G-Quadruplexes as Molecular Targets
RNA has long been considered to be the nucleic acid that is richest in terms of structural
diversity. A quick glance at the structures of tRNAs, ribosomes and the RNA
components of nucleoprotein complexes such as telomerase leaves one almost bewildered
by the range of secondary structural motifs. DNA, by comparison, requires the
uniformity of the Watson–Crick duplex structure for compaction of information within
the human genome. Nevertheless, there are exceptions to this uniformity, because certain
sequences can form unique secondary DNA structures. Most notably, intermittent runs of
guanines can form G-quadruplex structures (Keniry, 2001), and the complementary runs
of cytosine can form i-motif structures (Gehring et al., 1993). G-quadruplexes have been
studied far more extensively than i-motifs, not only because they were discovered first,
but also because they readily form under physiological conditions; whereas formation of
i-motif structures requires acidic conditions, which makes their physiological relevance
less likely, but still possible. G-quadruplex structures can vary in a number of different ways, including strand
stoichiometry and strand orientation (Figure 1A), but there is greatest variation at the
ends, which if capped by looping structures can consist of any of the four bases in loop
sizes of 2 to about 6 bases. The interconversion between double- or single-stranded DNA
and G-quadruplex in cells is dependent upon chaperone proteins, such as the ?-subunit of
the telomere binding protein from Oxytricha (Fang & Cech, 1993), that facilitate G-
quadruplex formation, as well as upon helicases such as Sgs1 from yeast (Sun et al.,
1999), which resolve these structures (Figure 1B). Proteins such as RAP-1 are also
known that bind to and stabilize these G-quadruplex structures (reviewed in Kerwin,
2000). The facile interconversion, at physiological conditions, between double- or single-
stranded DNA and G-quadruplex structures, together with the known occurrence of
binding or chaperone proteins, makes these secondary DNA structures attractive
candidates for biological signaling molecules, based upon their unique molecular
recognition properties in contrast to duplex DNA (reviewed in Han & Hurley, 2000). In
many respects, the folding of single-stranded DNA into G-quadruplex structures is
analogous to the folding of peptides into proteins, as the primary base sequence is the
main determinant of the folded G-quadruplex structure. However, as the genetic
information encoded by DNA must be passed on to the next generation, a mechanism for
interconversion between secondary DNA and duplex DNA structures must be available.
This requirement for interconversion between the different forms of DNA points to a
potential weakness in the system, as secondary "trapped" DNA structures might disrupt
signaling mechanisms or prevent replication (Figure 1B). The original purpose for the design of G-quadruplex-interactive molecules was
inhibition of telomerase by sequestration of the single-stranded DNA primer as a G-
quadruplex structure, thus eliminating the substrate required for the reverse transcriptase
activity of telomerase (Sun et al., 1997). This approach to inhibition of telomerase is in
contrast to that involving catalytic inhibition of the enzyme. Both approaches to
inhibition of telomerase have inherent problems. The selectivity imparted by the absence
of a requirement for telomerase from all but germ-line cells in normal tissues gives rise to
the anticipated selective toxicity to cancer cells that uniquely require this activity for
telomere maintenance and survival; however, the prolonged time for sufficient telomere
erosion is potentially a major therapeutic problem (Damm et al., 2001). In contrast,
telomere disruption by stabilization of G-quadruplex structures lacks the selectivity
associated with the unique requirement for telomerase in cancer cells and depends instead
upon whether or not telomere disruption is more detrimental to cancer cells than to
normal cells. As yet, data are lacking that would define such differences, although it is
likely that the onset of events triggered by telomere disruption is much faster than those
mediated by telomerase inhibition. So, strategies that depend purely upon disruption of
telomeres lack a solid rationale for selective therapeutics for cancer. Nevertheless,
encouraging data are emerging that shows that despite a lack of significant telomere
shortening, which would result from the inhibition of telomerase, telomeric disruption,
presumably through trapping of G-quadruplex structures, results in short-term biological
effects such as formation of anaphase bridges, apoptosis, and cell death (Figure 1) (Duan
et al., 2001). A recent report from the Greider laboratory (Hemann et al., 2001), which
demonstrates that it is the shortest telomere, not average telomere length, that determines
chromosomal stability and cell viability, has important implications for both strategies.
Cancer cells with just one abnormally short telomere will presumably be made more
sensitive than normal cells to catalytic inhibitors of telomerase. They may also be more
sensitive to G-quadruplex-interactive compounds if telomere modification by these
agents leads to degradation of the telomere. It remains to be seen whether clinical
efficacy will result from these strategies. G-quadruplex-forming sequences are also found in a number of transcriptional
regulatory regions of important oncogenes, including c-myc, c-myb, c-fos and c-abl
(Simonsson et al., 1998). Because of the polypurine–polypyrimidine nature of these
duplex sequences, which contain four or more runs of clusters of three or more guanines
on the purine-rich strand, they often show a single-stranded character and hence are
nuclease hypersensitivity regions. In the c-myc promoter, the purine- and pyrimidine-
rich strands bind transcription factors (CNBP and hnRNP) required for transcriptional
activation (Simonsson et al., 1998). As these elements can also form G-quadruplex and i-
motif structures, it is possible that these secondary DNA structures inactivate
transcription, and their conversion to the duplex region is required for transcriptional
activation. G-quadruplex-stabilizing molecules that would prevent this transition would
therefore inactivate c-myc expression. As down-regulation of c-myc expression in tumor
cells by only 30% leads to a dramatic reduction to ras and raf transformation (Bazarov et
al., 2001), the selective stabilization of G-quadruplex structures in the promoter regions
of c-myc should lead to selective effects on cancer cells. Bazarov, A. V. et al. A modest reduction in c-myc expression has minimal effects on cell
growth and apoptosis but dramatically reduces susceptibility to ras and raf
transformation. Cancer Res. 61, 1178–1186 (2001). Damm, D. et al. A highly selective telomerase inhibitor limiting human cancer cell
proliferation. EMBO J. 20, 6958–6968 (2001). Duan, W. et al. Design and synthesis of fluoroquinophenoxazines that interact with G-
quadruplexes and their biological effects. Molecular Cancer Therapeutics 1, 103–
120 (2001). Fang, G. & Cech, T. R. The ?-subunit of Oxytricha telomere-binding protein promotes
G-quartet formation by telomeric DNA. Cell 74, 875–885 (1993). Gehring, K., Leroy, J. L. & Gueron, M. A tetrameric DNA structure with protonated
cytosine·cytosine base pairs. Nature 363, 561–565 (1993). Han, H. & Hurley, L. H. G-Quadruplex DNA: a potential target for anti-cancer drug
design. Trends Pharm. Sci. 21, 136–142 (2000). Hemann, M. T., Strong, M. A., Hao, L.-Y. & Greider, C. W. The shortest telomere, not
average telomere length, is critical for cell viability and chromosome stability. Cell
107, 67–77 (2001). Keniry, M. A. Quadruplex structures in nucleic acids. Biopolymers (Nucleic Acid
Sciences) 56, 123–146 (2001). Kerwin, S. M. G-Quadruplex DNA as a target for drug design. Curr. Pharmaceut. Des.
6, 441–471 (2000). Simonsson, T., Pecinka, P. & Kubista, M. DNA tetraplex formation in the control region
of c-myc. Nucleic Acids Res. 26, 1167–1172 (1998). Sun, D. et al. Inhibition of human telomerase by a G-quadruplex-interactive compound.
J. Med. Chem. 40, 2113–2116 (1997). Sun, H., Bennett, R. J. & Maizels, N. The Saccharomyces cerevisiae Sgs1 helicase
efficiently unwinds G-G paired DNAs. Nucleic Acids Res. 27, 1978–1984 (1999).
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