Rational design of pyrrolobenzodiazepine derivatives
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Kaliszczak2009.doc (13.18Mb)
Date
25/11/2009Author
Kaliszczak, Maciej
Metadata
Abstract
Pyrrolobenzodiazepine (PBD) derivatives interact with the minor-groove of
DNA to form mono-adducts (monomers) or cross-links (dimers). They show
remarkable activity in vitro and in vivo in a wide range of tumour types and one
dimer, SJG-136 is currently in clinical development. Preclinical studies have shown
that SJG-136 is a P-gp substrate limiting its anti-tumour activity. The work
presented in this thesis identifies key physicochemical properties influencing both
the interaction of PBDs with ABC transporters P-gp, MRP1 and BCRP and their
growth inhibitory potency. A testable hypothesis for further optimisation of PBDs is
proposed.
The biological activity of 4 dimers and 12 monomers was assessed using
several in vitro models presenting differential expression of ABC transporters.
Biological endpoints were the growth inhibitory effect determined using a
sulforhodamine B assay and γ-H2AX foci formation. In addition PBD transport was
evaluated using a Caco-2 transwell assay.
P-gp substrate specificity was restricted to dimers. The MW, the number of
(N+O) atoms (>8), a polar surface area (>75 Ǻ2) and hydrogen bonding energy (>10)
could discriminate substrates among the PBDs. P-gp polymorphism was also
evaluated. The mutation in position 2677 (G/T) was associated with reduced
sensitivity to the PBDs. When combined mutations in position 3435/2677 were
linked, the transporter abrogated this apparent gain of function. The impact of MRP1
was identified for all dimers and 1/12 monomers. In addition, the cooperative role of
glutathione in the resistance mediated by MRP1 to the PBDs was revealed. The
presence of a carbonyl moiety at the extremity was shown to discriminate the
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substrate for MRP1 among the monomers. A structure-activity-relationship study
showed that negatively charged (N+O) atoms and a greater number of aromatic rings
confer greater dependency to BCRP. BCRP polymorphism was also evaluated. The
T482 mutant was associated with an increase in drug transport.
The cytotoxicity of the PBDs correlated to the interaction of the DNA as measured
by ΔTm. Compounds, being non surface active, with a greater polar surface area and
number of aromatic rings and a lower solvent accessible surface area were associated
with a greater cytotoxicity. Van-der-waals energy and the electrostatic forces were
identified in silico as predictable features involved in the DNA binding. New PBDs
were designed and were predicted to be associated with a greater affinity for DNA
and with minimal interaction with ABC transporters.