P-GLYCOPROTEIN AND DRUG TRANSPORT

Michael M. GottesmanDeputy Director for Intramural ResearchNational Institutes of Health

January 16, 2003

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

**

Reduced apoptosisAltered cell cycle checkpointsIncreased metabolism of drugsIncreased or altered targetsIncreased repair of damageCompartmentalization

Mechanisms of resistance to anti-cancer drugs

Decreaseduptake

Increasedefflux

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

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

MRP1

MXR

MDR1

TM Domain TM DomainATP binding ATP binding

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

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)

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

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

- + - --- ---

+ + - --- ---

+ + + +- ----

+ + - --- ---

+ + + --- ---

- - -++ ----

- - --+ ----

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.

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

2

51

9

26

1

54

5

27

2

24

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

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)

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

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

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

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 substratesdirectly 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 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

Vanadate-trapping can be used to dissect intermediates

E•ATP E•ADP + Pi

+ Vi E•ADP-Vi

(trapped conformation)

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

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)

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

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 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

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

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