Small Molecule-Mediated Allosteric Activation of the Base Excision Repair Enzyme 8-Oxoguanine DNA Glycosylase: Impact on Mitochondrial Function

Abstract

8-Oxoguanine DNA glycosylase (OGG1) initiates base excision repair of the oxidative DNA damage product 8-oxoguanine. OGG1 is bifunctional; catalyzing glycosyl bond cleavage, followed by phosphodiester backbone incision via a β-elimination apurinic lyase reaction. The product from the glycosylase reaction, 8-oxoguanine, and its analogues, 8-bromoguanine and 8-aminoguanine, trigger the rate-limiting AP lyase reaction. The precise activation mechanism remains unclear. The product-assisted catalysis hypothesis suggests that 8-oxoguanine and analogues bind at the product recognition (PR) pocket to enhance strand cleavage as catalytic bases. Alternatively, they may allosterically activate OGG1 by binding outside of the PR pocket to induce an active-site conformational change to accelerate apurinic lyase. Herein, steady-state kinetic analyses demonstrated random binding of substrate and activator. 9-Deazaguanine, which can’t function as a substrate-competent base, activated OGG1, albeit with a lower Emax value than 8-bromoguanine and 8-aminoguanine. Random compound screening identified small molecules with Emax values similar to 8-bromoGua. Paraquat-induced mitochondrial dysfunction was attenuated by several small molecule OGG1 activators; benefits included enhanced mitochondrial membrane and DNA integrity, less cytochrome c translocation, ATP preservation, and mitochondrial membrane dynamics. Our results support an allosteric mechanism of OGG1 and not product-assisted catalysis. OGG1 small molecule activators may improve mitochondrial function in oxidative stress-related diseases.

Nucleobase catalysts for the enzymatic activation of 8-oxoguanine DNA glycosylase 1

“…Based on substrate similarity we screened an in-house library of diverse modified nucleobases and discovered 8-methylpurines as potent OGG1 organocatalytic switches (3)(4)(5). Additional classes covered thioguanine analogues including FDA-approved drugs (1, 2), as well as 6-amino-substituted guanines (6)(7)(8)(9) but not adenines or 9-substituted nucleobases. Assay Details in Methods and Material.…”

“…[5,6] OGG1 is the enzyme responsible for the removal of 8-oxoG and the literature suggests that targeting of OGG1 function may be a viable strategy to counter-act these effects. [7,8] In addition to an allosteric mechanism, [9] pharmacological OGG1 activation has been reported to act by enhancing an otherwise rudimentary AP-lyase activity. [10] This AP-lyase activity is most likely controlled by product-assisted catalysis; a distinct mode of action compared to classic allosteric regulation.…”

“…We counter-screened all active compounds for DNA intercalation, auto-fluorescence and APE1 interaction. Among the primary hits were the FDA approved drugs thioguanine 1 and azathioprine 2 (Figure 1, Figure S1), 8-substituted thioguanines 3-5 and compounds combining guanine with amines in the 6-position (6)(7)(8)(9). Based on these screening results, we initially directed our attention towards the hits within the 6-thioguanine series.…”

OGG1 competitive inhibitors show important off-target effects by directly inhibiting efflux pumps and disturbing mitotic progression

“…Taken together, the above-mentioned features of the 8-oxoG-OGG1 pair lead to the idea that modulating OGG1 catalytic activity may be a promising intervention for cancer treatment. Therefore, a lot of efforts have been made to identify small-molecules able to either inhibit (Donley et al, 2015;Visnes et al, 2018a;Tahara et al, 2018) or enhance (Michel et al, 2022;Tian et al, 2022) OGG1 catalytic activity. OGG1 inhibitors were identified by screening libraries composed of several thousand of small chemical compounds that were evaluated for their ability to inhibit the in vitro DNA glycosylase activity of purified OGG1.…”

Nucleobase catalysts for the enzymatic activation of 8-oxoguanine DNA glycosylase 1

“…While competitive inhibition of OGG1 [16,17] may also influence protein levels through NEDD4Lmediated protein degradation, [18] pharmacological 2 OGG1 activation has so far only been reported through enhancement of an otherwise rudimentary AP-lyase activity. [19][20][21] This AP-lyase activity is most likely controlled via product-assisted catalysis, [22] although an allosteric mechanism appears to be an additional possibility. [21] In the former process, excised 8-oxoG acts as a weak chemical base and abstracts a proton from the Schiff base intermediate formed between the abasic site and the side chain of OGG1 active site Lys249.…”