In this study, we investigated the potential role of the multidrug efflux transporters ABCB1 and ABCG2, which are two of the most common mechanisms of acquired resistance to anticancer drugs, on the efficacy of citarinostat in human cancer cells. We discovered that the overexpression of ABCB1 or ABCG2 significantly reduced the sensitivity of human cancer cells to citarinostat. We demonstrated that the intracellular accumulation of citarinostat and its activity against HDAC6 were substantially reduced by the drug transport function of ABCB1 and ABCG2, which could be restored by treatment with an established inhibitor of ABCB1 or ABCG2, respectively. In conclusion, our results revealed a novel mechanism by which ABCB1 and ABCG2 actively transport citarinostat away from targeting HDAC6 in cancer cells. Our results suggest that the co-administration of citarinostat with a non-toxic modulator of ABCB1 and ABCG2 may optimize its therapeutic application in the clinic.

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.

An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites.

Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad-spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild-type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3-4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild-type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.

Licochalcone A Selectively Resensitizes ABCG2-Overexpressing Multidrug-Resistant Cancer Cells to Chemotherapeutic Drugs.

The overexpression of the ATP-binding cassette (ABC) transporter ABCG2 has been linked to clinical multidrug resistance in solid tumors and blood cancers, which remains a significant obstacle to successful cancer chemotherapy. For years, the potential modulatory effect of bioactive compounds derived from natural sources on ABCG2-mediated multidrug resistance has been investigated, as they are inherently well tolerated and offer a broad range of chemical scaffolds. Licochalcone A (LCA), a natural chalcone isolated from the root of Glycyrrhiza inflata, is known to possess a broad spectrum of biological and pharmacological activities, including pro-apoptotic and antiproliferative effects in various cancer cell lines. In this study, the chemosensitization effect of LCA was examined in ABCG2-overexpressing multidrug-resistant cancer cells. Experimental data demonstrated that LCA inhibits the drug transport function of ABCG2 and reverses ABCG2-mediated multidrug resistance in human multidrug-resistant cancer cell lines in a concentration-dependent manner. Results of LCA-stimulated ABCG2 ATPase activity and the in silico docking analysis of LCA to the inward-open conformation of human ABCG2 suggest that LCA binds ABCG2 in the transmembrane substrate-binding pocket. This study provides evidence that LCA should be further evaluated as a modulator of ABCG2 in drug combination therapy trials against ABCG2-expressing drug-resistant tumors.

ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators.

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37-80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

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.

A High-Throughput Screen of a Library of Therapeutics Identifies Cytotoxic Substrates of P-glycoprotein.

The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit both brain penetration and oral bioavailability of many chemotherapy drugs. Although US Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter the literature. In response, we developed a high-throughput screen to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected subline KB-8-5-11 that overexpresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversibility of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified, including 55 newly identified compounds. Substrates were confirmed using an orthogonal killing assay against human embryonic kidney-293 cells overexpressing P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor), AT9283, (Janus kinase 2/3 inhibitor), ispinesib (kinesin spindle protein inhibitor), gedatolisib (PKI-587, phosphoinositide 3-kinase/mammalian target of rampamycin inhibitor), GSK-690693 (AKT inhibitor), and KW-2478 (heat-shock protein 90 inhibitor) were substrates. In addition, we assessed direct ATPase stimulation. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693, and gedatolisib, whereas ispinesib, AT7519, and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. These data identified compounds whose oral bioavailability or brain penetration may be affected by P-gp. SIGNIFICANCE STATEMENT: The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to be expressed at barrier sites, where it acts to limit oral bioavailability and brain penetration of substrates. In order to identify novel compounds that are transported by P-gp, we developed a high-throughput screen using the KB-3-1 cancer cell line and its colchicine-selected subline KB-8-5-11. We screened the Mechanism Interrogation Plate (MIPE) library, the National Center for Advancing Translational Science (NCATS) pharmaceutical collection (NPC), the NCATS Pharmacologically Active Chemical Toolbox (NPACT), and a kinase inhibitor library comprising 977 compounds, for a total of 10,804 compounds. Of the 10,804 compounds screened, a total of 90 substrates were identified of which 55 were novel. P-gp expression may adversely affect the oral bioavailability or brain penetration of these compounds.

Synthesis and Characterization of Bodipy-FL-Cyclosporine A as a Substrate for Multidrug Resistance-Linked P-Glycoprotein (ABCB1).

Fluorescent conjugates of drugs can be used to study cellular functions and pharmacology. These compounds interact with proteins as substrates or inhibitors, helping in the development of unique fluorescence-based methods to study in vivo localization and molecular mechanisms. P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette (ABC) transporter that effluxes most anticancer drugs from cells, contributing to the development of drug resistance. To study the transport function of P-gp, we synthesized a Bodipy-labeled fluorescent conjugate of cyclosporine A (BD-CsA). After synthesis and characterization of its chemical purity, BD-CsA was compared with the commonly used 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-CsA probe. In flow cytometry assays, the fluorescence intensity of BD-CsA was almost 10 times greater than that of NBD-CsA, enabling us to use significantly lower concentrations of BD-CsA to achieve the same fluorescence levels. We found that BD-CsA is recognized as a transport substrate by both human and mouse P-gp. The rate of efflux of BD-CsA by human P-gp is comparable to that of NBD-CsA. The transport of BD-CsA was inhibited by tariquidar, with similar IC50 values to those for NBD-CsA. BD-CsA and NBD-CsA both partially inhibited the ATPase activity of P-gp with similar IC50 values. In silico docking of BD-CsA and NBD-CsA to the human P-gp structure indicates that they both bind in the drug-binding pocket with similar docking scores and possibly interact with similar residues. Thus, we demonstrate that BD-CsA is a sensitive fluorescent substrate of P-gp that can be used to efficiently study the transporter's localization and function in vitro and in vivo. SIGNIFICANCE STATEMENT: The goal of this study was to develop an effective probe to study drug transport by P-glycoprotein (P-gp). Fluorophore-conjugated substrates are useful to study the P-gp transport mechanism, structural characteristics, and development of its inhibitors. Cyclosporine A (CsA), a cyclic peptide comprising 11 amino acids, is a known substrate of P-gp. P-gp affects CsA pharmacokinetics and interactions with other coadministered drugs, especially during transplant surgeries and treatment of autoimmune disorders, when CsA is given as an immunosuppressive agent. We synthesized and characterized Bodipy-FL-CsA as an avid fluorescent substrate that can be used to study the function of P-gp both in vitro and in vivo. We demonstrate that Bodipy-FL-conjugation does not affect the properties of CsA as a P-gp substrate.