Apoptotic endonucleases, also called “cell death endonucleases”, are initially recognized group of enzymes capable of cleaving the phosphodiester bonds into its complete destruction to oligo- and mononucleotides. They are known to be involved in various types of cell death including apoptosis, necrosis, autophagy, anoikis, and mitotic catastrophe characterized by nuclear DNA fragmentation. This endonuclease-mediated DNA fragmentation is commonly observed in various injuries induced by toxicity, hypoxia and radiation. Nine apoptotic endonucleases have been identified; deoxyribonuclease I (DNase I), DNase γ, DNase X, DNase I-like 2 (DNase 1L2), DNase II, DNase II-like acidic DNase (DLAD), L-DNase II, endonuclease G (EndoG), and caspase-activated DNase (CAD). DNase I and/or EndoG are the most active and abundant apoptotic endonucleases in many tissues and species including human. Genetic inactivation of these two endonucleases caused no pathologic phenotype but showed protective effect on cells and tissues subjected to various injuries. In addition, improvement of DNA transfection efficiency was achieved by genetic inactivation of DNase I or EndoG. However, neither inhibitors nor high-throughput methods for screening of high-volume chemical libraries in search of endonuclease inhibitors are available. We developed the first of a kind high-throughput DNase assay based on a near infrared fluorescence (NIRF) oligonucleotide probe. The assay is highly sensitive to DNase I and EndoG, very reliable (Z’≥0.5), operationally simple, and has low operator, intra- and inter-assay variabilities. The assay was used to screen a chemical library, and several potential DNase I and EndoG inhibitors were identified. After subsequent characterization and comparison, two hit DNase I inhibitors (IG-17 and JR-132) and two hit EndoG inhibitors (PNR-3-80 and PNR-3-82) were selected, confirmed by a secondary assay, and characterized by the mechanisms as competitive or uncompetitive inhibitors, respectively. These compounds are more specific and effective DNase I and EndoG inhibitors than all previously known inhibitors of these endonucleases. The inhibitors protected against Cisplatin-induced kidney cell death and Docetaxel-induced prostate cell death in vitro. Two inhibitors, IG-17 and PNR-3-82, suppressed endonuclease activity in live mice at 5 mg/kg as determined by an intravital quantitative imaging using the same NIRF probe. All four of inhibitors were non-toxic at concentrations up to 25 mg/kg administered subcutaneously in vivo as measured by fourteen blood markers of organs’ function. Moreover, the protection of kidney from Cisplatin-induced kidney toxicity in vivo after injection of IG-17 or PNR-3-82 at 5 mg/kg was observed. In addition to kidney protection, we were first to demonstrate that DNase I and EndoG are responsible for degradation of naked plasmid DNA in blood plasma. IG-17 and PNR-3-82 enhanced in vitro transfection efficiency in cultured cells and in vivo gene expression in various mouse organs. Therefore, the new compounds are the first clinically-relevant candidates for amelioration of tissue injury and promotion of gene delivery.
|Advisor:||Basnakian, Alexei G.|
|Commitee:||Crooks, Peter A., Peterson, Eric C., Reis, Roberts JS, Rhee, Sung|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 76/09(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Toxicology, Surgery, Pharmacology|
|Keywords:||Apoptotic endonucleases, DNase I, Endonuclease inactivation|
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