THE ROLE OF THE ABC TRANSPORTERS DRUG RESISTANCE AND PHARMACOLOGY

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THE ROLE OF THE ABC TRANSPORTERS DRUG RESISTANCE
AND PHARMACOLOGY

• Michael Dean

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ATP-BINDING CASSETTE (ABC) GENES
-Use ATP to pump compounds across
membranes. -Many ABC genes are found in all
living organisms. -Pump from cytoplasm to outside
the cell or into organelles in eukaryotes. -Involv
ed in chemotherapy drug resistance and many
genetic diseases.
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CF SLIDE
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ABC GENE STRUCTURE
ATP
TM
Half-transporter Full transporter
-ABC genes are organized either as half
transporters These form hetero- or
homodimers. -Or, they can be organized as full
molecules with the entire transporter in one
transcript.
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HUMAN ABC GENES
1 2 3 4 5 6 7
8 9 10 11 12 13
A B C D E F G
14 15 16 17 18 19 20 21
22 X Y
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ABC GENES TRANSPORTERS MEDIATE CHEMOTHERAPY DRUG
RESISTANCE
ABCB1/MDR1
ABCC1/MRP1
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MITOXANTRONE ACCUMULATION IN RESISTANT CELLS
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OVEREXPRESSION OF ABCG2
ABCG2-
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ABC PROTEIN INHIBITORS TO BLOCK DRUG RESISTANCE
X
ABCB1/MDR1
X
ABCC1/MRP1
X
ABCG2
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ABC DRUG TRANSPORTERS
Gene Substrates Inhibitors ABCB1/PGP colchicin
e, doxorubicin, VP16, Verapamil,
PSC833 adriamycin, vinblastine, digoxin
ABCC1/MRP doxorubicin, daunorubicin, VP16,
Cyclosporin A, V-104 colchicines, etoposide,
rhodamine ABCC2 Vinblastine, sulfinpyrazone,
ABCC3 Methotrexate, etoposide ABCC4 Nucleosid
e monophosphates ABCG2/MXR Mitoxantrone,
topotecan, CPT-11, Fumitremorgin C,
rhodamine GF120918
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P-Glycoprotein
Pgp Substrates Adriamycin Daunorubicin Epirubicin
Paclitaxel Docetaxel Vincristine Vinblastine VP-16
Mitoxantrone Actinomycin D
Pgp Inhibitors Verapamil Quinidine Quinine Cyclos
porine A PSC 833 VX-710 LY335979 R101933 OC144-093
XR9576
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Pgp Substrates in Initial Therapy Drugs of
Choice According to The Medical Letter
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Pgp inhibitorsSteadily improving

• 1st generation agents
• hampered by lack of potency or toxicity
• verapamil, quinidine, cyclosporine A
• 2nd generation agents
• hampered by PK interaction, dose reductions,
• and interpatient variability
• PSC 833 (Valspodar), VX-710 (Biricodar)

• 3rd generation agents
• nontoxic, potent, and minimal PK interaction
• XR9576, LY335979, OC144-093, R101933

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Laniquidar
Zosuquidar
Tariquidar
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Rhodamine Efflux from CD56 Circulating Cells is
Still Blocked 48 h after a Single Dose of XR-9576
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Hepatic Retention of Sestamibi
Pre-XR9576
Post-XR9576
Sestamibi cpm/px/mCi
Sestamibi cpm/px/mCi
Surrogate assay
Time min
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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
• Irinotecan (SN-38) and novel camptothecins
• STI 571 (Gleevec)
• Flavopiridol
• Depsipeptide
• Gemcitabine
• Antiretroviral agents e.g., Nelfinavir

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ABCG2 Substrate Specificity
Pgp substrates Vincristine Vinblastine Taxol Colch
icine VP-16
MRP substrates Vincristine VP-16 Calcein
Flavopiridol
Overlap with Pgp and MRP
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ABCG2 Expression in Cancer
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ABCG2 INHIBITORS BIOAVAILABILITY
ABCG2 is highly expressed in the intestine and
inhibitors could increase the bioavailability of
many compounds. ABCG2 is plays a role in
maintaining the blood-brain barrier. Inhibitors
could be crucial to efforts to treat brain
tumors.
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FUMITREMORGIN C ANALOGS
FTC is a potent and specific inhibitor, but has
toxicity. The analogues are well tolerated in
mice.
Allen JD, et al.Mol Cancer Ther. 2002 1417-25.
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ABCG2 INHIBITORS INCREASE ORAL AVAILABILITY OF
TOPOTECAN
Schinkel and colleagues
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483 - 499
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ABCG2 substrate specificity is affected by amino
acid 482
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Cross-resistance studies with HEK 293-MXR
transfectants
482G and 482T are better mitoxantrone
pumps than wild type 482R
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ABCG2 mutation alters substrate selectivity
pcDNA3-10
S1-M1-80
482T-10
482G-2
482R-2
482G-1
482R-5
482T-7
ABCG2
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SNPs in ABCG2 sequence
aa
nt
Non-coding SNP Exon 1 C91T Exon
1 A175G Exon 16 G2597T Missense SNP Exon
2 G238A Val12Met Exon 5 C625A Gln141Lys Exon
16 G2062A Asp620Asn Silent SNP Exon
9 G1302A Glu366Glu Exon 12 A1629G Leu475Leu No
SNP Exon 3, 4, 6, 7, 8, 10, 11, 13, 14,
15 Intronic variants near Exons 2, 6, 7(5),
7(3), 11, 12, 14
aa 482 in Exon 12 no SNP found
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Single Nucleotide Polymorphism in ABCG2
D620N
V12M
Q141K
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3D STRUCTURE OF AN ABC PROTEIN
Chang and Roth Science (1991).
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TM PEPTIDE INHIBITORS
Peptide to TM domain
Peptide integrates Into protein and inhibits
-Successfully applied to GPCRs, ABCB1 and
ABCG2. -N Tarasova, C. Michejda
NCI-Frederick. JBC 27434911 (1999).
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SIDE POPULATION CELLS

• Using a cell sorter and the Hoechst 33342 dye a
  poorly staining population highly enriched in
  stem cells can be isolated.
• These cells fail to stain because they express
  drug transporters, especially ABCG2.

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Characteristic Hoechst 33342 dye staining
profiles of two primary neuroblastoma specimens
from patients with relapsed disease
Hirschmann-Jax, C. et al. (2004) Proc. Natl.
Acad. Sci. USA 101, 14228-14233
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SP cells from neuroblastoma cell lines become SP
and non-SP cells, whereas non-SP cells remain
non-SP (illustrated for SK-N-SH)
Hirschmann-Jax, C. et al. (2004) Proc. Natl.
Acad. Sci. USA 101, 14228-14233
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EXISTENCE OF SP CELLS IN ESTABLISHED CANCER CELL
LINES
Kondo, Toru et al. (2004) Proc. Natl. Acad. Sci.
USA 101, 781-786
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STEM (SP) CELLS IN CELL LINES

• C6 Rat Glioma 0.4
• HS 683 Human Glioma 2.0
• B104 Rat Neuroblastoma 0.4
• MCF7 Human Breast 2.0
• HeLa Human Cervical Carcinoma 1.2
• SK-BR-3 Human Breast adenocarcinoma 0.2
• SK-OV-3 Human Ovarian adenocarcinoma 1.2
• NCI-H146 Human Small cell lung 0.8
• RD-ES Human Ewing sarcoma 0.7
• U-2OS Human Osteosarcoma 0
• SaOS-2 Human Osteosarcoma 0
• A204 Human Rhabdomyosarcoma 0

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TUMOR THERAPY
-Therapies may seem to be working and the tumor
disappears, but the tumor stem cells may grow
back. -Studies of the bulk tumor dont tell us
about the critical cells, the stem cells.
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CONCLUSIONS

• ABCG2/MXR is an important player in the delivery
  of drugs to patients, and may play a role in
  leukemias and other tumors.
• While some promising ABCG2 inhibitors are
  available, the lesson from PGP studies is that we
  need a wide array of reagents for in vitro, in
  vivo and clinical tests.
• Cell lines that efflux fluorescent ABCG2
  substrates are available for a HTS assay.
• ABC inhibitors may have important applications in
  the elimination of cancer stem cells