ABSTRACT
The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Benzimidazoles/pharmacology , DNA Glycosylases/antagonists & inhibitors , Enzyme Inhibitors/therapeutic use , Gene Expression/drug effects , Inflammation/drug therapy , Piperidines/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Benzimidazoles/therapeutic use , DNA Glycosylases/metabolism , DNA Repair/drug effects , DNA Repair/genetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Gene Knockout Techniques , Guanine/analogs & derivatives , Guanine/antagonists & inhibitors , Guanine/metabolism , HEK293 Cells , Humans , Inflammation/genetics , Jurkat Cells , Mice , Mice, Mutant Strains , NF-kappa B/genetics , NF-kappa B/metabolism , Piperidines/therapeutic use , Promoter Regions, Genetic , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Human cystatin C (hCC) is a small cysteine protease inhibitor whose oligomerization by propagated domain swapping is linked to certain neurological disorders. One of the ways to prevent hCC dimerization and fibrillogenesis is to enable its interaction with a proper antibody. Herein, the sites of interaction of hCC with dimer-preventing mouse monoclonal anti-hCC antibodies Cyst28 are studied and compared with the binding sites found for mAb Cyst10 that has almost no effect on hCC dimerization. In addition, hCC epitopes in complexes with native polyclonal antibodies extracted from human serum were studied. The results obtained with hydrogen-deuterium exchange mass spectrometry (HDX MS) were compared with the previous findings made using the excision/extraction MS approach. The main results from the two complementary MS-based approaches are found to be in agreement with each other, with some differences being attributed to the specificity of each method. The findings of the current studies may be important for future design of hCC dimerization inhibitors.
