Post on 19-Jan-2016
SS 2008 lecture 10
Biological Sequence Analysis1
V10 Pharmacogenomics of P-Glycoprotein
Review of lecture V9 .. Paper on Pharmagenomics of PGP
SS 2008 lecture 10
Biological Sequence Analysis2
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
Drug resistance of cells
There are three major mechanisms of drug resistance in cells:
(1) decreased uptake of water-soluble drugs such as folate antagonists,
nucleoside analogues and cisplatin, which require transporters to enter
cells;
(2) various changes in cells that affect the capacity of cytotoxic drugs to kill
cells, including alterations in cell cycle, increased repair of DNA damage,
reduced apoptosis and altered metabolism of drugs; and
(3) increased energy-dependent efflux of hydrophobic drugs that can easily
enter the cells by diffusion through the plasma membrane.
SS 2008 lecture 10
Biological Sequence Analysis3
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
Drug resistance of cells
Of these mechanisms,
the one that is most commonly encountered in the laboratory
is the increased efflux of a broad class of hydrophobic cytotoxic drugs
that is mediated by one of a family of energy-dependent transporters,
known as ATP-binding cassette (ABC) transporters.
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Biological Sequence Analysis4
human ABC transporters
Based on sequence homology 48 different ABC transporters grouped into seven
subfamilies have been defined in the human genome.
Their functions range from export of cholesterol (ABCA1) to regulation of
chloride current (ABCC7-CFTR).
They also play roles in the absorption, distribution, and excretion of
pharmacological compounds.
P-glycoprotein (ABCB1 or MDR1): transports neutral or positively charged
hydrophobic compounds.
ABCC subfamily: also transports organic anions.
Szakács et al. Cancer Cell 6, 129 (2004)
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Biological Sequence Analysis5
Pharmacological roles of ABC transporters
ATP-binding cassette (ABC) transporters act to prevent the absorption of orally ingested or airborne toxins, xenobiotics or drugs.
Highly sensitive compartments, such as the brain, foetus or testes are protected by additional barriers. Enterohepatic circulation, as well asthe excretion of compounds, is regulated by ABC transporters in the liver, gastrointestinal (GI) tract and the kidney. Although the systemic localization of ABC transporters at absorptive barriers provides an effective means to protect against dietary toxins, it also decreases the bioavailability of orally administered drugs and reduces drug disposition to physiological sanctuaries. BBB, blood–brain barrier; BCSFB, blood–cerebrospinal fluid barrier; CSF, cerebrospinal fluid.
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
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Biological Sequence Analysis6
ABC transporters as drug exporters
Given the high degree of similarity of ABCs equences, however, it seems
plausible that additional members may also be drug exporters and thus be
associated with decreased sensitivity of cancer anticancer drugs.
To explore that proposition, Szakács et al. wanted to characterize ABC gene
expression in a set of cancer cells whose responses to a large number of
compounds are known and whose molecular characteristics have been
cataloged.
Szakács et al. Cancer Cell 6, 129 (2004)
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Biological Sequence Analysis7
NCI-60
data set for a panel of 60 human cancer cell lines (the NCI-60) used by the
Developmental Therapeutics Program (DTP) of the National Cancer Institute
(NCI) to screen >100,000 chemical compounds since 1990.
Included among the 60 cell lines are leukemias, melanomas, and cancers of
ovarian, breast, prostate, lung, renal, colon, and central nervous system origin.
Patterns of drug activity across the cell lines and patterns of cell sensitivity
across the set of tested drugs have been shown to contain detailed information
on mechanisms of action and resistance.
In addition to this pharmacological characterization, the NCI-60 cells have been
more extensively profiled at the DNA, mRNA, protein, and functional levels than
any other set of cells in existence.
Szakács et al. Cancer Cell 6, 129 (2004)
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NCI-60 @ Sanger Institute
The Sanger institute is sequencing the NCI-60 for mutations in known human
cancer genes.
The cell lines were supplied by the NCI/NIH Developmental Therpeutics
Program.
The coding exons and immediate flanking intron sequences of selected genes
from the Cancer Gene Census have been PCR amplified and sequenced.
The results of this work have been entered in the COSMIC database and web
site.
„All cancers arise as a result of the acquisition of a series of fixed DNA
sequence abnormalities, mutations, many of which ultimately confer a growth
advantage upon the cells in which they have occurred.“
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Biological Sequence Analysis9
ABC transporter expression
ABC transporter gene expression in the NCI-60 human cancer cell panel. The clustered image map shows patterns of gene expression assessed by real-time RT-PCR. Red and blue indicate high and low expression, respectively.
The hierarchical clustering on each axis was done using the average-linkage algorithm with 1/r as the distance metric, where r is the Pearson’s correlation coefficient, after subtracting row and column means.
Inset: highlights ABC transporters characteristically expressed in melanoma cells.
Szakács et al. Cancer Cell 6, 129 (2004)
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Biological Sequence Analysis10
Substrates and inhibitors of ABC transporters
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
Green: drug-gene pairs in which this ABC transporter was found to be overexpressed inthese cell lines selected for resistance to the respective drugRed: resistant cells overexpressing a single ABC transporter often show characteristic cross-resistance to other, structurally unrelated drugs.
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Biological Sequence Analysis11
Figure legend
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
Overlapping substrate specificities of the human ATP-binding cassette (ABC) transporters
confering drug resistance to cancer cells.
A single drug can be exported by several ABC transporters (rows), and each ABC
transporter can confer characteristic resistance patterns to cells (columns).
To determine which ABC transporters are involved in multidrug resistance (MDR), two
different experimental procedures are common. Cells could be selected in increasing
concentrations of a cytotoxic drug, which could result in the increased expression of a
specific ABC transporter (see green boxes representing drug–gene pairs in which an ABC
transporter was found to be overexpressed in cell lines selected for resistance to the
respective drug).
Resistant cells overexpressing a single ABC transporter often show characteristic cross-
resistance to other, structurally unrelated, drugs (red boxes).
Some ABC transporters were found to confer drug resistance only in transfection studies, in
which cells are engineered to overexpress a given transporter. On transfection, cells
become resistant to compounds that are substrates for transport (red boxes). White boxes
denote unexplored or absent drug–gene relationships.
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Biological Sequence Analysis12
Relationship between drug sensitivity and ABCB1 expression in the NCI-60 for a set of 118 drugs of putatively known mechanism of action.
Blue : known ABCB1 substrates; red : compounds shown in previous studies not to be substrates of ABCB1; black bars : compounds for which data were not available from the literature.
The drug names listed at the top and bottom are commonly used, representative agents from the classes shown by red and blue bars.
Szakács et al. Cancer Cell 6, 129 (2004)
Prediction for 118 drugs with known action
The activity pattern of known substratesof ABCB1 is negatively correlated with the level of B1 expression.Other substances that are not transportedby MDR1 are noncorrelated or positivelycorrelated.
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Biological Sequence Analysis13
Verification of novel ABCB1 substrates by follow-up studies.
A: Scatter plot showing the correlation (r) of ABCB1 expression with sensitivity of
the 60 cells to NSC 363997.
B: MTT assay dose-response curves for treatment of KB-3-1 parental cancer cells
and the selected resistant variant KBV1 with increasing concentrations
of NSC 363997.
KB-3-1: a human carcinoma cell line
KB-V1: a multidrug resistant derivative of KB-3-1 that overexpresses MDR1-PGp
PSC is an MDR1 antagonist. Szakács et al. Cancer Cell 6, 129 (2004)
Predictions for 1400 further compounds
Relevant for test!
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Biological Sequence Analysis14
Prediction from the NCI-60 data of new
substrates for ABCC2-MRP2 and
ABCC11-MRP8, then validation of the
predictions by MTT assay
Szakács et al. Cancer Cell 6, 129 (2004)
Further predictions of PGP substrates
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Biological Sequence Analysis15
Prediction of 1 positively correlated compound!
Prediction from the NCI-60 data, followed
by independent verification,
that the toxicity of NSC 73306 is
potentiated, rather than inhibited,
by expression of ABCB1
Szakács et al. Cancer Cell 6, 129 (2004)
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Conclusions
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
An ultimate goal in cancer therapy is to devise individually tailored treatment that
targets growth-promoting pathways and circumvents drug resistance.
In considering how to go about cataloguing important mechanisms of drug
resistance in cancer, it makes sense to begin by focusing on the family of ABC
transporters, as they are widely expressed in cancer cells and their capacity
to confer drug resistance has been established, at least in vitro.
Pgp represents one of the best-studied mechanisms of resistance to hydrophobic
anticancer drugs.
It remains to be seen whether other ABC transporters will emerge as culprits for
treatment failure.
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Biological Sequence Analysis17
Drug design to block ABC transporters
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
„Despite the clear rationale for the use of inhibitors of ABC transporters,
especially of Pgp, the development of these products and demonstration of their
efficacy has been slow.
With a lack of marketable products, pharmaceutical companies have begun to
lose interest. Only a few compounds are currently in clinical trials, as the
development of most of the inhibitors (including valspodar (PSC-833),
dexniguldipine, dextroverapamil and biricodar (VX-710)) has been discontinued.
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Biological Sequence Analysis18
Drug design to block ABC transporters
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
The bottleneck seems to be the unwelcome inhibition of ABC transporters at
pharmacologically important locations. However, as more and more information
about pharmacokinetic effects accumulate, new-generation inhibitors become
more specific and potent (as shown through careful Pgp measurements and
surrogate biological markers of Pgp inhibition).
Ultimately, we anticipate that the efficacy of ABC transporter modulation will be
established in a subset of human cancers. A clear-cut demonstration of the
effectiveness of targeting Pgp will result in renewed interest and the development
of further ABC transporter inhibitors will follow suit.“