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Transcript of P-GLYCOPROTEIN AND DRUG TRANSPORT Michael M. Gottesman Deputy Director for Intramural Research...
![Page 1: P-GLYCOPROTEIN AND DRUG TRANSPORT Michael M. Gottesman Deputy Director for Intramural Research National Institutes of Health January 16, 2003.](https://reader036.fdocuments.in/reader036/viewer/2022062421/56649e375503460f94b27ad2/html5/thumbnails/1.jpg)
P-GLYCOPROTEIN AND DRUG TRANSPORT
Michael M. GottesmanDeputy Director for Intramural ResearchNational Institutes of Health
January 16, 2003
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What is the Scope of the Problem?Estimated New Cancer Cases & Deaths, 2001
CA Cancer J Clin.51:23, 2001
**Vast majority of deaths due to chemoresistance
**
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Reduced apoptosisAltered cell cycle checkpointsIncreased metabolism of drugsIncreased or altered targetsIncreased repair of damageCompartmentalization
Mechanisms of resistance to anti-cancer drugs
Decreaseduptake
Increasedefflux
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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
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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
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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
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Structural Organization of an ABC Transporter
Transmembrane Domain
ATP-Binding Domain \/\/\
R
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ABC transporters: Domain organization
MRP1
MXR
MDR1
TM Domain TM DomainATP binding ATP binding
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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
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Phylogenetic Tree of the Human ABC Genes
Dean. Genome Res 11:1156, 2001
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Human diseases associated with an ABC Transporter
Disease Transporter
Cancer 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)
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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
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Chemotherapeutic Substrates for the MRP Family of ABC Transporters
Borst, BBA 1461:347-357, 1999
MRP1MRP2MRP3MRP4MRP5MRP6MRP7MRP8MRP9
+ + + ++- ---
OA VP-16 ADR VCR CPT MTX 6MP GEM
- + - --- ---
+ + - --- ---
+ + + +- ----
+ + - --- ---
+ + + --- ---
- - -++ ----
- - --+ ----
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RT-PCR and Microarray Analysis of ABC Transporters
• NCI 60 cancer cell lines with known sensitivity to >1000 different drugs
• Specific cell lines selected for drug resistance
• Cancers from patients
• Stem cells--changes during differentiation
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Real Time RT-PCR
-Specificity-Sensitivity-High dynamic range -Quantitative results
Crossing Point
The position of the log-linear phase contains quantitative information: as the copy number of the template increases, the log-linear phase shifts to lower cycle numbers.
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30
8
49
8
12
5
29
5
18
7
55
1
23
8
26
9
24
4
56
6
43
0
20
4
50
2
39
8
19
9
40
5
34
1
47
6
38
9
22
1
51
4
52
5
24
3
19
3
35
7
31
9 26
5
31
7
29
8
40
6
40
6
AB
C-B
1
AB
C-B
2
AB
C-B
3
AB
C-B
4
AB
C-B
11
AB
C-C
1
AB
C-C
2
AB
C-C
3
AB
C-C
4
AB
C-C
5
AB
C-C
6
AB
C-A
1
AB
C-A
3
AB
C-A
4
AB
C-A
5
AB
C- G
1
AB
C- G
4
AB
C-C
7
AB
C-C
7
AB
C-C
9
AB
C-C
11
AB
C-D
1
AB
C-D
4
AB
C- G
2
PX
R
AB
C-B
1
AB
C-A
6
ga
pd
h
ga
pd
h
AB
C-G
5
500 bp…………
PB
DG
AB
C-B
3
AB
C-C
8
AB
C-G
8
AB
C-B
6
AB
C-F
1
AB
C-A
10
AB
C-B
6
AB
C-D
3
AB
C-B
9
AB
C-A
5
AB
C-A
7
AB
C-B
7
AB
C-F
3
AB
C- A
8
AB
C- B
8
AB
CB
5
AB
C-A
12
AB
C-C
12
AB
C-D
2
AB
C-E
1
AB
C- C
12
AB
C-G
5
MA
RK
ER
AB
C-F
2
AB
C-A
2
500 bp
29
8
48
0
21
2
28
3
23
3
26
8
28
2
27
7
38
5
25
6
51
4
36
2
25
4
38
1
33
4
23
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51
9
26
1
54
5
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2
34
1
33
23
23
44
6
AB
C-B
10
GS
T-P
i
B-A
ct i
n
PB
GD
PX
R
RT-PCR of the 48 ABC transporters
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15
20
25
30
35
40
45
15 20 25 30 35 40 45
MDR1
MDR1(3’-UTR)
Actin
GST-
Cp KB-8-5 MDR
Cp KB-3-1
Real Time RT-PCR data KB-3-1 vs KB-8-5 (MDR)
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cns -0510snb75 cns -0510sf539 co-0514colo205 ov -0501wowcar3 le-0520hl60 co-0514ht29 br-0521mb231 le-0823MOLT4 le-0828CCRMCRF mel -0905SKMEL2 me-0904MALME3M br-0905MDAN me-0904M14 me-0827SKMEL28BIS me-0506wuaac co-0513h2298 ns-0503nih460 le-0823USKMEL5 br-020909MDAMB435 me-0823UACC62 me-0506wlox br-0520hs578t pc-0503pc3 br-0904BT49N cns -0906SF268 br-0520t47d br-0506mcf7 ov -0501wowcar4 ov -0502wowcar5 ns-0903H23 ov -0522IGROV1 co-0514hct15 br?-0506mcf7AR br?-0506mcf7AR ki-0503caki co-0514sw620 ns-020909EKVX ki-0903A498 ki-0521786o ki-0823OU31 ki-0521tk10 le-0520k562 ki-020906RXF393 ki-0904ACHN ns-0502whop62 cns -0510sf295 ns-020909H522M ov -020907OVCAR8 le-20910-8226 ns-020909H322M ov -020911SKOV3 co-0826WKM2 ns-20910-549 ns-020910HOP92 cns -020909SNB19
B4
A5-B
A5-A
B2
C5
D4
C4
B5
C2
D1
G1
A6-A
C6
PXR
G4-A
C7(3627)
B11
G5-B
A4
B3
G8
A9
YWHAZ
C1
B1
B1
B8
C3
A1
A10
A7
B10
F3
B6
B9
C9-B
C9-A
A8
G1
A3
C10
B3
A2
C12
D2
A12
G4-B
A6-B
C8
GAPDH
GAPDH
G2
G5-A
F1
D3
B7
E1
F2
C11
C7 (555)
PBDG
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Conclusions from RT-PCR Analysis
• Some ABC transporters are expressed at higher levels in some cancer cell lines than others
• Transporter expression is clustered in some cell lines, suggesting coordinate regulation
• Profiles of ABC transporter expression allow clustering of cancers by type
• Expression of some transporters is strongly associated with resistance to certain drugs (e.g., ABC B1 and paclitaxel analogs)
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ABC transporter-Toxi-Chip©
• In collaboration with Cyndi Afshar and colleagues at NIEHS
• Contains unique probes for 48 ABC transporters, plus detoxifying enzymes plus 20,000 human cDNAs
• Less quantitative than RT-PCR but specific and reliable for ABC transporters
• Being used to screen cell lines selected for MDR
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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
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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
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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?
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OUT
MEMBRANE
IN
+
AT
P
AT
P
+
+
P-glycoprotein removes hydrophobic substratesdirectly from the plasma membrane
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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
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Stoichiometry of ATP molecules hydrolyzed to substrate molecules transported
Pump Substrate Rat io(ATP hydrolysis:
Tr ansport)
Histidine permease Histidine 2:1Oligopeptide permease Oligopeptides 2:1P-glycoprotein Vinblastine 2-3:1
Rhodamine 123 2:1FoF1 H+ 1:4VoV1 H+ 1:2Na+/K+ AT Pase Na+, K+ 1:3:2Ca+ AT Pase Ca+ 1:2
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Vanadate-trapping can be used to dissect intermediates
E•ATP E•ADP + Pi
+ Vi E•ADP-Vi
(trapped conformation)
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P-glycoprotein in the vanadate-trapped conformationshows reduced affinity for the substrate IAAP
IAA
P in
corp
ora
ted
(p
mo
les/
pm
ole
Pg
p)
IAAP [nM]
213
120C
ON
AT
P
Vi
AT
P+
Vi
8A
zAT
P
8A
zAT
P+
V
i
Control
AMPPNP +Vi
ATP + Vi
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0 5 10 150
2
4
6
Recovery of IAAP binding to P-glycoprotein in transition state conformation requires ATP hydrolysis
IAA
P in
co
rpo
rati
on
(a
rbit
rary
un
its
)
ATP+Mg+2
ATP+EDTAAMPPNP
ATP+VO4
Time (min)
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Catalytic Cycle of P-glycoprotein
ADP
ADP
ATPDrug
First Hydrolysis for drug transport
P i, DD
ADP·P iATP
D
ATP
ADP
ADP·P i
Second Hydrolysis for resetting the conformation
ATPADP
Ambudkar, 2002
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Physiologic Role of P-glycoprotein
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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
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Polymorphisms in the MDR1 gene
• 5 common coding polymorphisms (Asn21Asp, Phe103Leu, Ser400Asn, Ala892Ser, Ala998Thr) have no demonstrable effect on drug transport function
• 1 polymorphism which doesn’t change coding sequence is linked to reduced expression in intestines and kidney (Siebenlist et al.). This results in increased absorption and decreased excretion of digoxin and fexofenadine.
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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
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Acute Leukemia:Influence of mdr-1 Expression on Remission Rate
Zhou, et al. Leukemia 6:879, 1992
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XR9576
OC144-093
LY335979
R101933
Newer Pgp Antagonists
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99mTc-Sestamibi Scan following XR-9576Diagnostic assay for Pgp detectionSurrogate assay for Pgp inhibition
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1 hour 2 hours 3 hours
BeforeXR9576
AfterXR9576
Renal Cell Carcinoma99mTc-Sestamibi Uptake in Left Thigh Metastasis
Effect of XR 9576
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Drugs of the Future: Substrates for Drug Transporters
• Work with NCI drug screen suggested that Pgp substrates numbered in the hundreds, if not thousands
• Newly approved agents and agents in the chemotherapy drug development pipeline are substrates for multidrug transporters– Depsipeptide (FR901228)
– STI 571 (Gleevec)
– Irinotecan (SN-38) and novel camptothecins
– Flavopiridol
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Conclusions
• Previous clinical trials aimed at inhibiting P-gp were limited by the need to reduce the dose of anticancer agents
• New inhibitors of P-gp are more potent and have reduced pharmacokinetic effects
• Surrogate studies have confirmed that these inhibitors are able to overcome P-gp in vivo
• Expanding numbers of ABC transporters offer potential as new mediators of drug resistance
• Classical agents, newer agents, and agents in development are likely to be substrates for drug efflux for an ABC transporter.
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Acknowledgements
• Jean-Philippe Annereau• Gergely Szakacs• Claudina Aleman• Chris Hrycyna• Saibal Dey• Chava Kimchi-Sarfaty
• Suresh Ambudkar– Zuben Sauna
• Ira Pastan• Tito Fojo• Susan Bates*• Michael Dean
*Special thanks for several
slides