title: Novel high-throughput deoxyribonuclease 1 assay.
abstract: Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z' ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.
authors.affiliation: Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Dae Song
authors.initials: DS
authors.lastname: Jang
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Narsimha R
authors.initials: NR
authors.lastname: Penthala
authors.affiliation: Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Eugene O
authors.initials: EO
authors.lastname: Apostolov
authors.affiliation: Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Tariq
authors.initials: T
authors.lastname: Fahmi
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Crooks
authors.affiliation: Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Renal Medicine Service, Central Arkansas Veterans Healthcare System, Little Rock, AR, USA basnakianalexeig@uams.edu.
authors.forename: Alexei G
authors.initials: AG
authors.lastname: Basnakian
mesh_terms: D000818:Animals; D002460:Cell Line; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057166:High-Throughput Screening Assays; D020128:Inhibitory Concentration 50; D051381:Rats; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D003850:Deoxyribonuclease I
keywords: DNase I; cisplatin; high-throughput assay; kidney; near-infrared fluorescence