ABCG2/BCRP: variants, transporter interaction profile of substrates and inhibitors.

INTRODUCTION: ABCG2 has a broad substrate specificity and is one of the most important efflux proteins modulating pharmacokinetics of drugs, nutrients and toxicokinetics of toxicants. ABCG2 is an important player in transporter-mediated drug-drug interactions (tDDI). Areas covered: The aims of the review are i) to cover transporter interaction profile of substrates and inhibitors that can be utilized to test interaction of drug candidates with ABCG2, ii) to highlight main characteristics of in vitro testing and iii) to describe the structural basis of the broad substrate specificity of the protein. Preclinical data utilizing Abcg2/Bcrp1 knockouts and clinical studies showing effect of ABCG2 c.421C>A polymorphism on pharmacokinetics of drugs have provided evidence for a broad array of drug substrates and support drug - ABCG2 interaction testing. A consensus on using rosuvastatin and sulfasalazine as intestinal substrates for clinical studies is in the formation. Other substrates relevant to the therapeutic area can be considered. Monolayer efflux assays and vesicular transport assays have been extensively utilized in vitro. Expert opinion: Clinical substrates display complex pharmacokinetics due to broad interaction profiles with multiple transporters and metabolic enzymes. Substrate-dependent inhibition has been observed for several inhibitors. Harmonization of in vitro and in vivo testing makes sense. However, rosuvastatin and sulfasalazine are not efficiently transported in either MDCKII or LLC-PK1-based monolayers. Caco-2 monolayer assays and vesicular transport assays are potential alternatives.

Inhibitor selectivity of CNTs and ENTs.

The concentrative nucleoside transporters (CNT; solute carrier family 28 (SLC28)) and the equilibrative nucleoside transporters (ENT; solute carrier family 29 (SLC29)) are important therapeutic targets but may also mediate toxicity or adverse events. To explore the relative role of the base and the monosaccharide moiety in inhibitor selectivity we selected compounds that either harbor an arabinose moiety or a cytosine moiety, as these groups had several commercially available drug members. The screening data showed that more compounds harboring a cytosine moiety displayed potent interactions with the CNTs than compounds harboring the arabinose moiety. In contrast, ENTs showed a preference for compounds with an arabinose moiety. The correlation between CNT1 and CNT3 was good as five of six compounds displayed IC50 values within the threefold threshold and one displayed a borderline 4-fold difference. For CNT1 and CNT2 as well as for CNT2 and CNT3 only two of six IC50 values correlated and one displayed a borderline 4-fold difference. Interestingly, of the six compounds that potently interacted with both ENT1 and ENT2 only nelarabine displayed selectivity. Our data show differences between inhibitor selectivities of CNTs and ENTs as well as differences within the CNT family members.

Species specificity profiling of rat and human organic cation/carnitine transporter Slc22a5/SLC22A5 (Octn2/OCTN2).

The purpose of this study was to characterize the uptake of carnitine, the physiological substrate, and the uptake of 3-(2,2,2-trimethylhydrazinium)propionate, a consensus substrate by rat Octn2 and human OCTN2 transporters as well as to characterize drug-mediated inhibition of l-carnitine uptake by the rat and human orthologs overexpressed in CHO-K1 cells. l-carnitine and 3-(2,2,2-trimethylhydrazinium)propionate were found to be a lower affinity substrate for rat Octn2 (KM = 32.66 ± 5.11 µM and 23.62 ± 4.99 µM respectively) than for human OCTN2 (KM = 3.08 ± 0.74 µM and 7.98 ± 0.63 µM). The intrinsic clearance (CLint) value for carnitine was higher for the human than for the rat transporter (22.82 ± 5.57 ml/min*mg vs 4.008 ± 0.675 ml/min*mg). For 3-(2,2,2-trimethylhydrazinium)propionate, in contrast, the CLint value for rat Octn2 was higher than for human OCTN2 (323.9 ± 72.8 ml/min*mg vs 65.11 ± 5.33 ml/min*mg). Furthermore, many pharmacologically important drugs were shown to affect l-carnitine transport by Octn2/OCTN2. The correlation between the IC50 datasets for the rat and human transporter resulted in an r value of 0.47 (p > 0.05). However, the greatest difference was less than seven-fold and 13 of 15 compounds yielded a difference less than 3-fold. Thus, the transporters from these two species showed an overlapping but somewhat different substrate and inhibitor specificity.

Investigating ABCB1-Mediated Drug-Drug Interactions: Considerations for In vitro and In vivo Assay Design.

BACKGROUND: ABCB1 is a key ABC efflux transporter modulating the pharmacokinetics of a large percentage of drugs. ABCB1 is also a site of transporter mediated drug-drug interactions (tDDI). It is the transporter most frequently tested for tDDIs both in vitro and in the clinic. OBJECTIVE: Understanding the limitations of various in vitro and in vivo models, therefore, is crucial. In this review we cover regulatory aspects of ABCB1 mediated drug transport as well as inhibition and the available models and methods. We also discuss protein structure and mechanistic aspects of transport as ABCB1 displays complex kinetics that involves multiple binding sites, potentiation of transport and probe-dependent IC50 values. RESULTS: Permeability of drugs both passive and mediated by transporters is also a covariate that modulates apparent kinetic values. Levels of expression as well as lipid composition of the expression system used in in vitro studies have also been acknowledged as determinates of transporter activity. ABCB1-mediated clinical tDDIs are often complex as multiple transporters as well as metabolic enzymes may play a role. This complexity often masks the role of ABCB1 in tDDIs. CONCLUSION: It is expected that utilization of in vitro data will further increase with the refinement of simulations. It is also anticipated that transporter humanized preclinical models have a significant impact and utility.

The multidrug resistance pump ABCB1 is a substrate for the ubiquitin ligase NEDD4-1.

The ATP Binding Cassette transporter ABCB1 can export the neurotoxic peptide ß-amyloid from endothelial cells that line the blood-brain barrier (BBB). This has the potential to lower cerebral levels of ß-amyloid, but ABCB1 expression in the BBB appears to be progressively reduced in patients with Alzheimer's disease. The surface density of many membrane proteins is regulated by ubiquitination catalyzed by ubiquitin E3 ligases. In brain capillaries of mice challenged with ß-amyloid ex vivo, we show that the level of the ubiquitin ligase Nedd4 increases concomitant with reduction in Abcb1. In vitro we show that human ABCB1 is a substrate for human NEDD4-1 ligase. Recombinant ABCB1 was purified from Sf21 insect cells and incubated with recombinant NEDD4-1 purified from Escherichia coli. The treated ABCB1 had reduced mobility on SDS-PAGE, and mass spectrometry identified eight lysine residues, K271, K272, K575, K685, K877, K885, K887 and K1062 that were ubiquitinated by NEDD4-1. Molecular modelling showed that all of the residues are exposed on the surface of the intracellular domains of ABCB1. K877, K885 and K887 in particular, are located in the intracellular loop of transmembrane helix 10 (TMH10) in close proximity, in the tertiary fold, to a putative NEDD4-1 binding site in the intracellular helix extending from TMH12 (PxY motif, residues 996-998). Transient expression of NEDD4-1 in HEK293 Flp-In cells stably expressing ABCB1 was shown to reduce the surface density of the transporter. Together, the data identify this ubiquitin ligase as a potential target for intervention in the pathophysiology of Alzheimer's disease.

The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle.

For a primary active pump, such as the human ATP-binding-cassette (ABC) transporter ABCB1, coupling of drug-binding by the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism, but is poorly understood at the biochemical level. Structure data suggest that signals are transduced through intracellular loops of the TMDs that slot into grooves on the NBDs. At the base of these grooves is the Q loop. We therefore mutated the eponymous glutamine in one or both NBD Q loops and measured the effect on conformation and function by using a conformation-sensitive antibody (UIC2) and a fluorescent drug (Bodipy-verapamil), respectively. We showed that the double mutant is trapped in the inward-open state, which binds the drug, but cannot couple to the ATPase cycle. Our data also describe marked redundancy within the transport mechanism, because single-Q-loop mutants are functional for Bodipy-verapamil transport. This result allowed us to elucidate transduction pathways from twin drug-binding cavities to the Q loops using point mutations to favor one cavity over the other. Together, the data show that the Q loop is the central flexion point where the aspect of the drug-binding cavities is coupled to the ATP catalytic cycle.

Structure and function of BCRP, a broad specificity transporter of xenobiotics and endobiotics.

The discovery and characterization of breast cancer resistance protein (BCRP) as an efflux transporter conferring multidrug resistance has set off a remarkable trajectory in the understanding of its role in physiology and disease. While the relevance in drug resistance and general pharmacokinetic properties quickly became apparent, the lack of a characteristic phenotype in genetically impaired animals and humans cast doubt on the physiological importance of this ATP-binding cassette family member, similarly to fellow multidrug transporters, despite well-known endogenous substrates. Later, high-performance genetic analyses and fine resolution tissue expression data forayed into unexpected territories concerning BCRP relevance, and ultimately, the rise of quantitative proteomics allows putting observed interactions into absolute frameworks for modeling and insight into interindividual and species differences. This overview summarizes existing knowledge on the BCRP transporter on molecular, tissue and system level, both in physiology and disease, and describes a selection of experimental procedures that are the most widely applied for the identification and characterization of substrate and inhibitor-type interactions.

Chlorothiazide is a substrate for the human uptake transporters OAT1 and OAT3.

The thiazide diuretic chlorothiazide is poorly metabolized, and is predominantly excreted via the kidneys. We have previously shown that chlorothiazide is transported by ATP-binding cassette transporter G2, suggesting a potential role for this transporter in apical efflux of chlorothiazide in the kidney. However, because of the poor passive permeability of the drug, it is likely that uptake transporters on the basolateral membrane are also involved to facilitate vectorial transport in the renal proximal tubule. Two suggested candidate transporters for this role are the human organic anion transporters, OAT1 and OAT3. By using mammalian cells stably expressing these transporters, we have demonstrated OAT1- and OAT3-dependent uptake of chlorothiazide with Michaelis constant values of 14.5 and 37.6 µM, respectively. Furthermore, we have found that probenecid, furosemide, and diclofenac inhibit chlorothiazide transport by OAT1 and OAT3, of which the probenecide-mediated inhibition may be of clinical importance. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1683-1687, 2013.

Canalicular ABC transporters and liver disease.

Bile is a complex mixture that includes bile salts, the membrane phospholipid phosphatidylcholine (PC), cholesterol and various endobiotic and xenobiotic toxins, each of which is secreted across the canalicular membrane of the hepatocyte by different ATP-binding cassette (ABC) transporters. The bile salts are essential for the emulsification of dietary fat and lipophilic vitamins. They are synthesized from cholesterol in the hepatocyte and their secretion by the bile salt export pump (BSEP or ABCB11) drives bile flow and is the starting point for the enterohepatic cycle. The detergent nature of bile salts that is key to their physiological role also means that they are inherently cytotoxic, and failure to secrete bile (intraheptic cholestasis) can precipitate severe liver disease and mortality. Such progressive familial intrahepatic cholestasis (PFIC) comes in three types of autosomal recessive disease. PFIC2 is caused by mutation to ABCB11. PFIC3 is caused by mutation of a closely related ABC transporter, ABCB4, which flops PC into the outerleaflet of the canalicular membrane. The flopped PC is extracted by the bile salts in the canaliculus to form a mixed micelle that reduces bile salt detergent activity. The third protein that is essential for bile flow from the hepatocyte is a member of a different class of transporter protein, a P-type ATPase, ATP8B1. Mutation of ATP8B1 causes PFIC1, but ATP8B1 does not transport a component of bile into the canaliculus. Data from different laboratories, published this year, suggests two different roles for ATP8B1 in the hepatocyte: a lipid flippase, that counterbalances the deleterious effects of ABCB4 on barrier function of the canalicular membrane; and an anchor of the actin cytoskeleton necessary to form the microvilli of the brush border. These latest discoveries are described, along with a spectrum of cholestatic disorders whose aetiologies lie in these and other transporters of the canalicular membrane.

Structure of ABC transporters.

ABC (ATP-binding cassette) transporters are primary active membrane proteins that translocate solutes (allocrites) across lipid bilayers. The prototypical ABC transporter consists of four domains: two cytoplasmic NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains). The NBDs, whose primary sequence is highly conserved throughout the superfamily, bind and hydrolyse ATP to power the transport cycle. The TMDs, whose primary sequence and protein fold can be quite disparate, form the translocation pathway across the membrane and generally (but not always) determine allocrite specificity. Structure determination of ABC proteins initially took advantage of the relative ease of expression and crystallization of the hydrophilic bacterial NBDs in isolation from the transporter complex, and revealed detailed information on the structural fold of these domains, the amino acids involved in the binding and hydrolysis of nucleotide, and the head-to-tail arrangement of the NBD-NBD dimer interface. More recently, several intact transporters have been crystallized and three types have, so far, been characterized: type I and II ABC importers, and ABC exporters. All three are present in prokaryotes, but only the ABC exporters appear to be present in eukaryotes. Their structural determination has provided insight into the mechanisms of energy and signal transduction between the NBDs and TMDs (i.e. between the ATP- and allocrite-binding sites) and, for some, the nature of the allocrite-binding site(s) within the TMDs. In this chapter, we focus primarily on the ABC exporters and describe the structural, biochemical and biophysical evidence for and against the controversial bellows-like mechanism proposed for allocrite efflux.

Substrate- and species-dependent inhibition of P-glycoprotein-mediated transport: implications for predicting in vivo drug interactions.

P-glycoprotein (P-gp)-based drug interactions are a major concern in the clinic and in preclinical drug development, especially with respect to the intestinal absorption of drugs and distribution of drugs across the blood-brain barrier. Thus, there is significant interest in developing in vitro (e.g., cell culture) and in vivo models (e.g., rodents) to predict such interactions. In order to generate accurate predictions from these models, however, an understanding of the magnitude of substrate- and species-dependent differences in P-gp inhibition is required. We have used a sensitive flow cytometry assay to measure the ability of various drugs to inhibit the initial rate of accumulation of two fluorescent drug analogs (probe substrates), 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s -indacene (BODIPY)-verapamil and BODIPY-prazosin, into Lewis lung carcinoma-porcine kidney 1 (LLC-PK1) cells expressing human or rat P-gp. The inhibition of P-gp-mediated efflux of these two fluorescent substrates by several drugs, including quinidine and itraconazole, was found to be substrate- and/or species-dependent. These data suggest that to provide accurate prediction of clinically significant P-gp drug interactions, multiple P-gp substrates will need to be used in both in vitro and in vivo (including human) drug interaction studies. In addition, extrapolation of P-gp-based drug interaction in rodents to humans must be conducted with caution.

Stereoselective effects of 4-hydroxynonenal in cultured mouse hepatocytes.

4-Hydroxynonenal (HNE) is produced from arachidonic acid or linoleic acid during oxidative stress. Although HNE is formed in tissues as a racemate, enantiospecific HNE effects have not been widely documented, nor considered. Therefore, a panel of cellular responses was compared after treatment with (R)-HNE, (S)-HNE, or racemic HNE. The phosphorylation status of Jun kinase (JNK) or Akt increased 28-fold or 2-3-fold, respectively, after treatment with 100 µM (S)-HNE and racemic HNE compared to (R)-HNE. In contrast, the increase in phosphorylation of MAPK was greatest for (R)-HNE. Caspase-3-dependent cleavage of the glutamate cysteine ligase (GCL) catalytic subunit and focal adhesion kinase (FAK) were greater in cells treated with (S)-HNE at 48 h. (S)-HNE also caused a greater number of subG1 nuclei, a hallmark of apoptosis, at 30 h after treatment. Together, the results demonstrate different dose- and time-dependent responses to (R)-HNE and (S)-HNE. The results further suggest that HNE enantiomers could differentially contribute to the progression of different diseases or contribute by different mechanisms.

Evidence for a Sav1866-like architecture for the human multidrug transporter P-glycoprotein.

The recently reported structures of the bacterial multidrug exporter Sav1866 suggest a domain architecture in which both nucleotide-binding domains (NBDs) of this ATP binding cassette (ABC) transporter contact both transmembrane domains (TMDs). Such a domain arrangement is particularly unexpected because it is not found in the structures of three solute importers BtuCD, HI1470/1, and ModBC from the same protein family. There is also no precedent for such an arrangement from biochemical studies with any ABC transporter. Sav1866 is homologous with the clinically relevant human P-glycoprotein (ABCB1). If the structure proposed for Sav1866 is physiologically relevant, the long intracellular loops of P-glycoprotein TMD2 should contact NBD1. We have tested this by using cysteine mutagenesis and chemical cross-linking to verify proximal relationships of the introduced sulfhydryls across the proposed interdomain interface. We report the first biochemical evidence in support of the domain arrangement proposed for the multidrug resistance class of ABC transporters. With a domain arrangement distinctly different from the three solute importers it seems likely that the TMDs of ABC importers and exporters have evolved different mechanisms to couple to common conformational changes at conserved NBDs.

Myf5 expression in satellite cells and spindles in adult muscle is controlled by separate genetic elements.

The myogenic regulatory factor Myf5 is integral to the initiation and control of skeletal muscle formation. In adult muscle, Myf5 is expressed in satellite cells, stem cells of mature muscle, but not in the myonuclei that sustain the myofibre. Using the Myf5(nlacZ/+) mouse, we now show that Myf5 is also constitutively expressed in muscle spindles-stretch-sensitive mechanoreceptors, while muscle denervation induces extensive reactivation of the Myf5 gene in myonuclei. To identify the elements involved in the regulation of Myf5 in adult muscle, we analysed reporter gene expression in a transgenic bacterial artificial chromosome (BAC) deletion series of the Mrf4/Myf5 locus. A BAC carrying 140 kb upstream of the Myf5 transcription start site was sufficient to drive all aspects of Myf5 expression in adult muscle. In contrast, BACs carrying 88 and 59 kb upstream were unable to drive consistent expression in satellite cells, although expression in muscle spindles and reactivation of the locus in myonuclei were retained. Therefore, as during development, multiple enhancers are required to generate the full expression pattern of Myf5 in the adult. Together, these observations show that elements controlling adult Myf5 expression are genetically separable and possibly distinct from those that control Myf5 during development. These studies are a first step towards identifying cognate transcription factors involved in muscle stem cell regulation.