Publications by authors named "N Osheroff"

223 Publications

1,2-Naphthoquinone as a Poison of Human Type II Topoisomerases.

Chem Res Toxicol 2021 Apr 24;34(4):1082-1090. Epub 2021 Mar 24.

VA Tennessee Valley Healthcare System, Nashville, Tennessee 37212, United States.

1,2-Naphthoquinone, a secondary metabolite of naphthalene, is an environmental pollutant found in diesel exhaust particles that displays cytotoxic and genotoxic properties. Because many quinones have been shown to act as topoisomerase II poisons, the effects of this compound on DNA cleavage mediated by human topoisomerase IIα and IIβ were examined. The compound increased the levels of double-stranded DNA breaks generated by both enzyme isoforms and did so better than a series of naphthoquinone derivatives. Furthermore, 1,2-naphthoquinone was a more efficacious poison against topoisomerase IIα than IIβ. Topoisomerase II poisons can be classified as interfacial (which interact noncovalently at the enzyme-DNA interface and increase DNA cleavage by blocking ligation) or covalent (which adduct the protein and increase DNA cleavage by closing the N-terminal gate of the enzyme). Therefore, experiments were performed to determine the mechanistic basis for the actions of 1,2-naphthoquinone. In contrast to results with etoposide (an interfacial poison), the activity of 1,2-naphthoquinone against topoisomerase IIα was abrogated in the presence of sulfhydryl and reducing agents. Moreover, the compound inhibited cleavage activity when incubated with the enzyme prior to the addition of DNA and induced virtually no cleavage with the catalytic core of the enzyme. It also induced stable covalent topoisomerase IIα-DNA cleavage complexes and was a partial inhibitor of DNA ligation. Findings were also consistent with 1,2-naphthoquinone acting as a covalent poison of topoisomerase IIβ; however, mechanistic studies with this isoform were less conclusive. Whereas the activity of 1,2-naphthoquinone was blocked in the presence of a sulfhydryl reagent, it was much less sensitive to the presence of a reducing agent. Furthermore, the reduced form of 1,2-naphthoquinone, 1,2-dihydroxynaphthalene, displayed high activity against the β isoform. Taken together, results suggest that 1,2-naphthoquinone increases topoisomerase II-mediated double-stranded DNA scission (at least in part) by acting as a covalent poison of the human type II enzymes.
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http://dx.doi.org/10.1021/acs.chemrestox.0c00492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058326PMC
April 2021

Novel, Potent, and Druglike Tetrahydroquinazoline Inhibitor That Is Highly Selective for Human Topoisomerase II α over β.

J Med Chem 2020 11 20;63(21):12873-12886. Epub 2020 Oct 20.

Laboratory of Molecular Modeling and Drug Discovery, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.

We disclose a novel class of 6-amino-tetrahydroquinazoline derivatives that inhibit human topoisomerase II (topoII), a validated target of anticancer drugs. In contrast to topoII-targeted drugs currently in clinical use, these compounds do not act as topoII poisons that enhance enzyme-mediated DNA cleavage, a mechanism that is linked to the development of secondary leukemias. Instead, these tetrahydroquinazolines block the topoII function with no evidence of DNA intercalation. We identified a potent lead compound [compound (ARN-21934) IC = 2 μM for inhibition of DNA relaxation, as compared to an IC = 120 μM for the anticancer drug etoposide] with excellent metabolic stability and solubility. This new compound also shows ~100-fold selectivity for topoIIα over topoβ, a broad antiproliferative activity toward cultured human cancer cells, a favorable in vivo pharmacokinetic profile, and the ability to penetrate the blood-brain barrier. Thus, ARN-21934 is a highly promising lead for the development of novel and potentially safer topoII-targeted anticancer drugs.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668297PMC
November 2020

Two-Dimensional Gel Electrophoresis to Resolve DNA Topoisomers.

Methods Mol Biol 2020 ;2119:15-24

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.

Agarose gel electrophoresis is one of the most straightforward techniques that can be used to differentiate between topoisomers of closed circular DNA molecules. Generally, the products of reactions that monitor the interconversion of DNA between negatively supercoiled and relaxed DNA or positively supercoiled and relaxed DNA can be resolved by one-dimensional gel electrophoresis. However, in more complex reactions that contain both positively and negatively supercoiled DNA, one-dimensional resolution is insufficient. In these cases, a second dimension of gel electrophoresis is necessary. This chapter describes the technique of two-dimensional agarose gel electrophoresis and how it can be used to resolve a spectrum of DNA topoisomers.
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http://dx.doi.org/10.1007/978-1-0716-0323-9_2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012906PMC
October 2020

Bimodal Actions of a Naphthyridone/Aminopiperidine-Based Antibacterial That Targets Gyrase and Topoisomerase IV.

Biochemistry 2019 11 28;58(44):4447-4455. Epub 2019 Oct 28.

VA Tennessee Valley Healthcare System , Nashville , Tennessee 37212 , United States.

Gyrase and topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore, it is critical to identify new agents that maintain activity against fluoroquinolone-resistant strains. One approach is to develop non-fluoroquinolone drugs that also target gyrase and topoisomerase IV but interact differently with the enzymes. This has led to the development of the "novel bacterial topoisomerase inhibitor" (NBTI) class of antibacterials. Despite the clinical potential of NBTIs, there is a relative paucity of data describing their mechanism of action against bacterial type II topoisomerases. Consequently, we characterized the activity of GSK126, a naphthyridone/aminopiperidine-based NBTI, against a variety of Gram-positive and Gram-negative bacterial type II topoisomerases, including gyrase from and gyrase and topoisomerase IV from and . GSK126 enhanced single-stranded DNA cleavage and suppressed double-stranded cleavage mediated by these enzymes. It was also a potent inhibitor of gyrase-catalyzed DNA supercoiling and topoisomerase IV-catalyzed decatenation. Thus, GSK126 displays a similar bimodal mechanism of action across a variety of species. In contrast, GSK126 displayed a variable ability to overcome fluoroquinolone resistance mutations across these same species. Our results suggest that NBTIs elicit their antibacterial effects by two different mechanisms: inhibition of gyrase/topoisomerase IV catalytic activity or enhancement of enzyme-mediated DNA cleavage. Furthermore, the relative importance of these two mechanisms appears to differ from species to species. Therefore, we propose that the mechanistic basis for the antibacterial properties of NBTIs is bimodal in nature.
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http://dx.doi.org/10.1021/acs.biochem.9b00805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450530PMC
November 2019

Topoisomerase II Is Crucial for Fork Convergence during Vertebrate Replication Termination.

Cell Rep 2019 10;29(2):422-436.e5

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

Termination of DNA replication occurs when two replication forks converge upon the same stretch of DNA. Resolution of topological stress by topoisomerases is crucial for fork convergence in bacteria and viruses, but it is unclear whether similar mechanisms operate during vertebrate termination. Using Xenopus egg extracts, we show that topoisomerase II (Top2) resolves topological stress to prevent converging forks from stalling during termination. Under these conditions, stalling arises due to an inability to unwind the final stretch of DNA ahead of each fork. By promoting fork convergence, Top2 facilitates all downstream events of termination. Converging forks ultimately overcome stalling independently of Top2, indicating that additional mechanisms support fork convergence. Top2 acts throughout replication to prevent the accumulation of topological stress that would otherwise stall converging forks. Thus, termination poses evolutionarily conserved topological problems that can be mitigated by careful execution of the earlier stages of replication.
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http://dx.doi.org/10.1016/j.celrep.2019.08.097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919565PMC
October 2019