Short Communication: Novel Di- and Triselenoesters as Effective Therapeutic Agents Inhibiting Multidrug Resistance Proteins in Breast Cancer Cells.

Breast cancer has the highest incidence rate among all malignancies worldwide. Its high mortality is mainly related to the occurrence of multidrug resistance, which significantly limits therapeutic options. In this regard, there is an urgent need to develop compounds that would overcome this phenomenon. There are few reports in the literature that selenium compounds can modulate the activity of P-glycoprotein (MDR1). Therefore, we performed in silico studies and evaluated the effects of the novel selenoesters EDAG-1 and EDAG-8 on BCRP, MDR1, and MRP1 resistance proteins in MCF-7 and MDA-MB-231 breast cancer cells. The cytometric analysis showed that the tested compounds (especially EDAG-8) are inhibitors of BCRP, MDR1, and MRP1 efflux pumps (more potent than the reference compounds-novobiocin, verapamil, and MK-571). An in silico study correlates with these results, suggesting that the compound with the lowest binding energy to these transporters (EDAG-8) has a more favorable spatial structure affecting its anticancer activity, making it a promising candidate in the development of a novel anticancer agent for future breast cancer therapy.

Brain Exposure to the Macrocyclic ALK Inhibitor Zotizalkib is Restricted by ABCB1, and Its Plasma Disposition is Affected by Mouse Carboxylesterase 1c.

Zotizalkib (TPX-0131), a fourth-generation macrocyclic anaplastic lymphoma kinase (ALK) inhibitor, is designed to overcome resistance due to secondary ALK mutations in non-small cell lung cancer (NSCLC). We here evaluated the pharmacokinetic roles of the ABCB1 (P-gp/MDR1) and ABCG2 (BCRP) efflux transporters, OATP1 influx transporters and the metabolizing enzymes CES1 and CYP3A in plasma and tissue disposition of zotizalkib after oral administration in relevant mouse models. Zotizalkib was efficiently transported by hABCB1 in vitro. In vivo, a significant ∼9-fold higher brain-to-plasma ratio was observed in Abcb1a/b-/- and Abcb1a/b;Abcg2-/- compared to wild-type mice. No change in brain disposition was observed in Abcg2-/- mice, suggesting that mAbcb1a/b markedly restricts the brain accumulation of zotizalkib. ABCB1-mediated efflux of zotizalkib was completely inhibited by elacridar, a dual ABCB1/ABCG2 inhibitor, increasing brain exposure without any signs of acute CNS-related toxicities. In Oatp1a/b-/- mice, no marked changes in plasma exposure or tissue-to-plasma ratios were observed, indicating that zotizalkib is not a substantial in vivo substrate for mOatp1a/b. Zotizalkib may further be metabolized by CYP3A4 but only noticeably at low plasma concentrations. In Ces1-/- mice, a 2.5-fold lower plasma exposure was seen compared to wild-type, without alterations in tissue distribution. This suggests increased plasma retention of zotizalkib by binding to the abundant mouse plasma Ces1c. Notably, the hepatic expression of human CES1 did not affect zotizalkib plasma exposure or tissue distribution. The obtained pharmacokinetic insights may be useful for the further development and optimization of therapeutic efficacy and safety of zotizalkib and related compact macrocyclic ALK inhibitors.

The Simultaneous Inhibition of Solute Carrier Family 6 Member 19 and Breast Cancer Resistance Protein Transporters Leads to an Increase of Indoxyl Sulfate (a Uremic Toxin) in Plasma and Kidney.

Solute carrier family 6 member 19 (SLC6A19) inhibitors are being studied as therapeutic agents for phenylketonuria. In this work, a potent SLC6A19 inhibitor (RA836) elevated rat kidney uremic toxin indoxyl sulfate (IDS) levels by intensity (arbitrary unit) of 13.7 ± 7.7 compared with vehicle 0.3 ± 0.1 (P = 0.01) as determined by tissue mass spectrometry imaging analysis. We hypothesized that increased plasma and kidney levels of IDS could be caused by the simultaneous inhibition of both Slc6a19 and a kidney IDS transporter responsible for excretion of IDS into urine. To test this, we first confirmed the formation of IDS through tryptophan metabolism by feeding rats a Trp-free diet. Inhibiting Slc6a19 with RA836 led to increased IDS in these rats. Next, RA836 and its key metabolites were evaluated in vitro for inhibiting kidney transporters such as organic anion transporter (OAT)1, OAT3, and breast cancer resistance protein (BCRP). RA836 inhibits BCRP with an IC50 of 0.045 µM but shows no significant inhibition of OAT1 or OAT3. Finally, RA836 analogs with either potent or no inhibition of SLC6A19 and/or BCRP were synthesized and administered to rats fed a normal diet. Plasma and kidney samples were collected to quantify IDS using liquid chromatography-mass spectrometry. Neither a SLC6A19 inactive but potent BCRP inhibitor nor a SLC6A19 active but weak BCRP inhibitor raised IDS levels, whereas compounds inhibiting both transporters caused IDS accumulation in rat plasma and kidney, supporting the hypothesis that rat Bcrp contributes to the excretion of IDS. In summary, we identified that inhibiting Slc6a19 increases IDS formation, while simultaneously inhibiting Bcrp results in IDS accumulation in the kidney and plasma. SIGNIFICANCE STATEMENT: This is the first publication to decipher the mechanism for accumulation of indoxyl sulfate (IDS) (a uremic toxin) in rats via inhibition of both Slc6a19 and Bcrp. Specifically, inhibition of Slc6a19 in the gastrointestinal track increases IDS formation, and inhibition of Bcrp in the kidney blocks IDS excretion. Therefore, we should avoid inhibiting both solute carrier family 6 member 19 and breast cancer resistance protein simultaneously in humans to prevent accumulation of IDS, a known risk factor for cardiovascular disease, psychic anxiety, and mortality in chronic kidney disease patients.

ATP-binding cassette transporter inhibitor potency and substrate drug affinity are critical determinants of successful drug delivery enhancement to the brain.

BACKGROUND: Pharmacotherapy for brain diseases is severely compromised by the blood-brain barrier (BBB). ABCB1 and ABCG2 are drug transporters that restrict drug entry into the brain and their inhibition can be used as a strategy to boost drug delivery and pharmacotherapy for brain diseases. METHODS: We employed elacridar and tariquidar in mice to explore the conditions for effective inhibition at the BBB. Abcg2;Abcb1a/b knockout (KO), Abcb1a/b KO, Abcg2 KO and wild-type (WT) mice received a 3 h i.p. infusion of a cocktail of 8 typical substrate drugs in combination with elacridar or tariquidar at a range of doses. Abcg2;Abcb1a/b KO mice were used as the reference for complete inhibition, while single KO mice were used to assess the potency to inhibit the remaining transporter. Brain and plasma drug levels were measured by LC-MS/MS. RESULTS: Complete inhibition of ABCB1 at the BBB is achieved when the elacridar plasma level reaches 1200 nM, whereas tariquidar requires at least 4000 nM. Inhibition of ABCG2 is more difficult. Elacridar inhibits ABCG2-mediated efflux of weak but not strong ABCG2 substrates. Strikingly, tariquidar does not enhance the brain uptake of any ABCG2-subtrate drug. Similarly, elacridar, but not tariquidar, was able to inhibit its own brain efflux in ABCG2-proficient mice. The plasma protein binding of elacridar and tariquidar was very high but similar in mouse and human plasma, facilitating the translation of mouse data to humans. CONCLUSIONS: This work shows that elacridar is an effective pharmacokinetic-enhancer for the brain delivery of ABCB1 and weaker ABCG2 substrate drugs when a plasma concentration of 1200 nM is exceeded.

Ticagrelor modestly raises plasma riboflavin concentration in humans and inhibits riboflavin transport by BCRP and MRP4.

Riboflavin (vitamin B2) has been proposed as a biomarker for breast cancer resistance protein (BCRP) activity. In recent studies in mice, cynomolgus monkeys, and humans, BCRP-inhibiting drugs increased the plasma concentration of riboflavin. We showed recently that ticagrelor inhibits BCRP and raises the plasma concentrations of the BCRP substrate rosuvastatin in healthy volunteers. In the same drug-drug interaction study, we now investigated whether ticagrelor affects the plasma concentrations of riboflavin. Intake of 90 mg ticagrelor increased the ratio between the peak plasma riboflavin concentration and the fasting riboflavin concentration before ticagrelor administration by 1.20-fold (90% confidence interval, 1.10-1.32; P = 0.006) compared to placebo. In vitro, riboflavin was transported by BCRP and multidrug-resistance-associated protein 4 (MRP4) but no clear transport was observed by MRP2, MRP3, or the P-glycoprotein. Moreover, ticagrelor inhibited the transport of riboflavin in BCRP- and MRP4-expressing membrane vesicles with unbound 50% inhibitory concentrations of 0.020 and 1.1 µM, respectively. Based on vesicle and tissue protein expression data, the small intestinal MRP4-mediated efflux clearance of riboflavin (1.2-1.4 nL/min/mg) was estimated to be similar to that mediated by BCRP (0.23-1.3 nL/min/mg). As MRP4 is expressed in the basolateral membrane of enterocytes, it may facilitate the absorption of riboflavin and impair the utility of riboflavin as a biomarker of intestinal BCRP. To conclude, ticagrelor modestly raises the plasma concentration of riboflavin probably by inhibiting intestinal BCRP. Inhibition of intestinal MRP4 may have reduced the absorption of riboflavin and limited the effect of ticagrelor on riboflavin levels.

The ATP-Binding Cassette Transporter-Mediated Efflux Transport of Ganciclovir at the Blood-Brain Barrier.

BACKGROUND AND OBJECTIVE: Recent studies have highlighted the key role of the ATP-binding cassette (ABC) transporters, including the P-glycoprotein (P-gp), the breast cancer resistance protein (BCRP), and the multi-drug resistance protein 4 (MRP4) in limiting the brain distribution of several antiviral agents. In this study, we investigated whether the inhibition of these transporters increases the permeability of the blood-brain barrier (BBB) to ganciclovir. METHODS: A microdialysis and high-performance liquid chromatographic method was developed to monitor the concentrations of unbound ganciclovir in the brain interstitial fluid and plasma, with and without the administration of ABC transporter inhibitors. Pharmacokinetic parameters, including the area under the plasma concentration-time curve from time 0 to time of the last measurable analyte concentration (AUC0-t,plasma), the area under the brain interstitial fluid concentration-time curve from time 0 to time of the last measurable analyte concentration (AUC0-t,brain), and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) were calculated. RESULTS: The mean AUC0-t,plasma, AUC0-t,brain, and Kp,uu,brain in rats who received ganciclovir (30 mg/kg, intraperitoneal) alone were 1090 min·µg/mL, 150 min·µg/mL, and 14%, respectively. After the administration of tariquidar (inhibitor of P-gp), Ko143 (inhibitor of BCRP), or MK-571 (inhibitor of MRP4), the Kp,uu,brain of ganciclovir increased to 31 ± 2.1%, 26 ± 1.3%, and 32 ± 2.0%, respectively. CONCLUSIONS: The findings of this study suggest that ABC transporters P-gp, BCRP, and MRP4 mediate the efflux of ganciclovir at the BBB and that the inhibition of these transporters facilitates the penetration of the BBB by ganciclovir.

HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer.

BACKGROUND: Resistance to chemotherapy is a major problem in the treatment of patients with triple-negative breast cancer (TNBC). Preclinical data suggest that TNBC is dependent on proteasomes; however, clinical observations indicate that the efficacy of proteasome inhibitors in TNBC may be limited, suggesting the need for combination therapies. METHODS: We compared bortezomib and carfilzomib and their combinations with nelfinavir and lopinavir in TNBC cell lines and primary cells with regard to their cytotoxic activity, functional proteasome inhibition, and induction of the unfolded protein response (UPR). Furthermore, we evaluated the involvement of sXBP1, ABCB1, and ABCG2 in the cytotoxic activity of drug combinations. RESULTS: Carfilzomib, via proteasome ß5 + ß2 inhibition, is more cytotoxic in TNBC than bortezomib, which inhibits ß5 + ß1 proteasome subunits. The cytotoxicity of carfilzomib was significantly potentiated by nelfinavir or lopinavir. Carfilzomib with lopinavir induced endoplasmic reticulum stress and pro-apoptotic UPR through the accumulation of excess proteasomal substrate protein in TNBC in vitro. Moreover, lopinavir increased the intracellular availability of carfilzomib by inhibiting carfilzomib export from cells that express high levels and activity of ABCB1, but not ABCG2. CONCLUSION: Proteasome inhibition by carfilzomib combined with nelfinavir/lopinavir represents a potential treatment option for TNBC, warranting further investigation.

Marein, a novel natural product for restoring chemo-sensitivity to cancer cells through competitive inhibition of ABCG2 function.

The pivotal roles of ATP-binding cassette (ABC) transporters in drug resistance have been widely appreciated. Here we report that marein, a natural product from Coreopsis tinctoria Nutt, is a potent chemo-sensitizer in drug resistant cancer cells overexpressing ABCG2 transporter. We demonstrate that marein can competitively inhibit efflux activity of ABCG2 protein and increase the intracellular accumulation of the chemotherapeutic drugs that belong to substrate of this transporter. We further show that marein can bind to the conserved amino acid residue F439 of ABCG2, a critical site for drug-substrate interaction. Moreover, marein can significantly sensitize the ABCG2-expressing tumor cells to chemotherapeutic drugs such as topotecan, mitoxantrone, and olaparib. This study reveals a novel role and mechanism of marein in modulating drug resistance, and may have important implications in treatment of cancers that are resistant to chemotherapeutic drugs that belong to the substrates of ABCG2 transporters.

Inhibition of human drug transporter activities by succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0-3.9 µM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 µM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 µM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.

The contradictory role of febuxostat in ABCG2 expression and potentiating hypericin-mediated photodynamic therapy in colorectal cancers.

Photodynamic Therapy (PDT) is an emerging method to treat colorectal cancers (CRC). Hypericin (HYP) is an effective mediator of PDT and the ABCG2 inhibitor, Febuxostat (FBX) could augment PDT. HT29 and HEK293 cells showed light dependant cytotoxic response to PDT in both 2D and 3D cell models. FBX co-treatment was not found to improve PDT cytotoxicity. Next, ABCG2 protein expression was observed in HT29 but not in HEK293 cells. However, ABCG2 gene expression analysis did not support protein expression results as ABCG2 gene expression results were found to be higher in HEK293 cells. Although HYP treatment was found to significantly reduce ABCG2 gene expression levels in both cell lines, FBX treatment partially restored ABCG2 gene expression. Our findings indicate that FBX co-treatment may not be suitable for augmenting HYP-mediated PDT in CRC but could potentially be useful for other applications.

One-step synthesis of diaryloxadiazoles as potent inhibitors of BCRP.

Aim: BCRP plays a major role in the efflux of cytotoxic molecules, limiting their antiproliferative activity. We aimed to design and synthesize new BCRP inhibitors to render cancerous tumors more sensitive toward anticancer agents. Materials & methods: Based on our previous work, we conceived potential BCRP inhibitors derived from 1,3,4-oxadiazoles bearing two substituted phenyl rings. Results: Evaluating 19 derivatives, we found that 2,5-diaryl-1,3,4-oxadiazoles possessing methoxy groups were the most active. The highest activity was recorded with derivatives bearing three methoxy groups. The most active compound (3j) was selective in inhibiting BCRP and nontoxic as evidenced by cellular tests. Conclusion: 3j is a promising BCRP inhibitor thanks to its synthetic accessibility and biological profile.

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The ABCB1 and ABCG2 efflux transporters limit brain disposition of the SYK inhibitors entospletinib and lanraplenib.

The highly selective Spleen Tyrosine Kinase (SYK) inhibitors entospletinib and lanraplenib disrupt kinase activity and inhibit immune cell functions. They are developed for treatment of B-cell malignancies and autoimmunity diseases. The impact of P-gp/ABCB1 and BCRP/ABCG2 efflux transporters, OATP1a/1b uptake transporters and CYP3A drug-metabolizing enzymes on the oral pharmacokinetics of these drugs was assessed using mouse models. Entospletinib and lanraplenib were orally administered simultaneously at moderate dosages (10 mg/kg each) to female mice to assess the possibility of examining two structurally and mechanistically similar drugs at the same time, while reducing the number of experimental animals and sample-processing workload. The plasma pharmacokinetics of both drugs were not substantially restricted by Abcb1 or Abcg2. The brain-to-plasma ratios of entospletinib in Abcb1a/b-/-, Abcg2-/- and Abcb1a/b;Abcg2-/- mice were 1.7-, 1.8- and 2.9-fold higher, respectively, compared to those in wild-type mice. For lanraplenib these brain-to-plasma ratios were 3.0-, 1.3- and 10.4-fold higher, respectively. This transporter-mediated restriction of brain penetration for both drugs could be almost fully inhibited by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, without signs of acute toxicity. Oatp1a/b and human CYP3A4 did not seem to affect the pharmacokinetics of entospletinib and lanraplenib, but mouse Cyp3a may limit lanraplenib plasma exposure. Unexpectedly, entospletinib and lanraplenib increased each other's plasma exposure by 2.6- to 2.9-fold, indicating a significant drug-drug interaction. This interaction was, however, unlikely to be mediated through any of the studied transporters or CYP3A. The obtained insights may perhaps help to further improve the safety and efficacy of entospletinib and lanraplenib.

High-throughput BCRP inhibitors screening system based on styrene maleic acid polymer membrane protein stabilization strategy and surface plasmon resonance biosensor.

Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The KD of xanthotoxin, bergapten, and naringenin were 5.14 µM, 4.57 µM, and 3.72 µM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy.

A Pilot Study To Assess the Suitability of Riboflavin As a Surrogate Marker of Breast Cancer Resistance Protein in Healthy Participants.

We recently showed that riboflavin is a selected substrate of breast cancer resistance protein (BCRP) over P-glycoprotein (P-gp) and demonstrated its prediction performance in preclinical drug-drug interaction (DDI) studies. The aim of this study was to investigate the suitability of riboflavin to assess BCRP inhibition in humans. First, we assessed the substrate potential of riboflavin toward other major drug transporters using established transfected cell systems. Riboflavin is a substrate for organic anion transporter (OAT)1, OAT3, and multidrug and toxin extrusion protein (MATE)2-K, with uptake ratios ranging from 2.69 to 11.6, but riboflavin is not a substrate of organic anion-transporting polypeptide (OATP)1B1, OATP1B3, organic cation transporter (OCT)2, and MATE1. The effects of BMS-986371, a potent in vitro inhibitor of BCRP (IC 50 0.40 µM), on the pharmacokinetics of riboflavin, isobutyryl carnitine, and arginine were then examined in healthy male adults (N = 14 or 16) after oral administration of methotrexate (MTX) (7.5 mg) and enteric-coated (EC) sulfasalazine (SSZ) (1000 mg) alone or in combination with BMS-986371 (150 mg). Oral administration of BMS-986371 increased the area under the plasma concentration-time curves (AUCs) of rosuvastatin and immediate-release (IR) SSZ to 1.38- and 1.51-fold, respectively, and significantly increased AUC(0-4h), AUC(0-24h), and C max of riboflavin by 1.25-, 1.14-, and 1.11-fold (P-values of 0.003, 0.009, and 0.025, respectively) compared with the MTX/SSZ EC alone group. In contrast, BMS-986371 did not significantly influence the AUC(0-24h) and C max values of isobutyryl carnitine and arginine (0.96- to 1.07-fold, respectively; P > 0.05). Overall, these data indicate that plasma riboflavin is a promising biomarker of BCRP that may offer a possibility to assess drug candidate as a BCRP modulator in early drug development. SIGNIFICANCE STATEMENT: Endogenous compounds that serve as biomarkers for clinical inhibition of breast cancer resistance protein (BCRP) are not currently available. This study provides the initial evidence that riboflavin is a promising BCRP biomarker in humans. For the first time, the value of leveraging the substrate of BCRP with acceptable prediction performance in clinical studies is shown. Additional clinical investigations with known BCRP inhibitors are needed to fully validate and showcase the utility of this biomarker.

Evaluation of ABT-751, a novel anti-mitotic agent able to overcome multi-drug resistance, in melanoma cells.

PURPOSE: Drug efflux transporter associated multi-drug resistance (MDR) is a potential limitation in the use of taxane chemotherapies for the treatment of metastatic melanoma. ABT-751 is an orally bioavailable microtubule-binding agent capable of overcoming MDR and proposed as an alternative to taxane-based therapies. METHODS: This study compares ABT-751 to taxanes in vitro, utilizing seven melanoma cell line models, publicly available gene expression and drug sensitivity databases, a lung cancer cell line model of MDR drug efflux transporter overexpression (DLKP-A), and drug efflux transporter ATPase assays. RESULTS: Melanoma cell lines exhibit a low but variable protein and RNA expression of drug efflux transporters P-gp, BCRP, and MDR3. Expression of P-gp and MDR3 correlates with sensitivity to taxanes, but not to ABT-751. The anti-proliferative IC50 profile of ABT-751 was higher than the taxanes docetaxel and paclitaxel in the melanoma cell line panel, but fell within clinically achievable parameters. ABT-751 IC50 was not impacted by P-gp-overexpression in DKLP-A cells, which display strong resistance to the P-gp substrate taxanes compared to DLKP parental controls. The addition of ABT-751 to paclitaxel treatment significantly decreased cell proliferation, suggesting some reversal of MDR. ATPase activity assays suggest that ABT-751 is a potential BCRP substrate, with the ability to inhibit P-gp ATPase activity. CONCLUSION: Our study confirms that ABT-751 is active against melanoma cell lines and models of MDR at physiologically relevant concentrations, it inhibits P-gp ATPase activity, and it may be a BCRP and/or MDR3 substrate. ABT-751 warrants further investigation alone or in tandem with other drug efflux transporter inhibitors for hard-to-treat MDR melanoma.

Structural Basis of the Allosteric Inhibition of Human ABCG2 by Nanobodies.

ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.

Metabolism-guided development of Ko143 analogs as ABCG2 inhibitors.

ATP-binding cassette subfamily G member 2 (ABCG2), an efflux transporter, is involved in multiple pathological processes. Ko143 is a potent ABCG2 inhibitor; however, it is quickly metabolized through carboxylesterase 1-mediated hydrolysis of its t-butyl ester moiety. The current work aimed to develop more metabolically stable ABCG2 inhibitors. Novel Ko143 analogs were designed and synthesized by replacing the unstable t-butyl ester moiety in Ko143 with an amide group. The synthesized Ko143 analogs were evaluated for their ABCG2 inhibitory activity, binding mode with ABCG2, cytotoxicity, and metabolic stability. We found that the amide modification of Ko143 led to metabolically stable ABCG2 inhibitors. Among these Ko143 analogs, K2 and K34 are promising candidates with favorable oral pharmacokinetic profiles in mice. In summary, we synthesized novel Ko143 analogs with improved metabolic stability, which can potentially be used as lead compounds for the future development of ABCG2 inhibitors.

Characterization of a Potent, Selective, and Safe Inhibitor, Ac15(Az8)2, in Reversing Multidrug Resistance Mediated by Breast Cancer Resistance Protein (BCRP/ABCG2).

Overexpression of breast cancer resistance transporter (BCRP/ABCG2) in cancers has been explained for the failure of chemotherapy in clinic. Inhibition of the transport activity of BCRP during chemotherapy should reverse multidrug resistance. In this study, a triazole-bridged flavonoid dimer Ac15(Az8)2 was identified as a potent, nontoxic, and selective BCRP inhibitor. Using BCRP-overexpressing cell lines, its EC50 for reversing BCRP-mediated topotecan resistance was 3 nM in MCF7/MX100 and 72 nM in S1M180 in vitro. Mechanistic studies revealed that Ac15(Az8)2 restored intracellular drug accumulation by inhibiting BCRP-ATPase activity and drug efflux. It did not down-regulate the cell surface BCRP level to enhance drug retention. It was not a transport substrate of BCRP and showed a non-competitive relationship with DOX in binding to BCRP. A pharmacokinetic study revealed that I.P. administration of 45 mg/kg of Ac15(Az8)2 resulted in plasma concentration above its EC50 (72 nM) for longer than 24 h. It increased the AUC of topotecan by 2-fold. In an in vivo model of BCRP-overexpressing S1M180 xenograft in Balb/c nude mice, it significantly reversed BCRP-mediated topotecan resistance and inhibited tumor growth by 40% with no serious body weight loss or death incidence. Moreover, it also increased the topotecan level in the S1M180 xenograft by 2-fold. Our results suggest that Ac15(Az8)2 is a promising candidate for further investigation into combination therapy for treating BCRP-overexpressing cancers.

Inhibitors of ABCB1 and ABCG2 overcame resistance to topoisomerase inhibitors in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is a highly aggressive disease with a poor prognosis. Although most patients initially respond to topoisomerase inhibitors, resistance rapidly emerges. The aim, therefore, is to overcome resistance to topoisomerase I (irinotecan) or II (etoposide) inhibitors in SCLCs. METHODS: To identify key factors in the chemoresistance of SCLCs, we established four cell lines resistant to etoposide or an active metabolite of irinotecan, SN-38, from SCLC cell lines and evaluated RNA profiles using parental and newly established cell lines. RESULTS: We found that the drug efflux protein, ATP-binding cassette sub-family B member 1 (ABCB1), was associated with resistance to etoposide, and ATP-binding cassette sub-family G member 2 (ABCG2) was associated with resistance to SN-38 by RNA sequencing. The inhibition of ABCB1 or ABCG2 in each resistant cell line induced synergistic apoptotic activity and promoted drug sensitivity in resistant SCLC cells. The ABC transporter inhibitors, elacridar and tariquidar, restored sensitivity to etoposide or SN-38 in in vitro and in vivo studies, and promoted apoptotic activity and G2-M arrest in resistant SCLC cells. CONCLUSIONS: ABC transporter inhibitors may be a promising therapeutic strategy for the purpose of overcoming resistance to topoisomerase inhibitors in patients with SCLC.

Inhibition of ABCG2 transporter by lapatinib enhances 5-aminolevulinic acid-mediated protoporphyrin IX fluorescence and photodynamic therapy response in human glioma cell lines.

5-Aminolevulinic acid (ALA) is an intraoperative molecular probe approved for fluorescence-guided resection (FGR) of high-grade gliomas to achieve maximal safe tumor resection. Although ALA has no fluorescence on its own, it is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) with red fluorescence for tumor detection and photosensitizing activity for photodynamic therapy (PDT). The preferential tumor accumulation of PpIX following ALA administration enables the use of ALA as a prodrug for PpIX FGR and PDT of gliomas. Since intracellular PpIX in tumor cells after ALA treatment is influenced by biological processes including PpIX bioconversion catalyzed by ferrochelatase (FECH) and PpIX efflux by ATP-binding cassette subfamily G member 2 (ABCG2), we determined the activity of FECH and ABCG2 in a panel of human glioma cell lines and correlated with intracellular and extracellular PpIX levels and PDT response. We found that glioma cell lines with ABCG2 activity exhibited the trend of low intracellular PpIX, high extracellular PpIX and low PDT response, whereas no particular correlation was seen with FECH activity. Inhibition of PpIX efflux with ABCG2 inhibitors was more effective in enhancing ALA-PpIX fluorescence and PDT response than blocking PpIX bioconversion with iron chelator deferoxamine. We also showed that a clinically used kinase inhibitor lapatinib could be repurposed for therapeutic enhancement of ALA due to its potent ABCG2 inhibitory activity. Our study reveals ABCG2 as an important biological determinant of PpIX fluorescence in glioma cells and suggests ABCG2 inhibition with lapatinib as a promising therapeutic enhancement approach.