Human ABCB1 Research Articles

Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and their Zebrafish Homologs at the Blood-Brain Barrier.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish where expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disrpution. We transfected HEK-293 cells with both NanoLuc and human ABCB1 or ABCG2, or their zebrafish homologs Abcb4 and Abcg2a, which are functionally homologous to human P-gp and ABCG2, respectively, and expressed at the zebrafish BBB. We evaluated the brightness of ten NanoLuc substrates, then screened the eight brightest for their ability to be effluxed by the ABC transporters. We identified one ABCB1 substrate, two Abcb4 substrates, six ABCG2 substrates, and four Abcg2a substrates. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. Significance Statement The ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

Structural insight into binding site access and ligand recognition by human ABCB1.

ABCB1 is a broad-spectrum efflux pump central to cellular drug handling and multidrug resistance in humans. However, its mechanisms of poly-specific substrate recognition and transport remain poorly resolved. Here we present cryo-EM structures of lipid embedded human ABCB1 in its apo, substrate-bound, inhibitor-bound, and nucleotide-trapped states at 3.4-3.9 Å resolution without using stabilizing antibodies or mutations and each revealing a distinct conformation. The substrate binding site is located within one half of the molecule and, in the apo state, is obstructed by transmembrane helix (TM) 4. Substrate and inhibitor binding are distinguished by major differences in TM arrangement and ligand binding chemistry, with TM4 playing a central role in all conformational transitions. Our data offer fundamental new insights into the role structural asymmetry, secondary structure breaks, and lipid interactions play in ABCB1 function and have far-reaching implications for ABCB1 inhibitor design and predicting its substrate binding profiles.

Differing temperature dependencies of functional homologs zebrafish Abcb4 and human ABCB1.

The ATP binding cassette (ABC) transporters human ABCB1 and zebrafish (Danio rerio) Abcb4 are functionally homologous multixenobiotic/multidrug (MXR/MDR) efflux transporters that confer the efflux of a broad range of diverse chemical compounds from the cell. As ATPases, the transporters utilize the energy released by ATP cleavage for protein conformation changes and concomitant active transport of substrate compounds. The temperatures, at which human ABCB1 and zebrafish Abcb4 need to function, can substantially differ: Whereas the ambient temperature of human ABCB1, which is that of the human body, is constant, zebrafish Abcb4 has to be active in a wider temperature range as the body temperature of zebrafish can considerably vary, depending on the ambient water temperature (18°C-40°C). Here, we examined the effect of temperature on the ATPase activities of recombinant human ABCB1 and zebrafish Abcb4 generated with the baculovirus expression system. Incubation temperatures for enzyme reactions were set to 37°C and 27°C, corresponding to the human body temperature and the cultivation temperature of zebrafish in our lab, respectively. For stimulation and inhibition of zebrafish Abcb4 and human ABCB1 ATPase activities verapamil and cyclosporin A were added at different concentrations and 50% effect concentrations (EC50) were determined. The different temperatures had a stronger effect on the human ABCB1 than on the zebrafish Abcb4 ATPase: Differences between EC50 values for verapamil at 37°C and 27°C, respectively, were 1.8-fold for human ABCB1 but only 1.2-fold for zebrafish Abcb4. Activation energies (Ea) of basal and verapamil-stimulated ATPases, calculated based on the Arrhenius equation, were 2-fold (basal) and 1.5-fold (verapamil-stimulated) higher for human ABCB1 than for zebrafish Abcb4. The differences between zebrafish Abcb4 and human ABCB1 ATPases in temperature sensitivity and activation energy could be important for the comparison of the functional properties of the two transporter proteins in the context of pharmaco-/toxicokinetics. Related to this, our finding that at equal reaction conditions the zebrafish Abcb4 ATPase activity tended to be generally higher than that of human ABCB1 may also be important, as this may point to a higher substrate compound transport rate of Abcb4.

ABCB1 attenuates brain exposure to the KRASG12C inhibitor opnurasib whereas binding to mouse carboxylesterase 1c influences its plasma exposure

Opnurasib (JDQ443) is a newly developed oral KRASG12C inhibitor, with a binding mechanism distinct from the registered KRASG12C inhibitors sotorasib and adagrasib. Phase I and II clinical trials for opnurasib in NSCLC are ongoing. We evaluated the pharmacokinetic roles of the ABCB1 (P-gp/MDR1) and ABCG2 (BCRP) efflux and OATP1 influx transporters, and of the metabolizing enzymes CYP3A and CES1 in plasma and tissue disposition of oral opnurasib, using genetically modified cell lines and mouse models. In vitro, opnurasib was potently transported by human (h)ABCB1 and slightly by mouse (m)Abcg2. In Abcb1a/b- and Abcb1a/b;Abcg2-deficient mice, a significant ∼100-fold increase in brain-to-plasma ratios was observed. Brain penetration was unchanged in Abcg2-/- mice. ABCB1 activity in the blood-brain barrier may therefore potentially limit the efficacy of opnurasib against brain metastases. The Abcb1a/b transporter activity could be almost completely reversed by co-administration of elacridar, a dual ABCB1/ABCG2 inhibitor, increasing the brain penetration without any behavioral or postural signs of acute CNS-related toxicity. No significant pharmacokinetic roles of the OATP1 transporters were observed. Transgenic human CYP3A4 did not substantially affect the plasma exposure of opnurasib, indicating that opnurasib is likely not a sensitive CYP3A4 substrate. Interestingly, Ces1-/- mice showed a 4-fold lower opnurasib plasma exposure compared to wild-type mice, whereas no strong effect was seen on the tissue distribution. Plasma Ces1c therefore likely binds opnurasib, increasing its retention in plasma. The obtained pharmacokinetic insights may be useful for further optimization of the clinical efficacy and safety of opnurasib, and might reveal potential drug-drug interaction risks.

Abstract LB258: ZDHHC20 is a potential target for overcoming ABCB1-mediated multidrug resisance (MDR) in cancer

Abstract ABCB1-mediated multidrug resistance (MDR) in cancer cells is an increasingly important cause of anticancer drug treatment failure. The occurrence of MDR in cancer reduces the effectiveness of antitumor drugs, which results in poor quality of life and increased mortality of patients, and creates a heavy burden for the patient's family and society. Hence, the reversal of ABCB1-mediated MDR in cancer is a highly urgent clinical need. Although reversal agents targeting ABCB1 have been developed and partially tested in clinical trials, their main mechanism limits to inhibit the function of ABCB1 efflux pumps, which lacks tumor tissue specificity and has poor clinical effects, and the development of ABCB1-based resistance reversal strategies has entered a bottleneck. ABCB1 mediates tumor MDR through drug efflux and lysosomal segregation, and intervention in the plasma membrane/lysosomal membrane localization of ABCB1 is a potential strategy to reverse drug resistance. Palmitoylation, as a post-translational lipid modification, plays an important role in protein anchoring, transport, and functional regulation, and is highly relevant to human tumors. In the present study, palmitoylation modification was first identified as a key post-translational lipid modification of ABCB1. ABCB1 is palmitoylated in its cytoplasmic domain, which is further stabilized by blocking its ubiquitination, consequently suppressing ABCB1 degradation by lysosomes. We identified palmitoyltransferase ZDHHC20 (DHHC20) as the main acetyltransferase required for the palmitoylation of ABCB1, and show that the inhibition of ABCB1 palmitoylation via 2-bromopalmitate (2-BP), or the silencing of DHHC20, decreases ABCB1 expression and overcomes MDR in vitro as well as in mice bearing SW620/Ad300 resistance cells. Moreover, Cysteine residues C787 was identified as the main palmitoylation cite in human ABCB1. Database and RNAseq analyses revealed that ZDHHC20 is highly expressed in tumor tissues and tumor MDR cells, which makes it a potential tumor tissue-specific target for overcoming ABCB1-mediated MDR in cancer. Our results firstly identified the novel post-translational modification (palmitoylation) of ABCB1 and provide a new research direction and theoretical basis for breaking through the bottleneck of the development of reversal agents for ABCB1-mediated MDR in cancer. Citation Format: Ning Ji, Xin Peng, Yi-nan Liu, Yuqi Yang, Zhe-Sheng Chen, Dexin Kong, Yueguo Li. ZDHHC20 is a potential target for overcoming ABCB1-mediated multidrug resisance (MDR) in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB258.

Abstract 7193: ABCB1-mediated chemotherapeutic drug resistance is reversed by a potent p53-MDM2 inhibitor NVP-HDM201

Abstract The failure of chemotherapy in cancer persists as a result of overexpression of various ATP-binding cassette (ABC) transporters. ABCB1 is one such transporter that actively pumps out chemotherapeutic drugs and it is one of the reasons for multidrug resistance (MDR). In the last few years, activation of p53 has been targeted as a therapeutic strategy in the treatment of cancers. Siremadlin (NVP-HDM201) is a potent and selective inhibitor of p53-MDM2 interaction. The objective of this study was to explore the ability of HDM201 to reverse tumor MDR attributable to overexpression of ABC transporters. According to our findings, HDM201 significantly increased the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates, such as colchicine and doxorubicin. We found that HDM201 reverses ABCB1-mediated MDR by interacting with ABCB1 and blocking its function without affecting the expression and cellular localization of ABCB1. Moreover, HDM201 promoted the accumulation of paclitaxel in cell lines that overexpress ABCB1. At both high and low doses, HDM201 increased the ATPase activity of ABCB1. The computational support for the cross-reactivity of this p53 inhibitor with human ABCB1 comes from docking simulation results that align with the binding conformation of HDM201 within the vast cavity of the transmembrane region of ABCB1. In conclusion, by blocking the transport function of ABCB1, HDM201 can reverse ABCB1-mediated MDR in vitro. Citation Format: Harsh Patel, Chaoyun Cai, Zhuoxun Wu, Qiuxu Teng, Hanli Li, Zhe-Sheng Chen. ABCB1-mediated chemotherapeutic drug resistance is reversed by a potent p53-MDM2 inhibitor NVP-HDM201 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7193.

Molecular Modeling Studies to Probe the Binding Hypothesis of Novel Lead Compounds against Multidrug Resistance Protein ABCB1.

The expression of drug efflux pump ABCB1/P-glycoprotein (P-gp), a transmembrane protein belonging to the ATP-binding cassette superfamily, is a leading cause of multidrug resistance (MDR). We previously curated a dataset of structurally diverse and selective inhibitors of ABCB1 to develop a pharmacophore model that was used to identify four novel compounds, which we showed to be potent and efficacious inhibitors of ABCB1. Here, we dock the inhibitors into a model structure of the human transporter and use molecular dynamics (MD) simulations to report the conformational dynamics of human ABCB1 induced by the binding of the inhibitors. The binding hypotheses are compared to the wider curated dataset and those previously reported in the literature. Protein-ligand interactions and MD simulations are in good agreement and, combined with LipE profiling, statistical and pharmacokinetic analyses, are indicative of potent and selective inhibition of ABCB1.

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.

Pharmacokinetics of the KRASG12C inhibitor adagrasib is limited by CYP3A and ABCB1, and influenced by binding to mouse plasma carboxylesterase 1c

Adagrasib (Krazati™) is the second FDA-approved specific KRASG12C inhibitor for non-small cell lung cancer (NSCLC) patients harboring this mutation. The impact of the drug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing enzymes CYP3A and carboxylesterase 1 (CES1) on the pharmacokinetics of oral adagrasib were studied using genetically modified mouse models. Adagrasib was potently transported by human ABCB1 and modestly by mouse Abcg2 in vitro. In Abcb1a/b-/- and Abcb1a/b;Abcg2-/- mice, the brain-to-plasma ratios were enhanced by 33- and 55-fold, respectively, compared to wild-type mice, whereas ratios in Abcg2-/- mice remained unchanged. The influence of ABC transporters was completely reversed by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, increasing the brain penetration in wild-type mice by 41-fold while no signs of acute CNS toxicity were observed. Tumor ABCB1 overexpression may thus confer adagrasib resistance. Whereas the ABC transporters did not affect adagrasib plasma exposure, CYP3A and Ces1 strongly impacted its apparent oral availability. The plasma AUC0–8 h was significantly enhanced by 2.3-fold in Cyp3a-/- compared to wild-type mice, and subsequently 4.3-fold reduced in transgenic CYP3A4 mice, indicating substantial CYP3A-mediated metabolism. Adagrasib plasma exposure was strongly reduced in Ces1-/- compared to wild-type mice, but tissue exposure was slightly increased, suggesting that adagrasib binds to plasma Ces1c in mice and is perhaps metabolized by Ces1. This binding could complicate interpretation of mouse studies, especially since humans lack circulating CES1 enzyme(s). Our results may be useful to further optimize the clinical safety and efficacy of adagrasib, and give more insight into potential drug-drug interactions risks.

Synergistic Inhibitory Effect of Quercetin and Cyanidin-3O-Sophoroside on ABCB1.

The human ABCB1 (P-glycoprotein, Pgp) protein is an active exporter expressed in the plasma membrane of cells forming biological barriers. In accordance with its broad substrate spectrum and tissue expression pattern, it affects the pharmacokinetics of numerous chemotherapeutic drugs and it is involved in unwanted drug-drug interactions leading to side effects or toxicities. When expressed in tumor tissues, it contributes to the development of chemotherapy resistance in malignancies. Therefore, the understanding of the molecular details of the ligand-ABCB1 interactions is of crucial importance. In a previous study, we found that quercetin (QUR) hampers both the transport and ATPase activity of ABCB1, while cyandin-3O-sophroside (C3S) stimulates the ATPase activity and causes only a weak inhibition of substrate transport. In the current study, when QUR and C3S were applied together, both a stronger ATPase inhibition and a robust decrease in substrate transport were observed, supporting their synergistic ABCB1 inhibitory effect. Similar to cyclosporine A, a potent ABCB1 inhibitor, co-treatment with QUR and C3S shifted the conformational equilibrium to the "inward-facing" conformer of ABCB1, as it was detected by the conformation-selective UIC2 mAb. To gain deeper insight into the molecular details of ligand-ABCB1 interactions, molecular docking experiments and MD simulations were also carried out. Our in silico studies support that QUR and C3S can bind simultaneously to ABCB1. The most favourable ligand-ABCB1 interaction is obtained when C3S binds to the central substrate binding site and QUR occupies the "access tunnel". Our results also highlight that the strong ABCB1 inhibitory effect of the combined treatment with QUR and C3S may be exploited in chemotherapy protocols for the treatment of multidrug-resistant tumors or for improving drug delivery through pharmacological barriers.

Assessment of the role of nucleoside transporters, P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in the placental transport of entecavir using in vitro, ex vivo, and in situ methods

The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.

In Vitro Screening and MD Simulations of Thiourea Derivatives against SARS-CoV-2 in Association with Multidrug Resistance ABCB1 Transporter.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) is considered a global public health concern since it causes high morbidity and mortality. Recently, it has been reported that repurposed anti-COVID-19 drugs might interact with multidrug resistance ABC transporter, particularly ABCB1. In the current study, a series of thiourea derivatives were screened as potential inhibitors against SARS-CoV-2 by targeting the attachment of receptor binding domain (RBD) of spike protein with ACE2 and their interaction with human ABCB1 has also been explored. The results indicated strong impairment of RBD-ACE2 attachment by BB IV-46 with a percentage inhibition of 95.73 ± 1.79% relative to the positive control, while BB V-19 was proven inactive with a percentage inhibition of 50.90 ± 0.84%. The same compound (BB IV-46) interacted with ABCB1 and potentially inhibited cell proliferation of P-gp overexpressing cell line with an IC50 value of 4.651 ± 0.06 μM. BB V-19, which was inactive against SARS-CoV-2, was inactive against ABCB1 with a higher IC50 value of 35.72 ± 0.09 μM. Furthermore, molecular dynamics simulations followed by binding free-energy analysis explored the binding interaction of BB IV-46 and BB V-19 to RBD region of spike protein of SARS-CoV-2. The results confirmed that compound BB IV-46 interacted strongly with RBD with a significant binding energy (-127.0 kJ/mol), while BB V-19 interacted weakly (-29.30 kJ/mol). The key interacting residues of the RBD involved in binding included Leu441, Lys444, and Tyr449. This study highlights the importance of BB IV-46 against SARS-CoV-2; however, further pharmacokinetic and pharmacodynamics studies are needed to be done.

Targeted co-delivery of paclitaxel and anti P-gp shRNA by low molecular weight PEI decorated with L-3,4-dihydroxyphenylalanine.

Co-delivery of small chemotherapeutic molecules and nucleic acid materials via targeted carriers has attracted great attention for treatment of resistant tumors and reducing adverse effects. In this study, a targeted carrier for co-delivery was prepared based on low-molecular weight polyethylenimine (LMW PEI). Paclitaxel (PTX) was covalently conjugated onto PEI via a succinate linker. The PEI conjugate was decorated with L-DOPA in order to target large neutral amino acid transporter-1 (LAT-1) that is over-expressed on various cancer cells. This PEI conjugate was complexed with human ABCB1 shRNA plasmid to down-regulate the expression of P-glycoprotein, as one of the major efflux pumps inducing resistance against chemotherapeutics. The formation of PEI conjugate enhanced the solubility of PTX and resulted in the condensation and protection of plasmid DNA in nanosized polyplexes. The results of targeted delivery into the cells demonstrated that PEI conjugate transferred the payloads to the cells over-expressing LAT-1 transporter, while the biological effects on the cells lacking the transporter was negligible. Also, shRNA-mediated down-regulation of P-gp led to the increase of toxic effects on the cells over-expressing P-gp. This study suggests a promising approach for co-delivery of small molecules and nucleic acid materials in a targeted manner for cancer therapy.

P-Glycoprotein (MDR1/ABCB1) Restricts Brain Accumulation of the Novel EGFR Inhibitor EAI045 and Oral Elacridar Coadministration Enhances Its Brain Accumulation and Oral Exposure.

EAI045 is a fourth-generation allosteric tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR). It targets T790M and C797S EGFR mutants in the treatment of non-small cell lung cancer (NSCLC). EAI045 and cetuximab combined induce tumor regression in mouse models of EGFR-mutant lung cancer. We investigated the pharmacokinetic roles of the multidrug efflux and uptake transporters ABCB1 (P-gp), ABCG2 (BCRP), and OATP1A/1B, and of the drug-metabolizing enzyme CYP3A in plasma and tissue distribution of EAI045 and its metabolites, using genetically modified mouse models. In vitro, EAI045 was a good transport substrate of human ABCB1. In vivo, oral EAI045 (20 mg/kg) was rapidly absorbed. Relative to wild-type mice, EAI045 brain-to-plasma ratios were increased 3.9-fold in Abcb1a/1b-/- and 4.8-fold in Abcb1a/1b;Abcg2-/- mice. However, in single Abcg2-/- mice they were unchanged. EAI045 oral availability was not markedly altered. Oral coadministration of elacridar, an ABCB1/ABCG2 inhibitor, increased the plasma AUC0–30min and brain-to-plasma ratios of EAI045 by 4.0-fold and 5.4-fold, respectively, in wild-type mice. EAI045 glucuronide showed an increased plasma AUC0–30min and a markedly decreased accumulation and tissue-to-plasma ratio in the small intestinal content when Abcb1a/1b and Abcg2 were absent. A large fraction of oral EAI045 was converted to its hydrolyzed metabolite PIA, but Abcb1a/1b, Abcg2, and Oatp1a/1b had little impact on PIA pharmacokinetics. Mouse Cyp3a knockout or transgenic human CYP3A4 overexpression did not significantly affect oral EAI045 pharmacokinetics. Our results show that blood–brain barrier ABCB1 can markedly limit EAI045 brain accumulation. Moreover, elacridar coadministration can effectively reverse this process.

Association between ABCB1 rs2235048 Polymorphism and THC Pharmacokinetics and Subjective Effects following Smoked Cannabis in Young Adults

Genetic influences on acute responses to psychoactive drugs may contribute to individual variability in addiction risk. ABCB1 is a human gene that encodes P-glycoprotein, an ATP-dependent efflux pump that may influence the pharmacokinetics of delta-9-tetrahydrocannabinol (THC), the primary psychoactive component of cannabis. Using data from 48 young adults (aged 19–25 years) reporting 1–4 days of cannabis use per week who completed a placebo-controlled human laboratory experiment, we tested the hypothesis that the rs2235048 polymorphism of ABCB1 would influence acute responses to smoked cannabis. C-allele carriers reported on average greater frequency of weekly cannabis use compared to the TT genotype carriers (TC/CC mean ± SEM = 2.74 ± 0.14, TT = 1.85 ± 0.24, p = 0.004). After smoking a single cannabis cigarette to their desired high, C-allele carriers had higher area-under-the-curve (AUC) of both THC metabolites (11-OH-THC TC/CC = 7.18 ± 9.64, TT = 3.28 ± 3.40, p = 0.05; THC-COOH TC/CC = 95.21 ± 116.12, TT = 45.92 ± 42.38, p = 0.043), and these results were impact by self-reported ethnicity. There were no significant differences in self-reported subjective drug effects except for a greater AUC of visual analogue scale rating of drug liking (TC/CC = 35,398.33 ± 37,233.72, TT = 15,895.56 ± 13,200.68, p = 0.017). Our preliminary findings suggest that further work in a larger sample should investigate whether human ABCB1 influences cannabis-related phenotypes and plays a role in the risk of developing a cannabis use disorder.

Structural insights into a novel collapsed confirmation of human ABCB1 in a lipid environment

Structural insights into a novel collapsed confirmation of human ABCB1 in a lipid environment

Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6.

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metal-free porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible “bulge” loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

ABCB1 restricts brain accumulation of the novel RORγ agonist cintirorgon, while OATP1A/1B and CYP3A limit its oral availability

Cintirorgon (LYC-55716), a first-in-class, small-molecule, oral selective RORγ agonist, has been developed as a new immuno-oncology drug for solid tumors. We here studied the functions of the ABCB1 and ABCG2 multidrug efflux transporters, the OATP1A/1B uptake transporters, and the drug-metabolizing CYP3A enzyme complex in cintirorgon pharmacokinetics using genetically modified mouse models. Cintirorgon was modestly transported by human ABCB1 and mouse Abcg2 in vitro. Upon oral administration at 40 mg/kg, net cintirorgon brain penetration was enhanced in Abcb1a/1b−/− (2.1-fold) and Abcb1a/1b;Abcg2−/− (2.7-fold) relative to wild-type mice. Deficiency of Oatp1a/1b led to a substantial (2.4-fold) increase in cintirorgon systemic exposure, with a corresponding (2.3-fold) decrease in hepatic distribution. However, these changes were not rescued in mice overexpressing human OATP1B1 or human OATP1B3 in liver, although this did partially reverse the altered cintirorgon glucuronide pharmacokinetics in Oatp1a/1b−/− mice. In Cyp3a−/− mice, the cintirorgon plasma AUC0-8h was 1.4-fold increased, and then decreased by 1.5-fold upon overexpression of transgenic human CYP3A4 in intestine and liver. Cintirorgon brain accumulation was thus markedly restricted by ABCB1. Mouse Oatp1a/1b mediated cintirorgon uptake into the liver, thus limiting its plasma exposure. Moreover, oral availability of cintirorgon was limited by CYP3A. These insights could help optimizing cintirorgon’s clinical application.

Role of the efflux transporters Abcb1 and Abcg2 in the brain distribution of olaparib in mice

Olaparib is a first-in-class poly (ADP-ribose) polymerase oral inhibitor used to treat various tumors. In this study, we clarified the roles of ABCB1/Abcb1 and ABCG2/Abcg2 transporters in restricting olaparib distribution to the brain. Olaparib was efficiently transported by human ABCG2, human ABCB1, and mouse Abcg2 in vitro. In the in vivo disposition study of olaparib using single or combination knockout mice, the systemic exposure of olaparib did not differ significantly between the strains over an 8-h period. However, the brain-to-plasma unbound concentration ratio of olaparib increased 5.6- and 8.1-fold in Abcb1a/1b and Abcb1a/1b;Abcg2 knockout mice, respectively, compared with wild-type mice. The Abcg2 single knockout mice exhibited a similar brain-to-plasma unbound concentration ratio to wild-type mice. Moreover, the brain distribution of olaparib could be modulated by the ABCB1/ABCG2 dual inhibitor elacridar to reach a similar degree of inhibition to Abcb1a/1b–/–. These findings suggest that olaparib is actively transported by both human and mouse ABCB1/Abcb1 and ABCG2/Abcg2; while Abcb1a/1b is a major determinant of olaparib brain penetration in mice, Abcg2 is likely to be a minor contributor. Concomitant treatment with temozolomide slightly increased the brain distribution of olaparib in mouse, but the clinical impact of the interaction was expected to be limited.

Annotation and Homology Modeling of the Multidrug Transport Protein P-Glycoprotein (ABCB1) of Equus Caballus

The function of ABC transporter proteins, such as ABCB1, is to transport substrates across cell membranes in many organs. ABCB1 can be found in multiple species, however, the sequence we annotated is derived from Equus caballus (horse). The objective of this research was to annotate and derive a structural homology model of the horse-derived P-glycoprotein. We classified the protein as a transmembrane ATP-binding cassette (ABC) protein ABCB1. Annotation of the sequence (which is not yet manually curated in NCBI) was carried out using various tools including BLAST, TMHMM, PFAM, HMM Logo, etc, and supports its designation as ABCB1 or P-glycoprotein of horse. The homology model was constructed using SWISS-MODEL based on the structure of human P-glycoprotein (PDB code: 6c0v). Three structures that share high sequence identity were chosen for analysis. Two homology models were prepared using the cryo-EM structures of human ABCB1 (PDB code: 6qex, % sequence identity) and human P-glycoprotein. The model using the structure of human P-glycoprotein as a template had the highest QMEAN score, Ramachandran favorability, and the most favorable Molprobity score. That model was evaluated using ProCheck and energy minimized using Chiron to give rise to the final model. Energy minimization resolved an unmodeled loop from Q625 to V691 and corrected other minor distortions. The clash ratio of the energy minimized model indicated that there are few clashes in the structure. Based on analysis of the structure validation parameters, the best homology model of equine P-glycoprotein was derived using the human P-glycoprotein as a template.