Journal: bioRxiv

Article Title: Non-DNA-damaging DNA-PK activation improving hearing and prolonging life due to NAD + and SIRT upregulation

doi: 10.1101/2025.04.18.649305

Figure Lengend Snippet: a, Separation of MA-5 R-enantiomer and S-enantiomer from MA-5 racemic body by the chiral column (upper). The chemical inversion/conversion of R -and S- enantiomer of MA-5 were not observed in a rhesus monkey administered each enantiomer (lower). b , Intracellular ATP measurement of HEI-OC1 cells with various concentration of MA-5, S-enantiomer and R-enantiomer at 0, 1, 3,10, 30 and 100nM for 6hr. Data were mean ± SE.* p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. c , The cell-protective effect of MA-5 S-enantiomer and R-enantiomer against cytotoxicity of BSO in skin fibroblasts from a MELAS patient. MELAS patient skin fibroblasts were cultured with BSO at 500μM through various concentrations of MA-5 S-enantiomer (left) or R-enantiomer (right) at 10, 30, 100, 300nM, 1 μM, 3μM 10 μM and 100 μM for 72hrs. Cell viability and cytotoxicity were measured by WST-8 assay. Data were mean ± SE.* p <0.05, ** p <0.01, *** p <0.001 vs 0.1% DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. d, Intracellular NAD + measurement of HEI-OC1 cells. HEI-OC1 cells were treated with 0.1 % DMSO or MA-5 racemate, MA-5 S-enantiomer and R- enantiomer at 3,10, 30 and 100nM for 6hr. Data were mean ± SE. * p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. e , Intracellular NAD + measurement of human iPS-induced inner ear cells. Human iPS-induced inner ear cells were treated with 0.1 % DMSO or MA-5 racemate, MA-5 S-enantiomer and R-enantiomer at 3,10, 30 and 100nM for 6hr. Data were mean ± SE. * p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. f, The effects of S-enantiomer on SIRT1 enzymatic activity. The effects of S- enantiomer on SIRT1 deacetylase activity in vitro at 10,30 and 100 μM (left). The effects of S-enantiomer on SIRT1 deacetylase activity in cell nuclear fraction extract from HEI-OC1 cells treated with S-enantiomer at 10 and 30 μM for 3hr (right). Data were mean ± SE.* p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. g, The acetylation of p53 (K305) was reduced by MA-5 S enantiomer. The effects of S-enantiomer on the acetylation of p53 (K305) in whole cell extraction from HEI-OC1 cells treated with MA-5 racemate and S-enantiomer at 10 and 30 μM for 24hr. h , The cellular localization of PARylation in HEI-OC1 cells treated with 0.1 % DMSO or MA-5 at 10μM for 24hr. (left). Poly-ADP ribosylation of PARP protein in HEI-OC1 cells treated with 0.1 % DMSO, MA-5 racemate, S-enantiomer and R-enantiomer at 30 100μM for 24hr. i , Immunocyto-staining of H2AX phosphorylated (γH2AX) of HEI-OC1 cells treated with 0.1 % DMSO, doxiorubicin (Dox) at 0.3μM, MA-5, S-enantiomer and R-enantiomer at 30 μM for 24hr. Representative (Upper). Florescence intensities of γH2AX immunocytostaining were measured (Lowere). Data were mean ± SE. p <0.05, ***: p <0.0001 vs. control. # p <0.05, #### p <0.0001 vs. Dox 0.3μM; statistics were calculated by One way anova Durnett’s multiple comparison test. j , Immunocytostaining of H2AX phosphorylated (γH2AX) of HEI-OC1 cells treated with 0.1 % DMSO, camptothecin (Capt) at 1μM, MA-5, S-enantiomer and R-enantiomer at 30 μM for 24hr. Representative (Upper). Florescence intensities of γH2AX immunocytostaining were measured (Lowere). Data were mean ± SE. p <0.05, ***: p <0.0001 vs. control. # p <0.05, #### p <0.0001 vs. Capt 1μM; statistics were calculated by One way anova Durnett’s multiple comparison test. k , The NAMPT enzymatic activity in vitro treated with MA-5, S-enantiomer and R- enantiomer at 3,10, 30 and 100nM for 1hr. Data were mean ± SE. * p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. l, The NAMPT enzymatic activity in vitro treated with S-enantiomer at 10 and 100μM with or without selective NAMPT inhibitor FK-866 at 10nM ( left ). Data were mean ± SE.* p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. The Inhibitory curve on NAMPT activity with various concentration of FK-866 at at 10, 20, 30, 40 and 50nM ( right ). The Inhibitory curve on NAMPT activity with FK-866 was shifted by addition of MA-5 S-enantiomer at 100 μM ( right ). * p <0.05, ** p <0.01 vs. FK-866 alone inhibitory curve; s statistics were calculated by unpaired two-side t-test. m , The binding between NAMNPT and MA-5, MA-5 S-enantiomer and R-enantiomer and FK-866 were quantitatively analyzed using Biolayer Interferometry (BLI). n , In silico study of Mitochonic acid 5 binding to NAMPT. (a) Possible binding sites on NAMPT of MA-5 S-enantiomer and R-enantiomer. (b) The binding interaction scores of MA-5 S-enantiomer and R-enantiomer at sites ( i ) and ( ii ). (c) The binding conformations of MA-5 S-enantiomer and R-enantiomer at sites ( i ) and ( ii ). (d) the conformational dynamics of ligand binding of MA-5 S-enantiomer and R-enantiomer. (e)

Article Snippet: Antibodies from the following companies were used for Western blot analysis: SIRT1 (1:1000 dilution, #9475, Cell Signaling), SIRT2 (1:1000 dilution, #12650, Cell Signaling), SIRT3 (1:1000 dilution, #5490, Cell Signaling), SIRT4 (1:1000 dilution, #69786, Cell Signaling), SIRT5(1:1000 dilution, #8782, Cell Signaling), SIRT6 (1:1000 dilution, #12486, Cell Signaling), SIRT7(1:1000 dilution, sc-365344,Santa Cruz), IMMT/Mitofilin (1:1000 dilution, 10179-1-AP, ProteinTech), p53 (1:1000 dilution, #2524, Cell Signaling), acertyl p53(K305) (1:1000 dilution, ab109396, abcam), Phospho-p53 (Ser15) (1:1000 dilution, #9284, Cell Signaling), TRIM28(KAP1)(1:10000 dilution, ab109287, abcam), 4H11 pTRIM28 (Ser 473) mouse mAb(1:1000 dilution, Gift from Dr .Shunsuke Chikuma), pTRIM28(KAP1) (Ser 824) (1:1000 dilution, ab133440, abcam), ATM (1:1000 dilution, #2873, Cell Signaling), pATM(Ser 1981)(1:1000 dilution, Invitrogen #14-9046-82), DNA-PKcs (1:1000 dilution, #4602, Cell Signaling), pDNA-PKcs(Ser 2056) (1:1000 dilution, ab18192, abcam), K48-linkage Specific Polyubiquitin (1:1000 dilution, #4289, Cell Signaling), FLAG® tag epitope(DYKDDDDK tag) (1:1000 dilution, F1804, Sigma Aldrich), MYC Tag (1:1000 dilution, #2278, Cell Signaling), PARP1 (1:1000 dilution, #9542, Cell Signaling), PARP1, acetyl Lys521 (1:1000 dilution, CSB-PA890185, CUSABIO), poly(ADP-ribose) polymer (pADPr) (1:1000 dilution, sc-56198, Santa Cruz), 53BP1 (1:1000 dilution, ab175933, Abcam), Phospho-53BP1 (Ser25) (1:1000 dilution, NB100-1803, Novus Biologicals), Acetyl-Histone H3 (Lys18) (1:1000 dilution, #9675, Cell Signaling), Phospho-Chk2 (Thr68) (1:1000 dilution, E-AB-20843, Elabscience), DNA PKcs (pS2056) (1:1000 dilution, ab124918, Abcam), GAPDH (1:1000 dilution, sc-32233, Santa Cruz), β-actin (1:500 dilution, sc-47778, Santa Cruz).

Techniques: Concentration Assay, Control, Comparison, Cell Culture, Activity Assay, Histone Deacetylase Assay, In Vitro, Extraction, Staining, Binding Assay, In Silico, Ligand Binding Assay