p-glycoprotein and drug transport
DESCRIPTION
P-GLYCOPROTEIN AND DRUG TRANSPORT. Michael M. Gottesman Chief, Laboratory of Cell Biology, NCI Deputy Director for Intramural Research National Institutes of Health. January 18, 2007. Estimated New Cancer Cases & Deaths, 2005. ** Vast majority of deaths due to chemotherapy resistance. - PowerPoint PPT PresentationTRANSCRIPT
P-GLYCOPROTEIN AND DRUG TRANSPORT
Michael M. GottesmanChief, Laboratory of Cell Biology, NCIDeputy Director for Intramural ResearchNational Institutes of Health
January 18, 2007
Estimated New Cancer Cases & Deaths, 2005
CA Cancer J Clin, 2005
Sites New Cases Deaths** %
All Sites 1,372,910 570,280 42%
Prostate 232,090 30,350 13%
Breast 212,930 40,870 19%
Digestive System 253,500 136,060 54%
Pancreas & Liver 59,730 47,220 79%
Lung and Bronchus 172,570 163,510 95%
Bladder 63,210 13,180 21%
Kidney and Renal Pelvis 36,160 12,660 35%
Genital System, female 79,480 28,910 36%
Lymphoma & Leukemia 98,550 43,180 44%
Brain & Nervous System 18,500 12,760 69%
**Vast majority of deaths due to chemotherapy resistance
Reduced apoptosisAltered cell cycle checkpointsIncreased metabolism of drugsIncreased or altered targetsIncreased repair of damageCompartmentalization
Mechanisms of resistance to anti-cancer drugs
Decreaseduptake
Increasedefflux
Drug Resistance in Cancer
• May affect multiple drugs used simultaneously: known as multidrug resistance (MDR)
• Affects all classes of drugs, including newly designed targetted drugs
• Just as oncogene targets have been catalogued, we need to enumerate all mechanisms of drug resistance in cancer to solve this problem and circumvent resistance
Ultimate Goal:
To use molecular analysis of human cancers to predict response to specific
therapy
To use this information to develop novel drugs to treat cancer
To learn more about cellular pharmacology and pharmacokinetics of drugs
Mechanisms of Resistance
How Drugs Get Into Cells
Diffusion Transport Endocytosis
e.g., immunotoxins
D
D
D D
D
vinblastine, doxorubicin
e.g., nucleoside analogs
e.g.,
D
D
Overexpressed ABC Transporters
Fewer Functional UptakeTransporters
Defective endocytosis
D
DD D
D
DD
D D DD
DIF
FU
SIO
N
D D
D
D
PgpD
DGS
MRPs
D
D
D
D
DD
D MXRDD
(ABCB1)(ABCG2)
ABCC1-C4
ATP-BINDING CASSETTE(N-terminal NBD of human Pgp)
A C B
Walker A ABC linker Walker B D-loop
Y GNSGCGKST LSGGQKQRIAIA ILLLD EA TSALD
390 427 556 620
Structural Organization of an ABC Transporter
Transmembrane Domain
ATP-Binding Domain \/\/\
R
ABC transporters: Domain organization
ABCC1
ABCG2
ABCB1
TM Domain TM DomainATP binding
ATP binding
600
OUT
MEMBRANE
IN
200
100
300
400
500
700
1
1100
1280
ATP SITE ATP SITE
800
900
1000 1200
POINT MUTATIONS ( ), PHOTOAFFINITY LABELED
REGIONS ( ), AND PHOSPHORYLATION SITES ( P )
P
P
P
P
A B
C
A B
C
Hypothetical Model of Human P-glycoprotein
Structure of E.coli BtuCD, a vitamin B12 transporter.
ATP binding domains
20 transmembrane helices
X-ray structure 3.2 A
Locher et al. Science. 2002
NH
N
N
N
OH
H
CH2CH3
COOCH3
CH2CH3
HOCH3
H3CO
H3COOC
COOCH3
CHOCH3
H O
OH
OCH3 O OH
OHO
H O
OH H
HNH2
H
H
H3C
O
N
L-Pro
D-Val O
L-Meval
Sar
C
L-Thr
C
L-Pro
D-Val
L-Thr
O
L-Meval
Sar
CH3 CH3
O
NH2
OO
C NH
OHC C
HC
O
O
HO
O
CH3
O
CH3
CH3
OH
O
O
O
CH2
C CH3
O
H3C
OC
O
H3C
OCH3
H3CO
H3CO
O
H3CONHCOCH3
H3CO
H3CO C
CN
CH(CH3)2
CH2
CH2
CH2
N
CH3
CH2
CH2
OCH3
OCH3
H3CO
HO
H3C
O OHH3C
O
ON
OO
HO
O OCH3
CH3
O
H3C
H3CO
CH3
H3C
CH3
Substrates and Reversing Agents of Pgp
Vinblastine
Daunorubicin
Colchicine
Verapamil
Taxol
Actinomycin D
Rapamycin
Questions about the mechanism of action of P-glycoprotein
• How does P-glycoprotein recognize so many different substrates?
• What do the two ATP binding cassettes do?
• How is substrate binding linked to ATP hydrolysis?
OUT
MEMBRANE
IN
+
AT
P
AT
P
+
+
P-glycoprotein removes hydrophobic substrates
directly from the plasma membrane
ATP sites in P-glycoprotein
• Both sites are essential; mutations in either site knock out transport function
• Sites work sequentially; only one site at a time binds and hydrolyzes ATP
• Stoichiometry of transport indicates that hydrolysis of two molecules of ATP are needed to transport one molecule of drug
Physiologic Role of P-glycoprotein
Lessons learned from mdr1a/mdr1b knockout mice (Berns, Schinkel, Borst)
• Mice are fully viable and fertile under controlled lab conditions
• Mice are very sensitive to toxic xenobiotics, especially those which are neurotoxic
• Pharmacokinetics of many different P-gp substrates altered: Vinca alkaloids, digoxin, fexofenadine, ivermectin--increased GI absorption, decreased kidney and liver excretion
Position of Common Polymorphisms in P-gp
Polymorphisms in the MDR1 gene
• 5 common coding polymorphisms (Asn21Asp, Phe103Leu, Ser400Asn, Ala892Ser, Ala998Thr) have no demonstrable effect on drug transport function
• 1 polymorphism (C3435T) which doesn’t change coding sequence is linked to altered function in intestines and kidney in some studies (Siebenlist et al.). It is part of a haplotype involving two other polymorphisms (C1236T and G2677T). All three insert rare codons in the place of more common ones. This results in increased absorption and decreased excretion of digoxin and fexofenadine.
MDRMDR1 wild-type, SNPs, and haplotypes show similar 1 wild-type, SNPs, and haplotypes show similar
P-gp total and surface expressionP-gp total and surface expression
100 101 102 103 104
Fluorescence Intensity
pTM1
MDR1
C1236T
G2677T
C3435T
C1236T-G2677T
C1236T-C3435T
G2677T-C3435T
C1236T-G2677T-C3435T
pTM
1
WT
X3
P-gp
MDRMDR1 wild-type and the haplotype 1 wild-type and the haplotype
(1236-2677-3435) exhibit similar (1236-2677-3435) exhibit similar
rhodamine 123 effluxrhodamine 123 efflux
100 101 102 103 104
Fluorescence Intensity
MDR1 WT
1236-2677-3435pTM1 control
MDRMDR1 wild-type and the haplotype (1236-2677-1 wild-type and the haplotype (1236-2677-
3435) do not exhibit similar 3435) do not exhibit similar
Bodipy-verapamil accumulationBodipy-verapamil accumulation
100 101 102 103 104
Fluorescence Intensity
pTM1WTX3
MDRMDR1 wild-type and the haplotype exhibit 1 wild-type and the haplotype exhibit
different patterns using rhodamine 123 efflux different patterns using rhodamine 123 efflux
with cyclosporin A with cyclosporin A reversing agentreversing agent
100 101 102 103 104
Fluorescence Intensity
pTM1 controlMDR1 WT
1236-2677-3435
5 M CsA
MDRMDR1 wild-type and haplotype show the same P-gp 1 wild-type and haplotype show the same P-gp
cell surface expression using MRK16 and 17F9, but cell surface expression using MRK16 and 17F9, but
not UIC2 - a conformational sensitive antibodynot UIC2 - a conformational sensitive antibody
pTM1pTM1
WTWT1236-2677-3435 1236-2677-3435
MDRMDR1 wild-type and haplotype show different1 wild-type and haplotype show different trypsinization patterns confirming altered trypsinization patterns confirming altered
conformationconformation
Conclusions We hypothesize that the use of a rare codon by the synonymous polymorphism affects the rhythm of co-translational folding and insertion of P-gp into the membrane. This may alter the structure of substrate and inhibitor interaction sites.
Synonymous polymorphisms (especially for transporters) should not be ruled out as having potential phenotypic significance, and synonymous mutations may lead to significant genetic disease.
Phylogenetic Tree of the Human ABC Genes
48 Human ABC
Transporters
Dean. Genome Res 11:1156, 2001
C2, B1, G2
C1
C2, B1, B4, B11, G2
C1, C3-5
B1, C1, C2, G2
C1
B1
G2
B1, C2, G2
C1
BLOOD
BRAIN
CS
F
GI TRACT
URINE
MILK
LIVER
FE
TU
S
B1, G2
oral
aerosol
LUNG
B1, C1,A3,G21
TESTIS
BTB
C1
KIDNEY
PLA
CE
NTA
cho
roid
ple
xus
BBB
ABC transporters determine oral bioavailability, excretion, penetration and protect the organism against airborne xenobiotics
Human diseases associated with an ABC Transporter
Disease TransporterCancer ABCB1 (MDR1), ABCC1 (MRP1), ABCG2
(MXR)
Cystic fibrosis ABCC7 (CFTR)
Stargardt disease & AMD ABCA4 (ABCR)
Tangier Disease and Familial HDL deficiency ABCA1 (ABC1)
Progressive familial intrahepatic cholestasis ABCB11 (SPGP), ABCB4
(MDR2)
Dubin-Johnson syndrome ABCC2 (MRP2)
Pseudoxanthoma elasticum ABCC6 (MRP6)
Persistent hypoglycemia of infancy ABCC8 (SUR1), ABCC9 (SUR2)
Sideroblastic anemia and ataxia ABCB7 (ABC7)
Adrenoleukodystrophy ABCD1 (ALD)
Sitosterolemia ABCG5, ABCG8
Immune deficiency ABCB2 (Tap1), ABCB3 (Tap2)
Pgp, MDR1
MRP2cMOAT
MRP4MOAT-B
MXR, BCRPABC-P
MRP1
MRP3MOAT-D
MRP5MOAT-C
MRP-6MOAT-E
ABC B1
ABC C1
ABC C2
ABC C3
ABC C6
ABC G2
ABC C4
ABC C5
ATP ATP
ATP
ATP ATP
ATP ATP
ATP ATP
ATP ATP
Neutral and cationic Organic compounds
GS-X and other conjugates,organic anions
GS-X and other conjugates,organic anions
GS-X conjugates, anti-Folates, bile acids, etoposide
Nucleoside analogs, methotrexate
Nucleoside analogs, cyclicnucleotides, organic anions
Anionic cyclic pentapeptide
Anthracyclines, mitoxantrone
Intestine, liver, kidney,Blood-brain barrier
Widespread
Intestine, liver, kidney
Pancreas, intestine, liver, kidney, adrenal
Prostrate, testis, ovary intestine, pancreas, lung
Widespread
Liver, kidney
Intestine, placenta, liver, breast
Common Names
SystematicName Structure Substrates Normal location
ABC transporters which are known to transport drugs
ATP ATP
ABC Transporters Confer Resistance to Anti-Cancer Drugs
ABC
A2
ABC
B1
ABC
C1
ABC
C2
ABC
C4
ABC
G2
ABC
B4
ABC
B11 AB
CC
3
ABC
C5
ABC
C6
ABC
C1
1
Vinca alkaloids VinblastineVincristine
Anthracyclines DaunorubicinDoxorubicinEpirubicin
Epipodophyllotoxins EtoposideTeniposide
Taxanes DocetaxelPaclitaxel
Kinase inhibitors GleevecImatinibFlavopiridol
Campthotecins Irinotecan (CPT-11)SN-38Topotecan
Thiopurines 6-mercaptopurine6-thioguanine5-FU
Other BisantreneCisplatinArseniteColchicineEstramustineMethotrexateMitoxantroneSaquinivirPMEAActinomycin-DAZTDigoxin
Confers resistance
Selected
Doesn’t transport
Can we discover new ABC transporter genes responsible for drug resistance?
• Use of Real Time (RT)-PCR to measure ABC mRNA levels for 48 ABC transporters in all 7 ABC families (A-G)
• Exploitation of the existing NCI-60 cell line database for which resistance to 100,000 different drugs is known
The NCI60 Cell Panel
LUNG (9): NCI-H23, NCI-H522, A549-ATCC, EKVX, NCI-H226, NCI-H332M, H460,
H0P62, HOP92
COLON (7): HT29, HCC-2998, HCT116, SW620, COLO205, HCT15, KM12
BREAST (8): MCF7, MCF7ADRr, MDAMB231, HS578T, MDAMB435, MDN, BT549, T47D
OVARIAN (6): OVCAR3, OVCAR4, OVCAR5, OVCAR8, IGROV1, SKOV3
LEUKEMIA (6): CCRFCEM, K562, MOLT4, HL60, RPMI8266, SR
RENAL (8): UO31, SN12C, A498, CAKI1, RXF393, 7860, ACHN, TK10
MELANOMA (8): LOXIMVI, MALME3M, SKMEL2, SKMEL5, SKMEL28, M14, UACC62, UACC257
PROSTATE (2): PC3, DU145
CNS (6): SNB19, SNB75, U251, SF268, SF295, SM539
Expression of ABC-transporters in the NCI60 panel.
Correlation of Matrices
Pinkel D, Nature Genetics 24, 208-9 (2000)
AB
C-t
rans
port
ers
Drugs
Conditions which must be met to correlate expression with resistance
• mRNA levels are quantitative and reflect levels of functional protein in cells
• Drug resistance data are accurate and quantitative
• Resistance is determined by levels of transporters, i.e., they are limiting
• Cells are different enough to give informative data
60 c
ells
Expression of an ABC-transporter Activity of a Drug
Correlation of expression and sensitivity
-5 0 5 10 15 -1.5 -1 -0.5 0 0.5 1 1.5 2
NSC 363997
-1
-0.5
0
0.5
1
-5 0 5 10 15ABCB1 expression (dCP)
Sen
sit
ivit
y (
dlo
gG
I50)
NSC 363997
-1
-0.5
0
0.5
1
-5 0 5 10 15ABCB1 expression (dCP)
Sen
sit
ivit
y (
dlo
gG
I50)
NSC 363997
0
20
40
60
80
100
120
140
0.1 1 10 100 1000 10000
concentration (uM)
ce
ll s
urv
iva
l (%
)
KB-3-1 (MDR1-)KB-V1 (MDR1+)KB-3-1+PSC 833KB-V1 +PSC 833
Inversely correlated compounds are ABCB1 substrates
NSC 363997
-1
-0.5
0
0.5
1
-5 0 5 10 15ABCB1 expression (dCP)
Sen
sit
ivit
y (
dlo
gG
I50)
KB-V1: MDR derivative of KB-3-1overexpressing P-gp
Inversely correlated compounds indicate potential substrates for 28
ABC transportersAnalysis of a dataset containing 1430 compounds at 99.99% bootstrap confidence interval (or equivalently a significance level of p<0.0001)
Substrate assignments have been confirmed for 4 of these transporters in transfected cell lines: ABCB1, ABCC2, ABCC4, and ABCC11
Search for MDR1-potentiated compounds in DTP’s database
-1
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1 -0 0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9 1
17K
Substrates
NSC 363997
-1
-0.5
0
0.5
1
-5 0 5 10 15ABCB1 expression (dCP)
Sen
sitiv
ity (d
logG
I50)
NSC 363997
0
20
40
60
80
100
120
140
0.1 1 10 100 1000 10000
concentration (uM)
ce
ll s
urv
iva
l (%
)
MDR1-potentiated compounds?
NSC 73306
The cytotoxicity of NSC 73306 is increased in KB-V1 cells
-1.5
-1
-0.5
0
0.5
1
1.5
-5 0 5 10 15
ABCB1 expression (dCP)
Sen
siti
vity
(d
log
GI5
0)
0
20
40
60
80
100
120
1 10 100 1000
Concentration (µM)
Cel
l su
rviv
al (
%)
r = 0.54
KB-3-1 (MDR1-) KB-V1 (MDR1+) KB-3-1+PSC 833 KB-V1 +PSC 833
NSC73306
NO
N N
NS
O-1
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1 -0 0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9 1
50 candidates7 thiosemicarbazones
Search for MDR1-potentiated compounds in DTP’s database
Loss of P-gp in NSC73306 Selected Cells
P-gp
GAPDH
1 2 3 4 5 6 7 8 9
Western Blot of Pgp-positive cells selected in 1ug/ml NSC 73306.
Selected: - + - + - + - + -
Cell lines: HCT15-2A ADR KBV1 DX5 / MES-SA
Potential Clinical Utility of Discovery of Compounds that Specifically Kill MDR1-Expressing Cells
Can be used in combination with standard chemotherapy to eliminate MDR1-
expressing cell populations
Role of P-glycoprotein in cancer
• Approximately 50% of human cancers express P-glycoprotein at levels sufficient to confer MDR
• Cancers which acquire expression of P-gp following treatment of the patient include leukemias, myeloma, lymphomas, breast, ovarian cancer; preliminary results with P-gp inhibitors suggest improved response to chemotherapy in some of these patients
• Cancers which express P-gp at time of diagnosis include colon, kidney, pancreas, liver; these do not respond to P-gp inhibitors alone and have other mechanisms of resistance
Acute Leukemia:Influence of mdr-1 Expression on Remission Rate
Zhou, et al. Leukemia 6:879, 1992
XR9576
OC144-093
LY335979
R101933
Newer Pgp Antagonists
99mTc-Sestamibi Scan following XR-9576Diagnostic assay for Pgp detectionSurrogate assay for Pgp inhibition
1 hour 2 hours 3 hours
BeforeXR9576
AfterXR9576
Renal Cell Carcinoma99mTc-Sestamibi Uptake in Left Thigh Metastasis
Effect of XR 9576
Balance of uptake and efflux determines drug Balance of uptake and efflux determines drug accumulation in cancer cellsaccumulation in cancer cells
SLC1 High affinity glutamate transporter SLC23 Nuclobase trasnporterGlutamate/neutral amino acid transporter
SLC2 Facilitated glucose transporter SLC24 Sodium/potassium/calcium exchanger
SLC3 Cystine, dibasic and neutral amino acid transporter SLC25 Mitochondrial carrierActivators of dibasic and neutral amino acid transport
SLC4 Sodium bicarbonate cotransporter SLC26 Sulfate transporter
SLC5 Sodium glucose cotransporter SLC27 Fatty acid transporter
SLC6 Neurotransmitter transporter SLC28 Sodium-coupled nucleoside trasnporter
SLC7 Cationic amino acid transporter SLC29 Nucleoside transporter
SLC8 Sodium/calcium exchanger SLC30 Zinc transporter
SLC9 Sodium/hydrogen exchanger SLC31 Copper transporter
SLC10 Sodium bile acid cotransporter SLC32 GABA vesicular trasnporter
SLC11 Proton-coupled divalent metal ion transporter SLC33 Acetyl-CoA transporter
SLC12 Potassium/chloride transporter SLC34 Sodium phosphate cotransporterSodium/potassium/chloride transporter
SLC13 Sodium/sulfate symporter SLC35 Nucleoside-sugar transporterSodium-dependent dicarboxylate transporter
SLC14 Urea transporter SLC36 Proton/amino acid symporter
SLC15 Oligopeptide transporter SLC37 Glycerol-3-phosphate transporterH+/peptide transporter
SLC16 Monocarboxylic acid transporter SLC38 Amino acid transport system N
SLC17 Sodium-dependent inorganic phosphate cotransporter SLC39 Zinc transporter
SLC18 Vesicular monoamine transporter SLC40 Iron-regulated transporter
SLC19 Folate transporter SLC41 MgtE Mg2+ transporterThiamine transporter
SLC20 Phosphate transporter SLC42
SLCO Organic anion transporting polypeptide SLC43 System L amino acid transporter
SLC22 Organic cation transporter Organic anion transporter SLC44 Choline transporter
SLC45 Putative sugar transporter
Solute Carrier (SLC) familiesSolute Carrier (SLC) families
SLCO familySLCO family
The SLCO gene family encodes the organic anion transporting polypeptides (OATPs)The SLCO gene family encodes the organic anion transporting polypeptides (OATPs)that mediate the sodium-independent transmembrane transport of a wide range of that mediate the sodium-independent transmembrane transport of a wide range of amphipathic compounds.amphipathic compounds.
Endogenous compounds: Endogenous compounds: bile salts, thyroid hormones, bile salts, thyroid hormones, conjugated steroids, conjugated steroids, prostanoidsprostanoids
Xenobiotics: Xenobiotics: HMG-CoA reductase inhibitorsHMG-CoA reductase inhibitors(pravastatin),(pravastatin),angiotensin converting enzyme angiotensin converting enzyme inhibitors (temocaprilat), inhibitors (temocaprilat), cardiac glycosides (digoxin), cardiac glycosides (digoxin), some antibiotics and anti-cancer drugssome antibiotics and anti-cancer drugsIncluding methotrexateIncluding methotrexate
Gene Symbol Protein Name Gene Locus
SLCO1A2 OATP1A2 12p12
SLCO1B1 OATP1B1 12p12
SLCO1B3 OATP1B3 12p12
SLCO1C1 OATP1C1 12p12.2
SLCO2A1 OATP2A1 3q21
SLCO2B1 OATP2B1 11q13
SLCO3A1 OATP3A1 15q26
SLCO4A1 OATP4A1 20q13.1
SLCO4C1 OATP4C1 5q21
SLCO5A1 OATP5A1 8q13.1
SLCO6A1 OATP6A1 5q21.2
• • Human: 11 membersHuman: 11 members• • SubstratesSubstrates
Hypothetical model of an SLCO family memberHypothetical model of an SLCO family member
ININ
OUTOUT
NHNH22
COOHCOOH
11 22 33 44 55 66 77 88 99 1010 1111 1212
Extracellular loop 5: Contains many conservedExtracellular loop 5: Contains many conserved cysteine residuescysteine residues
SLC22 familySLC22 familyThe SLC22 gene family encodes the organic cation transporters (OCTs),The SLC22 gene family encodes the organic cation transporters (OCTs),the organic zwitterion/carnitine transporters (OCTNs), and the organic zwitterion/carnitine transporters (OCTNs), and the organic anion transporters (OATs) .the organic anion transporters (OATs) .
OCTs; Uniporters that mediate facilitated diffusion in either directionOCTs; Uniporters that mediate facilitated diffusion in either direction
• • SubstratesSubstratesGene Symbol Protein Name Gene Locus
SLC22A1 OCT1 6q26
SLC22A2 OCT2 6q26
SLC22A3 OCT3 6q26-27
SLC22A4 OCTN1 5q23.3
SLC22A5 OCTN2 5q23.3
SLC22A6 OAT1 11q12.3
SLC22A7 OAT2 6q21.1-2
SLC22A8 OAT3 11q12.3
SLC22A9 UST3 11q12.3
SLC22A10 OAT5 11q12.3
SLC22A11 OAT4 11q13.1
SLC22A12 URAT1 11q13.1
SLC22A13 ORCTL3 3q22.2
SLC22A14 ORCTL4 3q22.2
SLC22A15 FLIPT1 1q13.1
SLC22A16 SLC22A16, CT2 6q21-22.1
SLC22A17 BOCT 14q11.2
SLC22A18 11p15.5
Endogenous compounds:Endogenous compounds:dopamine, norepinephrine, serotonin, dopamine, norepinephrine, serotonin, choline, L-carnitine, choline, L-carnitine, prostanoids, histamine, prostanoids, histamine, urateurate
Xenobiotics:Xenobiotics:tetraethylammonium (TEA),tetraethylammonium (TEA),1-methyl-4-phenylpyridinium (MPP) 1-methyl-4-phenylpyridinium (MPP) verapamil, quinidine, verapamil, quinidine, cimetidine, aciclovir, cimetidine, aciclovir, some anti-cancer drugs includingsome anti-cancer drugs includingdoxorubicindoxorubicin
• • Human: 18 membersHuman: 18 members
OCTN2; NaOCTN2; Na++/L-carnitine cotransporter/L-carnitine cotransporter OAT1,2, and URAT1; Anion exchangersOAT1,2, and URAT1; Anion exchangers
Hypothetical model of an SLC22 family memberHypothetical model of an SLC22 family member
M G SRH
F
EG
IYDH
VG
HF
GR F
QR
V
L
YFI CAFQ N
IS C
GIH YLA SVFM G
VTP
HHV
C
R
PPG
NV
S
Q
V
V
F
H
AML IQPL FMF G
V L L G
S V T
F G Y F
V V L W
A T S S
S MF
LF
GIA
AAF
AV
FF LAMV ASG Y
LV
V
G FVY VM
EF I
AS
VHL
HSF
FAV
GT
LLV
ALT
GYL
V L Y QM IL
STV
TV PF IL
C CW VL
LT
VWL
IWF T
GS LG FY
SFS
LN
SVN
L LN LF
LL
GV VEI PA YT FVC
IA
VL
AY SL
FC SAL A
CGV
VMV I
IL GV
V TAM V
GKF
AIG
A AFG
L IYL
YTA
IV
RSL
AVG
S GS
MV
CRL
ASI
LAP
FI F IPQ
L FV GTM
AL LS GVL
T L
N
H
S
NW S L
E
D
T
G
A
L
L
S
S
G
Q
K
D
YV T
VQL
Q
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T
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G
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RK
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S
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R
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AA
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MK
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RT W W
P
ET
PF
WLLSEGRYEEA
Q
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KW
NR
AS
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L
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VS
N SP
TE
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KH
NL S Y
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SITK
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G NE
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R
T
P
QK H
Y
EL Y P
T
S
V
D
L SS
I
W
K
LP
E
T
LG
KR
L
A
TTW
E
E
AAK
L
E
S
E
N
E
SK
S
SK
LLLT
T
N
NSGLE
K
T
EAI
TP
RDSGLG
E
ININ
OUTOUT
COOHCOOHNHNH22
MEMBRANEMEMBRANE
N-glycosylation sitesN-glycosylation sites
phosphorylation sitesphosphorylation sites
PP
PP
PP
PP
PP
PP
PP
121211111010998877665544332211
Which SLCO/SLC22?Which SLCO/SLC22?
What kind What kind of drug?of drug?
??
Which SLCs transport which drugs?Which SLCs transport which drugs?
GoalGoal
Evaluate the contribution of the SLCO and SLC22Evaluate the contribution of the SLCO and SLC22families to sensitivity / resistance of cancer cells families to sensitivity / resistance of cancer cells to anti-cancer drugs (Mitsunori Okabe) to anti-cancer drugs (Mitsunori Okabe)
LE:MOLT-4 LE:CCRF-CEM BR:BT-549 LE:HL-60(TB) BR:T-47D BR:MCF7 LC:NCI-H23 OV:OVCAR-3 LC:HOP-62 RE:ACHN UK:NCI/ADR/REST OV:OVCAR-8 LE:RPMI-8226 LC:NCI-H322M ME:UACC-257 ME:UACC-62 BR:MDA-MB-435 ME:MDA-N ME:SK-MEL-5 ME:SK-MEL-28
CO:HCT-15 LC:NCI-H460 ME:M14 ME:MALME-3M PR:PC-3 LC:A549/ATCC CO:KM12 CO:COLO205 CO:HCC-2998 LC:EKVX CO:HT-29 OV:OVCAR-5 CNS:SF-539 BR:MDA-MB-231 OV:SK-OV-3 OV:IGROV1 OV:OVCAR-4 ME:LOX IMVI CO:HCT-116 PR:DU-145 RE:SN12C LC:NCI-H522 CNS:SF-295 LC:HOP-92 CNS:U251 CNS:SF-268 CNS:SNB-19 BR:HS578T LE:K-562 LE:SR ME:SK-MEL-2 CO:SW-620 RE:A498 RE:TK-10 RE:CAKI-1 RE:UO-31 RE:786-O RE:RKF-393 CNS:SNB-75 LC:NCI-H226
SL
C2
2A
16
SL
CO
1A
2 S
LC
O4
C1
SL
CO
6A
1 S
LC
22
A1
3 S
LC
22
A1
4 S
LC
22
A1
7 S
LC
O2
A1
SL
C2
2A
15
SL
C2
2A
1 S
LC
O3
A1
SL
CO
5A
1 S
LC
22
A2
SL
C2
2A
4 S
LC
22
A5
SL
CO
4A
1 S
LC
22
A3
SL
C2
2A
11
SL
CO
2B
1
SL
CO
1C
1 S
LC
22
A1
8 S
LC
O1
B1
SL
CO
1B
3
Gene expression of SLCO and SLC22 family Gene expression of SLCO and SLC22 family members in the NCI-60 cancer cell linesmembers in the NCI-60 cancer cell lines
MelanomaMelanoma
Ce
ll lin
es
(cl
us
tere
d)
Ce
ll lin
es
(cl
us
tere
d)
Genes (clustered)Genes (clustered)((http://discover.nci.nih.gov/)http://discover.nci.nih.gov/)
CIMminer toolCIMminer tool
Identify potential SLC substratesIdentify potential SLC substrates
No SLC22A1 SLC22A2 SLC22A3 SLC22A4 SLC22A5 SLC22A11 SLC22A13 SLC22A14 SLC22A15 SLC22A1656410 0.31217732 -0.07922665 0.01277731 -0.25626202 -0.11116942 -0.16991988 0.27970117 0.15262371 0.04266599 -0.0028094626980 0.20009471 -0.08423846 0.01047526 -0.28229259 -0.13295532 -0.14912004 0.24659229 0.14142082 -0.02224928 -0.01106568
178248 -0.01507493 -0.0846674 0.1190922 -0.27668413 -0.04673201 0.11972923 -0.05616468 -0.24427015 -0.07291911 0.1688569195466 0.07319021 -0.07096717 0.18991411 -0.20421528 0.12819997 0.09301066 0.04023063 -0.28915386 -0.09753227 0.26907311
338947 0.17943592 -0.00029695 0.19533586 -0.26363152 0.13937002 0.17526069 0.0534773 -0.06514511 0.03364348 0.20313293353451 0.09638496 -0.14111469 0.02619936 -0.34772056 -0.01369285 0.08497674 -0.06917942 -0.18202563 -0.00952047 0.08992747409962 -0.08244515 -0.21080187 -0.15570814 -0.20334821 -0.04049964 0.03969127 -0.13160843 -0.27856725 -0.09750883 -0.03308029
79037 -0.15510329 0.00497603 0.16738755 0.02417695 0.08814805 0.14143012 -0.15207161 -0.19843043 0.17115279 0.0230839295441 -0.28299966 -0.01651927 -0.22579738 -0.17433283 -0.21193358 0.08740468 -0.26883833 -0.37316925 -0.21976171 0.31868305
102627 0.24685948 -0.00297079 -0.07304859 -0.24306023 -0.07519329 -0.07974952 0.10506973 -0.04338648 0.1039498 -0.02915382172112 0.00687864 -0.14196825 -0.09801817 -0.30875849 -0.10355957 0.06178361 0.05996666 -0.0492571 -0.00880085 -0.22512389
750 0.01585302 -0.15782512 -0.01050464 -0.4233501 0.0655575 0.05630849 0.14366774 0.03143881 0.13185695 0.1235665373754 0.0995236 -0.08061869 -0.13685131 -0.0812715 -0.07529458 0.03938285 -0.09128572 -0.10562111 0.11098002 -0.23770077
329680 0.24326009 -0.01434041 -0.00293244 -0.2215489 0.00781541 -0.0520558 0.14030933 -0.00526727 0.07251901 -0.06462474241240 -0.07648128 -0.13504995 -0.10393863 -0.34965424 -0.29698125 0.08875319 -0.16326486 -0.21674884 -0.23658795 -0.15364288344007 0.32940521 0.01011578 0.09487956 -0.1363134 0.07264565 -0.06301899 0.21128643 0.09368316 0.06886771 -0.06167068
6396 0.32206526 -0.09113615 0.04614285 -0.17986804 0.09128611 -0.07629671 0.20805053 0.03572174 0.05108767 -0.1188058425154 0.17677655 -0.1009669 -0.11250678 -0.17537532 -0.02528812 -0.02990033 0.04092447 -0.04759487 -0.00867606 -0.0817457
3088 0.29476437 -0.07795825 0.06226617 -0.14582716 0.14513495 -0.04098284 0.20406937 0.0626445 0.09910024 -0.07631051132313 0.2929453 0.05949831 0.18097669 -0.12424337 0.12322171 -0.01137185 0.24998519 0.06417567 0.08728851 -0.04696567348948 -0.01567599 -0.10210551 -0.10516261 -0.24520482 -0.15843057 0.00579575 -0.12642202 -0.17629083 -0.13596812 0.05857327256927 0.20004228 -0.20457051 0.09529207 0.16213038 -0.0603284 0.0431725 -0.00473075 0.03685661 0.0542746 -0.15841662
8806 0.34062197 -0.10224266 0.12998615 -0.13607443 0.09621835 0.00448254 0.1699378 0.03689995 0.12734735 -0.170581934462 0.31033258 -0.00991989 0.14005752 -0.14407666 0.14790855 -0.0434418 0.24477246 0.09082246 0.08441141 -0.07358331
182986 0.2057699 0.05300584 0.01437008 -0.25716146 -0.04783325 0.03479687 0.1543121 0.00542664 -0.11403819 -0.12018293296934 0.40118577 0.00397631 0.09759492 -0.07882788 0.09217189 -0.06622195 0.22798063 0.12950106 0.07502988 -0.16476242
9706 0.3367041 -0.07467914 0.09676359 -0.18022744 0.10261581 -0.05111491 0.23602735 0.06709457 0.03636947 -0.06900054167780 0.1511137 0.08974341 0.27162644 0.02893386 0.10592946 0.04915439 0.38376921 0.10730114 0.03582396 -0.024566119875 0.1314655 -0.20077808 0.05020688 -0.30423579 -0.0538852 0.09643678 -0.01174728 -0.1877992 -0.17947647 -0.17062449271674 0.03737928 0.11199002 0.1380524 0.22814923 0.09837968 -0.21022019 0.18599476 0.16405408 0.03246818 0.06692455
762 0.26931833 0.07922469 0.06705649 -0.01216443 0.07214326 -0.14221338 0.22698494 0.16534235 0.20182256 -0.14212125363812 0.01920794 0.06797754 0.20290475 0.27274677 0.08361875 -0.22303773 0.20203483 0.09694936 0.19403964 0.07750761135758 0.34566772 0.06457149 0.18624227 -0.17245132 0.03705162 -0.04864249 0.21978252 0.0705508 0.04555308 -0.01885809142982 -0.30132846 -0.04961211 -0.40774749 -0.16735972 -0.23478639 -0.44754151 -0.10263985 0.0861766 -0.21647399 0.31512134268242 -0.03111173 -0.09026632 0.0292751 -0.05616953 0.09514301 -0.15290427 -0.00422928 0.01461663 0.02866662 0.27669639366140 -0.10230102 -0.11237962 -0.33303766 -0.05572318 -0.01947022 -0.18113378 -0.16653512 -0.03761973 -0.09364184 0.13808111354646 -0.14159331 -0.23959418 -0.24381955 -0.43261557 -0.29127483 -0.2074447 0.02445994 0.03928138 -0.09949527 0.24634083357704 0.09228953 -0.18960826 -0.09742641 -0.42896113 -0.31255915 0.08874825 -0.11687304 -0.105092 -0.01132418 0.02779931623017 0.19066312 0.10130317 0.26516772 0.09761822 0.04277312 0.17855879 0.0057357 -0.10823575 0.22301875 -0.08521279134033 -0.028183 0.07648013 0.088901 0.18855637 0.24541171 -0.20947493 0.16372427 -0.07161708 -0.0512064 0.06099377132483 0.00823386 -0.02026076 0.06526897 0.1170598 0.20460909 -0.13658513 0.10683826 -0.11501508 0.01596625 0.16650215139105 0.13992632 -0.01519515 0.21049867 0.08932196 0.11910089 0.01117849 0.17102076 -0.0672376 0.15611205 -0.06796642184692 0.11494162 0.09040631 0.25054614 0.16586092 0.3671631 -0.05211129 0.18045749 -0.06904104 0.08960311 0.06143595
740 0.17220173 -0.04706001 0.04272407 0.05322555 0.26465396 -0.12890495 0.14286561 -0.05751532 0.14515769 -0.07680592174121 0.08859456 0.08663771 0.11162185 0.10833265 0.33921093 -0.15395855 0.13726555 -0.03630281 0.12546976 -0.09240559633713 0.10060497 -0.00763367 0.10015476 0.0330221 0.25861666 -0.23678128 0.22985189 0.07632871 -0.05509181 0.08420467352122 0.05468834 -0.07006252 0.10756031 0.12536417 0.17967799 -0.21498757 0.26261878 0.00814011 -0.0030376 -0.00367388
71851 0.00264011 -0.13828438 -0.23604076 -0.06409913 0.02948754 -0.18959522 -0.08709868 -0.03364047 0.11284721 0.09290321264880 -0.03190124 -0.16731582 0.04599249 -0.08930947 -0.02612686 -0.14924734 0.15553427 0.07884698 0.01504856 0.03161256
752 0.12566842 -0.02217756 -0.07453801 -0.05254569 0.10819465 -0.14251062 -0.00665221 0.01221643 0.19741467 -0.012836471261 0.05358824 -0.05948424 -0.19554451 -0.0204256 0.05106906 -0.1675372 0.0106988 -0.02978017 0.06285586 0.06305114
102816 -0.13354172 0.10621608 -0.20463875 -0.02259074 -0.13060899 -0.00522806 -0.1324949 -0.01709785 -0.09344114 0.153224381895 -0.00336981 0.05944225 -0.18926973 -0.16262158 -0.0279619 -0.05111089 -0.06384145 -0.16647486 -0.07345308 0.03057923
51143 0.09829376 0.0366927 -0.023303 -0.03145896 0.15312589 -0.06487475 0.15024292 -0.01749896 0.04472674 0.0287300532065 0.22999828 0.01776262 -0.0010186 -0.03766753 0.22907909 -0.06455021 0.09628891 -0.02251131 0.13045578 -0.24208198
148958 -0.2159127 0.02242544 -0.23366456 0.1745705 0.00797055 -0.22269535 0.17931039 0.19088128 -0.0984987 0.0697534819893 0.0280928 0.02668993 0.01886694 0.19939823 0.13836069 -0.30770682 0.29839631 0.2739511 0.01691879 0.11495102
145668 0.2414292 0.07042995 0.21683871 -0.01377446 0.2889849 -0.10430719 0.27214909 0.04731599 0.01365392 -0.0285735563878 0.29626253 -0.03672156 0.21772645 -0.09781067 0.22779141 -0.10141165 0.2682707 0.05737141 0.06584463 -0.02054463
303812 0.20760002 0.03102897 0.02823929 -0.19682699 0.18342991 -0.05901632 0.16597435 -0.04147881 0.0405126 -0.09827336755 0.14851905 -0.1011958 0.09087916 0.02980126 0.10525255 -0.07722664 0.08019081 -0.01946858 -0.09360843 -0.09269728
27640 0.22484065 0.02858565 0.144474 -0.15571351 0.0970196 -0.16266704 0.23645664 0.01584071 0.12592863 0.04061405330500 0.16771084 0.07505492 0.15839491 0.23739503 0.02239966 0.23211833 0.00747635 0.01398885 0.16016823 -0.28631566109229 0.09443206 -0.07878944 -0.10399581 -0.10162667 0.15358725 -0.12674536 0.06656105 0.02371226 0.19739804 0.05288218224131 0.02699534 -0.06999014 0.13483353 0.05471999 0.06203865 -0.03847498 0.08201501 -0.13924624 -0.11668013 0.13978862126771 0.31091335 -0.15453651 0.18097035 -0.02298393 0.13113325 -0.05114184 0.01551746 -0.09635 0.2465094 -0.06373961153353 -0.18044122 -0.2171951 -0.18609754 -0.06582655 -0.14075303 -0.14984365 -0.05217044 -0.19568919 0.03139297 0.02127899143095 0.25704003 -0.18464542 -0.03415697 -0.03737292 0.1856477 -0.08585136 0.01540035 -0.06324033 0.12435387 0.06763215163501 -0.1225667 -0.06976144 -0.17946072 -0.0133142 -0.14106504 -0.16597936 -0.14775691 -0.12781237 0.03939838 0.10881219
GenesGenes
Dru
gs
Dru
gs
0
40
80
120
160
-0.7 -0.5 -0.3 -0.1 0.2 0.4 0.6
Pearson coefficientPearson coefficient
Nu
mb
er o
f d
rug
sN
um
ber
of
dru
gs
R2 = 0.4823
-0.9
-0.6
-0.3
0
0.3
0.6
0 5 10 15
Gene expression (dCP)Gene expression (dCP)
Sen
siti
vity
(d
log
GI
Sen
siti
vity
(d
log
GI 5
050))
By correlating the expression profile with the growthBy correlating the expression profile with the growth inhibitory profile, candidate anti-cancer drug substratesinhibitory profile, candidate anti-cancer drug substrates of the SLCO and SLC22 family members can be identified.of the SLCO and SLC22 family members can be identified.
This revealed that most of the SLCO and SLC22 family This revealed that most of the SLCO and SLC22 family members are expressed at some level in cancer cell lines.members are expressed at some level in cancer cell lines.
Summary of SLCO and SLC22 TransportersSummary of SLCO and SLC22 Transporters
The expression profile of the SLCO and SLC22 family The expression profile of the SLCO and SLC22 family members in the NCI-60 cancer cell lines was established by members in the NCI-60 cancer cell lines was established by real-time quantitative RT-PCR.real-time quantitative RT-PCR.
Conclusions
• It is possible to associate up to 28 specific ABC transporters (and possibly several SLC and SLCO transporters) with resistance/sensitivity to specific drugs by correlating mRNA expression with resistance patterns of cancer cell lines
• This means that in many (but not all) cases, quantitative measurement of mRNA levels in cancer cells may be sufficient to predict drug-resistance to specific drugs
• However, reliable determination of drug-resistance using molecular approaches still requires a complete list of limiting mechanisms of resistance and confirmation using clinical samples
Unexpected Conclusions
*A haplotype in the P-gp gene consisting of synonymous polymorphisms changes specificity of P-gp by altering conformation. This has clinical implications.
*Drugs that specifically kill P-gp-expressing cells may be exploited to improve treatment of drug-resistant cancers
Acknowledgements
• Gergely Szakacs
• Jean-Philippe Annereau
• Kevin Chen
• Joe Ludwig
• Jill Paterson
• Mitsunori Okabe
• Matthew Hall
• Chava Kimchi-Sarfaty
• Jung-Mi Oh
• Suresh Ambudkar– Zuben Sauna– InWha Kim– Anna Calcagno
• Ira Pastan• John Weinstein• Carol Cardarelli• Takaaki Abe• Joe Covey• Henry Fales