AgiAl Nuclear Research Publishing House Vol. 2 (2015), Article ID 101178, 20 pages doi:10.11131/2015/101178 http://www.agialpress.com/

Review Article

Nuclear Receptors in Drug Metabolism, Drug Response and Drug Interactions

Chandra Prakash1,2, Baltazar Zuniga1,3, Chung Seog Song1, Shoulei Jiang1, Jodie Cropper1, Sulgi Park1, and Bandana Chatterjee1,4

1Department of Molecular Medicine/Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, Texas Research Park, 15355 Lambda Drive, San Antonio, Texas 78245, USA 2William Carey University College of Osteopathic Medicine, 498 Tucsan Ave, Hattiesburg, Mississipi 39401, USA 3University of Texas at Austin, 2100 Comal Street, Austin, Texas 78712, USA 4South Texas Veterans Health Care System, Audie L Murphy VA Hospital, 7400 Merton Minter Boulevard, San Antonio, Texas 78229, USA Corresponding Author: Bandana Chatterjee; email: [email protected]

Received 26 July 2015; Accepted 28 November 2015

Editor: William Baldwin

Copyright © 2015 Chandra Prakash et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract. Orally delivered small-molecule therapeutics are metabolized in the liver and intestine by phase I and phase II drug- metabolizing (DMEs), and transport proteins coordinate drug influx (phase 0) and drug/drug-metabolite efflux (phase III). involved in drug metabolism and disposition are induced by xenobiotic-activated nuclear receptors (NRs), i.e. PXR () and CAR (constitutive androstane receptor), and by the 1훼, 25-dihydroxy vitamin D3-activated (VDR), due to transactivation of xenobiotic-response elements (XREs) present in phase 0-III genes. Additional NRs, like HNF4-훼, FXR, LXR-훼 play important roles in drug metabolism in certain settings, such as in relation to and metabolism. The phase I enzymes CYP3A4/A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, CYP1A2, CYP2C8, CYP2A6, CYP2J2, and CYP2E1 metabolize >90% of all prescription drugs, and phase II conjugation of hydrophilic functional groups (with/without phase I modification) facilitates drug clearance. The conjugation step is mediated by broad-specificity like UGTs, SULTs, GSTs. This review delves into our current understanding of PXR/CAR/VDR-mediated regulation of DME and transporter expression, as well as effects of single nucleotide polymorphism (SNP) and epigenome (specified by promoter methylation, histone modification, microRNAs, long non coding RNAs) on the expression of PXR/CAR/VDR and phase 0-III mediators, and their impacts on variable drug response. Therapeutic agents that target epigenetic regulation and the molecular basis and consequences (overdosing, underdosing, or beneficial outcome) of drug-drug/drug-food/drug-herb interactions are also discussed. Precision medicine requires understanding of a drug’s impact on DME and transporter activity and their NR-regulated expression in order to achieve optimal drug efficacy without adverse drug reactions. In future drug screening, new tools such as humanized mouse models and microfluidic organs-on-chips, which mimic the physiology of a multicellular environment, will likely replace the current cell-based workflow.

Keywords: Nuclear receptors, PXR, CAR, Xenobiotic-response element, induction, Phase 0-III mediators, Genetic polymorphism, Epigenetics, Drug interactions, Drug screening 2 Research

1. Introduction clearance. Evaluation of drug-drug interactions (DDI), which result from changes in the level or activity of DMEs and/or Drug metabolism, which occurs primarily in the liver and transporters due to a second drug, is an essential component intestine, refers to the enzymatic modification and sub- of drug development workflow. sequent disposal of medicinally active compounds, origi- In this review, we describe various classes of DMEs and nating either endogenously (as steroids, neurotransmitters, transporters, present an overview of the molecular underpin- metabolic products like bile acids) or exogenously (as natural nings for NR-mediated genetic and epigenetic regulation of products or synthetic/semi-synthetic chemicals). Upon con- ADME genes and consider roles for various NRs (especially version to hydrophilic metabolites, drugs are eliminated from PXR/CAR/VDR) and their target genes in differential drug the body following biliary excretion and renal clearance by response. Illustrative examples highlighting critical roles glomerular filtration and tubular secretion. Drug metabolism of xenosensing NRs, DMEs and transporters in DDI are is also integral to the biotransformation of pro-drugs to also examined. Finally, we discuss current drug-screening pharmaco-active agents. Drug metabolism and disposition platforms and their potential future improvements. is coordinated by an array of liver- and intestine-expressed drug-metabolizing enzymes (DMEs) and drug-transporting proteins whose tissue abundance is transcriptionally regu- 2. Drug Metabolizing Enzymes (DMEs) and lated by specific nuclear receptors (NRs), which are - Drug Transporters activated transcription factors [1]. Of the 48 distinct receptors comprising the NR super- The four phases of drug metabolism entail cellular uptake of family in humans, pregnane X receptor (PXR, NR1I2) therapeutic molecules (phase 0); their enzymatic oxidation and constitutive androstane receptor (CAR, NR1I3) are (phase I) and conjugation (phase II), and efflux of drug primary transcriptional regulators of the genes involved in metabolites for clearance (phase III). PXR and CAR activate the metabolism and elimination of drugs/drug metabolites genes involved in all four phases. General steps in drug [2–4]. PXR and CAR are generically referred as xenobiotic metabolism and elimination are shown in Figure 1. NRs, since structurally diverse drugs and environmental xenobiotics activate these two NRs. PXR and CAR are 2.1. Phase 0 uptake proteins. Basolaterally located uptake also activated by a number of endobiotics (steroids, sterols, proteins guide cellular entry of drugs from circulation; drug retinoids, thyroid hormones, bile acids). In addition, PXR influx can be a rate-limiting step for drug metabolism and and CAR activation can result from receptor phosphorylation clearance [12, 15]. All uptake proteins are members of the by various kinases that are activated in response to drug- solute carrier (SLC) of which there are more mediated induction of specific intracellular signal cascades; than 300 members grouped under 52 subfamilies. Liver, in this case, drugs may not directly interact with the intestine and kidneys are major sites of SLC expression. xenobiotic NRs [5]. Vitamin D receptor (VDR, NR1I1), Most SLC proteins localize to cell membrane, although beyond its classic role in calcium and phosphate , some may localize to mitochondria and other organelles. has the ability to transcriptionally induce drug transporters Proteins from nineteen SLC gene subfamilies have drug and DMEs, especially in the enterocytes of intestinal tissue uptake activity. Most significant gene families of uptake [6, 7]. In certain contexts, additional NRs, such as the transporters are SLC15 (oligopeptide transporter), SLC22 bile acid-activated farnesoid X receptor (FXR, NR1H4); (organic anion/cation/zwitterion transporter), SLCO (organic oxysterol-activated (LXR-훼, NR1H3); fatty anion transporting polypeptide) and SLC47 (organic cation acid/eicosanoid-activated peroxisome proliferator activated transporter) [13, 14]. For example, OCT1 is a SLC22A1 receptor (PPAR-훼, NR1C1), and retinoid-related orphan encoded organic cation uniporter involved in the influx of receptors (ROR-훼, ROR-훾) regulate genes linked to drug the antiviral agent acyclovir, ganciclovir and the anti-diabetic absorption, distribution, metabolism and excretion (ADME) drug metformin. Drug substrates for proteins encoded by [8]. Hepatocyte nuclear factor (HNF4-훼, NR2A1), a member SLC15, SLC22, SLCO, and SLC47 families have been of the NR superfamily, plays a synergizing role in the PXR- described [13–15]. SLCs either serve as channels (uniporter) and CAR-mediated transactivation of DME- and transporter- to guide drug diffusion down an electrochemical gradient, encoding genes [9–11]. or drive drug transport against a diffusion gradient that is Altered activities of polymorphic variants of NRs and coupled to the symport or antiport of small inorganic or ADME-related gene products account for variable response organic ions. to prescription medicine between individuals. Amino acid changes due to nucleotide polymorphisms in the coding region can influence protein stability or activity, while poly- 2.2. Phase I DMEs. Heme-containing cytochrome P450s morphism at upstream, downstream or intragenic regulatory (CYPs), flavin-containing monooxygenases, monoamine loci can alter NR-mediated ADME gene transactivation. oxidases and xanthine oxidase/aldehyde oxidases are exam- Epigenetic, transcriptional and posttranslational regulation ples of phase I DMEs, which generally localize to the of xenobiotic NRs can further impact drug metabolism and endoplasmic reticulum of cells. CYP enzymes play the most

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Xenobiotics Pollutants Natural products

Uptake 1 transporter Drug-bound receptor

2 Nuclear receptor 4 Coactivator End metabolites CYTOPLASM cleared via biliary 3 NUCLEUS excretion and/or Efflux urinary excretion transporter XRE Gene 10 9 5 5 5 Drug metabolism Phase I Phase II Phase III product Metabolizing Metabolizing Efflux 6 enzymes enzymes Transporters 8

7

Figure 1: Steps involved in drug metabolism and disposition. (1) Uptake transporter mediates drug entry into the cell. (2) Drug activates xeno-sensing NR in the cytoplasm or nucleus. (3) NR binds to XREs in target genes that are involved in drug metabolism and clearance. (4) Coactivator association with the DNA-bound NR and a cascade of activating steps, which culminate in gene transcription for DMEs, transporters. (5) Expression of phase 0-III mediators. (6) Phase I adds water-soluble functional groups to the drug structure. (7) A phase II conjugative adds hydrophilic groups to drug/drug metabolite. (8) Phase III efflux transporter moves to plasma membrane. (9) Transporter- assisted drug efflux. (10) Drug clearance through biliary and urinary excretion.

prominent role in phase I metabolism. Liver is the first within the subfamily. As an example, for CYP2D6*1, pass and primary site of phase I metabolism, along with the *1 allele is wild type CYP2D6 with normal activity. the gastrointestinal tract, kidneys, skin, and lung serving Additional alleles of CYP2D6, marked with higher numbers as additional sites; most tissues, however, express phase preceded by *, exhibit aberrant functions [17]. CYPs are I DMEs. Addition or exposure of polar functional groups expressed in practically all tissues, with liver exhibiting (e.g., -OH, hydroxyl; -COOH, carboxyl; -NH2, amine; -SH, the highest abundance and expressing largest number of sulfhydryl) to drug substrates enhances their bioavailability individual CYPs. Enzymes of the CYP-1, -2, and -3 families and solubility and promotes pro-drug biotransformation. metabolize majority of drugs and nondrug xenobiotics. The Polar groups also arise by reduction of a ketone or aldehyde fraction of clinical drugs that are substrates for individual group to an alcohol; oxidation of an alcohol to an acidic CYPs is schematically presented as Figure 2. CYP3A4, the group; hydrolysis of esters and amides; reduction of azo most abundant CYP enzyme in human liver, acts on the and nitro groups; oxidative dealkylation of N-alkyl, O- greatest number of drugs and other xenobiotics. CYP2D6, alkyl, and S-alkyl groups. When sufficiently polar, phase I although present at lower abundance, metabolizes numerous metabolites can be pumped out of cells without additional drugs. Substrate specificity is narrower for other members of modification. the CYP family that are expressed in hepatic and extrahepatic CYPs are products of a multigene family, which for tissues. They target endogenous substrates like sterols, fatty humans include 57 CYP genes [16]. Individual CYP is acids, eicosanoids and vitamins. A comprehensive list specified by the family (with an Arabic numeral), then the of drug substrates for CYPs has been reported ([18]; subfamily (with a letter) followed by the isozyme within http://www.pharmacologyweekly.com/cytochrome-cyp- the subfamily (with another Arabic numeral) and the allele p450-enzyme-medication-herbs-substrates, updated May, number (with a preceding asterisk) of an individual gene 2015).

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CYP2D6 CYP2B6 20% CYP2C8 7.2% 4.7% CYP2A6 CYP2C19 3.4% 6.8%

CYP2C9 CYP1A2 12.8% 8.9% CYP3A4/5 30.2%

CYP2J2 CYP2E1 3% 3%

Figure 2: Percentage of all prescription drugs metabolized in human liver by a particular CYP enzyme. (adapted from [16]).

2.3. Phase II DMEs. Broad-specificity phase II transferases blood is assisted by ABC transporters such as the multi- catalyze conjugative reactions. Common phase II modifi- drug resistance associated proteins MRP3, MRP4, MRP5 cations are glucuronidation by UDP-glucuronyltransferase and, also, by the ATP-independent OST훼/OST훽 complex (UGT), sulfonation by sulfotransferase (SULT), glutathiony- that functions as an organic solute and steroid transporter. lation by glutathione S-transferase (GST), acetylation by N- OST훼 contains seven transmembrane domains and OST훽 acetyl transferase (NAT) and methylation by methyltrans- has a single transmembrane domain [20]; neither is part ferase (MT). For any given transferase family, individual of the ABC transporter superfamily. MATE (multidrug family members show predilection for a distinct set of and toxin extrusion) efflux transporters are H+-coupled substrates. Cofactors of phase II transferases react with antiporters, which transport structurally unrelated organic functional groups that are either part of the parent drug or cations out of cells. They are members of the solute carrier generated by phase I modification. In contrast to the enhanced subfamily SLC47, expressed primarily in the liver and potency of many phase I metabolites, phase II modified drug kidney, and they localize at apical membranes of renal metabolites normally exhibit diminished function. PXR- and tubular epithelia and bile canaliculi. MATE1 (product of CAR-mediated gene regulation for a number of phase II SLC47A1) mediates extrusion of organic cations into urine transferases has been studied [9, 19]. and bile. In the human kidney, the uptake transporter OCT2 (organic cation transporter, SLC22A2 encoded) promotes the import of cationic drugs (such as metformin, cisplatin, 2.4. Phase III efflux proteins. Members of the ATP binding imatinib) from the blood at the basolateral membrane of the cassette (ABC) superfamily, encoded by the ABCB, ABCC, proximal tubule epithelial cells. MATE-1 and the isoform and ABCG gene subfamilies, are broad-specificity exporters MATE-2K mediate secretion of cationic drugs across the that pump drugs out of cells using energy from ATP brush-border membrane into the proximal tubule lumen hydrolysis. In hepatocytes, efflux proteins reside either in [21]. canalicular/apical membranes or in blood-facing basolat- eral/sinusoidal membranes, guiding drugs, endobiotics and their metabolites for biliary excretion and efflux into systemic 3. Nuclear Receptors (NRs), Response circulation. Multidrug-resistance associated proteins MRP2 Elements, Gene Regulation by (ABCC2), the bile salt export pump BSEP (ABCB11), PXR/CAR/VDR the breast cancer resistance protein BCRP (ABCG2) are examples of ABC cassette family transporters which medi- NRs, upon association with DNA response elements, induce ate apical efflux of drugs, steroids, bile acids and their a cascade of protein-protein interactions that lead to the conjugates. P-glycoprotein (MDR1, ABCB1) is an apical assembly of multiple classes of regulatory proteins (coac- membrane transporter in hepatocytes [13]. Basolateral efflux tivators, corepressors, histone modifiers, chromatin remod- of unconjugated and phase II-conjugated drugs, steroids, eling complex) at the NR-bound chromatin region. Signal prostaglandin and bile acids from hepatocytes into sinusoidal transmission from the coregulator assembly to the basal

AgiAl Publishing House | http://www.agialpress.com/ Nuclear Receptor Research 5 transcription machinery via a multi-protein mediator com- 3’ end of DBD. A variable F domain follows the LBD E plex culminates in altered RNA polymerase II activity and domain in some but not all NRs. X-ray crystallography, cryo transcriptional response of NR-regulated genes. electron microscopy and solution structure determination The NR superfamily of ligand-activated transcription by various methods including small-angle X-ray scattering factors in humans is defined by 48 receptors grouped into and hydrogen-deuterium exchange, revealed DBD and LBD four classes (Type I-IV) based on the nature of activating lig- structures of several NRs, such as the first and second ands, preferred sequence organization of NR-binding DNA zinc finger modules and DNA-binding specificity motif of response elements in target genes and dimerization partner of DBD; receptor dimerization motif; twelve 훼-helices of LBD the activated NR [22, 23]. Type I NRs reside in the cytoplasm and ligand-induced helix-12 repositioning that creates an in an inactive state in association with chaperone proteins. interaction surface for coactivator or corepressor recruitment They are activated upon binding cognate steroid hormone [27–29]. ligands, translocated to the nuclear compartment and bind DNA elements cognate to Type I-III NRs constitute target gene response elements as homodimers to mediate repeats of the half-site consensus sequence RG(G/T)TCA (R: gene regulation. Type II receptors, such as the vitamin purine), configured as a direct repeat (DR), inverted repeat D receptor (VDR), are activated by nonsteroid endocrine (IR), or everted repeat (ER) and separated by a varying ligands (1훼,25-dihydroxy vitamin D3 (1,25-D3, in short) number of nucleotides. Type I NRs recognize IR3-type for VDR; retinoic acid-all trans, for RAR-훼/-훽/-훾; thyroid palindromic elements; Type II and III NRs recognize specific hormone for TR-훼/-훽). Several Type II receptors are acti- repeat motifs of the consensus half site. Type IV NRs bind vated by intracrine ligands (e.g. bile acids activating FXR- to a single hexamer consensus RG(G/T)TCA, which may 훼; oxysterols activating LXR-훼/-훽; fatty acids/eicosanoids contain a short preferred sequence 5’ to the hexameric site activating PPAR-훼/-훾/-훿). Type II NRs in an inactive state [26]. remain tethered to corepressors as heterodimers with the Preferred response elements for PXR, CAR and VDR obligate partner (RXR). Exchange of are 3- or 4- nucleotide spaced direct repeats (DR3, DR4), corepressors for coactivators initiates activation of ligand- as concluded from in vitro DNA-binding studies and bound Type II NRs. PXR and CAR, comprising the Type response element-induced promoter activity in transfected III subgroup, are transported from cytoplasm to the nucleus cells. Numerous PXR/CAR/VDR target genes are also upon activation by chemical inducers. Nuclear PXR and CAR found to contain ER or IR motifs as response elements. bind to DNA response elements as dimers with RXR to set the Nevertheless, genome-wide chromatin immunoprecipitation stage for subsequent regulation of target gene transcription. (ChIP) and deep sequencing of immunoprecipitated DNAs Activation of CAR in most cases entails a ligand-binding (ChIP-Seq) identified DR4 as the most frequent PXR- independent mechanism, as reported for phenobarbital-like associated recruitment sites in mouse liver [30]. DR4 in chemicals, which induce dephosphorylation of CAR at the is a preferred DNA- for the threonine-38, thereby activating CAR and promoting its CAR/RXR heterodimer as well, as recently observed in a nuclear translocation. Direct ligand binding and activation of modified yeast one- hybrid assay [31]. DR3 is the prevalent CAR has also been reported for some xenobiotic compounds VDR-binding site at genomic regions that contain primary [24, 25]. Type IV NRs (e.g., LRH1, NGF1-B/NUR77, RORs) VDR target genes. These genomic regions are induced bind to DNA elements as a monomer, homodimer, or even for chromatin opening in response to 1,25-D3 signaling as a heterodimer, partnering with RXR or another member [32]. of the same subfamily [26]. Although PXR, CAR and, to a significant extent VDR, are primary regulators of drug metabolism and disposition, NRs from all four classes are 3.1. Regulation of PXR, CAR, VDR expression. PXR and known to influence drug/xenobiotic response, as discussed CAR are the primary mediators of transcription regulation under Section 3.3. of ADME relevant genes. Pathological conditions nega- tively impact drug metabolism due to reduction of PXR For all NRs, the primary structure specifies a com- and CAR activity. As an example, CYP3A4 expression is mon generalized organization based on functional domains suppressed by inflammation in part due to interference of [2, 23]. The highly variable amino-terminal A/B domain inflammation-activated NF-휅B with PXR’s transactivation harbors constitutively active transactivation function (AF- function, since the p65 subunit of NF-휅B was found to disrupt 1) and multiple autonomous transactivation domains. This DNA binding of the PXR/RXR훼 complex in the CYP3A4 is followed by a DNA-binding domain (DBD, C domain), gene [33]. Reduced PXR and CAR activity impairs drug through which an activated NR binds to a DNA response metabolism under conditions of hepatic steatosis as well, element. The ligand-binding domain (LBD, E domain) at since SREBP-1 (sterol regulatory element binding protein- the carboxyl end encompasses the activation function-2 1), activated in hepatocytes by - stimulated LXR- region (AF-2). A less conserved flexible hinge domain (D) 훼, prevented p160 coactivator interaction with CAR or PXR, connects DBD and LBD. The hinge region contains a nuclear which curtailed phenobarbital-induced, PXR/CAR-mediated localization signal (NLS) sequence, which extends to the CYP3A4/CYP2B6 gene transactivation [34].

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PXR and CAR is regulated by many SULT2A1 (for the sulfotransferase enzyme, isoform A1, transcription factors including various NRs [25, 35, 36]. subfamily-2) are briefly described. Cholic acid-activated FXR robustly induced the mouse The phenobarbital responsive enhancer module (PBREM) Pxr gene in the liver via four FXR-binding elements in the human UGT1A1 includes three CAR-responsive XREs in the Pxr promoter [37]. HNF4훼 regulates xenobiotic that are required for the optimal induction of UGT1A1 response in mice during fetal liver development through by phenobarbital (PB) [50, 51]. Protein-DNA interaction, Pxr gene activation [38]. GR regulated rat Pxr pro- analyzed by electrophoretic gel mobility shift assay (EMSA), moter in transfected primary hepatocytes and in hep- revealed that CAR binds as a monomer to one of the atoma cells [39]. Human PXR expression in liver is functional XREs in the UGT1A1 PBREM, and similar to transcriptionally regulated by PPAR훼 [35] and HNF4-훼 the CAR/RXR dimer, the DNA-bound CAR monomer can [40]. Expression of CAR is induced by agonist ligands interact with coactivators and corepressors. Furthermore, for GR, PPAR훼 and functional binding sites for these binding of the monomeric CAR or CAR/RXR dimer to XRE NRs as well as a binding site for HNF4훼 were identi- is most favored when the hexamer repeat of the response fied in the upstream sequence of the CAR promoter [25, element is preceded at the 5’ end by the dinucleotide AG. 41]. Furthermore, in animal studies, CAR mRNA expres- Arginine residues at positions 90 and 91, located within sion was induced by fasting and calorie restriction [41]. the carboxy-terminal extension of CAR’s DBD, mediate the Additional mechanisms entailing genetic polymorphism, dinucleotide-dependent binding preference [50]. changes in the epigenetic landscape, post-transcriptional XRE-dependent and PXR- and CAR-mediated induction regulation by micro RNAs, and functional modulation of human SULT2A1 was investigated in our laboratory [9]. through posttranslational modification (PTM) can have major Preferred substrates for SULT2A1 are bile acids and dehy- impacts on the expression and activity of PXR and CAR. droepiandrosterone (DHEA) – the latter is the steroid pre- These examples are discussed under Sections 4.1 and cursor for testosterone and dihydrotestosterone. A major role 4.2. for SULT2A1 in the enterohepatic tissue is to promote bile VDR, upon activation by cognate ligands (i.e., 1,25- acid clearance as the sulfate conjugate. Notably, the prostate D3 and lithocholic acid, LCA), can also induce ADME cancer drug Zytiga® (abiraterone acetate) is hydrolyzed relevant genes, especially in the intestine. Examples for in vivo to the therapeutic metabolite abiraterone, which is VDR-mediated induction of DMEs and drug transporters in cleared from the body after conversion by SUL2A1 to the 1,25-D3- or LCA-treated cells include CYP3A4, CYP2B6, inactive abiraterone sulfate, and by CYP3A4 to the inactive CYP2C9 [6, 42], SULT2A1 [43], OATP1A2 [44], ABCA1 N-oxide abiraterone, which is then converted to a sulfated [45], and MRP3, MRP2 [46]. Crystal structures of VDR derivative (PubChem database, CID 132971). SULT2A1 bound to LCA- and 3-ketoLCA have been determined expression is induced by VDR and the bile acid receptor [47]. Seasonal differences in intestinal CYP3A4 levels are FXR as well, which is in keeping with its role in bile acid attributed to season-related fluctuations in sunlight exposure homeostasis [43, 52]. A PXR/CAR- responsive composite that lead to variations in serum levels of 25-hydroxy-D3 and XRE in the human SULT2A1 promoter and a synergizing 1,25-D3 [48]. role of HNF4-훼 in XRE- induced SULT2A1 expression is Transcription of the VDR gene is under auto-regulation; described below and is summarized schematically in Figure 1,25-D3 can increase VDR gene expression. Various 3. endocrine factors including parathyroid hormone, retinoic acid, and glucocorticoids also regulate VDR expression [49]. Like PXR/CAR, VDR expression/activity is influenced by 3.2.1. A composite XRE and HNF4-α-responsive DR1 gene polymorphism, micro RNAs and by post-translational element in the human SULT2A1 promoter. Induction of the modification, as discussed in Section 4. SULT2A1 promoter by ligand-activated PXR and CAR in transfected liver and intestinal cells was shown to be mediated by an upstream xenobiotic-responsive composite element 3.2. Xenobiotic response element (XRE). At the chromatin (XRE). Specific interaction of XRE with PXR/RXR훼 and level, XREs serve as sensors of xenobiotic (or endobiotic) CAR/RXR훼 was demonstrated by DNAse1 footprinting signals by recruiting activated PXR/RXR and CAR/RXR to and EMSA. The XRE from -190 to -131 positions, was target genes. XRE activation is demonstrated by its activity defined by an inverted repeat and a direct repeat of the in cis to induce promoter-directed reporter gene expression in AG(G/T)TCA element, which are configured as IR2 transfected cells. Screening for XRE activation by synthetic (-190AACGCAAGCTCA- GATGACCCCTAA-167) and or semi-synthetic chemicals is an integral part of the work- DR4 (-55GATAAGTTCATGATTGCTCAACATC-131) flow for drug development. XREs also help identify other [9]. XRE-mediated stimulation required both IR2 and regulatory factors, which modulate PXR- and CAR-mediated DR4 elements; neither by itself was sufficient to cause expression of phase 0-III mediators. XREs in the phase robust SULT2A1 promoter induction. Thus XRE is a II DME genes UGT1A1 (for the glucuronidating enzyme composite element. The composite XRE spanning -190 to UDP glucuronosyltransferase, isoform A1, subfamily-1) and -131 positions stimulated a heterologous promoter. Point

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Xenobiotic- Xenobiotic- activated CAR activated PXR

RXRα RXRα

Nucleus PXR/CAR PXR/CAR RXRα RXRα +1 HNF4-α -30

SULT2A1 promoter IR-2 DR-4 DR-1 Composite XRE HNF4-α element at -190/-131 upstream of -30

XRE DR1 interaction

Figure 3: Induction of the human SULT2A1 promoter by PXR, CAR and a synergizing effect of HNF4-훼. Schema showing a PXR- and CAR-binding composite XRE comprised of IR2 and DR4 elements, and an HNF4-훼-binding DR1 element located downstream of XRE. Dotted, upward arrows signify promoter induction. Interaction between DR1-bound HNF4-훼 and XRE-bound PXR/RXR, CAR/RXR has an synergistic effect (triple upward arrows) on SULT2A1 induction. (based on results described in [9]).

mutations in the XRE prevented its interaction with PXR mRNA Normal Chow Chow + cholic acid and CAR and abrogated induction of the SULT2A1 and the levels heterologous thymidine kinase promoter. HNF4-훼 plays a modifying role in the PXR- Sult2A1 and CAR-mediated target gene transcription, since HNF4-훼 potentiated PXR- and CAR-mediated transactivation of the SULT2A1 promoter. A DR1 element (-63GTGACATGCTGGGACAAGGTTAAAGATCG-35) β-actin in the SULT2A1 gene promoter, located upstream of -30 nucleotide position, serves as an HNF4-훼-binding element. A schema on the regulation of SULT2A1 by PXR and CAR Figure 4: SULT2A1 mRNA induction by cholic acid in mouse via the composite XRE, and the synergizing influence liver. Sult2A1 mRNAs in mouse livers were assayed by semi- of DR1-bound HNF4-훼 on xenobiotic-induced SULT2A1 quantitative RT-PCR. Cholic acid, a primary bile acid, was added expression is presented as schema in Figure 3. to diet at 1% w/w. Data are for 3 individual mice (6-month-old, male) from the control and experimental group. Levels of 훽-actin mRNAs served as the normalization control (B. Chatterjee & CS 3.2.2. Sult2A1 induction by FXR via an IR0 element. Song, unpublished). Apart from xenobiotic chemicals, bile acid overload induces SULT2A1 expression. For example, Sult2A1 mRNAs were induced in the mouse liver when animals were fed a cholic as several other FXR target genes has been demonstrated acid containing diet (Figure 4), and bile acid activated [55]. The FXR/LRH-1/SHP axis plays a key role in bile acid FXR robustly induced the Sult2A1 promoter through an homeostasis, as discussed in the next section. FXR-bound IR0 element [52]. However, IR1 is the most In summary, above examples of XREs demonstrate that abundantly encountered FXR-responsive element. An IR1 PXR, CAR, FXR bind a variety of repeat motifs of the element drives FXR-mediated transactivation of ABCB11, consensus half site RG(G/T)TCA to induce genes involved the gene for the human bile salt export pump [53]. A in drug metabolism and disposition. number of other repeat motifs of the half site RG(G/T)TCA including DR1, ER6, ER8 are known to be FXR-responsive functional elements in FXR target genes. Assessment of 3.3. NR, a drug target for diseases from disrupted bile genome-wide FXR binding in the mouse hepatic chromatin acid/cholesterol homeostasis. Bile acid synthesis is the pri- showed an IR1-type sequence as the preferred chromatin mary pathway for cholesterol catabolism in liver, accounting occupancy site for FXR in vivo [54]. FXR-occupied IR1 sites for ∼50% of daily cholesterol turnover. Cholesterol overload, are frequently juxtaposed to a hexameric half-site consensus the underlying cause for cholesterol stone, results from sequence, which binds a monomeric NR such as LRH-1 (liver insufficient bile acid synthesis when bile acid saturation with receptor homolog-1). Positive interplay between FXR and cholesterol leads to the formation of cholesterol stone. On LRH-1 for the gene encoding the small heterodimer partner the other hand, bile acid accumulation leads to cholestasis (SHP), which is an atypical NR devoid of a DBD, as well due to reduction or stoppage of bile flow. Oral bile acid

AgiAl Publishing House | http://www.agialpress.com/ 8 Nuclear Receptor Research therapy is given to patients with cholesterol stones, and 4. Genetics, Epigenetics and Interindividual ursodeoxycholic acid is used to treat cholestasis of pregnancy Differences in Drug Response and primary biliary cirrhosis (PBC), the autoimmune disease causing bile duct destruction. FXR and other NRs, such as 4.1. Gene polymorphism and NR-regulated variable LRH-1, HNF4-훼, LXR-훼, SHP, PXR, and VDR maintain DME/drug transporter activity. Single nucleotide bile acid/cholesterol homeostasis [55, 56]. polymorphism (SNP) at regulatory loci of ADME related CYP7A1 (cholesterol 7훼 hydroxylase) is the rate-limiting genes, or non synonymous SNPs in the coding region of NR enzyme for bile acid production from the catabolic break- itself, alter NR-mediated DME/transporter expression. A down of cholesterol. HNF4-훼 and LRH-1 are positive regu- non-coding SNP at an HNF4-훼 binding site in the CYP2B6 lators of CYP7A1 expression. Some aspects of CYP7A1 reg- promoter contributes to the interindividual variations in ulation are, however, species-specific—a prominent example CYP2B6 expression [61], and a common African haplotype being the positive regulation of the basal expression of for an SNP at a PXR-binding enhancer in GSTA (encoding CYP7A1 by oxysterol-activated LXR-훼 in the rodent liver but glutathione S-transferase A) causes hypersensitivity for not in human liver, since the LXR-binding site in the human GSTA induction by the human PXR ligand rifampin [62]. promoter is mutated [57]. Toxic accumulation of bile acids, CYP2D6, which metabolizes a large number of drugs on the other hand, is prevented by FXR-imposed negative including antidepressants and 훽 blockers, shows wide feedback regulation of CYP7A1. In this case, bile acid interindividual differences in expression and activity. An activated FXR induces SHP [58], and interference from SHP HNF4-훼 variant having reduced binding to the CYP2D6 due to protein-protein interaction inhibits positive regulation promoter and causing decreased CYP2D6 expression has of the CYP7A1 promoter by LRH-1 (an NR activated by been identified. The variant HNF4-훼 arises from a non phospholipids) [56]. SHP also interferes with the stimulatory synonymous SNP, which yields glycine → aspartic acid interaction between HNF4-훼 and the coactivator PGC1-훼 substitution at the position 60 (G60D). Compared to this (peroxisome proliferator activated receptor 훾 coactivator 1- variant, the wild-type HNF4훼 genotype is associated 훼) on the CYP7A1 promoter [55]. In the ileum part of with higher CYP2D6 activity in the human liver [10, 63]. intestine, SHP plays a role in the CYP7A1 repression by the The G60D HNF4-훼 appears at low frequency in Asian fibroblast growth factor-19 which, like SHP, is induced by populations; it has not been detected in Africans or FXR [56]. PXR blocks CYP7A1 expression by disrupting the Caucasians [63]. Variable CYP2D6 expression also results PGC1훼 → HNF4 stimulatory axis [59]. Thus, like FXR, PXR from gene amplification that ranges from 3 to 13 gene copies. also regulates bile acid homeostasis upon activation by drugs CYP2D6 deficiency is an autosomal recessive trait in ∼7% and certain bile acids. Drugs targeting FXR, SHP, LRH-1, Caucasians and ∼1% Orientals, making these individuals PXR and HNF4-훼 have therapeutic potential against liver poor metabolizers of CYP2D6 drug substrates [64]. and biliary disorders. Small molecules that augment SHP Pharmacogenomic tests for CYP2D6 variants are common activity may robustly reduce CYP7A1 expression to prevent practice for assessing the appropriateness and efficacy of a bile acid overload. Small molecules, which elevate LRH-1 CYP2D6 drug substrate. Interindividual differences in drug activity or increase PGC1-훼 ↔ HNF4-훼 interaction, would response are managed by dosage adjustment based on the be useful in enhancing CYP7A1 expression, which then patient’s pharmacogenetic profile. would promote cholesterol breakdown and reduce cholesterol The basal level of CYP3A4 in the liver varies up to 60- build up. fold between individuals, although SNPs in coding sequences Apart from FXR, TGR5, a transmembrane G protein and regulatory loci of CYP3A4 do not explain this variability coupled receptor, mediates bile acid signaling. TGR5 is [65]. Association analysis suggests that nonsynonymous located in intestinal epithelium, Kupffer cells, sinusoidal SNPs of PXR and FOXA2 (aka HNF3-훽, a liver-enriched endothelium and bile duct cells. Both TGR5 and FXR ) contribute to CYP3A4 variation in the are hotly pursued drug targets for diseases of errant bile human liver, since the mRNA expression level for CYP3A4 acid and cholesterol metabolism [55]. The athero-protective in the human liver significantly relates to SNPs of PXR and effect of LXR-훼 arises in part from the LXR-훼-mediated FOXA2, and PXR expression itself is regulated by FOXA2. induction of efflux transporters in resident macrophages Binding sites for FOXA2 and PXR in the human CYP3A4 of the arterial wall, and this in turn promotes cholesterol distal promoter were identified [66]. VDR polymorphism efflux and reverse cholesterol transport to the liver and accounts for disparate intestinal CYP3A4 levels and variable intestinal tissue and subsequent removal of cholesterol as first pass intestinal absorption and metabolism of CYP3A4- part of excreta. Therefore, small molecule activators of LXR- targeted drugs [48]. 훼 may normalize cholesterol homeostasis. Finally, VDR FXR, which regulates the expression of many uptake and can regulate bile acid and cholesterol homeostasis, since efflux transporters, shows a common non-coding -1G>T agonist-activated VDR promotes cholesterol catabolism polymorphism, where T replaces G at the -1 position of by repressing SHP and increasing CYP7A1 expression the translation start site causing reduced FXR expression. [60]. The FXR-1G>T SNP is associated with increased efficacy

AgiAl Publishing House | http://www.agialpress.com/ Nuclear Receptor Research 9 of the statin drug rosuvastatin in lowering hepatic choles- when 5mC methylation occurs within long stretches of CpG terol biosynthesis, thus affording greater LDL-cholesterol repeats (CpG island) at proximal promoters, although 5C- response [67]. Rosuvastain remains un-metabolized in hep- methylation at low CpG density (CpG shores) or even at atocytes and ABCG2 (BCRP), an apical ABC cassette efflux single CpG sites can mark reduced gene expression. Of transporter, plays a major role in the biliary clearance 3 major DNA methyltransferases (DNMTs) in mammals, of rosuvastatin. ABCC2 (MRP2) and possibly ABCC11 DNMT1 is the maintenance methyltransferase; DNMT-3a (BSEP) also contribute to rosuvastatin disposition from and -3b are de novo enzymes, essential for the genome- human liver. Mechanistically, low expression of the variant wide methylation of DNA following embryo implantation. FXR accounts for reduced expression of the transporters Gene repression by DNA hypermethylation is aided by the ABCG2, ABCC2, ABCC11, which leads to a blockade in the interaction of DNMTs with the polycomb repressor complex biliary clearance of rosuvastatin and longer residency of the (PRC2), especially with EZH2 (Enhancer of Zeste homolog drug in hepatocytes – hence a more potent effect of this statin 2), the histone methyltransferase component of PRC2 on hypercholesterolemic patients who carry the FXR-1G>T [75]. Cancer development is associated with genome-wide SNP [67]. DNA hypomethylation, which activates proto-oncogenes. A large number of SNPs for PXR (NR1I2)- and CAR For many tumor suppressors, site-specific hypermethylation (NR1I3)- encoding genes are known, several of which are contributes to gene silencing [75]. 5-hydroxymethylcytosine associated with altered expression and/or function of these (5hmc) modification of DNA, on the other hand, is an receptors [68, 69]. For the NR1I2 SNP 63396C>T, located activation mark, linked to active gene transcription [76]. in a putative transcription factor binding site, the 63396T Notably, the paternal sperm DNA methylation pattern has variant associates with elevated PXR expression, increased been linked to autism risks in an autism-dense cohort [77]. CYP3A4 expression and decreased plasma levels of the Extensive interindividual differences in the genome-wide CYP3A4 substrate atazanavir (an anti-retroviral drug). Nat- DNA methylation pattern have been reported [78]. ural PXR variants, which harbor single amino acid changes, Acquired drug resistance has been linked to altered DNA confer altered transactivation response of the CYP3A4 pro- methylation of NRs and NR-regulated ADME genes, as moter [65]. Among the 22 naturally occurring splice variants observed in i) drug-induced demethylation of MDR1 and of CAR, some are nonfunctional due to nonsense mutations. BCRP, which leads to their overexpression causing multidrug For the CAR (NR1I3) SNP rs2307424C>T, the T allele is resistance (MDR) of cancer cells [79, 80]; ii) drug-induced associated with a low plasma level of the anti-retroviral drug methylation of the (ER-훼) encoding ESR1 , which is a CYP2B6 and CYP3A4 substrate [70]. gene promoter, causing reduced ER-훼 expression and tamox- Extensive VDR gene polymorphism has been reported [71], ifen resistance in breast cancer [81]; and iii) resistance to and it has been reported that intestinal CYP3A4 expression progesterone therapy in endometrial cancer due to reduced levels are functions of VDR polymorphisms [48]. expression caused by enhanced methylation of the gene encoding isoform A (PR-A) [82]. 4.2. Epigenetic machinery and drug response. Roles for Methylation of the PXR gene promoter attenuated PXR DNA methylation, histone modification and microRNAs in expression and reduced CYP3A4 expression in colon cancer the regulation of a large number of mediators of phase 0- cells [83]. In colon and endometrial cancers, the VDR gene III processes and their NR regulators (PXR, VDR, HNF4- is aberrantly methylated [83]; differential methylation of 훼) have been reported [72, 73]. Epigenetic factors confer PXR and FXR at CpG promoter sites has been reported in heritable changes in chromatin structure and function, caused cholestatic pregnancy versus normal healthy pregnancy [84]. by mechanisms other than DNA sequence alteration at the A role for DNA methylation in the expression of a number coding or non-coding region of a gene. An integral role of DMEs and drug transporters has been reviewed [73, of epigenetics in health and disease is revealed by the 85]. A few representative examples are discussed here. 1) tragic history of the synthetic estrogen diethyl stilbesterol CYP3A4/5/7 expression is dependent upon the methylation (DES) as a birth control pill. In utero DES exposure caused status of these genes, since their expression was altered vaginal tumors and breast cancer in adult females. In mice, when human hepatoma cells were treated with 5-aza-2′- DES altered gene-specific DNA methylation, expression of deoxycytidine (a DNA demethylating agent). CYP3A4 induc- epigenetic enzymes (DNMT3A, MBD2, HDAC2, EZH2), tion is associated with reduced 5mC at CpG-rich regions and the abundance of HOTAIR, a lncRNA [74]. Epigenetic located at or near the binding sites for PXR, CAR, and systems are briefly discussed and current information on their VDR, which are well-known regulators of CYP3A4 [73]. 2) roles in drug metabolism and drug response is presented. Altered CYP1A1 expression in response to cigarette smoking is associated with changes in the methylation status of 4.2.1. DNA methylation, ADME gene activity, interindividual CYP1A1. 3) Development stage-dependent CYP2E1 expres- differences. DNA methylation at the 5’ cytosine of the CpG sion is influenced by the methylation status of this gene. sequence (5mC) is an epigenetic mark for gene activity 4) Phase II genes including UGT1A1, GSTP1, SULT1A1 [75]. Gene repression is linked to hypermethylated promoters and genes for efflux transporters like MDR1, BCRP and

AgiAl Publishing House | http://www.agialpress.com/ 10 Nuclear Receptor Research members of the OATP family of uptake transporters are neonatal CAR activation were significantly more sensitive to epigenetically regulated due to DNA methylation [86– low TCPOBOP concentrations for Cyp gene induction than 89]. hepatocytes from control mice lacking neonatal exposure to this inducer [92]. Also, the possibility remains that the long- lasting stability and biological potency of TCPOBOP in vivo, 4.2.2. Histone marks; impact on NR-regulated ADME with activity persisting in mouse liver for 6 months or more genes. Post-translational modification (PTM) of histones [93], contributed to the hepatic Cyp induction in adult mice (methylation, acetylation, phosphorylation, ubiquitinylation, when receiving a single dose of TCPOBOP as neonates. sumoylation, ADP- ribophosphorylation and several other PXR-mediated CYP3A4 induction was regulated by PRMT1, modifications), especially acetylation and methylation at the which methylated histone H4 at arginine-3 that is located amino-terminal histone tails for histone H3 and H4, are within a PXR-responsive chromatin region in the CYP3A4 well-characterized epigenetic signatures that influence gene gene [94]. In a rat model of chronic kidney disease, reduced activity. PTMs are also known for histone H2A and H2B Cyp2C and Cyp3A expression, and associated reduction in and the linker histone H1. More than 10 different PTMs at PXR and HNF4-훼 binding to cognate sites in the Cyp2C11 ∼80 sites on histone tails, histone core domains and on the and Cyp3A2 promoters, was accompanied by reduced histone H1 linker histone have been identified [90]. Gene-activating H4 acetylation at the Cyp3A2 promoter regulatory region and histone marks include H4 lysine-16 acetylation (H4K16ac); reduced histone H3 acetylation at the PXR- and HNF4-훼- H3 trimethylation at lysine-4 (H3K4me3) and lysine 36 bound regulatory loci of Cyp2C11 and Cyp3A2 promoters (H3K36me3), and H3 phosphorylation at serine-10. Among [95]. repression marks, trimethylated histone H3 at lysine-9, Given the reversible nature of chromatin modifications lysine-27 and lysine-20 are most well characterized [91]. His- by DNA methylation and histone PTM, drugs targeting tone deacetylases (HDACs, subgrouped as class I to IV), his- epigenetic enzymes (“epi drugs”), especially DNMT, HAT, tone acetyltransferases (HATs), histone methyltransferases HDAC, HMT and HDM, are being developed. 5-azacytidine (HMTs) and histone (HDMs) are important (Azacitidine) and 5-aza-2’-deoxycytidine (Decitabine) are drug targets. Numerous lysine-specific HMTs (SET7/9, nucleoside analogs and DNA demethylating agents that are MLL, EZ2H2) and lysine-specific HDMs (LSD1/KDM- clinically used against myelodysplastic syndromes, chronic 1, JMJD2A/KDM4A, JARID1A/KDM5A) have been char- myelomonocytic leukaemia and acute myeloid leukaemia. acterized. Arginine methylation of histones mediated by Second-generation DNA de-methylating agents (SGI-110, protein arginine methyltransferases (PRMTs) also regulates CP-4200) are under development [96]. gene activity, as seen for histone H3 Arg-17, H4 Arg-3. Valproic acid, a class I HDAC inhibitor and an anti- Histone marks are the “codes” that are “read” by chromatin convulsant, activates CAR and PXR to induce CYP3A4, remodelers (such as SWI/SNF containing complexes) and CYP2B6 and MDR1 expression [97–99]. Valproic acid also histone- modifying enzyme complexes (such as PRC2) in enhances tissue sensitivity to estrogen and progesterone order to prepare chromatin for positive or negative tran- by potentiating estrogen receptor (ER) and progesterone scriptional response. Enzymes that mark histones through receptor (PR) activity due to HDAC1 inhibition [100]. PTM are “writers”; those involved in removing histone Vorinostat (a hydroxamate) and romidepsine (a depsipeptide) marks are “erasers”; and protein/enzyme complexes, which are orally administered pan-HDAC inhibitors, which are recognize histone codes, are “readers”. Crosstalk of HMTs used in combination therapy with chemotherapeutics like with DNMT1 influences epigenetic regulation [75]. paclitaxel, doxorubicin. Vorinostat resistance is thought to Among the ADMEs whose genes are known to be develop from increased expression of efflux transporters, regulated by histone modification include the phase I DMEs since MDR1, BCRP, and MRPs were detected at elevated CYP3A4, CYP2E1, phase II DMEs SULT2B1, UGT1A1, levels in vorinostat-treated cells [73]. Inhibition of ABC the efflux transporters MDR1, BCRP, the OATP family transporters in this case may improve vorinostat’s therapeutic of uptake transporters and the SLC5A5 encoded iodine efficacy. Epi drugs, which target histone methylation, are at uptake transporter sodium/iodide symporter [73, 86]. Histone various stages of development [101]. modification can have long- lasting effects on ADME genes. For example, the CAR target genes Cyp2b10 and Cyp2c37, when neonatally exposed to the CAR ligand TCPOBOP, 4.2.3. Non-coding RNA-mediated regulation of PXR, CAR, remained induced in adult mouse livers; as a result, adult VDR and ADME gene expression. Non-coding RNAs (ncR- mice were much less sensitive to the Cyp2b10 substrate zox- NAs), best characterized for micro RNAs, are integrally azolamine [92]. H3K4 methylation and H3K9 demethylation linked to epigenetic machinery. Transcripts of more than at CAR-responsive elements in the Cyp genes, detected in 90% of the human genome represent ncRNAs, many of the neonatal liver, persisted in the adult mouse liver. It was which regulate gene expression at transcriptional and post- concluded that the early-life methylation status of histone H3 transcriptional levels. Short (<30 nucleotides) ncRNAs, best played a role in the Cyp gene induction in the adult liver, characterized for micro RNAs (miRNAs), and long ncRNAs since hepatocytes isolated from the livers of mice receiving (lnc RNAs) with >200-nucleotide lengths are two major

AgiAl Publishing House | http://www.agialpress.com/ Nuclear Receptor Research 11 categories of ncRNAs. The list for micro RNAs, which co-administered drug. By activating PXR and CAR, the influence ADME gene expression, is steadily growing [83, interfering drug renders changes in one or more components 102, 103]. of the drug metabolizing and disposition machinery. DDI is The miRbase lists as many as 2555 unique mature human assessed quantitatively by the pharmacokinetic parameters miRNAs (database version 20; June 2013 release). Base Cmax, which refers to the peak plasma drug concentration pairing of the miRNA nucleotide sequence with a cognate at post-dosing; and AUC (area under the time-plasma drug sequence in the 3’ untranslated region (3’-UTR) of a target concentration curve), which defines total serum drug levels messenger RNA (mRNA) within the RNA-induced silencing over time. DDI has three possible outcomes: i) overdosing complex leads to either mRNA degradation (in the case and potential toxicity due to increased half-life of a target of a perfectly complemented base pairing), or translational drug caused by one or more of the following – excessive suppression of the target mRNA when base pairing is not pro-drug bioactivation; attenuated DME activity; increased 100% complementary. A single miRNA can target 3’UTRs uptake activity and reduced efflux activity of transporters; of multiple messenger RNAs. ii) underdosing resulting in low drug efficacy, which is Studies in cell culture show that the messenger RNAs for due to reduced drug uptake and/or reduced bioactivation; ADME related genes are targeted directly or, via upstream enhanced metabolism and/or accelerated drug efflux; iii) a regulatory NR and other transcription factors, by one or more boost in medicinal potency. CYP-mediated DDI led to the miRNAs. Regulation of CYP1B1 and CYP3A4 mRNAs withdrawal of numerous drugs from clinical use, such as by miR-27b; CYP2E1 mRNA by miR-378; the MDR1 terfenadine (the antihistamine Seldane®) and cerivastatin transporter by miR-451; and the BCRP transporter by miR- (a cholesterol-lowering statin). Dietary ingredients (e.g., 328, miR-519C, miR-520h underscores the impact that furanocoumarins in grapefruit juice) or phytochemicals miRNAs may have on drug metabolism and disposition, in medicinal herbs (e.g., hyperforin in St John’s Wort) provided these miRNA-dependent regulations are upheld in can modulate a drug’s efficacy and engender potentially vivo. While miR-27b directly regulates CYP3A4 expression, fatal drug-food and drug-herb interactions. CYP3A4/3A5 the VDR level is regulated by this micro RNA as well, and CYP2D6 are most frequent participants in DDI [18, so that miR-27b can both directly and indirectly influence 110]. CYP3A4 expression. PXR expression is regulated also by Desirable outcomes may also result from drug interac- miR-148a. The miRNA-dependent regulation of several tions, as seen in the hepato-protective effect of ginger extracts epigenetic enzymes including DNMTs, HDACs, EZH2, and against diverse drugs including high-dose acetaminophen epigenetic enzymes that regulate miRNA-specifying genes [111]. DDI is not a concern for peptide or antibody based (which produce miRNA precursor transcripts) have been therapeutics, since they do not activate PXR and CAR. reported [104, 105]. Such cross talks provide a miRNA- Recently approved PCSK9 inhibitors are antibody-based dependent additional regulatory cascades that may alter drugs, which aid in LDL-cholesterol clearance from circula- DME/transporter expression. The abundance of specific tion by preventing PCSK9-mediated degradation of the LDL miRNAs may predict drug response, since miR-21 levels receptor [112]. in pancreatic cancer biopsies correlated with gemcitabine responsiveness, and ectopic miR-21 expression caused gem- citabine resistance in pancreatic cancer cells [106]. 5.2. Linking xenobiotic NRs to drug interactions. Associ- A lncRNA known as AIR is indirectly involved in the inac- ation of the xenobiotic NR activity with clinical DDI has tivation of the mouse organic cation transporter (OCT) genes been reported [1, 113, 114]. Orally delivered drugs, which Slc22a2 and Slc22a3, since AIR plays a role in silencing are CYP3A4 and/or MDR1 transporter substrates, can exhibit the Igf2R gene cluster and Slc22a2 and Slc22a3 are located markedly altered pharmacokinetics in response to rifampicin within this cluster [107]. LncLSTR, a recently reported co-administration. For example, increased enterohepatic liver-enriched lncRNA, is a regulator of Cyp8b1, which expression of CYP3A4, triggered by the long-term treatment is involved in bile acid biosynthesis [108]. The lncRNAs with the human PXR agonist rifampicin caused a 96% PCA3 and PCGEM1 are elevated in human prostate cancer. decrease in the oral bioavailability of the CYP3A4 substrate PCGEM1 coactivates activities of the (S)-verapamil and loss of the anti-hypertensive effect of this and cMYC oncoprotein. [109]. Whether lncRNAs directly drug in patients [115]. Cyp2C9 induction by rifampicin- regulate ADME-relevant genes remains to be determined. activated PXR reduced plasma concentrations of CYP2C9 substrates such as warfarin (anticoagulant) and sulfonylurea (antidiabetic) [116]. Binding and activation of PXR by 5. Drug Interactions: A Role for hyperforin, a bioactive component of St. John’s Wort, leads Xenosensing NRs to the transcriptional induction of CYP3A4 and widely prevalent clinical DDI due to increased metabolism and 5.1. Drug-drug, drug-food, drug-herb interaction. Drug- hence decreased efficacy of numerous drugs including oral drug interaction (DDI) reflects changes in target drug contraceptives, the immune suppressant drug cyclosporine pharmacokinetics or bioavailability in the presence of a and the anti-HIV protease inhibitor indinavir [117].

AgiAl Publishing House | http://www.agialpress.com/ 12 Nuclear Receptor Research

Apart from acting as direct ligands, certain drugs induce CAR-responsive functional XREs are present in genes for phosphorylation of PXR and CAR by activating signal many drug transporters including OATPs and various efflux pathways that lead to activation of kinases such as PKA, transporters [126]. PKC, CDK2, CDK5, and p70S6K [5]. One such PXR activator is forskolin, a diterpene constituent of the Indian 5.3.2. Prostate cancer, ZYTIGAⓇ, DDI with rifampicin. plant C. forskohlii, which is used for the treatment of glau- Zytiga® (abiraterone acetate), the anti-androgen drug against coma, asthma and various other diseases. Forskolin induces recurrent metastatic prostate cancer, is a CYP3A4 and PXR phosphorylation through PKA activation, and enhances SULT2A1 substrate. In a DDI trial, serum Zytiga® exposure PXR-coactivator interaction upon its direct binding to the decreased by 55% in the presence of rifampin, indicating PXR LBD [118]. Additionally, forskolin is a constituent of a need for higher dosage of this drug when a PXR acti- an herbal mixture marketed over-the-counter for weight loss. vator is co-administered [127]. DDI is likely caused by DDI/drug-herb interaction may interfere with forskolin’s increased CYP3A4 and SULT2A1 expression by rifampicin- therapeutic value. activated PXR, since inhibition of CYP3A4 activity by co- In the case of CAR, metformin induces phosphorylation of administered ketoconazole (a strong CYP3A4 inhibitor) did this xenosensing NR at threonine-38, mediated by activated not significantly alter Zytiga® pharmacokinetics (Clinical AMPK and the MAP kinase ERK1/2. Thr-38 phosphoryla- Pharmacol 12.3; FDA Drug Safety Reporting, 2015). tion restricts nuclear translocation of CAR and disruption of coactivator-CAR interaction, thereby preventing CAR- mediated induction of target genes such as CYP2B6 [25]. 5.4. Drug-food, drug-herb interactions As a result, co-administration of metformin is known to cause altered pharmacokinetics for CYP2B6 drug substrates 5.4.1. Grapefruit juice, CYP3A4, drug transporters, [119]. Reciprocally, reduced CYP2B6 activity may render PXR/CAR. Grapefruit and several other citrus fruits contain a negative impact on the renal clearance of metformin as furanocoumarins in addition to other phytochemicals. a result of reduced expression of the renal OCT2/MATE Furanocoumarins, which inhibit OATPBs and CYP3A4, transporter system. Pronounced DDI may be expected as a elevate the bioavailability of CYP3A4/OATPB substrates result of such negative interplays. including cyclosporine, midazolam, calcium channel Additional examples below underscore how PXR and blockers and certain statins [128]. Although in humans CAR may play roles in clinical DDI events. Several reviews furanocoumarins predominantly inhibit CYP3A4 activity, on NR-regulated drug interactions provide further elabora- Cyp 1, 2, 3 expression and activity in mice was induced tions on this subject [15, 114, 120, 121]. by isopimpinellin (a furanocoumarin) in a Pxr- and Car- dependent manner [129]. To settle whether species specificity explains such differences, effects of furanocoumarins on 5.3. Drug-drug interaction PXR and CAR activity should be re-assessed in humanized PXR-CAR-CYP3A4/5 mice where human counterparts 5.3.1. Statin-induced myopathy, DDI: a likely role for NRs. replace rodent Pxr, Car and Cyp genes [130, 131]. Uptake transporters of the OATP family are predominantly involved in the hepatic import of statins [15], and common variants in SLCO1B1, which encodes OATP1B1, are strongly 5.4.2. Pomegranate juice, SULT2A1, ZytigaⓇ activity. Puni- linked to an increased risk for statin-induced myopathy [122]. calagin, a polyphenol constituent of pomegranate, impairs As an example of adverse DDI, cyclosporine A, which sulfoconjugation of drugs in the intestine [132], which competitively inhibits OATP-mediated hepatic statin uptake, leads to reduced clearance and thus overdosing of orally caused skeletal muscle statin overload and muscle damage delivered Zytiga® (abiraterone acetate) which, as a CYP3A4 upon co-administration with pitavastatin or rosuvastatin. and SULT2A1 substrate, is normally metabolized to abi- Extreme statin overload is linked to the fatal condition of raterone sulfate and N-oxide abiraterone sulfate. Inhibition rhabdomyolysis [123]. of CYP2C9 by punicalagin has also been reported. It is not known whether this polyphenol influences PXR or CAR Various statins, however, differ significantly in pharma- activity. cokinetic characteristics due to differences in ADME. It is, therefore, conceivable that additional to altered uptake activity, the PXR-/CAR-regulated expression of uptake trans- 5.4.3. St. John’s Wort, PXR, CAR, CYP3A4. Hyperforin, porters may influence the hepatic uptake of some form of which confers the anti-depressant activity of St. John’s wort, statins. Indeed, long-term treatment of rifampin reduced is a ligand for human PXR and CAR [117, 133]. Hyperforin- atorvastatin bioavailability due to induced expression of activated PXR/CAR induces CYP3A4, other CYP genes CYP3A4 and efflux transporters by rifampin-activated PXR (CYP2B6, CYP2C9, CYP2C19), as well as MDR1. Acute [124], whereas short-term rifampicin administration caused rejection of transplanted hearts in patients due to self- inhibition of OATP-mediated hepatic uptake of atorvastatin medication with St. John’s Wort is an example of serious and caused elevated AUC for this statin [125]. PXR- and drug-herb interactions. Rejection was caused by a drop in

AgiAl Publishing House | http://www.agialpress.com/ Nuclear Receptor Research 13 plasma levels of cyclosporine, which is a CYP3A4 and genes are replaced by corresponding human genes, are better MDR1 substrate [134]. suited for drug testing since these models provide in vivo relevance and they approximate as human surrogates. The 5.4.4. Garlic, CYP2C9, Warfarin. Garlic extracts suppressed humanized PXR-CAR-CYP3A4/3A7 mouse strain is com- CYP2C9 mRNA expression and activity in the human mercially available. A new hPXR-hCAR-hCYP3A4/3A7- hepatocyte-derived Fa2N-4 cell line; furthermore, garlic hCYP2C9-hCYP2D6 mouse strain, with human PXR and extract can competitively inhibit CYP2C9 activity [135]. CAR genes substituted for the rodent Pxr and Car genes Increased systemic exposure of CYP2C9 drug substrates and the gene clusters Cyp3a, Cyp2c and Cyp2d replaced by such as warfarin in the presence of garlic extract has been counterpart human genes, has been reported [145]. reported. Reduced warfarin metabolism may enhance the In the not-to-distant future, microfluidic organs-on-chips possibility for uncontrolled bleeding. Since the diallyl sulfide may be adopted as a preferred platform for drug testing, in garlic extracts can activate CAR [136], CYP2C9 gene replacing animal models. In a microfluidic device, live suppression may be driven by a CAR-dependent mechanism. cells on chips, organized in continuously perfused cham- bers, mimic the complex multicellular environment so that bioavailability, efficacy and toxicity of test molecules could 5.4.5. Protection from acetaminophen toxicity by gar- be assessed in a context which, in part, recapitulates human lic extracts: a role for CAR-induced SULT. The hepato- tissue and organ physiology [146, 147]. The future drug protective effect of organo-sulfers in garlic extracts against discovery pipeline may also include a workflow that assesses acetaminophen-induced liver injury is due to two mecha- drug-induced PTM profiles of PXR and CAR determined nisms: 1) reduction of hepatic CYP2E1 expression and inhi- through liquid chromatography-coupled-tandem mass spec- bition of CYP2E1- mediated acetaminophen biotransforma- trometry, and examines how PTM alters PXR/CAR activity tion to a toxic metabolite [137]; 2) increased acetaminophen using an approach similar to that reported recently for PXR clearance as a sulfate metabolite by SULT activity. It has [148]. been reported that CAR, activated by diallyl sulfide (a garlic constituent), promotes acetaminophen conversion to a sulfated metabolite by inducing SULT2A1 and other SULTs (SULT1A1, SULT1A3/4, SULT1E1) [138, 139]. Reduced 6. Summary and Perspectives build up of acetaminophen prevents GSTpi induction by PXR and CAR, the two nuclear receptors that are acti- acetaminophen-activated CAR. The net result is diminished vated by drugs and other xenobiotics, coordinate both oxidative stress from glutathione depletion and consequent metabolism of orally administered drugs in the liver and reduction of oxidant-induced liver injury [140]. intestine and excretion of drug metabolites by mediating Additional NRs can potentially generate drug interactions. transcriptional induction of genes encoding phase I/phase VDR-mediated regulation of DMEs and transporters and II drug-metabolizing enzymes (DMEs) and transporters a modifier role of HNF4 in the expression of ADME- which regulate drug influx (phase 0) and efflux (phase relevant genes have been reported [6, 9, 43, 126, 141]. III) of drug metabolites. Phase 0-III mediators are also Whether long-term use of vitamin D supplements would induced by ligand-activated VDR, especially in the entero- cause adverse drug interactions should be evaluated. Drug cytes of intestine. Additional nuclear receptors, especially interaction from activated (GR) is a FXR, HNF4-훼, LRH-1 and SHP regulate expression of distinct possibility, since ligand-activated GR induces CAR the enzymes and transporters involved in cholesterol and and PXR expression; a GR-responsive element has been bile acid homeostasis. More than 90% of all known drugs identified in the CAR gene promoter [142]. Dexamethasone, are metabolized by a subset of cytochrome P450s (CYPs) a synthetic glucocorticoid, promotes nuclear translocation of – CYP3A4/3A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, CAR and PXR and induces PXR/CAR target genes [142, CYP1A2, CYP2C8, CYP2A6, CYP2J2, and CYP2E1. In 143]. Ketoconazole, an anti-fungal agent and GR antagonist, the human liver and intestinal epithelium, CYP3A4 and prevented rifampin- and phenobarbital-mediated PXR/CAR its functionally indistinguishable isoform CYP3A5 are the activation and induction of their target genes [144]. Thus, most abundant CYP enzymes and together, they metabolize under ketoconazole co-medication, a primary drug may more than half of all prescription medicines. Overdosing respond with altered pharmacokinetics. or underdosing leading to drug toxicity or reduced drug efficacy, respectively, is the consequence of interference from 5.5. Platforms for screening drug candidates. Early assess- a co-administered second drug (DDI, i.e. drug-drug inter- ment of drug candidates can avoid late-stage failure of clini- action) or from a dietary or herbal agent (drug-food/drug- cal trials due to DDI and help minimize costs for developing herb interaction). Adverse (or beneficial) drug interaction and marketing a new drug. Candidate drugs are routinely results from i) enhanced gene transactivation for DMEs or screened in a cell based workflow for their impact on DME transporters due to PXR/CAR activation by the interfering activity and PXR/CAR-mediated transactivation of XREs. drug or other agent; and/or ii) altered DME or transporter Humanized mouse models, where Pxr, Car and Cyp rodent activity. In order to minimize late-stage failure of clinical

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Screening Drug Candidates for CYP Activation and Drug Interactions

Traditional workflow Emerging workflow

High throughput Primary hepatocyte (to test for CYP Microfluidic organ-on-a chip culture activation)

Incubtion with recombinant Cross screening CYPs, or CYPs from to assess drug Organ-on-a chip liver microsomes interactions

Mice In-vivo Study Organ-on-a chip (Preclinical model) (non-transgenic) Humanized mouse (transgenic)

Clinical trials (phase 1, 2, 3)

FDA Approval

Figure 5: Overview of drug-screening platforms: Candidate drugs are screened for effects on the activity and expression of a select set of CYPs (e.g., CYP3A4, and several other CYPs). Workflow for traditional screening (shown at left) relies on cell-based high throughput assay to identify and narrow down candidates with potential for optimal drug activity. Microfluidic organ-on-a chip constitutes an emerging technology that may replace cell-based screening as the primary assay platform. In cross screening, cells are co-administered with a test drug and a second drug or a non-drug xenobiotic agent (such as a medicinal herb or a foodstuff) in order to reveal drug-drug or drug-herb or drug-food interactions. Subsequently, drugs are tested in mice. A humanized mouse model (transgenic mice with human PXR, CAR and CYP genes replacing the counterpart rodent genes) can serve as a human surrogate for the examination of drug interactions in the preclinical stage of drug screening. trials, an essential routine at early stages of drug develop- it can also be due to SNPs of PXR/CAR/VDR/HNF4- ment is to evaluate candidate molecules for effects on the 훼 that lead to variable expression or activity of these activities and expression of a select set of CYP isozymes; nuclear receptors. An epigenome signature is specified by for PXR and CAR activation and for DDI. Humanized DNA methylation, chromatin histone marks for transcription mouse strains, as in hPXR-hCAR-hCYP3A4-hCYP3A7 mice activation/repression (largely defined by lysine acetylation (available commercially), or recently reported hPXR-hCAR- and lysine/arginine methylation of the amino-terminal tails hCYP3A4/3A7-hCYP2C9-hCYP2D6 mice, may replace a of H3 and H4 histones), and by non-coding regulatory RNAs cell-based workflow for screening candidate drugs. A human- (microRNAs, long non-coding RNAs). The signature can ized mouse model provides human-like drug metabolism have a profound impact on drug metabolism and disposition machinery and in vivo relevance. A microfluidic organ-on-a due to changes in PXR/CAR/VDR mediated transactiva- chip platform, which mimics human physiology at tissue and tion of phase 0-III genes. The epigenome landscape also organ levels, may be used in the near future as a preferred contributes to interindividual variations in drug response, alternative to animal models for screening drug candidates since such a landscape is shaped by endogenous regulatory (Figure 5). molecules and exogenous factors that are as varied as Disparate drug response among individuals results from lifestyle, food habits, pollution and psychological disposi- altered activity or expression of DMEs/transporters due to tion. single nucleotide polymorphisms (SNPs) in coding regions An integrated scheme linking genetic and epigenetic or in PXR-/CAR-/VDR/HNF4-훼-regulated genomic loci; factors to drug metabolism/disposition, and interindividual

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NR-Regulated Drug Metabolism and Disposition Transcriptional Regulation Post-Translational Modification Drug Herb/Food (Phosphorylation, Ubiquitination, Drug (autoregulation: regulation by Sumoylation, Acetylation) other TFs and various NRs) B A B

NR(PXR, CAR, VDR)

A A A A

D Phase 0 Phase I Phase II Phase III

C E EE E E

Micro RNAs LncRNAs C Histone modification DNA Methylation C A C C

XRE C additional regulatory D loci (SNP) Genetic polymorphism Phase 0-III mediators (SNP) (DMEs, Transporters) CYP, PXR, CAR, VDR D F F Interaction Altered expression Inter-individual or activity differences in drug response TF = Transcription factor

Figure 6: NR-mediated regulation of drug metabolism, drug disposition: control at multiple steps. Transcriptional regulation primarily dictates NR expression and its cellular abundance (box at the upper right corner). Post-translational modification modulates NR stability and NR activity (box at upper left corner). (A) Drugs activate xenobiotic NRs (PXR, CAR), which in turn modulate the expression of phase 0-III mediators via induction of XREs. Ligand-activated VDR also induces DME and transporter expression. (B) Drug-drug, drug-herb, drug-food interactions cause altered NR expression/activity leading to altered expression of DME/transporter. An interfering agent (such as a second drug or a dietary constituent) may also modulate DME/transporter activity via competitive or . (C) Histone modification and DNA methylation modulate NR expression; they also modulate NR-regulated DME/transporter expression due to epigenetic changes at or near XREs. (D) SNP at an XRE or at an alternate regulatory locus of phase 0-III genes leads to a change in the NR interaction with the response element, which alters DME and transporter expression. SNP in coding regions of PXR/CAR/VDR/HNF4-훼, DMEs or transporters can alter the activity or cellular abundance of these proteins/enzymes. (E) Micro RNAs and long non-coding RNAs (lncRNAs) regulate the cellular abundance of NRs and mediators of phase 0-III processes. (F) Interindividual differences in drug response stem from SNP at an XRE, at another NR-interacting regulatory locus of the target gene, or at the coding region of NRs (PXR/CAR/VDR/HNF4-훼) or phase 0-III mediators.

variations in drug response is presented in Figure 6. In PXR, pregnane X receptor; CAR, constitutive androstane the era of personalized medicine, all of these regulatory receptor; VDR, vitamin D receptor; FXR, farnesoid X factors must be taken into consideration before deciding receptor; LXR, liver X receptor; CYP, cytochrome P450; on a medicinal regimen that provides optimal therapeutic DME, drug-metabolizing enzyme; ADME, absorption, dis- efficacy and minimal toxicity, while preventing adverse drug tribution, metabolism, excretion; DDI, drug-drug interac- reactions. tion; PTM, post-translational modification; MDR, multi- drug resistance; ABC, ATP-binding cassette; HDAC, histone Abbreviations deacetylase; HAT, histone acetyltransferase; HMT, his- tone methyltansferase; HMD, histone ; DNMT, NR, nuclear receptor; DBD, DNA-binding domain; LBD, DNA methyltransferase; SNP, single nucleotide polymor- ligand-binding domain; XRE, xenobiotic response element, phism.

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