Transcript Computational Biology

V10 Pharmacogenomics of P-Glycoprotein
Review of lecture V9 .. Paper on Pharmagenomics of PGP
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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.
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
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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.
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
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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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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 as
the 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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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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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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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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Substrates and inhibitors of ABC transporters
Green: drug-gene pairs in which this ABC transporter was found to be overexpressed in
these cell lines selected for resistance to the respective drug
Red: resistant cells overexpressing a single ABC transporter often show characteristic
cross-resistance to other, structurally unrelated drugs.
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Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
Figure legend
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 crossresistance 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.
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
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Prediction for 118 drugs with known action
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.
The activity pattern of known substrates
of ABCB1 is negatively correlated with
the level of B1 expression.
Other substances that are not transported
by MDR1 are noncorrelated or positively
correlated.
Szakács et al. Cancer Cell 6, 129 (2004)
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Predictions for 1400 further compounds
Relevant for test!
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)
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Further predictions of PGP substrates
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)
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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
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.
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
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Drug design to block ABC transporters
„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.
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
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Drug design to block ABC transporters
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.“
Szakács et al. Nat. Rev. Drug Disc. 5, 219 (2006)
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SS 2008
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