In Silico Design and Biological Evaluation of Benzofused Polyamides Targeting G-Quadruplex DNA Structures

Student thesis: Doctoral ThesisDoctor of Philosophy


Guanine-rich nucleic acids can fold into distinctive four-stranded G-quadruplex structures which are found in telomeric DNA repeats as well as in sequences in the promoter and other regulatory regions of genes, especially those involved in cellular proliferation. Small molecules that can selectively bind and stabilize the G-quadruplex structure have become of significant interest to researchers, and are gaining momentum as a possible new class of anticancer agents.

This project was based on a previously reported series of novel biaryl polyamides with significant selectively toward G-quadruplexes compared to duplex DNA, and with modest selectivity between different quadruplex types. Using a distamycin scaffold as a starting point, biaryl building blocks were introduced in place of pyrroles to switch preference from duplex to quadruplex DNA. This alteration in shape ensured that the molecules had low affinity for duplex DNA while increasing their interaction with a G-quadruplex structure since the ligands have similar 3D structures. The main aim of this project was to modify the structure of the previously reported biaryl polyamides (with the help of a molecular modelling based approach) through the incorporation of benzofused building blocks to improve their affinity for G-quadruplexes, while further reducing their affinity for duplex DNA, thereby enhancing their selectivity for quadruplex DNA.

A small, focused benzofused-polyamide library (18 molecules) was initially synthesized and evaluated for the ability of members to stabilize G-quadruplex structures using a FRET-based DNA thermal denaturation assay and molecular dynamics (MD) simulations. However, these compounds failed to stabilize the F21T (human telomeric G-quadruplex), c-Kit quadruplexes and Bcl-2 quadruplexes, and MD simulations suggested that the shape of the molecules required further modification to facilitate quadruplex interaction. A second library of molecules (43 in total) was then designed and synthesized using a molecular modelling-based approach. In this series, the shape of the polyamide fragment was changed, while retaining the original scaffold, by introducing benzofused moieties with 3,5-substitutions. Evaluation of these molecules in the same FRET assay showed a notable increase in stabilization of the F21T quadruplex for many library members. For example, compounds 4.93 and 4.71 stabilized the quadruplex by 15°C and 17°C, respectively (at 1 μM concentration), while showing insignificant affinity for duplex DNA. Moreover a third set of benzofused polyamides (9 in total) has been synthesized by the addition of two consecutive benzofused moieties instead of three consecutive benzofused moieties for the ideal length of G-quadruplex DNA targeting ligand molecules. These molecules were also evaluated by the same FRET-based DNA thermal denaturation assay. The overall data showed that benzofused polyamides made of three consecutive benzofused moieties had a specific curvature which improved their G-quadruplex interacting capacities compared to those with two consecutive benzofused moieties.

Cytotoxicity studies were undertaken on MDA-MB-231 and HeLa cell lines and some library members are active at the low micromolar level. Molecule 4.45 has emerged as a lead compound, possessing a cytotoxicity of 40nM in MDA-MB-231.

Given their low molecular weight (between 422-646 Daltons), reasonable water solubility and good cellular penetration properties compared to other known G-quadruplex inhibitors which are mostly non-drug-like, molecules of this type have the potential to be developed into both reagents that can probe DNA structure and into novel quadruplex-targeting therapeutic agents.
Date of Award2016
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorDavid Thurston (Supervisor) & Miraz Rahman (Supervisor)

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