Transcript No Slide Title

P-GLYCOPROTEIN AND DRUG
TRANSPORT
Michael M. Gottesman
Deputy Director for Intramural Research
National Institutes of Health
January 16, 2003
What is the Scope of the Problem?
Estimated New Cancer Cases & Deaths, 2001
Site
New
Cases
All Sites
Oral Cavity & P harynx
Digestive System
Respiratory System
Bones, Joints, & Soft Tissues
Breast
Prostate
Genital System
Urinary System
Endocrine System
Brain & Nervous System
Lymphoma & Leukemia
Other & Unspecified
**
Deaths
1,268,000 553,400
30,100
7,800
235,700 131,300
184,600 162,500
11,600
5,800
193,700
40,600
198,100
31,500
88,700
58,500
87,500
25,000
21,400
2,300
17,200
13,100
95,100
49,100
31,400
36,200
%
44%
26%
56%
88%
50%
21%
16%
66%
29%
11%
76%
52%
**Vast majority of deaths due to chemoresistance
CA Cancer J Clin.51:23, 2001
Mechanisms of resistance to anticancer drugs
Decreased
uptake
Reduced apoptosis
Altered cell cycle checkpoints
Increased metabolism of drugs
Increased or altered targets
Increased repair of damage
Compartmentalization
Increased
efflux
How Drugs Get Into Cells
Diffusion
Transport
Endocytosis
D
D
D
D
D
e.g., vinblastine,
doxorubicin
D
e.g., nucleoside
analogs
D
e.g., immunotoxins
DIFFUSION
D DD
D
D
D
(ABCB1)
D
D
Pgp
D
D
D
D
DD
D
D
D
D
(ABCG2)
D
D
GS
D
MRPs
D
ABCC1-C4
DD
MXR
D
D
ATP-BINDING CASSETTE
(N-terminal NBD of human Pgp)
390
427
556
A
Walker A
Y GNSGCGKST
C
ABC linker
620
B
Walker B D-loop
LSGGQKQRIAIA ILLLD EA TSALD
Structural Organization of an ABC Transporter
Transmembrane Domain
R
\/\/\
ATP-Binding Domain
ABC transporters: Domain organization
MDR1
TM Domain
MRP1
MXR
ATP binding
TM Domain
ATP binding
Structure of E.coli BtuCD, a vitamin B12 transporter.
Locher et al. Science. 2002
20 transmembrane helices
ATP binding domains
X-ray structure 3.2 A
Phylogenetic Tree
of the Human
ABC Genes
Dean. Genome Res 11:1156, 2001
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)
ABC transporters which are known to transport drugs
Common
Names
Systematic
Name
Pgp, MDR1
ABC B1
Structure
ATP
MRP1
ABC C2
MRP3
MOAT-D
ABC C3
MRP4
MOAT-B
ABC C4
MRP5
MOAT-C
ABC C5
MRP-6
MOAT-E
ABC C6
MXR, BCRP
ABC-P
ABC G2
ATP
ATP
ATP
ATP
ATP
Normal location
ATP
Neutral and cationic
Organic compounds
Intestine, liver, kidney,
Blood-brain barrier
Widespread
ATP
GS-X and other conjugates,
organic anions
GS-X and other conjugates,
organic anions
Intestine, liver, kidney
ATP
GS-X conjugates, antiFolates, bile acids, etoposide
Pancreas, intestine, liver,
kidney, adrenal
ATP
Nucleoside analogs,
methotrexate
Prostrate, testis, ovary
intestine, pancreas, lung
Widespread
ATP
Nucleoside analogs, cyclic
nucleotides, organic anions
Anionic cyclic pentapeptide
Liver, kidney
ABC C1
MRP2
cMOAT
Substrates
ATP
ATP
Anthracyclines, mitoxantrone
Intestine, placenta, liver,
breast
Chemotherapeutic Substrates for the MRP
Family of ABC Transporters
OA VP-16 ADR VCR CPT MTX 6MP GEM
MRP1
MRP2
MRP3
MRP4
MRP5
MRP6
MRP7
MRP8
MRP9
+
+
+
+
+
-
Borst, BBA 1461:347-357, 1999
+
+
+
-
+
+
-
+
+
-
+
-
+
+
+
+
-
+
+
-
+
-
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
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.
RT-PCR of the 48 ABC transporters
…………
500 bp
500 bp
Real Time RT-PCR data KB-3-1 vs KB-8-5 (MDR)
Cp KB-8-5
MDR
45
40
35
GST-p
30
Actin
25
MDR1
20
MDR1(3’-UTR)
15
15
20
25
30
35
40
45
Cp KB-3-1
A5-A
B2
C5
D4
C4
B5
C2
D1
G1
B4
A5-B
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
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
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)
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
Hypothetical Model of Human P-glycoprotein
100
OUT
200
MEMBRANE
IN
ATP SITE
300
A
ATP SITE
B
1000
700
A
B
1200
P
400
C
C
800
P
600
P
900
1100
P
1
500
POINT MUTATIONS ( ),
PHOTOAFFINITY LABELED
REGIONS (
), AND
PHOSPHORYLATION SITES ( P )
1280
Substrates and Reversing Agents of Pgp
OCH3
OH
N
Sar
H3 CO
L-Pro
H3 CO
D-Val
H
N
N
C
C
N
Colchicine
NH 2
O
CH3
CH 3
Actinomycin D
O
O
OH
C
O
NH
H
C
C
H
CHOCH3
O
C
C
O
O
CH 3
H 3C
OH
HO
CH 3
O
CH 3
O
OH
O
H3 CO
CH3
O
HO
OH
O
C
CH 2
O
H3C
CH 3
O
N
Taxol
H
O
H3C
H3 CO
H3 CO
Daunorubicin
O
HO
OCH 3
H 3C
OCH3
CN
H
OH
H3 CO
O
O
H
O
H3C
O
O
H 3C
H
OH
L-T
O hr
O
H 3C
NH 2
O
NHCOCH3
Vinblastine
H
D-Val
O
COOCH 3
COOCH3
HO
CH3
H
O
O
CH 2CH 3
OCH 3 O
L-Meval
L-Thr
H3 CO
O
L-Pro
CH 2CH 3
N
H
H3 COOC
H3 CO
Sar
L-Meval
C
CH3
C C C N
H2 H2 H2
CH(CH3) 2
C C
H2 H2
Verapamil
OCH3
CH 3
CH3
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?
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
Stoichiometry of ATP molecules hydrolyzed to substrate
molecules transported
Pump
Substrate
Ratio
(ATP hydr oly sis:
Tr ans por t)
Histidine permease
Oligopeptide permease
P-glycoprotein
FoF1
VoV1
Na+/K+ ATPase
Ca+ ATPase
Histidine
Oligopeptides
Vinblastine
Rhodamine 123
H+
H+
Na+, K+
Ca+
2:1
2:1
2-3:1
2:1
1:4
1:2
1:3:2
1:2
Vanadate-trapping can be used to
dissect intermediates
E•ATP
E•ADP + Pi
+ Vi
E•ADP-Vi
(trapped conformation)
8AzATP
+ Vi
8AzATP
ATP+Vi
Vi
ATP
CON
P-glycoprotein in the vanadate-trapped conformation
shows reduced affinity for the substrate IAAP
213
IAAP incorporated
(pmoles/ pmole Pgp)
120
Control
AMPPNP +Vi
ATP + Vi
IAAP [nM]
Recovery of IAAP binding to P-glycoprotein in transition
state conformation requires ATP hydrolysis
(arbitrary units)
IAAP incorporation
6
ATP+Mg+2
4
ATP+EDTA
AMPPNP
ATP+VO4
2
0
0
5
Time (min)
10
15
Catalytic Cycle of P-glycoprotein
P i, D
ATP
Drug
D
D
ATP
ADP·P i
ADP
First Hydrolysis for
drug transport
ADP
ADP
ATP
ADP
ADP·P
i
ATP
Second Hydrolysis for
resetting the conformation
Ambudkar, 2002
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
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.
Role of P-glycoprotein in cancer
• Approximately 50% of human cancers express Pglycoprotein 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
Newer Pgp Antagonists
R101933
OC144-093
LY335979
XR9576
99mTc-Sestamibi
Scan following XR-9576
Diagnostic assay for Pgp detection
Surrogate assay for Pgp inhibition
Renal Cell Carcinoma
99mTc-Sestamibi Uptake in Left Thigh Metastasis
Effect of XR 9576
Before
XR9576
1 hour
After
XR9576
2 hours
3 hours
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
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.
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