Structure and Function of ABC Transporters in Health and Disease

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1 Structure and Function of ABC Transporters in Health and Disease Michael M. Gottesman, M.D. Chief, Laboratory of Cell Biology Center for Cancer Research, NCI National Institutes of Health, DHHS Clinical harmacology, January 5, 2012 Drug Resistance in Cancer May affect multiple drugs used simultaneously: known as multidrug resistance (MDR) Affects all classes of drugs, including newly designed targeted 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 Ultimate Goals 1. Molecular analysis of human cancers to predict response to therapy 2. Use this information to develop novel drugs to treat cancer and new imaging modalities for cancer 3. To learn more about cellular pharmacology and pharmacokinetics of drugs 1

Mechanisms of resistance to anti-cancer drugs Decreased Uptake-- 100 s of Solute carriers Reduced apoptosis Altered cell cycle checkpoints and/or growth pathways Increased

Important role in multidrug resistance in cancer and in pathogens Important role in drug pharmacokinetics (uptake, distribution, and excretion) Important role in drug toxicity

of transport proteins known. Currently, more than 2000 members have been identified.

2 Mechanisms of resistance to anti-cancer drugs Decreased Uptake s of Solute carriers Reduced apoptosis Altered cell cycle checkpoints and/or growth pathways Increased metabolism of drugs Increased or altered targets Increased repair of damage Compartmentalization Increased Efflux--48 ABC transporters Why study multidrug transporters? Important role in multidrug resistance in cancer and in pathogens Important role in drug pharmacokinetics (uptake, distribution, and excretion) Important role in drug toxicity Key role in development (stem cells, morphogenesis) To learn about the biology of all transport systems -Binding Cassette (ABC) Transporter Superfamily One of the largest family of transport proteins known. Currently, more than 2000 members have been identified. Transport substrates include-- ions, sugars, glycans, phospholipids, cholesterol, peptides, proteins, toxins, antibiotics, and hydrophobic natural product anticancer drugs. Structurally, consist of various combinations of -binding cassettes and segments with 6 trans-membrane domains. 2

choroid plexus 1/5/2012 C. elegans Arabidopsis Dichtyostelium Yeast The Eukaryotic ABCome 57 ABC-family genes Mosquito Bee Human Chimp Drosophila Macaque Dog Chicken Sea squirt Sea urchin.

Dean 48 Human ABC Genes ABCC (12) ABCD (4) ABCD1 ABCC7_2 ABCD2 ABCC1_2 ABCC8_ 2 ABCD3 ABCC3_2 ABCC9_2 ABCC2_2 ABCC11_ 2 ABCC4_2 ABCD4 ABCC6_2 ABCC12_2 ABCC10_2 ABCC7_1 ABCC5_2 ABCB7 ABCB (11) ABCE

3 choroid plexus 1/5/2012 C. elegans Arabidopsis Dichtyostelium Yeast The Eukaryotic ABCome 57 ABC-family genes Mosquito Bee Human Chimp Drosophila Macaque Dog Chicken Sea squirt Sea urchin. falciparum Mouse Rat Opossum Frog. yoelii Fugu Tetraodon Zebrafish From M. Dean 48 Human ABC Genes ABCC (12) ABCD (4) ABCD1 ABCC7_2 ABCD2 ABCC1_2 ABCC8_ 2 ABCD3 ABCC3_2 ABCC9_2 ABCC2_2 ABCC11_ 2 ABCC4_2 ABCD4 ABCC6_2 ABCC12_2 ABCC10_2 ABCC7_1 ABCC5_2 ABCB7 ABCB (11) ABCE (1) ABCC4_1 ABCC10_1 ABCC9_1 ABCC8_1 ABCC5_1 ABCC12_1 ABCC11_1 ABCC6_1 ABCC2_1 ABCC3_1 ABCC1_1 ABCB6 ABCB2 ABCB3 ABCB9 ABCB1_1 ABCB4_1 ABCB5_1 ABCB11_1 ABCB1_2 ABCB4_2 ABCB5_2 ABCB11_2 ABCB10 ABCE1_1 ABCE1_2 ABCF1_2 ABCB8 ABCA1_1 ABCA2_ ABCA7_1 ABCF (3) ABCF3_2 ABCA4_1 ABCA3_1 ABCF2_2 ABCA12_1 ABCA13_1 ABCF2_1 ABCA8_1 ABCF3_1 ABCA9_1 ABCA10_1 ABCF1_1 ABCA6_1 ABCA5_1 ABCG4 ABCG1 ABCA1_2 ABCG5 ABCA4_2 ABCG8 ABCG2 ABCA2_2 ABCA5_2 ABCA9_2 ABCA7_2 ABCA13_2 ABCA3_2 ABCA6_2 ABCA8_2 ABCA10_2 ABCA12_2 ABCG (5) ABCA (12) The Clustal W program was used to make the alignment of the NBDs and the tree was built by using the MEGA program -- By Mike Dean, NCI ABC transporters determine oral bioavailability, excretion, penetration and protect the organism against airborne xenobiotics BLOOD C2, B1, B4, B11, G2 G2 B1 C1 C1, C3-5 GI TRACT C1 oral aerosol LIVER C2, B1, G2 FETUS LUNG B1, C1, A3,G21 B1, G2 C1 C1 3

4 Human diseases associated with an ABC Transporter Disease Transporter Cancer ABCB1, ABCC1, ABCG2 Cystic fibrosis ABCC7 (CFTR) Stargardt disease & AMD ABCA4 (ABCR) Tangier Disease (HDL deficiency) ABCA1 (ABC1) rogressive familial intrahepatic cholestasis ABCB11(SG), ABCB4 (MDR2) Dubin-Johnson syndrome ABCC2 (MR2) seudoxanthoma elasticum ABCC6 (MR6) ersistent hypoglycemia of infancy, neonatal diabetes ABCC8 (SUR1), ABCC9 (SUR2) Sideroblastic anemia and ataxia ABCB7 (ABC7) Adrenoleukodystrophy ABCD1 (ALD) Sitosterolemia ABCG5, ABCG8 Immune deficiency ABCB2 (Tap1), ABCB3 (Tap2) ABC transporters that confer MDR: Domain organization ABCB1 TM Domain binding TM Domain binding ABCC1 ABCG2 Overlapping substrate specificity of ABCB1, ABCG2 and ABCC1 ABCB1 aclitaxel Colchicine Verapamil razosin Topotecan Bisantrene Dihydropyridines H33342 Fluo-3-AM Calcein-AM Vinblastine Doxorubicin Mitoxantrone Daunorubicin Etoposide Nilotinib heophorbide A Sulfasalazine Flavopiridol ABCG2 Calcein LTC4 NEM-GS Estrone- 3-sulfate Methotrexate ABCC1 4

Multiple ABC Transporters Confer Resistance to Anti-Cancer Drugs Confers resistance Selected Hypothetical Model of Human - glycoprotein 100 In Membrane 200 Out 300 SITE SITE A B 700 1000 1200 A B 400

5 Multiple ABC Transporters Confer Resistance to Anti-Cancer Drugs Confers resistance Selected Hypothetical Model of Human - glycoprotein 100 In Membrane 200 Out 300 SITE SITE A B A B 400 C 800 C OINT MUTATIONS ( ), HOTOAFFINITY LABELED REGIONS ( ), AND HOSHORYLATION SITES ( ) -glycoprotein removes hydrophobic substrates directly from the plasma membrane + Out Membrane + + In 5

sufficient to confer MDR Cancers which acquire expression of -gp following treatment of the patient include leukemias, myeloma, lymphomas, breast, ovarian cancer; preliminary results with -gp

6 Atomic models of the structures of -gp Mouse -gp at 3.8Å (Aller and Chang) Human -gp model based on Sav1866 (Xia) hysiologic Role of -glycoprotein Role of -glycoprotein in cancer Approximately 50% of human cancers express -glycoprotein at levels sufficient to confer MDR Cancers which acquire expression of -gp following treatment of the patient include leukemias, myeloma, lymphomas, breast, ovarian cancer; preliminary results with -gp inhibitors suggest improved response to chemotherapy in some of these patients Cancers which express -gp at time of diagnosis include colon, kidney, pancreas, liver; these do not respond to -gp inhibitors alone and have other mechanisms of resistance Animal models with human cancer xenografts and BRCA1- driven mouse mammary cancers show role for -gp in MDR (ajic et al., Cancer Res. 69, , 2009) 6

PET Imaging of P-gp: an efflux transporter that protects brain but also causes drug resistance

PET Imaging of P-gp: an efflux transporter that protects brain but also causes drug resistance Robert B. Innis, MD, PhD Molecular Imaging Branch National Inst. Mental Health 1 Outline of Talk 1. P-glycoprotein