Biology Reference
In-Depth Information
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FIGURE 6.3
Protein
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protein interactions in the NER pathway focusing on damage recognition proteins and incision nucleases.
proteins, the interactions can be driven by primarily
ionic interactions between the sugar phosphate back-
bone of the DNA and hydrophobic interactions with
the nitrogenous bases occurring in the major grove or
with single-stranded DNA.
Both RPA and XPA have been the subject of extensive
investigation with respect to DNA damage recognition
in the NER pathway. The studies have ranged from basic
enzymology to kinetic analysis and extensive muta-
tional analysis. While RPA is an essential protein, the
rationale for targeting this protein stems from its role
in DNA replication and demonstration that knockdown
can induce apoptosis and sensitize cells to DNA
damaging agents.
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In addition, mutations in RPA
have been linked to the development of cancer
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and
thus inhibition of RPA is likely to inhibit the growth of
cancer cells and ultimately induce cell death. XPA's
only known role is in NER and thus reduced activity
of this protein can sensitize cells to cisplatin therapy,
which has been demonstrated in numerous cell culture
models. In addition, the extreme sensitivity of testicular
cancer cells to platinum-based therapies has been attrib-
uted, in part, to the low level of XPA.
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Compared to
other NER proteins, XPA is not highly expressed and
may be one of the limiting factors in the repair pathway
and thus targeting this protein holds great potential.
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All of these studies over the past decades have resulted
in a great depth of understanding of the protein
RPAwas first targeted in a high throughput screen of
the NCI diversity set of compounds and small molecules
were identified that inhibited the protein
DNA interac-
tion and inhibited excision of a cisplatin-damaged DNA
in a reconstituted NER assay.
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More recently the ability
of derivatives of these original compounds to inhibit
RPA was demonstrated in cell culture models of lung
and ovarian cancer (submitted). In addition, screening
of larger commercial libraries identified a new series of
compounds that inhibit the RPA
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DNA interaction and
extensive characterization of this class revealed single
agent activity as well as synergy with cisplatin in
a NSCLC model.
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High throughput screening for XPA inhibitors has
been somewhat less successful though recently an
in silico
screen resulted in the identification of a series
of compounds capable of inhibiting interaction of XPA
with DNA.
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The NMR structure of the minimal DNA
binding domain of XPA was used to dock 200,000
compounds and multiple hits were identified though
only one class was validated in an
in vitro
assay. The
challenge of demonstrating
in vivo
activity for XPA
inhibitors remains.
Both DDB1/2 and the XPC-Rad23B complex bind
DNA and are involved in damage recognition. These
proteins appear to be required for only a subset of
NER repair reactions. XPC-Rad23B is only necessary
for GG-NER and in the absence of this protein TC-
NER can proceed normally. DDB, on the other hand, is
involved in recognition of a subset of DNA adducts
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DNA
interactions and have contributed to the development
of inhibitors of these interactions.
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