Targeting Twist: Single-molecule insights into supercoiled DNA-topoisomerase interactions for drug discovery

Topoisomerases are enzymes, present in all organisms from humans to bacteria, which are essential for life due to their ability to untangle knotted and twisted DNA. The knotting and twisting of our DNA occurs as 2 metres of our DNA is folded into the cell nucleus; much narrower than the width of a human hair. This is exacerbated by the molecular machinery in our cells which travel along our DNA, pulling it apart and manipulating it, in a compact environment. Without topoisomerases, our DNA becomes irreversibly knotted and twisted, and the cell will die. For this reason, key therapeutics such as anticancer and antibiotic drugs target human and bacterial topoisomerases respectively either killing cancerous or bacterial cells.

The pharmaceutical pipeline is continually working both to improve the therapeutics that we have, and to create novel therapeutics. To improve existing therapeutics, we require better understanding of how they work. There remains much room for development in this field. Anti-cancer therapeutics that target topoisomerases currently suffer from a lack of selectivity. Further to this, there remain a number of unexplored antibacterial targets, which have significant potential, but are as yet untargeted.

I propose to use single molecule techniques such as Atomic Force Microscopy, where a needle moves up and down as it is scanned over a surface, building up a picture of what the surface looks like, line by line, (much like a record player reads out a record by feeling the contours of its surface). These will be complemented by faster single molecule techniques, such as magnetic tweezers where a piece of DNA is tethered to a magnetic ball at one end, and a glass surface at the other. The DNA is twisted into a more complex state, by rotating the magnetic ball. The length of DNA is determined by measuring the height of the ball. This gives a very fast readout of how topoisomerases are able to untwist DNA, and how this process can be disrupted by drugs such as anti-cancer therapeutics and antibiotics. The aim of the project is to improve our understanding of how topoisomerases untwist DNA, and how this is prevented by topoisomerase inhibitors, to aid in the development of new or improved anti-cancer drugs and antibiotics.

Topoisomerases are molecular machines that perform vital rearrangement of DNA, unknotting and decatenating it in order that transcription machinery can pass along genomic DNA unhindered. Topoisomerases are targeted by antibiotic and anti-cancer therapeutics that trap them on DNA, reducing their activity. This mechanism of action, discovered for topoisomerase inhibitors, also extends to PARP inhibitors used in anti-cancer therapies, and may be applied to target transcription factors. There remains much room for development in this field; anti-cancer therapeutics that target topoisomerases currently suffer from a lack of selectivity, and there remain a number of unexplored antibacterial targets (including topoisomerases I and III), which have significant potential, but are as yet untargeted. This is due to both a lack of structural information and fundamental gaps in our knowledge of their mechanism of action. Using single molecule techniques, I will provide valuable insight into the mechanism of action for topoisomerases and their inhibitors, with the aim of informing the rational design of novel therapeutics.

This work will bring together an international multidisciplinary collaboration. Using Atomic Force Microscopy techniques I have pioneered, I will determine the conformation of DNA molecules, as they interact with topoisomerases, at double-helical resolution in aqueous solution. Using magnetic tweezers and single molecule fluorescence, I will interrogate topoisomerase-DNA interactions at millisecond time scales, providing dynamic insights into the binding of these enzymes, and how this is modulated by novel therapeutics such as anticancer drugs and antibiotics. These pioneering studies will allow for the determination of the supercoiling dependence of topoisomerases, and provide insight into the mechanism of topoisomerase-DNA interactions, and how these are modulated by topoisomerase inhibitors, a key class of therapeutics.