Research Report 2000 - MDC

RNA Chemistry
Eckart Matthes
Two target inhibitors of human
telomerase (hTERT)
Telomerase is an unique
ribonucleoprotein polymerase using
its RNA component as a template for
the synthesis of multiples of telomeric
repeats onto the end of replicating
chromosomes. The extension
mechanism of telomerase compensates
for the loss of telomeric DNA
associated with each round of DNA
replication. However, most somatic
cells lack telomerase and so the
telomere length reduction is thought
to limit their proliferative capacity and
to lead to cellular senescence.
On the other hand, an activation of
telomerase seems to be required for
the sustained growth potential of
malignant tumor cells, stem cells of
renewable tissues and germ cells.
Telomerase activity was detected in
85-95 % of advanced malignant
tumors.
These findings make telomerase an
attractive target for anti-neoplastic
drugs. One promising target might be
the RNA molecule of telomerase
which is an intregral part of the
enzyme. Indeed, it has been shown
that oligonucleotides (ODNs)
covering the template site of RNA are
able to control the growth of tumor
cells.
Telomeric DNA has been suggested
to bind not only to the template RNA
but seems also to be attached via its
5´-end to a telomerase protein site
called the primer binding site. We
have found that this protein site is an
appropriate target for inhibition of
telomerase. This possibility emerged
from our investigations of differently
modified ODNs. Of these,
phosphorothioate-modified ODNs
(PS-ODNs) were found to be the most
122
efficient inhibitors compared with
other oligomers including peptide
nucleic acids (PNA). We found that
telomerase protein, rather than its
RNA, is the target of PS-ODNs, a
property which has proved to more
length- than sequence-dependent. This
mode of action seems to lead to a
higher efficiency of PS-ODNs
compared with antisense oligomers
targeting telomerase RNA. The
concentration required for 50 %
inhibition of telomerase in HL-60
cell-lysates was found to be in the
nanomolar range.
To increase the selectivity of PS-
ODN, we designed chimeric ODNs
(cODNs) which are extended at the
3´-end by an oligomer hybridizing
effectively with the subsequent
template region of RNA. Furthermore,
such cODNs address two different
targets of telomerase and might be
more efficient. Most of these cODNs
have been synthesised by our group
and optimized by length-variations
(10-20mers) of the PS-part and by
length- (5 and 11 mers) and structuralmodifications
of of the antisense part
(e.g. 2´-methoxy, 2´-methoxyethoxy,
phosphoramidate). PS-PNA chimeric
oligomers were provided by Dr. E.
Uhlmann, Hoechst Marion Roussel.
Our results show that cODN are more
effective than pure PS-ODN and
inhibit human telomerase in the
subnanomolar range. Complexed with
lipofectin, cODNs can be taken up by
U87 glioblastoma cells and effectively
inhibit telomerase. The concentration
required for 50 % inhibition of
telomerase inside U87 cells is 0.05 -
0.3 µM.
Therefore, we consider our cODNs to
be useful candidates for in vivo
applications to investigate the
consequences of permanent inhibition
of telomerase on the growth of human
tumors in nude mice.
Selected Publications
von Janta-Lipinski, M., Costisella, B.,
Ochs, H., Hübscher, U., Hafkemeyer,
P., and Matthes, E. (1998) Newly
synthesized L-enantiomers of 3´fluoro-modified
β-2´-deoxynucleoside
5´-triphosphates inhibit hepatitis B
DNA polymerases but not the five
cellular DNA polymerases α, β, γ, δ,
and ε nor HIV-1 reverse transcriptase.
J. Med. Chem. 41, 2040-2046.
Theil, F., Ballschuh, S., Flatau, S., von
Janta-Lipinski, M., and Matthes, E.
(1998) Chemoenzymatic synthesis of
a new type of enantiomerically pure
carbocyclic nucleoside analogues with
strong inhibitory effects on terminal
deoxynucleotidyl transferase. Bioorg.
& Med Chem. 41, 701-706.
Matthes, E., and Lehmann, Ch. (1999)
Telomerase protein rather than its
RNA is the target of
phosphorothioate-modified oligonucleotides.
Nucleic Acids Res. 27,
1152-1158.
Krayevsky, A. A., Dyatkina, N. B.,
Semizarov, D. G., Victorova, L. S.,
Shirokova, E. A., Theil, F., von Janta-
Lipinski, M., Gosselin, G., and
Imbach, J.-L. (1999) Reasons and
limits of substrate activity of modified
L-dNTP in DNA biosynthesis.
Nucleosides & Nucleotides 18, 863-
864.
von Janta-Lipinski, M., Gaertner, K.,
Lehmann, C., Scherr, H., Schildt, J.,
and Matthes, E. (1999) Protein and
RNA of human telomerase as targets
for modified oligonucleotides.
Nucleosides & Nucleotides 18, 1719-
1720.
Patent Application
Matthes, E., and von Janta-Lipinski,
M. Chimeric oligonucleotides and the
use of thereof PCT-Application:
PCT/DE98/01216; 4th May, 1998.
Structure of the Group
Group leader
Dr. Eckart Matthes
Scientists
Dr. Martin von Janta-Lipinski
Dr. Dieter Bärwolff
Technical assistants
Mike Antonius
Klaus Gaertner
Christine Lehmann
Helga Scheer
Jürgen Schildt
Marianne Stulich