New Cornell analysis is offering a recent view into the methods a standard chemotherapy agent, etoposide, stalls and poisons the important enzymes that permit most cancers cells to flourish.
The findings, from the lab of Michelle Wang, the James Gilbert White Distinguished Professor of the Physical Sciences and Howard Hughes Medical Institute Investigator within the College of Arts and Sciences, will advance the research of a spread of most cancers inhibitors. The methods developed by the group may even allow the creation of delicate screening instruments for figuring out drug mechanisms that may enhance affected person remedy.
The group’s paper, “Etoposide Promotes DNA Loop Trapping and Barrier Formation by Topoisomerase II,” was printed 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 quite a lot of cancers. Etoposide succeeds by concentrating on Type IIA eukaryotic topoisomerases, enzymes – also referred to as topo IIs – that allow the replication of most cancers cells.
At the middle of that replication course of are the lengthy, entangled, helical coiled-strands of DNA. In order for 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 sort of rope trick that relaxes the supercoiled DNA by reducing 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 carried out with out damaging the DNA’s delicate genetic construction – an unimaginable, and extremely quick, feat of biology that occurs within the physique roughly 300 billion occasions a day.
Etoposide’s nice advantage is that it might stabilize a DNA double-stranded break earlier than something is reconnected, and thereby prevents the most cancers cell from replicating. However, the intricacies of how etoposide interacts with DNA’s construction have remained murky.
We usually ask: What is one of the simplest ways to check molecular machineries that happen on DNA? To perceive how these enzymes work, we wish to mimic what is likely to be taking place within the cell. Motor proteins pull on the DNA or apply a pressure on the DNA. So we mentioned, OK, we will apply a pressure and see what occurs.”
Michelle Wang, the James Gilbert White Distinguished Professor of the Physical Sciences and Howard Hughes Medical Institute Investigator within the College of Arts and Sciences
Wang’s lab used three completely different single-molecule manipulation methods to watch etoposide’s impact on three topo IIs, which had been offered by collaborators led by professor James Berger of Johns Hopkins University: yeast topoisomerase II, human topoisomerase II alpha and human topoisomerase II beta.
“DNA topology, conceptually and by way of torsional mechanical properties, is admittedly onerous for individuals to understand,” Wang mentioned. “There had been only a few methods to check it. But we occur to have simply the appropriate instruments. And the rationale we’ve the appropriate instruments is as a result of for the final 20 years, we have been engaged on creating them. These instruments and this downside simply occurred to converge on the proper time.”
First, the researchers used optical tweezers to stretch DNA into numerous configurations, demonstrating how etoposide compacts, releases and breaks it, and creates DNA loops. This loop-trapping conduct shocked 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 powerful roadblock of DNA-processing machineries.
Their third approach is a model of magnetic tweezers during which they twisted DNA with a certain topo II and watched the topo II calm down the DNA at a gradual fee. When they added etoposide, they discovered the chemical staggered this sample, introducing pauses that correlate with the trapping of supercoiled loops.
By capturing the other ways etoposide enhances these actions and interferes with topo II operate, the researchers now have a quantitative system for characterizing how different topoisomerase medicine behave.
“I believe this offers us a set of instruments that will permit us to check many alternative sorts of topoisomerases and different kinds of medication in a really complete method,” Wang mentioned. “Everything we do mimics what occurs in vivo. We simply do it in a mechanically managed trend. This is why 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 University School of Medicine.
The analysis was supported by the National Institutes of Health and the Howard Hughes Medical Institute.
Source:
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.