Modulating the activity of OGG1 using small molecules to target the oxidative DNA damage response in cancer and inflammation

Sammanfattning: The production of reactive oxygen species (ROS) is increased in several pathological conditions including cancer and inflammation. Multiple lines of evidence suggest that ROS are involved in signaling events that promote tumorigenesis and inflammatory responses. If redox homeostasis is not maintained, high levels of ROS can induce oxidative DNA damage which is primarily repaired by base excision repair (BER). 8-oxoguanine DNA glycosylase 1 (OGG1) is a key DNA glycosylase that eliminates 8-oxo-7,8-dihydroxyguanine (8-oxoG) when present opposite to cytosine in duplex DNA to initiate BER. Recently, there has been a great interest in targeting the DNA damage response as an anti-cancer approach. In this respect, OGG1 has gathered particular attention for its established role in BER in addition to its newly identified functions in modulating gene transcription. This has motivated this thesis work aiming at studying the validity of OGG1 as a drug target in clinically relevant treatment strategies for cancer and inflammation. In Paper I, we reported the development of TH5487, a potent pharmacologically active OGG1 inhibitor. In addition, we provided proof of concept that inhibiting OGG1 represents a novel anti-inflammatory strategy. We show that TH5487 engages with OGG1 reducing its activity and DNA binding capacity in in vitro assays. Notably, TH5487 impairs NF-κB binding to the promoter regions of proinflammatory cytokines. This results in suppression of proinflammatory gene expression in cells stimulated with tumor necrosis factor-alpha (TNF-α) or lipopolysaccharide (LPS). Importantly, we found that TH5487 is well-tolerated in vivo, where it reduces the expression of inflammatory mediators and perturbs neutrophil infiltration in mice lungs. Thus, targeting OGG1 can be a potential beneficial strategy to treat inflammatory conditions. In Paper II, we sought to characterize TH5487 regarding its effect on genomic 8-oxoG accumulation. Moreover, we studied OGG1 recruitment kinetics to regions of DNA damage as well as OGG1-chromatin dynamics after TH5487 treatment. We show that TH5487 impairs the repair of potassium bromate induced 8-oxoG lesions and results in fewer incisions. The inhibitor treatment alters both OGG1 recruitment kinetics and OGG1- chromatin binding as evident by the results of laser microirradiation experiments and fluorescence recovery after photobleaching (FRAP) assays respectively indicating that TH5487 interferes with OGG1 recruitment and activity in cells. Paper III validates OGG1 as a potential target for cancer therapy. We reported the crystal structure of human OGG1 in complex with TH5487 showing that the inhibitor targets the active site of OGG1. We found that TH5487 treatment is selectively toxic to a large panel of cancers cells but not to normal immortalized cells. We show that TH5487 treatment induces replication stress as demonstrated by accumulation of phosphorylated gH2AX in S-phase cells. Furthermore, it significantly reduces the replication fork speed. Importantly, TH5487 treatment downregulates a set of DNA replication genes altering the cellular transcriptional profile which contributes to replication stress. TH5487 was not found to reduce tumor growth in xenograft mouse models, probably due to binding to serum albumin proteins. This warrants the development of new formulations with an improved pharmacokinetic profile. In Paper IV, we show that NEIL1 and NEIL2 can potentially compensate for OGG1 inhibition. The recruitment of NEIL1—and to a lesser extent NEIL2—to sites of DNA damage is altered in TH5487-treated cells. In addition, NEIL1 and NEIL2 are more tightly bound to chromatin in oxidatively stressed cells after OGG1 depletion and inhibition. Importantly, we observe a higher level of genomic 8-oxoG lesions in NEIL1- and NEIL2- siRNA depleted cells treated with TH5478 suggesting a potential backup function for NEIL1 and NEIL2 after OGG1 inhibition. In Paper V, we elucidated the mechanism of action of a small-molecule OGG1 activator in vitro and in cellulo. We demonstrated that in the presence of TH10785, OGG1 efficiently processes abasic sites by a new activity not found in native OGG1. Cells treated with TH10785 become more dependent on PNKP to complete the repair process. This novel concept of small-molecule activation paves the way to potentially establish new enzymatic functions in DNA repair enzymes, potentiate weak functions or recover lost ones through chemical intervention

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