ACKNOWLEDGMENTS

1
CRYSTALLOGRAPHIC STUDIES OF COMPLEXES OF DNA
WITH POTENTIAL ANTI-CANCER DRUGS S.C.M.
Teixeira, J.H. Thorpe, B.C. Gale, C.J.
Cardin Chemistry Department, University of
Reading, UK.
DNA-targeting agents are being extensively
studied for their cytotoxic properties and
potential medical applications. The structures
here described are binary complexes of DNA with
intercalators. These compounds are acridine
derivatives containing a fused tricyclic
aromatic moiety and a cationic carboxamide
side-chain that interacts with the DNA through
H-bonds. Although many studies have been and are
being done on these compounds, a lot is yet
unknown about the mechanisms involved. The
studies described here aim at providing a
structural basis for the interpretation of the
different cytotoxic activities shown by the
drugs, as well as at supporting drug design.
DACA3
BISDACA
Carboxamide side-chain of one of the drugs
conformations (orange) H-bonding to a phosphate
oxygen on the DNA backbone. (2Fo-Fc) maps 1s
contours are shown in pink, 0.8s in blue.

• STRUCTURE HIGHLIGHTS
• Cobalt ions stabilise the flipped-out bases (see
  Figures in DACA3).
• Novel intercalation mode although the size of
  the linker chain should allow for intercalation
  conforming with the nearest-neighbour exclusion
  principle, one single drug intercalates between
  two duplexes.
• This work has been submitted for publication
• (Teixeira et al., 2002).

• STRUCTURE HIGHLIGHTS
• Disordered Mg ion found in the pseudo-Holliday
  junction (see Figure in 9AMINO-DACA)
• Drug shows alternate conformations. Carboxamide
  side-chain for one of the drugs is not observed
  (higher disorder).

9AMINO-DACA
5Br-9AMINO-DACA

• STRUCTURE HIGHLIGHTS
• The drug shows alternate conformations. For one
  of the conformations the carboxamide side chain
  is not visible in the electron density maps (as
  happened with the DACA3-DNA complex). For the
  other conformation the side chain H-binds to the
  N7 of a neighbour Guanine base.
• 2001 Cytosine seems to show less disorder up to
  the sugar ring of the nucleotide, that has
  alternate conformations. Although the base cannot
  be seen in the maps, it should be present in the
  same cavity as the disordered carboxamide
  side-chain (also not possible to model into the
  maps) of one of the drugs conformations.

5-Br-9AMINO -DACA
RESULTS AND DISCUSSION The drugs in the studies
here described come as the result of many studies
on structure/activity relationships as well as
DNA-binding kinetics (see for example 4),
through which it has been determined that these
compounds bind selectively to CG-rich sequences
and the 4-position of the carboxamide chain is
optimal to increase cytotoxicity. With the
exception of DACA3, all drugs here mentioned have
shown cytotoxicity in vivo. BISDACA has shown
cytotoxicity against a wide range of tumour cells
in culture (Wakelin, L.P.G. and Denny, W.A.,
unpublished observations) and it seems to form a
more stable complex with DNA than the parental
monomer. It is thought that the cytotoxicity of
these drugs is due to their role in stabilising
the transient complex of topoisomerases (I and/or
II) with DNA the so-called cleavable complex.
This complex is reversible once the drug is
dissociated 5, so the drug residence time is an
important factor. A characteristic of all the
structures here shown is the disorder of the drug
in the intercalation cavity. The atomic
resolution structure of the complex with
5Br-9amino-DACA shows that even with high data
quality it is very difficult to fully resolve the
drug positions, particularly the carboxamide
side-chains. In the structures with DACA3,
9amino-DACA and BISDACA the DNA forms a
pseudo-Holliday junction (see 9amino-DACAs
picture). DNA junctions play important roles in
the normal physiology of cells during DNA
replication, DNA repair, recombination, and viral
integration, for example, making them potential
targets for the development of novel anti-tumor,
antiviral and antibacterial agents. Importantly,
it has been demonstrated that human DNA
topoisomerase IIb binds to four-way junction DNA
6, strengthening the proposal that in vivo
topoisomerases preferentially interact with DNA
crossovers, cruciforms and hairpins.
Disordered Thymine

• STRUCTURE HIGHLIGHTS
• Anisotropic refinement to atomic resolution
  reveals structural details a disordered thymine
  and the disordered drugs intercalating in a
  pseudo-infinite helical stacking.
• The carboxamide side-chains show high
  displacements.
• The N1-CD1OD1 atoms in the carboxamide
  side-chain show non-planar geometry for both
  drugs. Although the standard deviations of the
  CD1-OD1 bond lengths were not small enough to
  confirm this, it is possible that this is due to
  steric effects, as has been observed before for
  other structures (see for example 7).

ACKNOWLEDGMENTS SCMT is grateful to the Chemistry
Department of the University of Reading, Xenova
Plc. and the Portuguese Foundation for Science
and Technology for funding, as well as the
Portuguese Gulbenkian Foundation for travel
support. BCG would like to thank the Association
of International Cancer Research for their
essential funding.
REFERENCES 1 Blackburn, G. M. and Gait, M.J.
(Editors), (1996). Nucleic Acids in Chemistry
and Biology, 2nd Edition, Oxford University
Press. 2 Thorpe, J.H., Hobbs, J.R., Todd, A.,
Denny, W.A., Charlton, P., Cardin, C.J., (2000).
Biochemistry, 39, 15055-15061. 3 Todd, A. K.,
Adams, A., Thorpe, J. H., Denny, W. A., Wakelin,
L. P. G., Cardin, C. J. (1999). J.Med. Chem., 42,
536-540. 4 Wakelin, L.P.G., Atwell, G.J.,
Rewcastle, G. W., Denny. W.A. (1987). J.
Med.Chem., 30, 855-861. 5 Hurley, L.H. (2002).
Nature Reviews Cancer, Vol. 2, No.3, 188-200.6
West, K. L. and Austin, C. A. (1999). Nucleic
Acids Res., 27, 984-992. 7 Dodson, E. , (1998).
Acta Cryst. , D54, 1109-118.
The pseudo-Holliday junctions observed in the
structures obtained are stabilised by the drug.
The length of the side chains seems sufficient to
allow for many possible interactions with the
oligonucleotides, which may be the reason behind
the high disorder observed. The structure with
BISDACA shows less disorder in the drug cavity
despite the high displacements observed. In this
case the drug mobility is restricted (both by the
linker chain between the two chromophores and by
the carboxamide side-chain) to form a structure
with a novel intercalation mode. It is likely
that less disordered structures are more stable
and have higher residence times of the drug in
the DNA (as happens with BISDACA). However, the
role of kinetics and the drug geometry within the
complex with DNA cannot be ignored. The
complexity and correlation of the factors that
can influence the activity of these compounds
require further work to support the design of
optimised drugs with high potency and specificity
in vivo. For this reason structural studies are
currently being done on intercalators and several
different oligonucleotide sequences.
XENOVA