An Update on ABCB1 Pharmacogenetics

Home , Pharmacogenomics

An update on ABCB1 pharmacogenetics: insights from a 3D model into the location and evolutionary conservation of residues corresponding to SNPs associated with drug pharmacokinetics

SJ Wolf1,6, M Bachtiar1,6, The human ABCB1 protein, (P-glycoprotein or MDR1) is a membrane-bound 1 2 3 glycoprotein that harnesses the energy of ATP hydrolysis to drive the J Wang , TS Sim , SS Chong unidirectional transport of substrates from the cytoplasm to the extracellular 1,4,5 and CGL Lee space. As a large range of therapeutic agents are known substrates of ABCB1 protein, its role in the onset of multidrug resistance has been the focus of 1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of much research. This role has been of particular interest in the field of Singapore, Singapore, Singapore; 2Department pharmacogenomics where genetic variation within the ABCB1 gene, of Microbiology, Yong Loo Lin School of Medicine, particularly in the form of single nucleotide polymorphisms (SNPs), is National University of Singapore, Singapore, believed to contribute to inter-individual variation in ABCB1 function and Singapore; 3Department of Pediatrics, Yong Loo Lin School of Medicine, National University of drug response. In this review we provide an update on the influence of Singapore, Singapore, Singapore; 4Division of coding region SNPs within the ABCB1 gene on drug pharmacokinetics. By Medical Sciences, National Cancer Centre, utilizing the crystal structure of the mouse ABCB1 homolog (Abcb1a), which Singapore, Singapore and 5Duke-NUS Graduate is 87% homologous to the human sequence, we accompany this discussion Medical School, Singapore, Singapore with a graphical representation of residue location for amino acids Correspondence: corresponding to human ABCB1 coding region SNPs. Also, an assessment Professor CGL Lee, Department of of residue conservation, which is calculated following multiple sequence Biochemistry, Division of Medical Sciences, alignment of 11 confirmed sequences of ABCB1 homologs, is presented and National Cancer Center, Level 6, Lab 5, 11 discussed. Superimposing a ‘heat map’ of residue homology to the Abcb1a Hospital Drive, Singapore 169610, Singapore. E-mail: [email protected] crystal structure has permitted additional insights into both the conservation of individual residues and the conservation of their immediate surroundings. Such graphical representation of residue location and conservation supple- ments this update of ABCB1 pharmacogenetics to help clarify the often confounding reports on the influence of ABCB1 polymorphisms on drug pharmacokinetics and response. The Pharmacogenomics Journal (2011) 11, 315–325; doi:10.1038/tpj.2011.16; published online 31 May 2011

Keywords: ABCB1; P-glycoprotein; MDR1; SNP; drug transporter; drug resistance

ABCB1 is involved in multidrug resistance and altered drug pharmacokinetics 6These authors contributed equally to this work. ABCB1 (P-glycoprotein or P-gp/MDR1) is a member of the ATP-binding cassette Received 17 August 2010; revised 16 March 2011; accepted 22 March 2011; published superfamily of proteins and is responsible for the unidirectional efflux of online 31 May 2011 substrates across cellular membranes.1 It has been widely associated with ABCB1 pharmacogenomics review SJ Wolf et al 316

multidrug resistance (MDR) during the treatment of various or altered mRNA production/processing via genetic diseases, including cancer, epilepsy, heart disease and polymorphisms may be significant. This is particularly true AIDS.2,3 The MDR phenotype may manifest itself through where alterations to substrate-binding sites or highly differences in drug efficacy or through more subtle effects conserved regions of the NBD potentially impact protein such as the onset of adverse side effects and altered drug– function and the efflux or pharmacokinetics of small drug interactions.4,5 molecules. It is important to note early that the role of ABCB1 in drug resistance and pharmacokinetics is often complicated by additional factors that may affect the proteins expression or Single nucleotide polymorphisms (SNPs) in the ABCB1 location. Drug pharmacokinetics may be altered for example gene may contribute to inter-individual differences following the activation of the transcription factor in drug response pregnane-X-receptor (PXR) and the subsequent upregula- tion of ABCB1.6 In this case, various small molecules In recent years, great interest has been generated with activate PXR causing it to form a heterodimer with the regards to the influence of genetic variation at the ABCB1 retinoid X receptor-a , which subsequently binds to response gene on the expression and function of the protein. The elements within the ABCB1 promoter region. Small mole- most common form of genetic variation is the SNP and, cules that activate PXR may not necessarily be substrates depending on the location of the SNP in the gene, the of ABCB1 themselves and it is through this mechanism mechanism by which their influence is exerted will differ. that drugs administered simultaneously with ABCB1 sub- SNPs located in the intronic or untranslated regions of the strates, may exacerbate the effect of ABCB1 on drug gene may alter mRNA generation, integrity or processing. resistance. Evidence also suggests that ABC proteins, if not Those located in the coding regions, however, may alter ABCB1 itself, localize to the lipid bilayers of intracellular protein structure directly, producing amino-acid substitu- vesicles where they facilitate the uptake of drug into tions (non-synonymous SNPs) or altering the mRNA lysosomes or endosomes for further processing.7,8 Cross sequence, and possibly its structure and stability, while specificity of substrates with alternative drug transporters is encoding the same amino acid (synonymous SNPs). The particularly important where these alternative transporters mechanisms driving SNP inheritance have been reviewed directly influence the intracellular concentrations of ABCB1 previously, specifically in relation to the ABC transport substrates. proteins.21,22 Although there is a wide consensus on the marginal effect The influence of SNPs may be manifested through the of genetic variants to the plasma pharmacokinetics of drugs, onset of adverse side effects, altered drug efficacy or varying degrees of evidence exist on the effect of drug multidrug resistance profile. The role of ABCB1 in drug transporter polymorphisms on intracellular or cerebrospinal resistance as well as its contribution to inter-individual fluid changes on drugs concentration.9 Moreover, the differences in drug response has received a growing amount contribution of ABCB1 to multidrug resistance is well of attention over the past decade leading to a number of established and this has been attributed to its overall comprehensive reviews on the topic.3,9,18,23 Despite this, structure and function, which has been studied exten- with the recent crystallization of the mammalian ABCB1,12 sively1,10 since its discovery in 197611 to the crystallization the discovery of additional SNPs at the ABCB1 locus and a of the first mammalian homolog, the mouse ABCB1 substantial increase in the number of association studies homolog Abcb1a.12 It is well known that substrates of involving ABCB1, an update on role of ABCB1 polymorph- ABCB1 have a wide range of structural and chemical isms in the pharmacokinetics of drugs is warranted. By diversity. These diverse small molecule substrates have been exploiting the high resolution mammalian Abcb1a crystal proposed to bind to residues located on the internal surface structure, we are now able to map coding region SNPs to of the substrate-binding region, which is essentially a large their corresponding positions within the 3D structure, open cavity composed of 12 transmembrane domains that providing greater insight into their respective locations span the lipid bilayer.1,12 Connected to these transmem- and relationships with both conserved regions, and neigh- brane domains are cytoplasmic nucleotide-binding domains boring residues, which may also correspond to coding (NBDs) where ATP binding and hydrolysis occurs,13,14 region SNPs. Hence in this review, in addition to an update facilitated by several highly conserved motifs15–18 including on ABCB1 pharmacogenetic association studies, insights the Walker A, Walker B, Signature C motif, A-loop, D-loop, from 3D modeling into the location and evolutionary H-loop and Q-loop. These motifs are common to most conservation of residues corresponding to SNPs associated ABCB1 homologs and alternative members of the ABC with drug pharmacokinetics are also discussed. A flash superfamily alike and it has been proposed that each movie showing the corresponding location on the 3D contributes to an ATP-binding pocket that forms during structure of the various SNPs associated with functional NBD dimerization.19 Substrate dissociation and efflux result differences is also provided (http://pfs.nus.edu.sg/demo_src/ from conformational changes in the protein that occurs abcb1.html). A detailed explanation on the methods of upon ATP binding.19,20 homology calculation and the tertiary location of SNPs With ABCB1 actively transporting such a wide range of based on the mouse Abcb1a protein crystal structure is substrates,18 the consequences of amino acid substitution, included as Supplementary Material.

The Pharmacogenomics Journal ABCB1 pharmacogenomics review SJ Wolf et al 317

ABCB1 is polymorphic and has been well conserved Table 2). All residues corresponding to sSNPs in ABCB1 are during evolution conserved between human and mouse (shaded blue in Table 1, Figure 1) whereas five residues (R442, L554, T558, S565 The human ABCB1 protein sequence is 87% identical to the and I598; #s8, #s11, #s12, #13 and #s14 respectively M. musculus homolog Abcb1a (Supplementary Figure 1). in Figure 2c) are conserved in all 11 organisms examined Eleven confirmed ABCB1 homologs from different organ- (Table 1). Of the 42 nsSNPs, 37 (88.1%) can be mapped to isms are available on NCBI’s Homologene resource (Supple- the M. musculus crystal structure based on a pair-wise mentary Table 1). Multiple sequence alignment using the alignment method using ClustalW algorithm (Table 1, ClustalW program24,25 (accessible at http://www.ebi.ac.uk/ Supplementary Figure 1).24,25 The major amino-acid alleles Tools/msa/clustalw2/) highlights that the MDR1 protein of 31 of the 37 nsSNPs (83.7%) are conserved between sequence is reasonably well conserved from E. coli to human and mouse (Table 1, Figure 1). Of these, just two humans, although it is only a half transporter in the lower residues (E566, and D1088; #ns10 and #ns28 in Figure 2b organisms (Sav1866 and MsbA) (Figure 1). In this manu- respectively) are conserved in all 11 organisms examined, script, SNP nomenclature begins with the prefix ‘E’, which whereas 12 are conserved in at least 7 of the 11 organisms indicates the exon location of the polymorphism, in the examined (Table 1). The global view of SNP architecture at ABCB1 gene. A total of 66 coding SNPs, including 24 the ABCB1 locus reveals that the majority of the amino acid synonymous (sSNPs—prefix ‘s’ in the Figures and Table) and substitution sites lie within the NBDs, close to the highly 42 non-synonymous (nsSNPs—prefix ‘ns’ in the Figures and conserved Walker A and Walker B motifs as well as in close Table) SNPs, at the human ABCB1 locus were found in the proximity to the ABC signature motif C (Figure 1, Figures 2b latest dbSNP database (Build 131) (Table 1 and Supplementary and c). It is observed that the NBDs have B6.2 SNPs per 100

Figure 1 Homology map derived from the multiple sequence alignment of 11 ABCB1 homologs. Black bar height indicates the number of identical residues between the 11 homologous sequences at the respective amino-acid site (between 11 and 0 identical residues may occur). The synonymous single nucleotide polymorphism and non-synonymous single nucleotide polymorphism (sSNPs and nsSNPs) whose corresponding residues can be mapped into the mouse protein are represented with blue and red dots, respectively. The C-terminal portion of the alignment where the bacterial half transporter sequences of Sav1866 and MsbA were repeated to produce a full sequence is marked with an arrow. This map also depicts the putative P-glycoprotein secondary structure shown in a linear chain format modified from the Protein Database (PDB). Conserved regions essential for protein function are highlighted both in the alignment and Abcb1a crystal structure.

The Pharmacogenomics Journal ABCB1 pharmacogenomics review SJ Wolf et al 318

Table 1 Genetic conservation of amino acids corresponding to ABCB1 coding region SNPs

Amino acid residue housing SNP a Mapped to Conservation (%) SNP (amino acid Abcb1a domain D. # rsNo H. C. l. M. G. C. A. S. S. E. P. Individual Regional 3 substitution) (internal/external melanoga Sapiens Familiaris Musculus gallus elegans thaliana pombe aureus coli falciparum structure surface) ster

1 - rs28381804 E3/49T>C (F17L) F17 W16 S16 Y17 Y35 F39 - A42 - - G11 -- 2 - rs41304191 E3/55C>T (L19L) L19 M18 M18 I19 G37 P41 - E44 - - L13 - - - 3 - rs76199854 E3/57G>A (L19L) L19 M18 M18 I19 G37 P41 - E44 - - L13 - - 4 - rs9282564 E3/61A>G (N21D) N21 K20 K20 N21 N39 K43 - H46 - - I15 - - 5 ns1 rs1202183 E5/131A>G (N44S) N44 N43 G43 S55 T70 T81 D16 D88 K10 A21 P50 18.2 38.7 6 ns2 rs41315618 E5/178A>C (I60L)TM1 I60 I59 I59 A71 I86 A97 G32 G104 K26 N37 L66 27.3 46.6 7 ns3 rs9282565 E5/239C>A (A80E) A80 A79 A79 V91 I106 I117 G52 Y124 N46 G57 N86 36.4 29.1 8 - rs35810889 E5/266C>T (M89T) - M89 F89 S85 T96 I112 ------9 ns4 rs61607171 E7/431T>C (I144T) TM2 I144 I145 I140 V152 N168 I176 A98 A172 L93 V97 L124 36.4 40.9 10 ns5 rs61122623 E7/502G>A (V168I) V168 V169 V164 A176 S192 S200 T122 A196 V117 T121 G148 54.5 46.2 11 s1 rs1128500 E8/540C>T (S180S) S180 S181 S176 S188 E204 S212 L136 N208 E129 E133 E160 27.3 42.0 12 ns6 rs60419673 E8/548A>G (N183S) TM3 N183 N184 N179 N191 K207 E215 Q139 Q211 K132 A136 S163 27.3 39.9 13 ns7 rs1128501 E8/554G>T (G185V) G185 G186 G181 G193 G209 G217 F141 G213 F134 S138 G165 45.5 40.9 14 s2 rs1128502 E8/555A>T (G185G) G185 G186 G181 G193 G209 G217 F141 G213 F134 S138 G165 45.5 40.9 15 s3 rs2235022 E9/729A>G (E243E) E243 E244 E239 E251 E267 E275 I199 Q271 R192 M196 S223 18.2 33.3 TM4 16 s4 rs28381867 E9/738G>A (A246A) A246 A247 A242 A254 R270 M278 E202 V274 A195 T199 Y226 9.1 34.5 17 ns8 rs36008564 E9/781A>G (I261V) C-NBD (Internal) I261 I262 I257 V267 I285 I293 V217 I289 I210 H214 I241 36.4 50.3 18 s5 rs80153317 E10/879T>C (I293I) TM5 I293 I294 I289 I301 L317 M325 L249 I321 R242 S246 F273 18.2 36.3

19 ns9 rs2229109 E12/1199G>A (S400N) N-NBD (Internal)S400 S401 S396 N408 T424 Q439 T355 V428 Q348 T350 H386 18.2 60.7

20 s6 rs1128503 E13/1236C>T (G412G) N-NBD (External) G412 G413 G408 G420 G436 K451 D367 N440 D359 N361 D398 27.3 55.9

21 s7 rs35068177 E13/1308A>G (T436T) T436 T437 T432 T44 I460 C475 V391 I464 L383 I385 I422 54.5 64.6 22 s8 rs41311775 E15/1326G>A (R442R) R442 R443 R438 R450 R466 R481 R397 R470 R389 R391 R428 100.0 54.5 23 s9 rs35633772 E15/1617C>T (I539I) I539 I540 I535 I547 I563 I578 I494 I576 L486 I489 I571 90.9 65.8 24 s10 rs60247941 E15/1632C>T (A544A) A544 A545 A540 A552 A568 A583 A499 A581 I491 A494 A576 63.6 65.1 N-NBD (Internal) 25 s11 rs2235012 E15/1662G>C (L554L) L554 L555 L550 L562 L578 L593 L509 L591 L501 L504 L586 100.0 73.6 26 s12 rs56871767 E15/1674G>A (T558T) T558 T559 T554 T566 T582 T597 T513 T595 T505 T508 T590 100.0 78.7 27 s13 rs59697741 E15/1695C>T (S565S) S565 S566 S561 S573 S589 S604 S520 S602 S512 S515 S597 100.0 75.4 28 ns10 rs28381902 E15/1696G>A (E566K) E566 E567 E562 E574 E590 E605 E521 E603 E513 E516 E598 100.0 76.6 29 ns11 rs28381914 E16/1777C>T (R593C) R593 R594 R589 R601 R617 R632 R548 R630 T540 E543 R627 54.5 67.3 N-NBD (External) 30 ns12 rs56107566 E16/1778G>A (R593H ) R593 R594 R589 R601 R617 R632 R548 R630 T540 E543 R627 54.5 67.3 31 s14 rs28381915 E16/1794C>T (I598I) I598 I599 I594 I606 I622 I637 I553 I635 I545 I548 I632 100.0 65.5 N-NBD (Internal) 32 ns13 rs2235036 E16/1795G>A (A599T) A599 A600 A595 A607 I623 V638 C554 V636 V546 V549 F633 54.5 63.6 33 ns14 rs57001392 E16/1837G>T (D613Y) N-NBD (External)D613 D614 D609 S621 R637 Q652 E568 N650 R560 N563 D677 45.5 60.5 34 - rs35657960 E17/1985T>C (L662R) L662 L663 L658 E671 M694 K698 E615 A708 - - E736 0.0 - 35 - rs35023033 E17/2005C>T (R669C)- R669 R670 R665 R678 I702 D705 S662 T715 - - N743 0.0 - 36 - rs59340265 E17/2037C>T (D679D) D679 D680 D675 N688 D712 N715 S632 N725 - - E753 9.1 - 37 ns15 rs41316450 E18/2207T>A (I736K) TM7 I736 I737 V732 I745 M779 I771 V682 I820 I37 L48 V814 72.7 36.7 38 ns16 rs77144566 E19/2281A>C (A761S) TM8 A761 V763 I757 A769 V802 I796 G706 I844 I647 G655 L836 63.6 42.1 39 ns17 rs41305517 E21/2398G>A (D800N) C-NBD (Internal)D800 D801 D796 D808 H841 D835 E745 D883 S108 P112 E875 9.1 45.4 40 ns18 rs2235039 E21/2401G>A (V801M) C-NBD (External)V801 V802 V797 M809 I842 V836 V746 V884 A109 V113 M876 63.6 47.6 41 ns19 rs2032581 E22/2485A>G (I829V) I829 I830 T825 T837 I870 T864 V744 S912 I135 S139 L904 45.5 30.2 42 s15 rs28381966 E22/2505A>G (V835V) V835 V836 V831 L843 T876 T870 L780 T918 N141 T145 F911 45.5 33.0 TM9 43 ns20 rs28381967 E22/2506A>G (I836V) I836 I837 I832 I844 V877 I871 L781 V919 I142 V146 F911 63.6 28.5 44 ns21 rs36105130 E22/2547A>G (I849M) I849 I850 I845 I857 I890 D884 I794 L932 I155 M159 M924 81.8 44.7 45 s16 rs9282563 E22/2650C>T (L884L) L884 L885 L880 K892 V925 M919 R829 E967 R190 K194 I959 27.3 31.2 TM10 46 ns22 rs2032582 E22/2677G>T/A (S893A/T) S893 A894 S889 A901 S934 C928 S838 S976 V199 V203 P982 45.5 35.7 47 ns23 rs56849127 E25/2975G>A (S992N) S992 S993 S988 S1000 T1035 L1027 G937 F1075 V298 T302 M1081 36.4 38.4 TM12 48 ns24 rs72552784 E25/2995G>A (A999T) A999 A1000 A995 A1007 A1042 Q1034 V944 T1082 T305 Q309 E1089 27.3 36.5 49 s17 rs2235044 E26/3084G>A (P1028P) P1028 P1029 P1024 P1036 - P1063 P973 V1111 P332 V334 I1117 0.0 33.0 50 ns25 rs28401798 E26/3151C>G (P1051A) P1051 P1052 P1047 K1059 E1093 Q1086 I996 K1135 P355 P357 P1142 18.2 57.6 C-NBD (External) 51 ns26 rs2707944 E26/3188G>C (G1063A) G1063 G1064 G1059 G1071 G1105 G1098 G1008 G1147 G367 G369 K1154 45.5 53.8 52 s18 rs2707943 E26/3189C>G (G1063G) G1063 G1064 G1059 G1071 G1105 G1098 G1008 G1147 G367 G369 K1154 45.5 53.8 53 ns27 rs74755520 E26/3222A>C (C1074W) C-NBD (Internal) C1074 C1075 C1070 C1082 C1116 C1109 S1019 C1158 G378 S380 S1165 63.6 67.8 54 ns28 rs57521326 E26/3262G>A (D1088N) D1088 D1089 D1084 D1096 D1130 D1123 D1033 D1172 D392 D394 D1179 100.0 53.9 55 ns29 rs41309225 E27/3295A>G (K1099E) K1099 K1100 K1095 I1107 S1141 C1134 R1044 V1183 H403 H405 I1237 18.2 38.5 C-NBD (External) 56 ns30 rs55852620 E27/3320A>C (Q1107P) Q1107 Q1108 Q1103 Q1115 E1149 T1142 R1052 N1191 G411 A413 R1245 27.3 43.7 57 ns31 rs35730308 E27/3322T>C (W1108R) W1108 Q1109 W1104 Q1116 H1150 N1143 S1053 D1192 S412 S414 D1246 27.3 43.5 58 s19 rs34748655 E27/3396C>T (A1132A) C-NBD (Internal) A1132 A1133 A1128 A1140 I1174 S1167 M1077 V1216 L436 A438 K1270 27.3 56.8 59 ns32 rs41309228 E27/3410G>T (S1137I ) S1137 S1138 S1133 S1145 P1179 A1172 S1082 S1220 P440 E443 - 18.2 41.8 C-NBD (External) 60 ns33 rs2229107 E27/3421T>A (S1141T) S1141 S1142 S1137 S1149 T1183 T1176 D1086 S1224 D444 R447 T1277 45.5 50.9 61 s20 rs1045642 E27/3435C>T (I1145I) C-NBD (Internal) I1145 I1146 I1141 I1153 V1187 I1180 I1090 M1228 V447 I450 V1281 72.7 57.6

62 ns34 rs59241388 E28/3502A>G (K1168E) K1168 R1169 R1164 R1176 R1210 R1203 C1113 L1251 E470 V473 N1304 27.3 61.9 C-NBD (External) 63 ns35 rs41309231 E29/3669A>T (E1223D) E1223 E1224 E1219 E1231 E1265 E1258 V1168 Q1306 K525 K528 D1359 27.3 60.2 64 s21 rs2235051 E29/3747C>G (G1249G) C-NBD (Internal) G1249 G1250 G1245 G1257 G1291 G1284 G1194 G1332 G551 G554 T1392 63.6 63.0 65 ns36 rs45456698 E29/3751G>A (V1251I) V1251 V1252 V1247 V1259 I1293 V1286 V1196 I1334 I553 I556 V1394 81.8 59.2 C-NBD (External) 66 ns37 rs35721439 E29/3767C>A (T1256K) T1256 T1257 T1252 T1264 T1298 D1291 N1201 T1339 T558 T561 T1399 72.7 59.4 aThe conservation of residues corresponding to all coding regions SNPs was obtained following multiple sequence alignment of 11 confirmed ABCB1 homolog protein sequences. For each species the amino acid, as well as its position in the corresponding sequence, is indicated. Please refer to the Supplementary Materials for a description of the regional conservation score. SNPs of particular pharmacogenetic interest are marked in bold and flanked by the same color that corresponds to the SNP ID in Figures 2–4. Highlighted in faded blue are the rows containing synonymous SNPs. Shaded in green is the sequence alignment obtained after re-using the same bacterial half-transporter sequence that were used for the first half of the alignment. The SNP nomenclature (that is #ns or #s) is similar to what were used throughout this article.

amino acids whereas the transmembrane domains have only Review of SNPs associated with protein function or B3.7 SNPs per 100 amino acids. In fact, one ns-SNP (E26/ expression 3222A4C (C1074W), ns#27 in Figure 2b) lies within the Walker A motif of the C-NBD itself (Table 1, Figure 1). Of the Two sSNPs and 12 nsSNPs have been associated 14 amino-acid substitution sites located outside of the NBDs with differences in function or expression of the ABCB1 five are located on the external surface of the protein and protein (Supplementary Table 2). Four of the 12 associated three on the internal surface of the substrate-binding cavity nsSNPs (E3/61A4G, E5/266C4T, E17/1985T4Cand (Figure 2). E17/2005C4T) cannot be mapped to the mouse crystal

The Pharmacogenomics Journal ABCB1 pharmacogenomics review SJ Wolf et al 319

structure as they lie in an uncrystallized region of the structure of Abcb1a. The individual conservation scores of protein. each residue in the list, as well as five residues on either side of these amino acids (based on the mRNA sequence), was The residues corresponding to SNPs E13/1236C4T, averaged and assigned to the corresponding residue as the E22/2677G4T/A and E27/3435C4T are varied in regional conservation score (Table 1). With this five amino- their evolutionary conservation and located in distant acids method, we were able to obtain a finer spread of data regions of the protein and demarcate the regions with strong difference in conservation scores as compared with using more residues Of the 14 SNPs associated with drug response or pharmaco- in the calculation. kinetics, three in particular have been researched SNP E13/1236C4T (#s6 in Figure 2c) correlates to a extensively.26–31 At the DNA level, these three SNPs are glycine residue located on the external surface of the N- regularly identified to be in high linkage disequilibrium29 terminal NBD (Figures 1 and 2c). It lies within a b-sheet and and are present at highly variable frequencies in different is located between the functional A-loop (10 amino acids populations (Supplementary Table 2). Two of these SNPs are away) and the Walker A motif (15 amino acids away). synonymous (E13/1236C4T (#s6 in Figure 2c) and E27/ Importantly, this polymorphism has an individual conser- 3435C4T (#s20 in Figure 2c)) whereas E22/2677G4T/A vation score of only 28% suggesting that variation in (#ns22 in Figure 2b) is a non-synonymous triallelic poly- residue properties have been tolerated during the evolution morphism that confers a Ser to Ala/Thr amino-acid of the protein (Table 1, Figure 3a). Other residues found substitution at residue position 893. When mapped to the at this position in homologous structures include lysine, crystal structure of Abcb1a, both synonymous SNPs are aspartic acid and asparagine (Table 1). Each residue differs found to lie in different regions of the protein (Figure 2c; see from glycine in their polarity and side chain charge and flash movie at http://pfs.nus.edu.sg/demo_src/abcb1.html), all three are more hydrophilic (À3.5 to À3.9 versus À0.4). located more than 50 A˚ of each other and have very This tolerance of variation is important given any effect different levels of conservation. from E13/1236C4T is likely to be exerted at the mRNA A full description of regional conservation score calcula- level. tion is presented in the Supplementary Material and in The other synonymous SNP, E27/3435C4T (I1145) Table 1. Briefly, a list of amino acids located within 10 A˚ of (#s20 in Figure 2c), is highly conserved (73%) across the the residue in question was obtained from the crystal 11 confirmed sequences of ABCB1 homologs (Table 1,

Figure 2 (a) A global view of residue conservation following the multiple sequence alignment of 11 ABCB1 homologs using the ClustalW algorithim (see Supplementary Table 1 and Table 1). A heat map with differing colors depending on the residue conservation score is shown (blue: low conservation, red: high conservation). (b) Global view of amino-acid residues corresponding to non-synonymous single nucleotide polymorphisms (SNPs) (prefixed as ‘ns’) and (c) synonymous SNPs (prefixed as ‘s’) within the human ABCB1 gene mapped to the mouse Abcb1a crystal structure (PDB: 3G5U). Identical residues between human and mouse are represented as blue (sSNPs) and red (nsSNPs) balls/fonts. Those residues that are not homologous are represented as green balls/fonts. The conserved Walker A (light blue), Walker B (pink) and signature C motifs (dark blue) are also highlighted as colored lines. Number labels correspond with the data/labels in Table 1 and Supplementary Table 2. The residue housing SNPs of pharmacogenomics interest are consistently highlighted by the same colors in Figures 2–4 and Table 1 and Supplementary Table 2.

The Pharmacogenomics Journal ABCB1 pharmacogenomics review SJ Wolf et al 320

Supplementary Table 1) and the isoleucine residue is Evidence in support of this hypothesis is, however, limited observed at a corresponding site in all confirmed ABCB1 and the majority of studies focus on the haplotype of homolog sequences (Supplementary Table 1) except that of this SNP together with SNPs E13/1236C4T (#s6 in Figure 2c) C. elegans, S. aureus and P. falciparum where a valine is and E22/2677G4T/A (#ns22 in Figure 2b), rather than present at the corresponding sites (Figure 1, Table 1). Ile1145 E27/3435C4T (I1145I) (#s20 in Figure 2c) on its own. is located in the internal regions of the C-terminal NBD Like E27/3435C4T, E22/2677G4T/A (#ns22 in Figure 2b) (Figure 2b). As both isoleucine and valine are non-polar, is also more conserved than the average conservation of its hydrophobic with neutral side chains, it suggests that immediate surroundings. This non-synonymous SNP con- preservation of these properties at this site may be fers one of two amino-acid substitutions at position 893 important during the evolution of the protein. Similar to (Ser893Ala/Thr). However, with an individual conservation the E13/1236C4T, the variability of SNP E27/3435C4T may score of 46% and a regional conservation score of 36%, it be influenced at the transcript level or perhaps the seems that both the residue and the region in which it lies methionine 1228 residue, which is observed in S. pombe may have undergone a degree of variation over the course of actually arises due to a mutation at the third nucleotide of evolution (Table 1, Figure 3b). Residues such as Ala, which is the isoleucine codon from ATC to ATG. As is evident from represented at the corresponding position in the ABCB1 Figure 3c, residue 1145 lies directly adjacent to the highly homologs of C. l. Familiaris and G. gullus, as well as Cys, conserved signature C motif, which is essential for protein which is found in the homologs of D. melanogaster, and Val, function, specifically the binding and hydrolysis of ATP.32,33 which is found in the bacterial homologs Sav1866 and Despite variation in their level of conservation, and residing MsbA at amino-acid positions 199 and 203, respectively, in opposing NBDs, the regional conservation scores for these (Table 1) all differ from Ser not only in side-chain polarity SNPs are highly similar at 56% and 58% for E13/1236C4T but also in residue hydropathy index: 1.8, 2.5, 4.2 and À0.8, and E27/3435C4T, respectively. With the overall regional respectively.37 conservation score 450%, it seems that the region in which To date there have been a vast number of reports these SNPs lie is slightly more conserved than the average of correlating the presence of these three SNPs with drug all other SNPs. response or disease progression (see Supplementary Table 3). It has recently been postulated that the sSNP E27/ Some reports highlight individual SNPs as being causative, 3435C4T (I1145I) (#s20 in Figure 2c) may alter the tertiary however, in the majority of cases it is the effect of SNP structure of P-gp, and subsequently its stability or expres- haplotypes that was assessed. Despite this, evidence for sion, when a ribosomal stall occurs during mRNA transla- the influence of E13/1236C4T, E22/2677G4T/A and E27/ tion. It is proposed that this ribosomal stall transpires from 3435C4T on various drug pharmacokinetics and response the presence of a rarer codon and alters the rate of mRNA remains controversial. Many contradictory results are translation and chaperone-facilitated protein folding.34–36 reported and as such no definitive conclusion has been

Figure 3 Location and conservation of (a) E13/1236C4T (G412G), (b) E22/2677G4T/A (S893A/T) and (c) E27/3435C4T (I1145I). Individual residue scores are mapped to the ribbon model with each colour representing a percentage of conservation where 100% is equal to 11 species expressing the same residue at the corresponding position. A heat map with differing colors depending on the conservation score is shown (blue: low conservation, red: high conservation).

The Pharmacogenomics Journal ABCB1 pharmacogenomics review SJ Wolf et al 321

presented to explain their influence on drug response. The individual conservation scores are 36, 27 and 45% for Evidence indicates genetic variation is most prominent I144, N183 and G185, respectively, (Figure 4a, Table 1) at the population level yet many studies fail to state the whereas the average of their regional conservation scores is genetic history of their populations, or present data B40%. In fact, only three residues lying within 10 A˚ of this obtained from cohorts of mixed race (see Supplementary SNP cluster possess an individual conservation of greater Table 3). There have been reviews that comprehensively than 80%. This suggests that some degree of residue covered results from association studies between ABCB1 variation has been tolerated at this site over the evolution genotype and protein functions or drug pharmacoki- of the protein. It would be interesting to evaluate if these netics.3,9,38 Drawing conclusions from comparisons of many two closeby SNPs also have similar roles in influencing studies is difficult, particularly in assessing the pharmaco- conformational changes between the catalytic sites and the genetics of ABCB1. With the availability of the highly drug binding sites or influence the role of G185V. homologous mouse 3D crystal structure, we are now able to The implication of these three SNPs in drug response assess the potential consequences ABCB1 SNPs to protein remains unclear as, to date, none of these SNPs have been structure or function. This could assist in the selection of investigated and no genotype information is available for SNPs that are truly functional for future association studies. these SNPs (Supplementary Table 2). It would thus be interesting to determine the frequency of these SNPs as well as their respective haplotypes in the various populations and The classic E8/554G4T (G185V) polymorphism that to determine if drug specificity can be influenced by this influences drug specificity resides in close 3D proximity cluster of SNPs. to two other non-synonymous polymorphisms in a region that is less evolutionary conserved The evolutionary non-conserved E12/1199G4A Although research into ABCB1 polymorphisms appears (S400N), which alters drug pharmacokinetics and to focus on E13/1236C4T, E22/2677G4T/A and E27/ response, resides in an evolutionary conserved region 3435C4T, it is important to note that a number of additional non-synonymous SNPs have been associated SNP E12/1199G4A (S400N) (#ns9 in Figure 2b), which with drug pharmacokinetics, drug response, protein expres- occurs at o4% in Caucasians and Africans but is not found sion and disease progression. In the majority of cases these in Asians (Supplementary Table 2) represents another nsSNP additional SNPs with established associations can be that has been associated with drug pharmacokinetics. mapped to the crystal structure of mouse Abcb1a. Through cell-based experiments using limited substrates, One of these SNPs, E8/554G4T (#ns7 in Figure 2b) confers Kimchi-Sarfaty et al.42 inferred that substrate specificity the classic amino-acid substitution G185V that was reported was not significantly influenced by the any of the non- more than 20 years ago to confer changes in drug specificity synonymous coding SNPs including S400N. Nonetheless, of vinblastine and colchicine.39 This glycine residue was using cell-based Quantitative Structure-Activity Relation- suggested to have a pivotal role in transmitting conforma- ship (QSAR) analysis, Sakurai et al.43 concluded that the tional changes between the catalytic sites and the drug effect of SNPs on the drug transport activity appears to binding sites.40 Combining molecular dynamics simulations depend on the substrates tested. Several other studies also with atomic detail homology modeling, it was further revealed that this SNP affects drug pharmacokinetics. proposed that this improved efflux involves a reduction in Cellular uptake and permeability of five HIV protease non-polar van der Waals forces, which would otherwise inhibitors, especially ritonavir and saquinavir, was increased secure colchicine to its binding site near or at residue 185. in cells carrying the minor allele.44 This SNP was also With the substitution of glycine for a more bulky valine reported to confer resistance to vinblastine, vincristine, residue, such interactions are removed, allowing for im- paclitaxel and etoposide, but not doxorubicin.45,46 Interest- proved dissociation and efflux rates.40,41 ingly, a novel T allele of this SNP (E12/1199G4A (S400N)) Interestingly, as shown in Figure 4a, in the crystal coding for the amino acid, isoleucine (I), was identified in structure, this SNP resides in close 3D proximity with a 2.3% of leukemia patients. Presence of this novel T allele was molecular distance of 10.9 and 5.2 A˚ to two other non- found to be associated with reduced resistance to vinblas- synonymous SNPs, namely E7/431T4C (I144T) (#ns4 in tine, vincristine, paclitaxel and doxorubicin compared with Figure 2b) and E8/548A4G (N183S) (#ns6 in Figure 2b), patients carrying the major G allele encoding for serine. On respectively. Nonetheless, as only one of many possible the other hand, patients carrying the more common minor A conformations of P-gp is represented in the mammalian allele encoding the asparagine amino acid exhibit increased Abcb1a crystal structure,12 there is a possibility that residues resistance to these drugs compared with those carrying the that may reside close to one another in one conformation of major G allele.47 In ovarian cancer patients treated with the 3D structure may not be close together in a different paclitaxel, the E12/1199G4A/T SNP affected progression-free conformation. Hence, we also mapped these SNPs to survival. Although only two patients with heterozygous (G/A) the ADP-bound half-transporter Sav1866 homolog from genotype were identified, their mean progression-free survival Staphylococcus aureus and found that the molecular distances was only 2 months compared with 19 months for patients are still similar (11.3 and 5.3 A˚ ) (Supplementary Figure 2). who are homozygous for the G allele.48

The Pharmacogenomics Journal ABCB1 pharmacogenomics review SJ Wolf et al 322

Figure 4 Location and conservation of (a) E8/554G4T (G185V), (b) E12/1199G4A (S400N), (c) E26/3151C4G (P1051A), (d) E27/3322T4C (W1108R), (e) E27/3421T4A (S1141T), (f) E29/3751G4A (V1251I). Individual residue scores are mapped to the ribbon model with each colour representing a percentage of conservation where 100% is equal to 11 species expressing the same residue at the corresponding position. A heat map with differing colors depending on the conservation score is shown (blue: low conservation, red: high conservation).

From the 3D crystal structure, this polymorphism (S400N) Majority of the other SNPs previously associated with was found to lie in the same turn region between two drug pharmacokinetics that could be mapped to the b-sheets as Gly412, which corresponds to the codon housing crystal structure reside at the outer surface of the the synonymous SNP E13/1236C4T (Figure 4b) (see C-terminal NBD flash movie at http://pfs.nus.edu.sg/demo_src/abcb1.html). This SNP resides next to the A-loop (Figures 1 and 4b), which Of the eight remaining SNPs to have been associated with has previously been identified as being crucial for ATP drug response or expression, four can be mapped to the binding and hydrolysis.15 The individual evolutionary mouse Abcb1a crystal structure based on pair-wise alignment conservation score for S400 is just 18% indicating it is (Supplementary Figure 1).24,25 These are E26/3151C4G poorly conserved, and differences in residue properties (P1051A) (#ns25), E27/3322T4C (W1108R) (#ns31), E27/ have been tolerated at this position over the course of 3421T4A (S1141T) (#ns33) and E29/3751G4A (V1251I) evolution (Table 1). Nonetheless, although serine is present (#ns36) (Figure 2b). It is interesting to note that each of these at the corresponding position only in human, canine and SNPs reside in the C-terminal NBD. These SNPs may have mouse, relatively conservative substitution of this residue functional significance if they reside at the surface of regions with other polar residues was observed in all other organ- of the protein that are potentially important such as the ATP- isms except for yeast where this residue is substituted with binding pockets, or reside in close proximity to such regions. the hydrophobic valine (Table 1). Interestingly, as depicted In addition, some parts of the outer surface of the protein in Figure 4b this evolutionary non-conserved residue may also have a crucial role in the dimerization of the NBDs, lies in between two completely conserved residues (con- either through their impact on ATP binding and hydrolysis, servation scores of 100%) and within a region of high or through the interactions with the opposing NBD. homology where the regional conservation score is 60.7% We highlight the E26/3151C4G (#ns25) SNP, which is (Table 1). translated to P1051A substitution (Figures 2b and 4c). This

The Pharmacogenomics Journal ABCB1 pharmacogenomics review SJ Wolf et al 323

substitution is reasonably well conserved and affects only the NBD (Figures 2b, 4d and 4e and flash movie http:// the hydropathy of the protein. Interestingly, this SNP is pfs.nus.edu.sg/demo_src/abcb1.html). Interestingly, SNP located at the surface of the C-terminal NBD that forms the E27/3421T4A (S1141T) (#ns33) occurs at reasonably high ATP-binding pocket. It resides within a turn region between frequency (44%) in the African population but is not found two opposing b-sheets in an almost similar fashion to that of in the non-African populations (Supplementary Table 2). It #ns9 E12/1199G4A (S400N) (Figure 4b), close to the well- is not a highly conserved residue and does not reside in conserved A-loop (Tyrosine 1040) and Walker A motif that is a highly conserved region of P-gp with an individual crucial for ATP activity.15,18 This residue has an individual conservation score of 45.5 and a regional conservation score conservation score of just 18.2% but have a regional of 50.9 (Table 1). Hence, it is likely that alteration in residue conservation score of B60% (Table 1, Figure 4c) suggesting properties at this site was tolerated during the evolution of that during the evolution of the protein, there is also the protein. No genotype information is available with considerable tolerance of variation at this residue but not of regards to the other SNP E27/3322T4C (W1108R) (#ns31). the surrounding residues. This SNP was found through the This SNP is even less conserved than SNP E27/3421T4A yeast-based assay to influence resistance to valinomycin (S1141T) (#ns33) and also less conserved than its immediate when it is in diplotype with E22/2677G4T/A.49 surroundings as it has an individual conservation score of Several additional SNPs have been highlighted in some only 27.3% and a regional conservation score of 43.5% studies for their influence on drug pharmacokinetics, which (Table 1, Figure 4e). Hence, these two SNPs, which were cannot be mapped to the crystal structure. These are speculated to contribute to the substrate discrimination E3/61A4G (N21D), E5/266C4T (M89T), E17/1985T4C activity of P-gp49 resides within the C-terminal NBD and are (L662R), E17/2005C4T (R669C). poorly conserved during the evolution of the protein. When in haplotype with the intensely studied SNPs SNP E29/3751G4A (V1251I) (#ns36) was found to E13/1236C4T (#s6), E22/2677G4T/A (#ns22) and increase BODIPY-FL-paclitaxel accumulation and modulate E27/3435C4T (#s20), SNP E3/61A4G (N21D), which the effect of cyclosporine A on the intracellular accumula- cannot be mapped to the 3D crystal structure, was reported tion of BODIPY-FL-paclitaxel transport.50 When mapped to to increase BODIPY-FL-paclitaxel accumulation and mod- the crystal structure, V1251I resides at the outer surface of ulate the effect of cyclosporine A on the intracellular the C-terminal NBD (Figures 2b and 4f). Notably, this SNP is accumulation of BODIPY-FL-paclitaxel transport.50 How- highly conserved throughout evolution with an individual ever, the N21D substitution was found not to influence score of 81.8% and only conservative changes were observed cyclosporine A pharmacokinetics in another study.51 In yet in 4 of the 11 species examined. Nonetheless, this highly another study, the N21D polymorphism was reported to conserved SNP resides in a region that is less conserved influence the trough but not the peak plasma levels (regional conservation score 59.2) suggesting that there concentration of methadone.52 is some tolerance of nucleotide changes in the vicinity of Using a yeast-based assay, SNP E27/3421T4A (S1141T) this SNP. (#ns33) and several SNPs that cannot be mapped to the Hence, of these several SNPs that had been previously crystal structure (E5/266C4T (M89T), E17/1985T4C associated with functional differences, most of them (L662R), E17/2005C4T (R669C)) were reported to increase (E26/3151C4G (P1051A) (#ns25), E27/3421T4A (S1141T) drug resistance to two or more drugs whereas SNP E27/ (#ns33) and E29/3751G4A (V1251I) (#ns36) reside at the 3322T4C (W1108R) (#ns31) decreased drug resistance.49 outer surface of the C-terminal NBD except for SNP E27/ Curiously, in another study, the E17/2005C4T (R669C) SNP 3322T4C (W1108R) (#ns31), which resides within the was reported to be associated with decreased resistance interior of the C-terminal NBD (Table 1, Figure 2b and flash to paclitaxel and etoposide in transfected LLC-PK1 cells.53 movie http://pfs.nus.edu.sg/demo_src/abcb1.html). Except The increased resistance by SNP E17/2005C4T (R669C) was for SNP E29/3751G4A (V1251I) (#ns36) with high indivi- found to be reversed by SNP E27/3322T4C (W1108R) dual conservation score (480%), the other three SNPs have (#ns31).49 In another study using HEK293T cells, SNP below average conservation scores (o46%). All these SNPs E27/3421T4A (S1141T) (#ns33) was reported to be less reside within 3D regions that are only moderately conserved sensitive to cyclosporine A inhibition of BODIPY-FL-pacli- throughout evolution (43–60%). taxel transport.50 It was suggested that as the resistance profiles of SNPs E27/3421T4A (S1141T) (#ns33), E27/3322T4C (W1108R) ABCB1 nsSNPs tend to reside in less evolutionary (#ns31) and diplotype E27/3322T4C (W1108R)- E17/ conserved residues 2005C4T (R669C)) display the largest variation across substrates, the region where S1141T (#ns33) and W1108R Interestingly, there seems to be some correlation between (#ns31) reside might contribute to the substrate discrimina- SNPs and the degree of residue conservation. As shown in tion activity of P-gp.49 Supplementary Figure 3A, greater percentage of nsSNPs (up The 3D crystal structure reveals that both S1141T (#ns33) to 12% of nsSNPs versus 7% of sSNPs) has low individual and W1108R (#ns31) reside at the C-terminal NBD of the residue conservation score (20–30%) suggesting that nsSNPs P-gp protein with S1141T (#ns33) residing on the outer tend to reside in less conserved amino acids. However, no surface and W1108R (#ns31) residing in the interior of clear correlation can be observed between the overall

The Pharmacogenomics Journal ABCB1 pharmacogenomics review SJ Wolf et al 324

presence of SNPs and the degree of regional conservation history. This is rarely achievable and remains a factor that score as SNPs are distributed throughout the conserved as must be addressed in future studies. well as non-conserved regions of the protein (Supplemen- As more SNPs are identified through the current state-of- tary Figure 3B). Nonetheless, although several SNPs were the-art Next Generation Sequencing technologies, it is found to reside close to highly conserved NBD regions, most pertinent to understand how changes in the sequences of of the mappable ABCB1 SNPs (39%) are associated with genes will impact the structure, and ultimately the function, amino acids with low individual and regional conservation of the proteins these genes encode. Depicting the location scores (Table 1). of residues corresponding to non-synonymous, as well as synonymous SNPs, and their evolutionary conservation, in the 3D crystal structure of mammalian P-gp provides a Concluding remarks more realistic and accurate method of SNP visualization than the more common 2D schematic diagrams. This review In conclusion, of the 66 coding SNPs found in the dbSNP highlights the feasibility of such visualization methods, database, B2 synonymous and 12 non-synonymous SNPs which may be extended for use in the interpretation of have previously been associated with functional differences. results from current association studies, and in allowing for Four of these SNPs cannot be mapped to the crystal structure an improved method of selection of exonic SNPs for of the protein. Of the remaining 10 previously associated functional validation assays. SNPs, which can be mapped to the 3D structure, none reside in the substrate-binding region highlighted by Aller et al.12 Only one SNP, E8/554G4T (G185) (#ns7) resides within the Conflict of interest membrane whereas another SNP (E22/2677G4T/A (S893A/ T) (#ns22)) resides in the region between the membrane and The authors declare no conflict of interest. the cytosolic N-terminal NBD in the crystal structure, although this residue is part of TM10 (Figure 2). The majority of the functionally associated SNPs are localized Acknowledgments at the NBD, particularly the external surface of C-terminal This work was supported by grants from the Academic Research NBD suggesting that this domain may have important roles Fund-Ministry of Education Tier 2 (MOET206B3105) to C.G. L. Lee in modulating ABCB1 function. Genotype data that are through the National University of Singapore; the National Medical available for 8 of these 14 previously associated SNPs Research Council (NMRC) (NMRC/0993/2005), Singapore to C.G.L. revealed that the frequencies of these SNPs differed greatly Lee through the National Cancer Centre, Singapore; as well as block among the various populations examined with SNP E27/ funding to C.G.L. Lee through the National Cancer Centre. 3421T4A (S1141T) (#ns33) occurring only in the African Americans (Supplementary Table 2). Except for synonymous References SNP E27/3435C4T (I1145I) (#s20) and non-synonymous SNPs E29/3751G4A (V1251I) (#ns36) and E26/3262G4A 1 Tusnady GE, Sarkadi B, Simon I, Varadi A. Membrane topology of (D1088N) (#ns28), these previously associated SNPs have human ABC proteins. FEBS Lett 2006; 580: 1017–1022. 2 Tate SK, Sisodiya SM. Multidrug resistance in epilepsy: a pharmacoge- below average individual conservation scores (o46%) and nomic update. Expert Opin Pharmacother 2007; 8: 1441–1449. only average regional 31 structure conservation scores (40– 3 Leschziner GD, Andrew T, Pirmohamed M, Johnson MR. ABCB1 60%) suggesting that there is some tolerance of variability in genotype and PGP expression, function and therapeutic drug response: these regions of the P-gp protein, perhaps facilitating a critical review and recommendations for future research. Pharmaco- genomics J 2007; 7: 154–179. differences in substrate specificity of the different poly- 4 Pal D, Mitra AK. MDR- and CYP3A4-mediated drug-drug interactions. morphs. J Neuroimmune Pharmacol 2006; 1: 323–339. The field of ABCB1 pharmacogenetics has grown con- 5 Vourvahis M, Kashuba AD. Mechanisms of pharmacokinetic and siderably in recent years. This is evident both in terms of the pharmacodynamic drug interactions associated with ritonavir-enhanced tipranavir. Pharmacotherapy 2007; 27: 888–909. number of studies associating ABCB1 polymorphisms with 6 Harmsen S, Meijerman I, Beijnen JH, Schellens JH. The role of nuclear drug pharmacokinetics, and also the number of SNPs being receptors in pharmacokinetic drug-drug interactions in oncology. identified in the gene. Despite the increase in association Cancer Treat Rev 2007; 33: 369–380. studies there is little clarification on the role ABCB1 SNPs 7 Chapuy B, Koch R, Radunski U, Corsham S, Cheong N, Inagaki N et al. Intracellular ABC transporter A3 confers multidrug resistance in have in drug pharmacokinetics or an individual’s response leukemia cells by lysosomal drug sequestration. Leukemia 2008; 22: to therapy. Although many studies attempt to investigate 1576–1586. the effect of SNPs in ABCB1 on the pharmacokinetics of 8 Rajagopal A, Simon SM. Subcellular localization and activity of multi- similar drugs, key differences in their experimental design drug resistance proteins. Mol Biol Cell 2003; 14: 3389–3399. 9 Cascorbi I. Role of pharmacogenetics of ATP-binding cassette transpor- confound the issue further. Most notably, disparities in ters in the pharmacokinetics of drugs. Pharmacol Ther 2006; 112: study cohorts make comparisons of multiple studies diffi- 457–473. cult. Inter-individual differences in drug response are most 10 Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette notable between populations of distinct genetic history and (ABC) transporter superfamily. Genome Res 2001; 11: 1156–1166. 11 Juliano RL, Ling V. A surface glycoprotein modulating drug permeability therefore true comparisons can only be made between in Chinese hamster ovary cell mutants. Biochim Biophys Acta 1976; 455: studies assessing patients of known, and equal, genetic 152–162.

The Pharmacogenomics Journal ABCB1 pharmacogenomics review SJ Wolf et al 325

12 Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R et al. Structure of 33 Tombline G, Bartholomew L, Gimi K, Tyndall GA, Senior AE. Synergy P-glycoprotein reveals a molecular basis for poly-specific drug binding. between conserved ABC signature Ser residues in P-glycoprotein Science 2009; 323: 1718–1722. catalysis. J Biol Chem 2004; 279: 5363–5373. 13 Higgins CF, Linton KJ. The ATP switch model for ABC transporters. 34 Fung KL, Gottesman MM. A synonymous polymorphism in a common Nat Struct Mol Biol 2004; 11: 918–926. MDR1 (ABCB1) haplotype shapes protein function. Biochim Biophys Acta 14 Sauna ZE, Smith MM, Muller M, Kerr KM, Ambudkar SV. The 2009; 1794: 860–871. mechanism of action of multidrug-resistance-linked P-glycoprotein. 35 Tsai CJ, Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM, J Bioenerg Biomembr 2001; 33: 481–491. Nussinov R. Synonymous mutations and ribosome stalling can lead to 15 Ambudkar SV, Kim IW, Xia D, Sauna ZE. The A-loop, a novel conserved altered folding pathways and distinct minima. J Mol Biol 2008; 383: aromatic acid subdomain upstream of the Walker A motif in 281–291. ABC transporters, is critical for ATP binding. FEBS Lett 2006; 580: 36 Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, 1049–1055. Ambudkar SV et al. A ‘silent’ polymorphism in the MDR1 gene changes 16 Gottesman MM, Pastan I, Ambudkar SV. P-glycoprotein and multidrug substrate specificity. Science 2007; 315: 525–528. resistance. Curr Opin Genet Dev 1996; 6: 610–617. 37 Kyte J, Doolittle RF. A simple method for displaying the hydropathic 17 Kim IW, Peng XH, Sauna ZE, FitzGerald PC, Xia D, Muller M et al. The character of a protein. J Mol Biol 1982; 157: 105–132. conserved tyrosine residues 401 and 1044 in ATP sites of human 38 Marzolini C, Paus E, Buclin T, Kim RB. Polymorphisms in human MDR1 P-glycoprotein are critical for ATP binding and hydrolysis: evidence for (P-glycoprotein): recent advances and clinical relevance. Clin Pharmacol a conserved subdomain, the A-loop in the ATP-binding cassette. Ther 2004; 75: 13–33. Biochemistry 2006; 45: 7605–7616. 39 Choi KH, Chen CJ, Kriegler M, Roninson IB. An altered pattern of 18 Sharom FJ. ABC multidrug transporters: structure, function and role in cross-resistance in multidrug-resistant human cells results from spontaneous chemoresistance. Pharmacogenomics 2008; 9: 105–127. mutations in the mdr1 (P-glycoprotein) gene. Cell 1988; 53: 519–529. 19 Sauna ZE, Ambudkar SV. About a switch: how P-glycoprotein (ABCB1) 40 Omote H, Figler RA, Polar MK, Al-Shawi MK. Improved energy coupling harnesses the energy of ATP binding and hydrolysis to do mechanical of human P-glycoprotein by the glycine 185 to valine mutation. work. Mol Cancer Ther 2007; 6: 13–23. Biochemistry 2004; 43: 3917–3928. 20 Sheps JA. Biochemistry. Through a mirror, differently. Science 2009; 41 Omote H, Al-Shawi MK. Interaction of transported drugs with the lipid 323: 1679–1680. bilayer and P-glycoprotein through a solvation exchange mechanism. 21 Wang Z, Wang J, Tantoso E, Wang B, Tai AY, Ooi LL et al. Signatures of Biophys J 2006; 90: 4046–4059. recent positive selection at the ATP-binding cassette drug transporter 42 Kimchi-Sarfaty C, Gribar JJ, Gottesman MM. Functional characterization superfamily gene loci. Hum Mol Genet 2007; 16: 1367–1380. of coding polymorphisms in the human MDR1 gene using a vaccinia 22 Wang Z, Wang J, Chong SS, Lee CGL. Mining potential virus expression system. Mol Pharmacol 2002; 62: 1–6. functionally significant polymorphisms at the ATP-binding- cassette 43 Sakurai A, Onishi Y, Hirano H, Seigneuret M, Obanayama K, Kim G et al. transporter genes. Curr Pharmacogenomics Personalized Med 2009; 7: Quantitative structure–activity relationship analysis and molecular 40–58. dynamics simulation to functionally validate nonsynonymous poly- 23 Zhou SF. Structure, function and regulation of P-glycoprotein morphisms of human ABC transporter ABCB1 (P-glycoprotein/MDR1). and its clinical relevance in drug disposition. Xenobiotica 2008; 38: Biochemistry 2007; 46: 7678–7693. 802–832. 44 Woodahl EL, Yang Z, Bui T, Shen DD, Ho RJ. MDR1 G1199A 24 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, polymorphism alters permeability of HIV protease inhibitors across McWilliam H et al. Clustal W and Clustal X version 2.0. Bioinformatics P-glycoprotein-expressing epithelial cells. AIDS 2005; 19: 1617–1625. 2007; 23: 2947–2948. 45 Woodahl EL, Crouthamel MH, Bui T, Shen DD, Ho RJ. MDR1 (ABCB1) 25 Thompson JD, Gibson TJ, Higgins DG. Multiple sequence alignment G1199A (Ser400Asn) polymorphism alters transepithelial permeability using ClustalW and ClustalX. Curr Protoc Bioinformatics 2002; Chapter 2, and sensitivity to anticancer agents. Cancer Chemother Pharmacol 2009; Unit 2.3. 64: 183–188. 26 Leschziner G, Zabaneh D, Pirmohamed M, Owen A, Rogers J, Coffey AJ 46 Woodahl EL, Yang Z, Bui T, Shen DD, Ho RJ. Multidrug resistance gene et al. Exon sequencing and high resolution haplotype analysis of ABC G1199A polymorphism alters efflux transport activity of P-glycoprotein. transporter genes implicated in drug resistance. Pharmacogenet J Pharmacol Exp Ther 2004; 310: 1199–1207. Genomics 2006; 16: 439–450. 47 Crouthamel MH, Wu D, Yang Z, Ho RJ. A novel MDR1 G1199T variant 27 Kim RB, Leake BF, Choo EF, Dresser GK, Kubba SV, Schwarz UI alters drug resistance and efflux transport activity of P-glycoprotein in et al. Identification of functionally variant MDR1 alleles among European recombinant Hek cells. J Pharm Sci 2006; 95: 2767–2777. Americans and African Americans. Clin Pharmacol Ther 2001; 70: 189–199. 48 Green H, Soderkvist P, Rosenberg P, Horvath G, Peterson C. ABCB1 28 Tang K, Wong LP, Lee EJ, Chong SS, Lee CG. Genomic evidence for G1199A polymorphism and ovarian cancer response to paclitaxel. recent positive selection at the human MDR1 gene locus. Hum Mol J Pharm Sci 2008; 97: 2045–2048. Genet 2004; 13: 783–797. 49 Jeong H, Herskowitz I, Kroetz DL, Rine J. Function-altering SNPs in the 29 Tang K, Ngoi SM, Gwee PC, Chua JM, Lee EJ, Chong SS et al. Distinct human multidrug transporter gene ABCB1 identified using a Sacchar- haplotype profiles and strong linkage disequilibrium at the MDR1 omyces-based assay. PLoS Genet 2007; 3: e39. multidrug transporter gene locus in three ethnic Asian populations. 50 Gow JM, Hodges LM, Chinn LW, Kroetz DL. Substrate-dependent Pharmacogenetics 2002; 12: 437–450. effects of human ABCB1 coding polymorphisms. J Pharmacol Exp Ther 30 Sai K, Kaniwa N, Itoda M, Saito Y, Hasegawa R, Komamura K et al. 2008; 325: 435–442. Haplotype analysis of ABCB1/MDR1 blocks in a Japanese population 51 Crettol S, Venetz JP, Fontana M, Aubert JD, Pascual M, Eap CB. CYP3A7, reveals genotype-dependent renal clearance of irinotecan. Pharmaco- CYP3A5, CYP3A4, and ABCB1 genetic polymorphisms, cyclosporine genetics 2003; 13: 741–757. concentration, and dose requirement in transplant recipients. Ther Drug 31 Kroetz DL, Pauli-Magnus C, Hodges LM, Huang CC, Kawamoto M, Monit 2008; 30: 689–699. Johns SJ et al. Sequence diversity and haplotype structure in the human 52 Crettol S, Deglon JJ, Besson J, Croquette-Krokar M, Hammig R, ABCB1 (MDR1, multidrug resistance transporter) gene. Pharmacoge- Gothuey I et al. ABCB1 and cytochrome P450 genotypes and netics 2003; 13: 481–494. phenotypes: influence on methadone plasma levels and 32 Leslie EM, Deeley RG, Cole SP. Multidrug resistance proteins: role of response to treatment. Clin Pharmacol Ther 2006; 80: 668–681. P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. 53 Liu L, Fan L, Peng X, Hu D, Zhou H. MDR1 C2005T polymorphism changes Toxicol Appl Pharmacol 2005; 204: 216–237. substrate specificity. Cancer Chemother Pharmacol 2010; 66: 617–623.

Supplementary Information accompanies the paper on the The Pharmacogenomics Journal website (http://www.nature.com/tpj)

The Pharmacogenomics Journal