1 / 64

P-GLYCOPROTEIN AND DRUG TRANSPORT

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.

emmet
Download Presentation

P-GLYCOPROTEIN AND DRUG TRANSPORT

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. Estimated New Cancer Cases & Deaths, 2005 **Vast majority of deaths due to chemotherapy resistance CA Cancer J Clin, 2005

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

  4. 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

  5. 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

  6. Mechanisms of Resistance Overexpressed ABC Transporters Fewer Functional UptakeTransporters Defective endocytosis

  7. Pgp DIFFUSION D D D D D D D D D D D D D D D D D D D D D D D D MXR GS MRPs (ABCB1) (ABCG2) D D ABCC1-C4

  8. ATP-BINDING CASSETTE(N-terminal NBD of human Pgp) 390 427 556 620 A C B Walker A ABC linker Walker B D-loop Y GNSGCGKST LSGGQKQRIAIA ILLLDEA TSALD

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

  10. ABCB1 TM Domain TM Domain ATP binding ATP binding ABCC1 ABCG2 ABC transporters: Domain organization

  11. 100 OUT MEMBRANE IN P P P P Hypothetical Model of Human P-glycoprotein 200 ATP SITE ATP SITE 300 1200 A 1000 A B B 700 C C 400 800 900 1100 600 1 500 POINT MUTATIONS ( ), PHOTOAFFINITY LABELED 1280 REGIONS ( ), AND PHOSPHORYLATION SITES ( P )

  12. 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

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

  14. 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?

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

  16. 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

  17. Physiologic Role of P-glycoprotein

  18. 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 liverexcretion

  19. Position of Common Polymorphisms in P-gp

  20. 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.

  21. pTM1 MDR1 C1236T G2677T C3435T C1236T-G2677T C1236T-C3435T G2677T-C3435T C1236T-G2677T-C3435T MDR1 wild-type, SNPs, and haplotypes show similar P-gp total and surface expression WT pTM1 X3 P-gp

  22. MDR1 wild-type and the haplotype (1236-2677-3435) exhibit similar rhodamine 123 efflux 1236-2677-3435 pTM1 control MDR1 WT

  23. MDR1 wild-type and the haplotype (1236-2677-3435) do not exhibit similar Bodipy-verapamil accumulation X3 pTM1 WT

  24. pTM1 control MDR1 WT 1236-2677-3435 MDR1 wild-type and the haplotype exhibit different patterns using rhodamine 123 efflux with cyclosporin A reversing agent 5 mM CsA

  25. MDR1 wild-type and haplotype show the same P-gp cell surface expression using MRK16 and 17F9, but not UIC2 - a conformational sensitive antibody pTM1 WT 1236-2677-3435

  26. MDR1 wild-type and haplotype show different trypsinization patterns confirming altered conformation

  27. 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.

  28. Phylogenetic Tree of the Human ABC Genes 48 Human ABC Transporters Dean. Genome Res 11:1156, 2001

  29. ABC transporters determine oral bioavailability, excretion, penetration and protect the organism against airborne xenobiotics B1, C1, C2, G2 MILK BRAIN BBB BLOOD G2 C2, B1, B4, B11, G2 B1 C1, C3-5 LIVER C1 PLACENTA GI TRACT C2, B1, G2 FETUS C1 oral CSF choroid plexus aerosol B1, C1, A3,G21 B1, G2 LUNG KIDNEY B1, C2, G2 BTB URINE TESTIS C1 C1

  30. Human diseases associated with an ABC Transporter DiseaseTransporter 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)

  31. ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ABC transporters which are known to transport drugs Common Names Systematic Name Structure Substrates Normal location Intestine, liver, kidney, Blood-brain barrier Neutral and cationic Organic compounds Pgp, MDR1 ABC B1 ATP ATP ABC C1 GS-X and other conjugates, organic anions Widespread MRP1 ATP MRP2 cMOAT GS-X and other conjugates, organic anions Intestine, liver, kidney ABC C2 GS-X conjugates, anti- Folates, bile acids, etoposide Pancreas, intestine, liver, kidney, adrenal MRP3 MOAT-D ABC C3 Prostrate, testis, ovary intestine, pancreas, lung MRP4 MOAT-B Nucleoside analogs, methotrexate ABC C4 Nucleoside analogs, cyclic nucleotides, organic anions MRP5 MOAT-C ABC C5 Widespread MRP-6 MOAT-E ABC C6 Anionic cyclic pentapeptide Liver, kidney MXR, BCRP ABC-P Intestine, placenta, liver, breast ABC G2 Anthracyclines, mitoxantrone

  32. ABC Transporters Confer Resistance to Anti-Cancer Drugs Confers resistance Selected Doesn’t transport

  33. 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

  34. 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

  35. Expression of ABC-transporters in the NCI60 panel .

  36. Correlation of Matrices ABC-transporters Drugs Pinkel D,Nature Genetics24, 208-9 (2000)

  37. 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

  38. Correlation of expression and sensitivity Expression of an ABC-transporter Activity of a Drug 60 cells

  39. Inversely correlated compounds are ABCB1 substrates KB-V1: MDR derivative of KB-3-1 overexpressing P-gp KB-3-1 (MDR1-) KB-V1 (MDR1+) KB-3-1+PSC 833 KB-V1 +PSC 833

  40. Inversely correlated compounds indicate potential substrates for 28 ABC transporters Analysis 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

  41. NSC 73306 Substrates MDR1-potentiated compounds? Search for MDR1-potentiated compounds in DTP’s database 17K

  42. The cytotoxicity of NSC 73306 is increased in KB-V1 cells r = 0.54 NSC73306 KB-3-1 (MDR1-) KB-V1 (MDR1+) KB-3-1 +PSC 833 KB-V1 +PSC 833

  43. Search for MDR1-potentiated compounds in DTP’s database 50 candidates 7 thiosemicarbazones

  44. P-gp GAPDH 1 2 3 4 5 6 7 8 9 Selected: - + - + - + - + - Cell lines: HCT15-2A ADR KBV1 DX5 / MES-SA Western Blot of Pgp-positive cells selected in 1ug/ml NSC 73306. Loss of P-gp in NSC73306 Selected Cells

  45. 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

  46. 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

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

  48. Newer Pgp Antagonists R101933 OC144-093 LY335979 XR9576

  49. 99mTc-Sestamibi Scan following XR-9576 Diagnostic assay for Pgp detection Surrogate assay for Pgp inhibition

  50. 1 hour 2 hours 3 hours Renal Cell Carcinoma 99mTc-Sestamibi Uptake in Left Thigh Metastasis Effect of XR 9576 Before XR9576 After XR9576

More Related