Peter Chiba

The bile salt export pump (BSEP/ABCB11) is responsible for the transport of bile salts from hepatocytes into bile canaliculi. Malfunction of this transporter results in progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2) and intrahepatic cholestasis of pregnancy (ICP). Over the past few years, several small molecular weight compounds have been identified, which hold the potential to treat these genetic diseases (chaperones and potentiators). As the treatment response is mutation-specific, genetic analysis of the patients and their families is required. Furthermore, some of the mutations are refractory to therapy, with the only remaining treatment option being liver transplantation. In this review, we will focus on the molecular structure of ABCB11, reported mutations involved in cholestasis and current treatment options for inherited BSEP deficiencies.

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site. ABC transporters are one of the largest membrane protein superfamilies, present in all organisms from archaea to humans. They transport a wide range of molecules including amino acids, sugars, vitamins, nucleotides, peptides, lipids, metabolites, antibiotics, and xenobiotics. ABC transporters energize substrate transport by hydrolyzing ATP in two symmetrically arranged nucleotide binding sites (NBSs). The human multidrug resistance transporter ABCB1 has two active NBSs, and it is generally believed that integrity and cooperation of both sites are needed for transport. Several human ABC transporters, such as the bile salt transporter ABCB11, have one degenerate NBS, which has significantly reduced ATPase activity. Interestingly, unilateral mutations affecting one of the two NBSs completely abolish the function of symmetrical ABC transporters. Here we engineered an ABCB1 variant with a degenerate, ABCB11-like NBS1, which can nevertheless transport substrates. Our results indicate that ABCB1 can mediate active transport with a single active site, questioning the validity of models assuming strictly alternating catalysis.

Human P‐glycoprotein (P‐gp) is a multispecific drug‐efflux transporter, which plays an important role in drug resistance and drug disposition. Recent cryo‐electron microscopy structures confirmed its rotationally symmetric architecture, which allows dual interaction with ATP and substrates. We here report the existence of two distinct, symmetry‐related outer gates. Experiments were aided by availability of the X‐ray structure of a homodimeric eukaryotic homolog of P‐gp from red alga (CmABCB1), which defined the role of an apical tyrosine residue (Y358) in outer gate formation. We mutated analogous tyrosine residues in each half of the human full‐length transporter (Y310, Y953) to alanine. These mutants were introduced in engineered transporters which bind rhodamine 123 in one of two symmetry‐related binding modes only. Outer gate dysfunction was detected by a loss of active transport characteristics, while these mutants retained the ability for outward downhill transport. Our data demonstrate that symmetric tyrosine residues Y310 and Y953 are involved in formation of two distinct symmetry‐related outer gates, which operate contingent on the rhodamine 123 binding mode. Hence, the rotationally symmetric architecture of P‐gp, which determines duality in ATP binding and rhodamine 123 interaction, also forms the basis for the existence of two independently operating outer gates.

P-glycoprotein (ABCB1) is an important component of barrier tissues that extrudes a wide range of chemically unrelated compounds. ABCB1 consists of two transmembrane domains forming the substrate binding and translocation domain, and of two cytoplasmic nucleotide binding domains (NBDs) that provide the energy by binding and hydrolyzing ATP. We analyzed the mechanistic and energetic properties of the NBD dimer via molecular dynamics simulations. We find that MgATP stabilizes the NBD dimer through strong attractive forces by serving as an interaction hub. The irreversible ATP hydrolysis step converts the chemical energy stored in the phosphate bonds of ATP into potential energy. Following ATP hydrolysis, interactions between the NBDs and the ATP hydrolysis products MgADP + P_i remain strong, mainly because Mg²⁺ forms stabilizing interactions with ADP and P_i. Despite these stabilizing interactions MgADP + P_i are unable to hold the dimer together, which becomes separated by avid interactions of MgADP + P_i with water. ATP binding to the open NBDs and ATP hydrolysis in the closed NBD dimer represent two steps of energy input, each leading to the formation of a high energy state. Relaxation from these high energy states occurs through conformational changes that push ABCB1 through the transport cycle.

Background: Psoralen (PSO), a major active component of Psoralea corylifolia, has been shown to overcome multidrug resistance in cancer. A drug carrier comprising a lipid-monolayer shell and a biodegradable polymer core for sustained delivery and improved efficacy of drug have exhibited great potential in efficient treatment of cancers. Methods: The PSO-loaded lipid polymer hybrid nanoparticles were prepared and characterized. In vitro cytotoxicity assay, cellular uptake, cell cycle analysis, detection of ROS level and mitochondrial membrane potential (ΔΨm) and western blot were performed. Results: The P-LPNs enhanced the cytotoxicity of doxorubicin (DOX) 17-fold compared to free DOX in multidrug resistant HepG2/ADR cells. Moreover, P-LPNs displayed pro-apoptotic activity, increased levels of ROS and depolarization of ΔΨm. In addition, there were no significant effects on cellular uptake of DOX, cell cycle arrest, or the expression of P-glycoprotein. Mechanistic studies suggested that P-LPNs enhanced DOX cytotoxicity by increased release of cytochrome c and enhanced caspase3 cleavage, causing apoptosis in HepG2/ADR cells. Conclusion: The lipid-polymer hybrid nanoparticles can be considered a powerful and promising drug delivery system for effective cancer chemotherapy.

Breast cancer is the leading cause of death from cancer in women worldwide. Chemotherapy represents one key treatment modality for the clinical management of breast cancer. However, ATP-binding cassette (ABC) transporter mediated active efflux of structurally and mechanistically different cytotoxic compounds results in multidrug resistance (MDR), eventually leading to failure of chemotherapy. The concept of combining anti-cancer drugs and transport inhibitors has been advocated as a concept for re-sensitization of resistant breast cancer to chemotherapy. Whether inhibition of efflux transporters may have the potential to improve therapeutic outcomes is discussed controversially. In this review we discuss challenges in the treatment of breast cancer, the role of MDR in development and the potential of natural products to overcome MDR.

Amphetamine abuse is a major public health concern for which there is currently no effective treatment. To develop effective treatments, the mechanisms by which amphetamine produces its abuse-related effects need to be fully understood. It is well known that amphetamine exerts its actions by targeting high-affinity transporters for monoamines, in particular the cocaine-sensitive dopamine transporter. Organic cation transporter 3 (OCT3) has recently been found to play an important role in regulating monoamine signaling. However, whether OCT3 contributes to the actions of amphetamine is unclear. We found that OCT3 is expressed in dopamine neurons. Then, applying a combination of in vivo, ex vivo, and in vitro approaches, we revealed that a substantial component of amphetamine’s actions is OCT3-dependent and cocaine insensitive. Our findings support OCT3 as a new player in the actions of amphetamine and encourage investigation of this transporter as a potential new target for the treatment of psychostimulant abuse.

The bile salt export pump (BSEP/ABCB11) transports bile salts from hepatocytes into bile canaliculi. Its malfunction is associated with severe liver disease. One reason for functional impairment of BSEP is systemic administration of drugs, which as a side effect inhibit the transporter. Therefore, drug candidates are routinely screened for potential interaction with this transporter. Hence, understanding the functional biology of BSEP is of key importance. In this study, we engineered the transporter to dissect interdomain communication paths. We introduced mutations in noncanonical and in conserved residues of either of the two nucleotide binding domains and determined the effect on BSEP basal and substrate-stimulated ATPase activity as well as on taurocholate transport. Replacement of the noncanonical methionine residue M584 (Walker B sequence of nucleotide binding site 1) by glutamate imparted hydrolysis competency to this site. Importantly, this mutation was able to sustain 15% of wild-type transport activity, when the catalytic glutamate of the canonical nucleotide binding site 2 was mutated to glutamine. Kinetic modeling of experimental results for the ensuing M584E/E1244Q mutant suggests that a transfer of hydrolytic capacity from the canonical to the noncanonical nucleotide binding site results in loss of active and adoption of facilitative characteristics. This facilitative transport is ATP-gated. To the best of our knowledge, this result is unprecedented in ATP-binding cassette proteins with one noncanonical nucleotide binding site. Our study promotes an understanding of the domain interplay in BSEP as a basis for exploration of drug interactions with this transporter.

Point mutations of ATP‐binding cassette (ABC) proteins are a common cause of human diseases. Available crystal structures indicate a similarity in the architecture of several members of this protein family. Their molecular architecture makes these proteins vulnerable to mutation, when critical structural elements are affected. The latter preferentially involve the two transmembrane domain (TMD)/nucleotide‐binding domain (NBD) interfaces (transmission interfaces), formation of which requires engagement of coupling helices of intracellular loops with NBDs. Both, formation of the active sites and engagement of the coupling helices, are contingent on correct positioning of ICLs 2 and 4 and thus an important prerequisite for proper folding. Here, we show that active site compounds are capable of rescuing P‐glycoprotein (P‐gp) mutants ∆Y490 and ∆Y1133 in a concentration‐dependent manner. These trafficking deficient mutations are located at the transmission interface in pseudosymmetric position to each other. In addition, the ability of propafenone analogs to correct folding correlates with their ability to inhibit transport of model substrates. This finding indicates that folding correction and transport inhibition by propafenone analogs are brought about by binding to the active sites. Furthermore, this study demonstrates an asymmetry in folding correction with cis‐flupentixol, which reflects the asymmetric binding properties of this modulator to P‐gp. Our results suggest a mechanistic model for corrector action in a model ABC transporter based on insights into the molecular architecture of these transporters.