New Cornell analysis is offering a recent view into the methods a standard chemotherapy agent, etoposide, stalls and poisons the important enzymes that enable most cancers cells to flourish.
The findings, from the lab of Michelle Wang, the James Gilbert White Distinguished Professor of the Bodily Sciences and Howard Hughes Medical Institute Investigator within the Faculty of Arts and Sciences, will advance the examine of a spread of most cancers inhibitors. The strategies developed by the group may even allow the creation of delicate screening instruments for figuring out drug mechanisms that may enhance affected person therapy.
The group’s paper, “Etoposide Promotes DNA Loop Trapping and Barrier Formation by Topoisomerase II,” was revealed Jan. 30 in Nature Chemical Biology. The co-lead authors are analysis specialist Tung Le and postdoctoral researcher Meiling Wu.
For 40 years, etoposide has been a trusted chemotherapeutic for treating a wide range of cancers. Etoposide succeeds by concentrating on Sort IIA eukaryotic topoisomerases, enzymes – also referred to as topo IIs – that allow the replication of most cancers cells.
On the middle of that replication course of are the lengthy, entangled, helical coiled-strands of DNA. To ensure that most cancers to unfold, these strands should be untangled, rotated and copied by motor proteins. Topo IIs are well-suited for the job. They carry out an elaborate form of rope trick that relaxes the supercoiled DNA by slicing it, in a short time passing one other DNA strand by means of its center, after which reconnecting the lower DNA again collectively. All of that’s finished with out damaging the DNA’s delicate genetic construction – an unbelievable, and extremely quick, feat of biology that occurs within the physique roughly 300 billion occasions a day.
Etoposide’s nice advantage is that it could actually stabilize a DNA double-stranded break earlier than something is reconnected, and thereby prevents the most cancers cell from replicating. Nevertheless, the intricacies of how etoposide interacts with DNA’s construction have remained murky.
We usually ask: What’s the easiest way to check molecular machineries that happen on DNA? To know how these enzymes work, we need to mimic what could be taking place within the cell. Motor proteins pull on the DNA or apply a pressure on the DNA. So we mentioned, OK, we are able to apply a pressure and see what occurs.”
Michelle Wang, the James Gilbert White Distinguished Professor of the Bodily Sciences and Howard Hughes Medical Institute Investigator within the Faculty of Arts and Sciences
Wang’s lab used three totally different single-molecule manipulation strategies to look at etoposide’s impact on three topo IIs, which have been supplied by collaborators led by professor James Berger of Johns Hopkins College: yeast topoisomerase II, human topoisomerase II alpha and human topoisomerase II beta.
“DNA topology, conceptually and when it comes to torsional mechanical properties, is basically exhausting for individuals to know,” Wang mentioned. “There have been only a few methods to check it. However we occur to have simply the correct instruments. And the rationale we’ve got the correct instruments is as a result of for the final 20 years, we have been engaged on creating them. These instruments and this drawback simply occurred to converge on the proper time.”
First, the researchers used optical tweezers to stretch DNA into varied configurations, demonstrating how etoposide compacts, releases and breaks it, and creates DNA loops. This loop-trapping habits stunned everybody because it revealed a brand new influence of etoposide that was not beforehand identified. It implies that etoposide may promote topo II to considerably alter DNA construction and topology in vivo.
Then the workforce used optical tweezers to unzip double-stranded DNA into two single strands for high-resolution mapping of protein interactions with the DNA, and so mimicked the motor elimination of a certain protein. The findings counsel that etoposide may convert topo II into a robust roadblock of DNA-processing machineries.
Their third approach is a model of magnetic tweezers by which they twisted DNA with a certain topo II and watched the topo II chill out the DNA at a gradual fee. After they added etoposide, they discovered the chemical staggered this sample, introducing pauses that correlate with the trapping of supercoiled loops.
By capturing the alternative ways etoposide enhances these actions and interferes with topo II perform, the researchers now have a quantitative system for characterizing how different topoisomerase medicine behave.
“I feel this provides us a set of instruments that might enable us to check many various sorts of topoisomerases and different kinds of medication in a really complete means,” Wang mentioned. “All the things we do mimics what occurs in vivo. We simply do it in a mechanically managed vogue. Because of this it is so highly effective.”
Co-authors embody doctoral pupil Neti Bhatt and analysis specialist James Inman; and James Berger and Joyce Lee of Johns Hopkins College Faculty of Drugs.
The analysis was supported by the Nationwide Institutes of Well being and the Howard Hughes Medical Institute.
Supply:
Journal reference:
Le, T.T., et al. (2023) Etoposide promotes DNA loop trapping and barrier formation by topoisomerase II. Nature Chemical Biology. doi.org/10.1038/s41589-022-01235-9.
