Small molecule-mediated allosteric activation of the base excision repair enzyme 8-oxoguanine DNA glycosylase and its 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-bromoguanine. 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.

Conflict of interest statement

WLR is chief scientific officer, serves on the board and owns stock in Luciole Pharmaceuticals, Inc, a startup company devoted to development of OGG1 activators. All other authors have no competing interests.

© 2022. The Author(s).

Figures

Figure 1

OGG1 catalytic mechanism. The panels describe the structural interactions between DNA containing 8-oxo-dG and OGG1 represented by the lys249 amino acid. In (a), the glycosylase reaction is displayed and in (b) the AP lyase one with the projected re-arrangements resulting from product assisted catalysis (i) or allosteric modification (ii).

Figure 2

Kinetic mechanisms of enzyme activation. In these kinetic schemes, the symbols represent: E, enzyme, S, substrate, A, activator, Km, Michaelis-Menton constant, Ka, activation constant, α, the coefficient of interaction between activator and substrate, and β, factor of kcat activation. Equations that describe these mechanisms of random binding, ordered with substrate binding first, and ordered with activator binding first are also provided where V is the maximal initial rate (kcatEt, in which Et is the total enzyme concentration). Further details are found in the Supplementary Table S2.

Figure 3

Activation of OGG1 by 8-bromoGua and 9-deazaGua. The term [S] refers to substrate A displayed in Supplementary Fig. S1. Values were obtained in duplicate from an independent experiment by varying substrate concentration over time. In (a) OGG1 (at 10 nM) initial rates were measured as a function of substrate A concentration listed on the x-axis in the presence of 8-bromoGua at 0.0 (×), 0.63 (♢), 2.5 (▽), 5 (△), 10 (◻), and 20 (○) μM. Kinetic parameters from analyzing the data using Eq. (2) were used to calculate the theoretical values (solid lines). (b) Bar plot of OGG1 activity in the presence of 200 μM 8-bromoGua and 9-deazaGua. Data were normalized to OGG1 activity in the presence of 200 μM 8-bromoGua. (c) Similarly, to panel (a), plots of OGG1 (at 10 nM) initial rates as a function of substrate A concentration (x-axis) in the presence of 9-deazaGua at 0.0 (●), 39 (+), 78 (×), 156 (♢), 313 (▽), 625 (△), 1250 (◻), and 2500 (○) μM. (d) Kinetic parameters from analyzing the data using Eq. (2) (Supplementary Table 2) were used to calculate the theoretical values (solid lines). Standard deviation of the fit of the lines provides the error terms.

Figure 4

19F NMR Binding Experiments. (a) Non 1H-decoupled 19F NMR spectra of compound VI in the presence of different concentrations of OGG1. Gradual line broadening of the 19F NMR resonance as OGG1 concentration is increased indicates an interaction with OGG1. (b) Non 1H-decoupled 19F NMR spectra of 100 μM compound VI in the absence of OGG1 (top), presence of 16 μM OGG1 (middle) and presence of 16 μM OGG1 and 100 μM compound VII (bottom). The line narrowing effect observed on the bottom spectrum of Compound VI when compared to the middle spectrum is indicative of the two compounds competing for the same binding site. (c) Bar graph of the average peak heights extracted from the 19F NMR spectra (N = 2) of compound VI obtained with various OGG1 concentrations shown in panel c (dark grey on the left) and in the presence of 16 μM OGG1 plus 100 μM of a displacement compound, VII-X (light grey on the right). For the displacement experiments, the peak height of compound VI can be observed to increase, reflecting a line narrowing of its NMR resonance.

Figure 5

Proposed sites for small molecule OGG1 activator binding. OGG1 (blue ribbon) shown with 8-oxo-dG (yellow spheres) and DNA oligonucleotide (gold) from the cross-liked complex (PDB ID 1HU0). Hot spots (A–G) that were identified using mixed probe molecular dynamics were explored via docking experiments. Three hot spots, (B), (E), and (G) could accommodate > 9 small molecule activators.

Figure 6

Effects of paraquat on mitochondrial membrane integrity, mtDNA damage and cytochrome c translocation. In (a) baseline values of mitochondrial integrity are noted by the punctate orange colors whereas in (b), paraquat exposure for 24 h elicits a diffuse characteristic in A549 cells. MitoTracker™ orange dye (overlaid orange) was used to demarcate mitochondria (red arrows) and Hoechst 33342 (blue) to denote the nucleus. (c) provides graphical representation of the mean values obtained from the imaging data from 3 independent experiments. Asterisks represent p < 0.0001 by 1-way ANOVA with Dunnet’s post hoc analyses. In (d), paraquat concentration-dependently damaged mtDNA as measured using two mitochondrial primers described in Methods. Values are depicted as percent of baseline control measurements. For (e–g), representative examples obtained from 3 independent experiments show the impact of compounds XI, XII and VII, respectively, on preventing mtDNA damage. For (h–j), changes in cytochrome c translocation are depicted in representative images using anti-cytochrome c-DyLight™-550 (yellow), CellMask™ Deep Red (red) and Hoechst 33342. In this case, OGG1 activators were applied 2 h prior to paraquat rather than 4 h in the other experiments. For (i) statistical analyses (ANOVA with Dunnett’s post-test), asterisks represent p < 0.0001 and for (j) PQ value was p < 0.0001 vs control while 30 and 50 µM of the test compound resulted in p = 0.0002 and 0.0003, respectively.

Figure 7

Paraquat-induced changes on mitochondrial dynamics. A549 cells were exposed to paraquat for 48 h and/or pre-treated with OGG1 activators for 4 h. Images were obtained and from these images graphical representations are displayed for (a) TOM20, (b) DRP1 and (c) MFN1. The dotted lines refer to the level of change induced by 0.6 mM paraquat. Values represent the average of two independent experiments in triplicate. Statistical significance was conducted and denoted as described in Fig. 6. For compound VII in (a), p = 0.042. In (b) p values from left to right side of the panel denoted by the asterisks equal 0.0138, 0.0023, 0.0002, < 0.0001, < 0.0001, 0.0242, 0.0002, 0.0003 and < 0.0001, respectively. For (c) the asterisks from left to right of the panel represent p-values of; 0.0099, 0.0498, 0.0183, 0.0045, 0.005 and < 0.0001, respectively.

Figure 8

Effects of paraquat on DNA Ligase III in the presence and absence of OGG1 activators. Panels (a–c) represent images obtained in the absence and presence of 0.6 and 1.0 mM paraquat for 48 h, respectively. Sample images depict changes in DNA ligase III content (overlaid orange) with overlaid Cell Mask™ Deep Red (red) and Hoechst 33342 (blue). The line = 40 microns. In (d), graphical representation of the changes resulting in DNA ligase III content after exposure to 0.6 mM paraquat with the dotted line used to depict the changes brought about by the presence of the OGG1 activators (4 h pre-treatment). Similar data were obtained with 24 h of paraquat treatment (data not shown). Values are representative of two independent experiments performed in triplicate. In (d) p-values denoted by the asterisks from left to right of the panel equal 0.0427, 0.0448, 0.0083, 0.0239 and < 0.0001, respectively.