The bis-naphthalimidopropyl drugs DMP8408, BNIPDaoct and BNIPDanon, have been developed as novel synthetic lead compounds in cancer therapy. Biochemical assays of these compounds have demonstrated high inhibitor activity (IC50=0.8-1.8μM) against cancer cells, which makes them therapeutically significant. However, knowledge on the structure of these compounds, their target DNA and the drug-DNA complexes is seriously lacking. This justifies the detailed structural and DNA binding studies of these drugs. Determination of the NMR structures of these drugs and their complexes should assist the screening process and enable the discovery of more target specific drugs in the near future.Towards the above goal, the detailed NMR study of DMP8408, BNIPDaoct and BNIPDanon drugs and their interaction with DNA was carried out. The high resolution NMR data and their in depth analysis helped to generate the first NMR structures of DMP8408, BNIPDaoct and BNIPDanon in solution state with a high degree of precision and low RMSD. The results show that the length of the linker chain plays a vital role in the structural and binding properties of these drug compounds, establishing pie- pie interactions between the two aromatic ends of the drugs. This in return facilitates specific binding to the DNA via intercalation.The classical, self-complementary DNA dodecamer sequence was chosen for drug binding studies. A high resolution NMR structure of the DNA was generated with a low RMSD (A) and detailed conformational analysis. NMR titrations of DMP8408, BNIPDaoct and BNIPDanon drugs with the 12mer DNA were carried out at 2 ̊C and 25 ̊C, and the effects induced have been monitored by measuring changes to the chemical shifts (1H and 31P) and patterns of intra and intermolecular NOEs. The results show that DNA binding was much stronger in the case of BNIPDaoct and BNIPDanon than DMP8408. Due to a shorter and rigid linker chain, DMP8408 binds to the DNA via the mono intercalation principle, whereas BNIPDaoct and BNIPDanon have a longer and flexible linker chain which facilitates their binding to the DNA via the neighbour exclusion bis-intercalation principle. In addition to the above, similar NMR titration spectra of selectively fluorinated DNA analogues were also measured to probe and provide additional support to the above drug binding results.Based on the above NMR investigation we conclude that BNIPDaoct shows high affinity towards the DNA followed by BNIPDanon and DMP8408 with preference for the major groove of the DNA. The DNA is significantly deformed upon binding to the drugs and this is manifested in the increased line width of the 1H and 31P resonances. It was also shown that the fluorinated DNA is able to distinguish between both the BNIPDaoct and BNIPDanon in its labelled and the unlabelled forms. Similarly, the drugs were able to bind to the DNA and show the same affinity and other effects despite the incorporation of the fluorine label on the DNA. The above mentioned NMR results support the synthetic and biophysical data in the literature and therefore provide new avenues for further research into anticancer drug discovery.
|Date of Award||31 Dec 2012|
- The University of Manchester
|Supervisor||Vasudevan Ramesh (Supervisor)|
- NMR, Anticancer Drugs, Bis-naphthalimidopropyl, Misbah Ghafoor