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 7 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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:562729 |
| Date | January 2009 |
| Creators | Kaliszczak, Maciej |
| Contributors | Jodrell, Duncan. ; Guichard, Sylvie M. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/4923 |
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