**5. Oxidative Damage Repair: Cancer Sensitization Strategy**

OGG1 is a human DNA glycosylase that initiates the repair of oxidized purine bases, mainly oxoG and formamidopyrimidines, thus being a functional analog of Fpg. Nevertheless, in its sequence and structure, OGG1 is completely different from Fpg (Table 1). Overexpression of *OGG1* in fibroblasts, pulmonary epithelial cells, and bone marrow protects them from the toxic effects of thiotepa, carmustine, and mafosfamide, which mainly yield *N*7-alkylated purines further hydrolyzed in the cell to formamidopyrimidine derivatives [163–165]. However, it is unclear whether normal OGG1 levels can reduce the toxicity of these drugs in tumor cells. A similar effect of OGG1 has been described for cisplatin and oxaliplatin [166], although the nature of the damage removed in this case is not entirely clear. Of the antitumor agents that produce oxidative DNA damage, *OGG1* downregulation or inhibition sensitizes cells to bleomycin [167] and ionizing radiation [168].

As with uracil incorporation and repair, the activity of OGG1 may not only safeguard cells from genomic damage but also potentiate the action of DNA-damaging agents, converting damaged bases to more cytotoxic strand breaks. For instance, OGG1 downregulation protects several cancer cell lines from β-lapachone, an NAD(P)H dehydrogenase (quinone 1)-dependent redox cycling drug that produces copious amounts of intracellular H2O<sup>2</sup> [169]. Hence, a strategy alternative to OGG1 inhibition may consist in saturating the BER capacity with oxidative lesions. In human cells, OGG1 together with mismatched adenine-DNA glycosylase MUTYH and nucleoside triphosphatase MTH1 (NUDT1) forms an analog of the GO system, which prevents the mutagenic effect of oxoG [170]. Recently, the knockdown or inhibition of MTH1, which hydrolyzes oxoG triphosphates and prevents their incorporation into growing DNA and RNA chains, was shown to be toxic to tumor cells, due to the accumulation of oxidized bases and DNA breaks [71,171,172]. Apparently, as in the case of PARP inhibitors, selective toxicity is due to the suppression of the last remaining pathway for the oxidative damage repair in cancer cells. Several low-molecular-weight MTH1 inhibitors were identified, including a clinically approved tyrosine kinase inhibitor, crizotinib [71,171,173] (Table 2). Interestingly, crizotinib possesses a chiral center that gives rise to *(R)*- and *(S)*- enantiomers, of which clinically used

*(R)*-crizotinib inhibits c-MET and ALK protein kinases, whereas the *(S)*-enantiomer preferentially binds to and inhibits MTH1 [71,174,175]. While it is still debated to which extent the cytotoxic activity of crizotinib and other MTH1 inhibitors is dependent on MTH1 and oxidative overload [172,176–178], most reports agree that oxidative damage is an important cell-killing factor, although its causes might not be limited to a direct suppression of MTH1 activity (recently reviewed in [179]). As of today, (*S*)-crizotinib is not used for patient treatment. However, another anti-cancer drug candidate, karonudib, was developed from previously found MTH1 inhibitors [172,180,181]. Presently, two Phase 1 clinical trials of karonudib are registered with the US National Library of Medicine Clinical Studies Database.

Inhibitors of OGG1 have been reported in the literature but have not yet reached clinical trials. Mechanism-based approaches had only limited success: both oxoG base and its analogs proved to be weak inhibitors [182,183], while substituted 2,6-diaminopurines performed slightly better [76]. Experimental and computational screening of small-molecule pharmacological libraries produced several hits that were expanded into inhibitors with submicromolar affinity, structurally unrelated to the OGG1 substrate [72–74] (Table 2). Combinatorial design based on the identified OGG1 and MTH1 inhibitors was used to obtain a compound with submicromolar affinity for both these enzymes [75] (Table 2).

Other DNA glycosylases that repair oxidative damage, including NEIL1, NEIL2, NEIL3, MUTYH, and NTHL1, have been targeted less successfully. Purine-analog library and general library screening produced several inhibitors for NEIL1, but their affinities were in the micromolar range, and the target selectivity was quite low when compared with the inhibition of other glycosylases [76,184] (Table 2). Fumarylacetoacetate was reported to inhibit NEIL1 and NEIL2 and to a lesser degree, OGG1 and NTHL1 (Table 2), but the structural reasons under this effect have not been established [77]. Moreover, these enzymes have low experimental support as targets for sensitization to antitumor therapy, although NEIL1 confers some resistance to ionizing radiation and antifolates [185,186].

#### **6. Oxidative Damage Repair: Unexpected Connections**

While DNA damage and its repair are well understood in the cancer paradigm, two unexpected connections of oxidative damage with other human pathologies emerged recently. OxoG and its repair by OGG1 are suspected to play a regulatory role in the inflammatory response. Several lines of evidence support this conclusion. *Ogg1*-null or -depleted mice show a significantly alleviated inflammatory response to many factors, including bacterial endotoxins, *Helicobacter* infection, foreign protein response, ragweed pollen grain extract-induced allergy, etc. [187–190]. Interestingly, however, the inflammation associated with UVB or pulmonary hyperoxia is enhanced rather than reduced in *Ogg1*−/<sup>−</sup> mice [191–193], suggesting that OGG1-dependent inflammation requires foreign antigens. After the excision of oxoG, the OGG1·oxoG complex can bind Ras family GTPases and facilitate the GDP-to-GTP exchange [194,195], which triggers the signaling pathway leading to the activation of NF-κB, the key pro-inflammatory transcription factor [196]. Moreover, OGG1 can bind oxidized G-rich promoters of pro-inflammatory genes in an enzymatically non-productive mode and facilitate their expression by attracting NF-κB [197–199]. A small-molecule OGG1 inhibitor, TH5487, was developed that competes with oxoG for binding and downregulates the inflammatory response in a mouse model [73] (Table 2). Although TH5487 has a 4-bromobenzimidazolone moiety, which is structurally similar to oxoG, the crystal structure of its complex with OGG1 [73] (Table 3) unexpectedly revealed that the oxoG-binding pocket is occupied by another moiety of TH5487, *p*-iodophenylacetamide, whereas 4-bromobenzimidazolone resides in a so-called *exo*-site, which normally binds undamaged G and serves as a transient binding site for oxoG on its way to the active site [200,201]. Thus, TH5487 functionally resembles bipartite inhibitors of UNG, simultaneously engaging two selective binding sites in the enzyme molecule. Such design may be employed to construct new potent inhibitors of OGG1 and other DNA glycosylases.

In addition, inhibition of OGG1 holds promise to prevent or delay the onset of Huntington's disease in risk groups. This hereditary condition, which belongs to the class of "trinucleotide repeat" genetic diseases, is caused by expansion of the (CAG)<sup>n</sup> repeat run in the *HTT* gene beyond the critical length of ~35 repeats [202]. Before becoming symptomatic, carriers of a pathogenic allele experience an explosive growth of the (CAG)<sup>n</sup> run up to several hundred repeats in the striatum at the early stage of the disease [203]. This expansion is triggered by the normal repair of oxoG initiated by OGG1 [204,205] and is likely caused by an imbalance of BER enzymes in this part of the brain, which leads to the accumulation of unprocessed repair intermediates [206]. In the Huntington's disease mouse model, *Ogg1* knockout suppresses the repeat number growth in the striatum and delays the onset of motor dysfunction [207]. Thus, in carriers of the pathogenic *HTT* allele, for whom the penetrance is inevitably 100%, inhibition of OGG1 may be a reasonable therapeutic strategy.
