Current Drug Targets - Volume 12, Issue 5, 2011

The superfamily of ABC transporters comprises 49 proteins in humans. They all use ATP hydrolysis as an energy source and share common structural features. In particular, the presence of 2 membrane-spanning domains made of 6 transmembrane segments and of 2 ATP binding cassettes is required to make them functional. Yet, the function of many of these proteins still needs to be elucidated and their substrates remain unknown, but for those that are best characterized, it is clear that they play a critical role in the maintenance of cell homeostasis, by modulating the cell concentration in xenobiotics or in physiological substrates. In the present issue, we will examine the role of selected transporters in this superfamily, to illustrate their importance in drug disposition or in physiopathology. We will also discuss therapeutic perspectives related to modulation of their activity. The figure illustrates the transporters we will focus on, together with the indication of their function and of the pathology associated to their dysfunction. Xenobiotic transporters catalyse the efflux of drugs, or metabolites thereof, out of the cells. In contrast to many other transporters, they all display very broad substrate specificity, recognizing their substrates based on physico-chemical properties rather than on specific molecular determinants, hence their appellation of ‘Multidrug Transporters’. These transporters belong to 3 families, namely ABCB for P-glycoprotein, ABCC for MRPs (Multidrug-resistance Related Proteins), and ABCG for the half transporter BCRP (Breast Cancer Resistance Protein). In this issue, we will review their specific role in modulating drug pharmacokinetics [1] or in conferring resistance to anticancer chemotherapy [2], and the interest of inhibitors to reverse their effect. We will also discuss for 2 of them (ABCB1 and ABCC2) how genetic polymorphisms can lead to variations in their transport capacity among individuals and therefore justify therapeutic monitoring of blood levels, or patient's genotyping for drugs with narrow therapeutic index [3]. Other ABC transporters rather transport physiological substrates that can be highly variable in their chemical nature (Chlorure ions for ABCC7 or cholesterol for ABCA1, for example). The physiological role of these transporters has been generally identified when discovering the molecular mechanism of the disease their defect can cause, which may explain why for most of them, it remains largely unknown. In this issue, we will describe how the dysfunction in ABCA1 or ABCG1, ABCB11, ABCC6, ABCC7, or ABCD transporters can lead to atherosclerosis [4], cholestatic liver disease [5], Pseudoxanthoma elasticum [6], Cystic Fibrosis [7], or adrenoleukodystrophy [8], respectively. We will also examine the strategies that are developed to correct these defects in a therapeutic perspective.....

Nine proteins of the ABC superfamily (P-glycoprotein, 7 MRPs and BCRP) are involved in multidrug transport. Being localised at the surface of endothelial or epithelial cells, they expel drugs back to the external medium (if located at the apical side [P-glycoprotein, BCRP, MRP2, MRP4 in the kidney]) or to the blood (if located at the basolateral side [MRP1, MRP3, MRP4, MRP5]), modulating thereby their absorption, distribution, and elimination. In the CNS, most transporters are oriented to expel drugs to the blood. Transporters also cooperate with Phase I/Phase II metabolism enzymes by eliminating drug metabolites. Their major features are (i) their capacity to recognize drugs belonging to unrelated pharmacological classes, and (ii) their redundancy, a single molecule being possibly substrate for different transporters. This ensures an efficient protection of the body against invasion by xenobiotics. Competition for transport is now characterized as a mechanism of interaction between co-administered drugs, one molecule limiting the transport of the other, which potentially affects bioavailability, distribution, and/or elimination. Again, this mechanism reinforces drug interactions mediated by cytochrome P450 inhibition, as many substrates of P-glycoprotein and CYP3A4 are common. Induction of the expression of genes coding for MDR transporters is another mechanism of drug interaction, which could affect all drug substrates of the up-regulated transporter. Overexpression of MDR transporters confers resistance to anticancer agents and other therapies. All together, these data justify why studying drug active transport should be part of the evaluation of new drugs, as recently recommended by the FDA.

ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are membrane proteins that couple the energy derived from ATP hydrolysis to efflux many chemically diverse compounds across the plasma membrane, thereby playing a critical and important physiological role in protecting cells from xenobiotics. These transporters are also implicated in the development of multidrug resistance (MDR) in cancer cells that have been treated with chemotherapeutics. One approach to blocking the efflux capability of an ABC transporter in a cell or tissue is inhibiting the activity of the transporters with a modulator. Since ABC transporter modulators can be used in combination with chemotherapeutics to increase the effective intracellular concentration of anticancer drugs, the possible impact of modulators of ABC drug transporters is of great clinical interest. Another possible clinical use of modulators that has recently attracted attention is their ability to increase oral bioavailability or increase tissue penetration of drugs transported by the transporters. Several preclinical and clinical studies have been performed to evaluate the feasibility and the safety of this approach. The primary focus of this review is to discuss progress made in recent years in the identification and applicability of compounds that may serve as ABC transporter modulators and the possible role of these compounds in altering the pharmacokinetics and pharmacodynamics of therapeutic drugs used in the clinic.

The ATP-binding cassette (ABC) transporter superfamily comprises membrane proteins that translocate a variety of substrates across extra- and intra-cellular membranes, and act as efflux proteins. ABC transporters are characterised by the presence of genetic polymorphisms mainly represented by single nucleotide polymorphisms (SNPs), some of which having an impact on their activity. Besides physiological substances, drugs are also substrates of some ABC transporters, mainly ABCB1, ABCC1, ABCC2, ABCC3 and ABCG2. Identifying the impact of these polymorphisms on the pharmacokinetics (PK) of these drugs may have important clinical implications, certainly for those characterised by a narrow therapeutic index and significant inter- and intra-patient PK variability. This review focuses specifically on ABCB1 and ABCC2 and critically analyses important publications dealing with the influence of ABCB1 and/or ABCC2 polymorphisms on drug disposition in humans. For different reasons discussed in this paper, the effect of ABCB1 and/or ABCC2 polymorphisms on drug concentrations in blood is not always easy to interpret and to correlate with pharmacological effects. In contrast, intracellular or target tissue drug concentrations appear more directly influenced by these polymorphisms, as illustrated with intralymphocyte concentrations for immunosupressants and antiretrovirals or with cerebrospinal fluid (CSF) concentrations for antiepileptics and antidepressants. Further research on intracellular and/or target tissue drug concentrations are still needed to better characterise the PK-PG (pharmacogenetics) relationship involving ABC transporters.

Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in arteries. Plasma high density lipoprotein (HDL) levels bear a strong independent inverse relationship with atherosclerotic cardiovascular disease. One central antiatherogenic role of HDL is believed to be its ability to remove excessive peripheral cholesterol back to the liver for subsequent catabolism and excretion, a physiologic process termed reverse cholesterol transport (RCT). Cholesterol efflux from macrophage foam cells, the initial step of RCT is the most relevant step with respect to atherosclerosis. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play crucial roles in the efflux of cellular cholesterol to HDL and its apolipoproteins. Moreover, ABCA1 and ABCG1 affect cellular inflammatory cytokine secretion by modulating cholesterol content in the plasma membrane and within intracellular compartments. In humans, ABCA1 mutations can cause a severe HDL-deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. Disrupting Abca1 or Abcg1 in mice promotes accumulation of excessive cholesterol in macrophages, and physiological manipulation of ABCA1 expression affects atherogenesis. Here we review recent advances in the role of ABCA1 and ABCG1 in HDL metabolism, macrophage cholesterol efflux, inflammation, and atherogenesis. Next, we summarize the structure, expression, and regulation of ABCA1 and ABCG1. Finally, we give an update on the progress and pitfalls of therapeutic approaches that target ABCA1 and ABCG1 to stimulate the flux of lipids through the RCT pathway.

Bile formation is a key function of the liver and is driven by active secretion of bile salts and other organic compounds into the biliary tree. Bile salts represent the major organic constituent of bile. They are released with bile into the small intestine, where they are almost quantitatively reabsorbed and transported via the portal circulation back to the liver. In the liver, they are taken up into hepatocytes and secreted into bile. This cycling between the liver and the small intestine is called enterohepatic circulation of bile salts. Bile salts are secreted from hepatocytes into the bile by the bile salt export pump BSEP. This step constitutes the rate-limiting step of handling of bile salts in the liver and is the major driving force of the enterohepatic circulation of bile salts. Improper functioning of BSEP leads to an accumulation of bile salts within hepatocytes, where bile salts become cytotoxic. If persistent, accumulation of bile salts in hepatocytes will lead to liver disease. This review summarizes the essential concepts of bile formation and the current knowledge of mechanisms known to impair BSEP function. Finally, it sets the current therapeutic approaches for cholestatic liver disease into perspective to the pathophysiologic mechanisms of impaired BSEP function.

The ABCC6 gene encodes an organic anion transporter protein, ABCC6/MRP6. Mutations in the gene cause a rare, recessive genetic disease, pseudoxanthoma elasticum, while the loss of one ABCC6 allele is a genetic risk factor in coronary artery disease. We review here the information available on gene structure, evolution as well as the present knowledge on its transcriptional regulation. We give a detailed description of the characteristics of the protein, and analyze the relationship between the distributions of missense disease-causing mutations in the predicted threedimensional structure of the transporter, which suggests functional importance of the domain-domain interactions. Though neither the physiological function of the protein nor its role in the pathobiology of the diseases are known, a current hypothesis that ABCC6 may be involved in the efflux of one form of Vitamin K from the liver is discussed. Finally, we analyze potential strategies how the gene can be targeted on the transcriptional level to increase protein expression in order to compensate for reduced activity. In addition, pharmacologic correction of trafficking-defect mutants or suppression of stop codon mutations as potential future therapeutic interventions are also reviewed.

Several novel compounds recently appeared as promising leads to develop effective drugs against the basic defect in Cystic fibrosis (CF) and the first rationale therapies for CF relying on the understanding of the basic defect started to hit the clinical setting. Most of these efforts are focused on correcting the F508del mutation (occurring in ∼90% of CF patients) which causes misfolding of the CF transmembrane conductance regulator (CFTR) protein, the intracellular retention of such abnormal conformation by the endoplasmic reticulum quality control and premature degradation, thus precluding CFTR from reaching the cell membrane where it normally functions as a cAMP-stimulated Cl- channel. Here, several rationale therapeutic strategies are briefly reviewed, namely, mutation-specific (or “CFTR-repairing”) approaches (with a particular focus on the cellular defect associated with F508del-CFTR), manipulation of other ionic (non-CFTR) conductances and gene therapy. Still more innovative strategies, such as manipulation of the proteostasis network, displacement of molecular chaperones, targeting mutant CFTR by in silico small-molecule screens and systems biology approaches are also discussed.

Peroxisomes are involved in a variety of metabolic processes, including β-oxidation of fatty acids, especially very long chain fatty acids. Three peroxisomal ABC proteins belonging to subfamily D have been identified in mammalian peroxisomes that have an important role in fatty acid metabolism. ABCD1/ALDP and ABCD2/ALDRP are suggested to be involved in the transport of very long chain acyl-CoA, and ABCD3/PMP70 is involved in the transport of long chain acyl-CoA. ABCD1 is known to be responsible for X-linked adrenoleukodystrophy (X-ALD); an inborn error of peroxisomal β-oxidation of very long chain fatty acids. X-ALD is characterized biochemically by the accumulation of very long chain fatty acids in all tissues, including the brain white matter. Progressive demyelination of the central nervous system and adrenal dysfunction have been observed. The pharmacological up-regulation of peroxisomal β- oxidation of very long chain fatty acids and the suppression of fatty acid elongation are important aspects of an optimal therapeutic approach. Attractive targets for the treatment of X-ALD patients include the ABCD2 as well as elongase that is involved in the elongation of very long chain fatty acids. In addition, stabilization of mutant ABCD1 that has retained some of its function might be another approach, since most of the mutant ABCD1s with a missense mutation are degraded rapidly by proteasomes before or after targeting to peroxisomes. Protection of the central nervous system against oxidative damage is also important in order to delay the progress of disease. We summarize recent pharmaceutical studies and consider the potential for future X-ALD therapies.

Calcium is a ubiquitous signal molecule and critical to cell function. Maintaining calcium homeostasis is essential to life. Calcium homeostasis is mediated by a number of plasma membrane and intracellular calcium channels and transporters that are driven by stimuli that allow rapid alterations in release and uptake [1]. A large electrochemical gradient is created across the plasma membrane as a result of the differential in calcium concentration between the extracellular and cytosolic domains. Therefore changes in intracellular calcium can occur rapidly as a result of calcium influx into cells through calcium channels or transporters in the plasma membrane. Internal stores can also contribute to oscillations in intracellular calcium through receptor mediated release and uptake. Changes in intracellular calcium mediate alterations in cell function through activation of calcium-dependent kinases and regulatory proteins.` The manipulation of genes encoding calcium channels and transporters has provided insight into the critical role ion transport plays in physiology. Mutations in calcium channels cause a variety of disorders. Mice that are deficient in calcium channel pore forming subunits are often not viable as calcium channels are critical for muscle function. In skeletal and cardiac muscle the ryanodine receptor contributes to elevation of intracellular calcium that is required for calcium binding to contractile regulatory proteins and muscle contraction. Mice lacking the skeletal muscle ryanodine receptor (RyR1) are lethal at birth due to respiratory failure and mutations in the RyR1 protein can cause severe disorders such as malignant hyperthermia and central core disease in skeletal muscle [2]. Mutations in the cardiac RyR2 are associated with stress-induced catecholaminergic polymorphic ventricular tachycardia in the heart [3]. In the heart contraction is initiated by calcium influx through the L-type Ca2+ channel. The L-type Ca2+ channel is also critical to cardiac excitation as it is responsible for the plateau phase (phase 2) of the action potential [4]. The pore forming and ion conducting α subunit (Cav1.2) plays a critical role in development as Cav1.2-/- transgenic mice are lethal before 14.5 days postcoitum [5]. Conversely over-expression of the Cav1.2 subunit leads to hypertrophy [6] and increased expression of the auxiliary β subunit leads to alterations in channel activity reminiscent of heart failure [7]. Ca2+ influx into cells is also regulated by the Na+/Ca2+ exchanger that exchanges Na+ ions for Ca2+ ions across the plasma membrane. Several isoforms of the exchanger have been identified including a mitochondrial isoform that is responsible for the regulation of calcium release from the mitochondria [8]. Recent evidence suggests the exchanger contributes to ischemic damage in the heart [9]. A recently discovered group of non-selective plasma membrane cation channels that conduct calcium and are activated by temperature, osmolarity, mechanical stress and noxious stimuli are the transient receptor potential (TRP) channels. A number of families of the channels have been identified but the main subfamilies are canonical (TRPC), vanilloid (TRPV) and melastatinrelated (TRPM) channels [10]. They are expressed in a number of cell types and have been linked to chronic inflammatory diseases in humans [10]. The importance of calcium is highlighted by the fact that many naturally occurring mutations in calcium transporting proteins are known to underlie human disorders including childhood absence epilepsy, familial hemiplegic migraine, spinocerebellar ataxia type 6 (a severe movement disorder), hypokalemic periodic paralysis and X-linked congenital stationary night blindness [11]. In this Hot Topic the importance of the RyR in regulating intracellular calcium in skeletal and cardiac muscle is discussed using insights from biophysical studies performed in lipid bilayers [12]. The calcium-permeable Canonical, Melastatin and Ankyrin type TRP channels are reviewed including their roles in asthma and stroke [13] and recent studies implicating the Na+/Ca2+ exchanger in myocardial injury are presented [9]. Finally the role of calcium transporting proteins including the Ltype Ca2+ channel in regulation of mitochondrial function during oxidative stress is examined [14]. Understanding how calcium regulatory proteins contribute to the development of pathology has facilitated the development of therapy aimed at preventing disease. The most recent developments in therapy are discussed in each review article.

The ryanodine receptor (RyR) calcium release channel is an essential intracellular ion channel that is central to Ca2+ signaling and contraction in the heart and skeletal muscle. The rapid release of Ca2+ from the internal sarcoplasmic reticulum Ca2+ stores through the RyR during excitation-contraction coupling is facilitated by the unique arrangement of the surface and sarcoplasmic reticulum membrane systems. Debilitating and sometimes fatal skeletal and cardiomyopathies result from changes in RyR activity that disrupt normal Ca2+ signaling. Such changes can be caused by point mutations in many different regions of the RyR protein or acquired as a result of stress associated with exercise, heart failure, age or drugs. In general, both inherited and acquired changes include an increase in RyR channel activity. Because of its central function, the RyR is a potential therapeutic target for the inherited disorders and many of the acquired disorders. The RyR is currently used as a therapeutic target in malignant hyperthermia where dantrolene is effective and to relieve ventricular arrhythmia and with the use of JTV519 and flecainide. These drugs show that the RyR is a valid therapeutic target, but have side effects that prevent their chronic use. Thus there is an urgent need for the development of skeletal and cardiac specific drugs to treat these diverse muscle disorders. In this review, we discuss the mutations that cause skeletal myopathies and cardiac arrhythmias and how these mutations pinpoint residues within the RyR protein that are functionally significant and might be developed as targets for therapeutic drugs.

Mammals contain 28 genes encoding Transient Receptor Potential (TRP) proteins. The proteins assemble into cationic channels, often with calcium permeability. Important roles in physiology and disease have emerged and so there is interest in whether the channels might be suitable therapeutic drug targets. Here we review selected members of three subfamilies of mammalian TRP channel (TRPC5, TRPM2 and TRPA1) that show relevance to sensing of adversity by cells and biological systems. Summarized are the cellular and tissue distributions, general properties, endogenous modulators, protein partners, cellular and tissue functions, therapeutic potential, and pharmacology. TRPC5 is stimulated by receptor agonists and other factors that include lipids and metal ions; it heteromultimerises with other TRPC proteins and is involved in cell movement and anxiety control. TRPM2 is activated by hydrogen peroxide; it is implicated in stress-related inflammatory, vascular and neurodegenerative conditions. TRPA1 is stimulated by a wide range of irritants including mustard oil and nicotine but also, controversially, noxious cold and mechanical pressure; it is implicated in pain and inflammatory responses, including in the airways. The channels have in common that they show polymodal stimulation, have activities that are enhanced by redox factors, are permeable to calcium, and are facilitated by elevations of intracellular calcium. Developing inhibitors of the channels could lead to new agents for a variety of conditions: for example, suppressing unwanted tissue remodeling, inflammation, pain and anxiety, and addressing problems relating to asthma and stroke.

The Na+/Ca2+ exchanger (NCX) is the main Ca2+ extrusion mechanism of the cardiac myocyte and thus is crucial for maintaining Ca2+ homeostasis. It is involved in the regulation of several parameters of cardiac excitation contraction coupling, such as cytosolic Ca2+ concentration, repolarization and contractility. Increased NCX activity has been identified as a mechanism promoting heart failure, cardiac ischemia and arrhythmia. Transgenic mice as well as pharmacological interventions have been used to support the idea of using NCX inhibition as a future pharmacological strategy to treat cardiovascular disease.

Calcium is a key determinant of cardiac excitation, contraction and relaxation. Cardiac excitation and contraction are powered by ATP that is synthesized within mitochondria via a calcium-dependent process known as oxidative phosphorylation. During this process oxygen molecules within the mitochondria are converted to superoxide. Under physiological conditions, low levels of ROS are required to maintain normal cellular function. This is achieved as a result of a balance between ROS formation and amelioration by antioxidants. Uninhibited increases in ROS production lead to oxidative stress. Large increases in ROS are associated with damage to mitochondria, DNA, proteins and lipids. In the heart this ultimately leads to apoptosis and loss of myocytes. However sub-lethal increases in ROS can activate hypertrophic signaling kinases and transcription factors including NFAT, CaMK and serine-threonine and tyrosine kinases. Calcium is also an important signaling molecule and a mediator of hypertrophic signaling pathways. ROS and calcium appear to participate as partners in pathological remodeling but their interaction and early mechanisms associated with the development of cardiac hypertrophy are poorly understood. An increase in cytoplasmic calcium can potentiate cellular oxidative stress via effects on mitochondrial metabolism. In addition oxidative stress can regulate the function of calcium channels and transporters. We discuss the evidence for calcium transporting proteins and the mitochondria in oxidative stress responses and propose sites to target in the prevention of cardiac hypertrophy.