1. P-GLYCOPROTEIN AND DRUG TRANSPORT Michael M. Gottesman Deputy Director for Intramural Research National Institutes of Health January 16, 2003

2. 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 **

3. Reduced apoptosis Altered cell cycle checkpoints Increased metabolism of drugs Increased or altered targets Increased repair of damage Compartmentalization Mechanisms of resistance to anti- cancer drugs Decreased uptake Increased efflux

5. D DDD D D D D D D D DIFFUSION D D D D Pgp D D GS MRPs D D D D D D D MXR DDDD (ABCB1) (ABCG2) ABCC1-C4

6. 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

7. Structural Organization of an ABC Transporter Transmembrane Domain ATP-Binding Domain \/\/\ R

8. ABC transporters: Domain organization MRP1 MXR MDR1 TM Domain ATP binding

9. Structure of E.coli BtuCD, a vitamin B 12 transporter. ATP binding domains 20 transmembrane helices X-ray structure 3.2 A Locher et al. Science. 2002

10. Phylogenetic Tree of the Human ABC Genes Dean. Genome Res 11:1156, 2001

11. 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 deficiencyABCB2 (Tap1), ABCB3 (Tap2)

12. Pgp, MDR1 MRP2 cMOAT MRP4 MOAT-B MXR, BCRP ABC-P MRP1 MRP3 MOAT-D MRP5 MOAT-C MRP-6 MOAT-E ABC B1 ABC C1 ABC C2 ABC C3 ABC C6 ABC G2 ABC C4 ABC C5 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, cyclic nucleotides, 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 Systematic Name StructureSubstratesNormal location ABC transporters which are known to transport drugs ATP

13. Chemotherapeutic Substrates for the MRP Family of ABC Transporters Borst, BBA 1461:347-357, 1999 MRP1 MRP2 MRP3 MRP4 MRP5 MRP6 MRP7 MRP8 MRP9 +++++----+++++---- OA VP-16 ADR VCR CPT MTX 6MP GEM -+--------+------- ++-------++------- ++++-----++++----- ++-------++------- +++------+++------ ---++-------++---- ----+--------+----

14. 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

15. 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.

16. 308498 125 295 187 551 238 269244566430 204 502398 199 405341 476 389 221 514 525 243 193 357 319 265 317 298 406 ABC-B1ABC-B2ABC-B3 ABC-B4 ABC-B11 ABC-C1 ABC-C2 ABC-C3ABC-C4ABC-C5ABC-C6 ABC-A1 ABC-A3ABC-A4 ABC-A5 ABC-G1ABC-G4 ABC-C7 ABC-C9 ABC-C11 ABC-D1 ABC-D4 ABC-G2 PXR ABC-B1 ABC-A6 gapdh ABC-G5 500 bp ………… PBDG ABC-B3 ABC-C8 ABC-G8 ABC-B6 ABC-F1 ABC-A10 ABC-B6ABC-D3 ABC-B9 ABC-A5ABC-A7ABC-B7 ABC-F3 ABC-A8ABC-B8 ABCB5 ABC-A12ABC-C12 ABC-D2 ABC-E1 ABC-C12 ABC-G5 MARKER ABC-F2 ABC-A2 500 bp 298 480212 283 233 268282 277385 256514362254 381334 232 519 261 545 272242 341 332 323 446 ABC-B10 GST-Pi B-Actin PBGD PXR RT-PCR of the 48 ABC transporters

17. 15 20 25 30 35 40 45 15202530354045 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)

18. B4A5-B A5-A B2 C5D4C4B5C2D1G1 A6-A C6PXR G4-AC7(3627) B11G5-BA4B3G8A9YWHAZC1B1 B8C3A1A10 A7 B10F3B6 B9C9-B C9-AA8G1A3C10B3A2C12D2A12G4-BA6-BC8GAPDH G2G5-AF1D3B7E1F2C11C7 (555)PBDG

19. 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)

20. 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

21. 600 OUT MEMBRANE IN 200 100 300 400 500 700 1 1100 1280 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

22. Substrates and Reversing Agents of Pgp Vinblastine Daunorubicin Colchicine Verapamil Taxol Actinomycin D Rapamycin

23. 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?

24. P-glycoprotein removes hydrophobic substrates directly from the plasma membrane

25. 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

26. Stoichiometry of ATP molecules hydrolyzed to substrate molecules transported

27. Vanadate-trapping can be used to dissect intermediates EATPEADP + Pi + ViEADP-Vi (trapped conformation)

28. P-glycoprotein in the vanadate-trapped conformation shows reduced affinity for the substrate IAAP IAAP incorporated (pmoles/ pmole Pgp) IAAP [nM] 213 120 CONATPVi ATP+Vi 8AzATP + Vi Control AMPPNP +Vi ATP + Vi

29. Recovery of IAAP binding to P-glycoprotein in transition state conformation requires ATP hydrolysis IAAP incorporation (arbitrary units) ATP+Mg +2 ATP+EDTA AMPPNP ATP+VO4 Time (min)

30. Catalytic Cycle of P-glycoprotein ADP ATP Drug First Hydrolysis for drug transport P i, D D ADP·P i ATP D ADP ADP·P i Second Hydrolysis for resetting the conformation ATP ADP Ambudkar, 2002

31. Physiologic Role of P-glycoprotein

32. 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

33. 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.

34. 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

35. Acute Leukemia: Influence of mdr-1 Expression on Remission Rate Zhou, et al. Leukemia 6:879, 1992

36. XR9576 OC144-093 LY335979 R101933 Newer Pgp Antagonists

37. 99m Tc-Sestamibi Scan following XR-9576 Diagnostic assay for Pgp detection Surrogate assay for Pgp inhibition

38. 1 hour2 hours3 hours Before XR9576 After XR9576 Renal Cell Carcinoma 99m Tc-Sestamibi Uptake in Left Thigh Metastasis Effect of XR 9576

39. 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

40. 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.

41. 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