A novel small molecule that alters shelterin integrity and triggers a DNA-damage response at telomeres

Article abstract of DOI:10.1021/ja805615w

We describe a novel synthetic small molecule which shows an unprecedented stabilization of the human telomeric G-quadruplex with high selectivity relative to double-stranded DNA. We report that this compound can be used in vitro to inhibit telomerase activity and to uncap human POT1 (protection of telomeres 1) from the telomeric G-overhang. We also show that the small molecule G-quadruplex binder induces a partial alteration of shelterin through POT1 uncapping from telomeres in human HT1080 cancer cells and the presence of γH2AX foci colocalized at telomeres. Copyright

Full text of DOI:10.1021/ja805615w

Published on Web 11/01/2008
A Novel Small Molecule That Alters Shelterin Integrity and Triggers a
DNA-Damage Response at Telomeres
Raphae¨l Rodriguez,† Sebastian Mu¨ller,† Justin A. Yeoman,† Chantal Trentesaux,‡
Jean-Franc¸ois Riou,*,‡ and Shankar Balasubramanian*,†
The UniVersity Chemical Laboratory, Lensfield Road, Cambridge, CB2 1EW, U.K., and Regulation et Dynamique
des Genomes, Museum National d’Histoire Naturelle, USM 503, INSERM U565, CNRS UMR 5153, Paris, France
Received July 18, 2008; E-mail: riou@mnhn.fr; sb10031@cam.ac.uk
The protection and maintenance of the genome in human cells
is critical. The cell uses signaling pathways that can down regulate
the cell cycle when DNA-damage occurs, giving an opportunity
for efficient repair before division.1 However, this raises the question
of how cells manage to distinguish the telomeres from DNA double-
strand breaks. A six-protein complex named “telosome” or “shel-
terin” (Figure 1) binds to the double-stranded telomeric DNA and
single-stranded telomeric G-overhang at the chromosome ends.2,3
The complex shelters telomeres from the DNA-damage response
machinery and protects chromosomes from shortening, nonho-
mologous end-joining, and homology-directed repair. Moreover,
the shelterin component POT1 (protection of telomeres 1)4 has been
shown to modulate the activity of telomerase,5 the enzyme capable
of conferring infinite proliferative capacity on cells by extension
of the G-overhang.6 It has been shown that the telomeric G-
overhang, when folded into G-quadruplexes, is resistant to extension
by telomerase,7 and that synthetic small molecules that stabilize
Figure 1. (a) The six-protein complex shelterin bound to telomeric DNA;
(b) structure of 1.
these structures can decrease the enzyme efficiency.8-10 Gomez
et al. showed that the potent G-quadruplex binding natural product
telomestatin induces apoptosis of cancer cells via a mechanism
we are aware of, accompanied by a high level of selectivity over
proposed to involve the uncapping of POT1 from telomeres.11
duplex DNA.16
Herein, we describe a novel synthetic small molecule (compound
We next explored the ability of 1 to uncap POT1 from telomeric
1, Figure 1), which exhibits unprecedented G-quadruplex stabiliza-
single-stranded DNA. The dissociation of a complex formed by
tion leading to an alteration of shelterin at the telomeres of human
POT1 and the telomeric DNA, in the presence of increasing amounts
cancer cells.
of small molecule, was evaluated by electrophoretic mobility shift
Compound 1 was designed following intensive research on the
assay on a native agarose gel.17 We found that 1 uncaps POT1
biology of G-quadruplex nucleic acids.12 The design rationale
from the DNA in a dose-dependent manner with an IC50(POT1) of
comprises certain structural features shared by known quadruplex
200 nM (Figure 2), the lowest reported value for a small molecule.
binding small molecules, with particular emphasis on an electron
In contrast, telomestatin exhibits an IC50(POT1) of 500 nM (see
rich aromatic surface, the potential for a flat conformation, and an
Supporting Information). We then assessed telomerase inhibition
ability to participate in hydrogen bonding.13 The small molecule
by direct assay using d(T2AG3)3 as primer (see Supporting In-
formation). Compound 1 exhibits an IC50(Telo) of 21 µM, which
represents a relatively weak inhibition considering the high ∆Tm
recorded. These results suggest that stabilization of the telomeric
is readily accessible in six synthetic steps that are easily scalable
and amenable to molecular diversity (see Supporting Information).
We first evaluated the potential for 1 to stabilize the telomeric
G-quadruplex by FRET-melting experiments.14 Compound 1 sta-
G-quadruplex by the small molecule has a stronger potential to
bilized the human telomeric G-quadruplex with a maximum ∆Tm
perturb the binding of a shelterin component to telomeric DNA
of 35 K in 60 mM K+ and 44 K in 100 mM Na+ at 0.18 and 0.34
than to inhibit extension of the DNA by telomerase.
µM compound, respectively. In contrast, the ligand-induced double-
To investigate whether 1 could uncap POT1 in cells, we used a
model human cancer cell line HT1080 modified to express a GFP-
POT1 fusion protein that colocalizes with TRF2 at telomeres.11
The incubation of HT1080GFP-POT1 cells with 1 µM of compound
1 for 72 h, conditions under which most cells were still viable,18
resulted in a strong disappearance of GFP signal associated with
telomeres compared to the untreated control as observed by fluo-
rescence microscopy (Figure 3). This result is consistent with the
uncapping of POT1 in vitro, and a model whereby the folding of
the telomeric G-overhang into quadruplexes induced by 1 leads to
POT1 uncapping from the telomeres in cells.
stranded DNA stabilization was negligible with a ∆Tm of 0.5 K in
60 mM K+ at 1 µM compound. It is noteworthy that the G-
quadruplex melting profile was almost unaffected by the presence
of 25 mol equiv of unlabeled double-stranded DNA competitor (see
Supporting Information). By comparison, the maximum ∆Tm
induced by telomestatin was 30 K in 60 mM K+ at 1.2 µM
compound.15 The data recorded for 1 represent the highest induced
shifts in melting temperature for the telomeric G-quadruplex that
† The University Chemical Laboratory.
‡ Museum National d’Histoire Naturelle.
9
15758 J. AM. CHEM. SOC. 2008, 130, 15758–15759
10.1021/ja805615w CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
a DNA-damage response. Compound 1 is therefore a small molecule
with considerable potential to dissect the biological processes
occurring at telomeres. Such studies are ongoing and will be
reported in due course.
Acknowledgment. We thank Cancer Research UK for pro-
gramme funding and for a studentship (S.M.), the BBSRC for a
studentship (J.A.Y.) and the “Ligue Nationale Contre le Cancer”
for financial support (C.T. and J.-F.R.). We also thank Dr. D. Gomez
for generously providing us with recombinant hPOT1.
Supporting Information Available: Experimental details for the
synthesis of 1, FRET-melting, direct telomerase extension assay, in
vitro POT1 uncapping assay, POT1 and γH2AX in cellulo experiments,
growth inhibition assay. This material is available free of charge via
the Internet at http://pubs.acs.org.
Figure 2. POT1 uncapping: inhibition of POT1 binding to the telomeric
sequence dG3(T2AG3)7 induced by 1 in vitro.
References
(1) Sancer, A.; Lindsey-Boltz, L. A.; Unsal-Kac¸maz, K.; Linn, S. Annu. ReV.
Biochem. 2004, 73, 39–85.
(2) Liu, D.; O’Connor, M. S.; Qin, J.; Songyang, Z. J. Biol. Chem. 2004, 279,
51338–51342.
(3) (a) de Lange, T. Genes DeV. 2005, 19, 2100–2110. (b) Palm, W.; de Lange,
T. Annu. ReV. Genet. 2008, 42, DOI: 10.1146/annurev.genet.41.110306.
130350.
(4) Baumann, P.; Cech, T. R. Science 2001, 292, 1171–1175.
(5) Wang, F.; Podell, E. R.; Zaug, A. J.; Yang, Y.; Baciu, P.; Cech, T. R.; Lei,
M. Nature 2007, 445, 506–510.
(6) Blackburn, E. H. Cell 2001, 106, 661–673.
(7) Zahler, A. M.; Williamson, J. R.; Cech, T. R.; Prescott, D. M. Nature 1991,
350, 718–720.
(8) Sun, D.; Thompson, B.; Cathers, B. E.; Salazar, M.; Kerwin, S. M.; Trent,
J. O.; Jenkins, T. C.; Neidle, S.; Hurley, L. H. J. Med. Chem. 1997, 40,
2113–2116.
(9) De Cian, A.; Lacroix, L.; Douarre, C.; Temime-Smaali, N.; Trentesaux,
C.; Riou, J.-F.; Mergny, J.-L. Biochimie 2008, 90, 131–155.
(10) De Cian, A.; Cristofari, G.; Reichenbach, P.; De Lomos, E.; Monchaud,
D.; Teulade-Fichou, M.-P.; Shin-ya, K.; Lacroix, L.; Lingner, J.; Mergny,
J.-L. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17347–17352.
(11) Gomez, D.; Wenner, T.; Brassert, B.; Douarre, C.; O’Donohue, M.-F.; El
Khoury, V.; Shin-ya, K.; Morjani, H.; Trentesaux, C.; Riou, J.-F. J. Biol.
Chem. 2006, 281, 38721–38729.
(12) (a) Granotier, C.; Pennarun, G.; Riou, L.; Hoffschir, F.; Gauthier, L. R.;
De Cian, A.; Gomez, D.; Mandine, E.; Riou, J.-F.; Mergny, J.-L.; Mailliet,
P.; Dutrillaux, B.; Boussin, F. D. Nucleic Acids Res. 2005, 33, 4182–4190.
(b) Jantos, K.; Rodriguez, R.; Ladame, S.; Shirude, P. S.; Balasubramanian,
S. J. Am. Chem. Soc. 2006, 128, 13662–13663. (c) Gonc¸alves, D. P. N.;
Rodriguez, R.; Balasubramanian, S.; Sanders, J. K. M. Chem. Commun.
2006, 4685–4687. (d) Rodriguez, R.; Pantos¸, G. D.; Gonc¸alves, D. P. N.;
Sanders, J. K. M.; Balasubramanian, S. Angew. Chem., Int. Ed. 2007, 46,
5405–5407. (e) Bejugam, M.; Sewitz, S.; Shirude, P. S.; Rodriguez, R.;
Shahid, R.; Balasubramanian, S. J. Am. Chem. Soc. 2007, 129, 12926–
12927. (f) Bugaut, A.; Jantos, K.; Wietor, J.-L.; Rodriguez, R.; Sanders,
J. K. M.; Balasubramanian, S. Angew. Chem., Int. Ed. 2008, 47, 2677–
2680. (g) Waller, Z. A. E.; Shirude, P. S.; Rodriguez, R.; Balasubramanian,
S. Chem. Commun. 2008, 1467–1469. (h) Fernando, H.; Rodriguez, R.;
Balasubramanian, S. Biochemistry 2008, 47, 9365–9371.
(13) Monchaud, D.; Teulade-Fichou, M.-P. Org. Biomol. Chem. 2008, 6, 627–
636.
(14) Mergny, J.-L.; Maurizot, J.-C. ChemBioChem 2001, 2, 124–132.
(15) Shirude, P. S.; Gillies, E. R.; Ladame, S.; Godde, F.; Shin-ya, K.; Huc, I.;
Balasubramanian, S. J. Am. Chem. Soc. 2007, 129, 11890–11891.
(16) Kieltyka, R.; Englebienne, P.; Fakhoury, J.; Autexier, C.; Moitessier, N.;
Sleiman, H. F. J. Am. Chem. Soc. 2008, 130, 10040–10041.
(17) Gomez, D.; O’Donohue, M.-F.; Wenner, T.; Douarre, C.; Macadre´, J.;
Koebel, P.; Giraud-Panis, M.-J.; Kaplan, H.; Kolkes, A.; Shin-ya, K.; Riou,
J.-F. Cancer Res. 2006, 66, 6908–6912.
(18) Trypan blue exclusion indicated a low level of cell death at up to 3 µM of
1 after 72 h incubation (<5%). However, a significant inhibition of cell
growth was observed with an IC50(GI) of 1.4 µM (see Supporting Informa-
tion).
Figure 3. Effect of 1 on HT1080GFP-POT1 cells: (a) Untreated control,
fluorescent GFP-POT1 (green); (b) after treatment with 1 (1 µM) for 72 h;
(c) γH2AX foci in cells treated with 1 (3 µM) for 24 h (red); (d)
colocalization of γH2AX foci (red) and GFP-POT1 (green) at telomeres
(marked with arrowheads). DAPI DNA staining (blue) throughout.
Dysfunctional telomeres that are no longer protected by shelterin
have been shown to activate the DNA-damage response machinery,
which can trigger cell cycle arrest, senescence and apoptosis.19 Such
dysfunction has been associated with the appearance of nuclear foci
of phosphorylated histone H2AX (γH2AX), an early DNA-damage
response marker. To evaluate the consequence of POT1 uncapping
from telomeres induced by 1, we performed γH2AX immunofluo-
rescence microscopy11 on cells incubated with 3 µM compound
for 24 h, conditions where POT1 is only partially removed from
telomeres. A strong increase in γH2AX foci compared to the
untreated control was observed in the nucleus, and partially co-
localized with GFP-POT1 at telomeres (Figure 3). This observation
suggests that 1 induces a DNA-damage response through the
removal of POT1 from telomeres.
In conclusion, we have described a novel synthetic small
molecule that stabilizes the folded human telomeric quadruplex with
an unprecedented induced shift in the melting temperature, and very
good selectivity relative to double-stranded DNA. We have shown
that the small molecule interacts with telomeres and alters the
integrity of shelterin in cells through POT1 uncapping resulting in
(19) (a) d’Adda di Fagagna, F.; Reaper, P. M.; Clay-Farrace, L.; Fiegler, H.;
Carr, P.; von Zglinicki, T.; Saretzki, G.; Carter, N. P.; Jackson, S. P. Nature
2003, 426, 194–198. (b) d’Adda di Fagagna, F. Nat. ReV. Cancer 2008, 8,
512–522.
JA805615W
9
J. AM. CHEM. SOC. VOL. 130, NO. 47, 2008 15759