Investigating impacts of the mycothiazole chemotype as a chemical probe for the study of mitochondrial function and aging

Abstract

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), MTZ, a newly employed ETC complex I inhibitor, exhibited higher cytotoxicity against cancer cell lines compared to certain non-cancer cell lines. Interestingly, 8-OAc demonstrated greater selectivity for cancer cells when compared to both MTZ and Rote, which has promising potential for anticancer therapeutic development. Furthermore, in vivo experiments with these small molecules utilizing a C. elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPR^MT) pathway, that extends lifespan of worms when applied in their adult stage. We also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilizes the transcription factors ATFS-1 and HSF1, which are involved in the UPR^MT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

Keywords: C. elegans; aging; cancer; mitochondria; stress.

Fig. 1

Cancer cells are more sensitive than non-cancer cells to mitochondrial complex I inhibitor MTZ and 8-OAc. (A) Cytotoxicity of (i) Huh-7 liver carcinoma cells and (ii) BJ fibroblast non-cancer cells by MTT assay after treatment with vehicle/DMSO or various concentrations of MTZ, 8-OAc, and Rote treatment for 24h. Data is presented as OD values normalized to the DMSO-treated control. Corresponding chart shows the IC50 values of cells. (B) Bar graphs represent the percentage of early apoptotic cells analyzed by flow cytometry with annexin V and propidium iodide double staining after treatment with DMSO or indicated concentration of MTZ, 8-OAc, and Rote for 24h. (C) Graphs show the percentage of ROS-producing cells analyzed by flow cytometry with DHE staining after treating Huh-7 and BJ with DMSO or MTZ, 8-OAc, and Rote for 24h. (D) Graphs showing the inhibition of oxygen consumption rate after treating the indicated cells with 10μM of MTZ, 8-OAc, or Rote for 24h in comparison with vehicle control DMSO. Bar graphs represent mean and standard deviation. All statistical analysis was performed by one-way ANOVA using GraphPad Prism 10. ns = not significant, * = p < 0.03; ** = p <0.002; *** = p<0.0002; **** = p< 0.0001

Fig. 2

Low concentration of MTZ and 8-OAc activate UPR^MT, but do not impact lifespan. (A) Representative images of wild-type N2 animals grown on DMSO controls or the indicated concentrations of MTZ, 8-OAc, or Rote from L1 for 3 days at 20 °C (i.e., day 1 adulthood for control conditions). (B) Seahorse analysis of mitochondrial respiration/OCR (pmol/min) of day 1 adult wild-type worms after growing them on indicated concentration of MTZ, 8-OAc or Rote from L1 until day 1 of adulthood. (C) Schematic representation of UPR^MT transcriptional reporter, hsp-6p::GFP. (D) Representative fluorescent micrograph of UPR^MT transcriptional reporter worms (hsp-6p::GFP) of day 1 adult wild-type animals grown on specified concentration of MTZ, 8-OAc, or Rote from L1. The corresponding bar graph shows the graphical representation of change in GFP intensity of the worms treated with indicated concentration of MTZ, 8-OAc or Rote compared to the control DMSO. Statistical analysis was performed by one-way ANOVA using GraphPad Prism 10. ns = not significant, * = p < 0.03; ** = p <0.002; *** = p<0.0002; **** = p< 0.0001 (E) Survival of wild-type N2 animals grown on DMSO control or indicated concentrations of MTZ, 8-OAc, or Rote from L1

Fig. 3

High concentrations of MTZ and 8-OAc promotes longevity. (A) Schematic of worms treated with high concentrations of compounds starting from day 1 of adulthood. (B) Seahorse analysis of mitochondrial respiration/OCR (pmol/min) of day 2 adult wild-type worms after growing them on 5 μM MTZ, 8-OAc, and Rote for 24 hours starting from day 1 of adulthood. (C) Survival of wild-type N2 animals grown on DMSO control or 5 μM MTZ, 8-OAc, or Rote from day 1 of adulthood. (D) Representative fluorescent micrograph of UPR^MT transcriptional reporter worms (hsp-6p::GFP) of day 2 adult animals grown on 5 μM MTZ, 8-OAc, or Rote for 24 h from day 1 of adulthood. The corresponding bar graph shows the graphical representation of change in GFP intensity of the worms treated with indicated concentration of MTZ, 8-OAc or Rote compared to the control DMSO. Statistical analysis was performed by one-way ANOVA using GraphPad Prism 10. ns = not significant, * = p < 0.03; ** = p <0.002; *** = p<0.0002; **** = p< 0.0001

Fig. 4

Transcriptomics analysis of MTZ, 8-OAc, and Rote. (A) Volcano plots representing the changes in gene expression in MTZ, 8-OAc, or Rote treated worms. For RNA-seq analysis, sterile glp-4(bn2) animals were grown at 22 °C until day 1 of adulthood and animals were moved onto compounds at day 1 and RNA was collected at day 2 of adulthood after 24 h of growth on compounds. Data was analyzed on 3 biological replicates for each condition. Red dots indicate significantly differentially expressed genes, while black dots indicate genes that are not significant. (B) Venn diagram representing the overlap of significant differentially expressed genes in indicated treatment conditions. Heatmap represents the expression profile of common genes for indicated treatment conditions. (C) Heatmap showing the expression profile of UPR^MT target genes found common for MTZ, 8-OAc, or Rote treated conditions. (D) Representative graphs indicating the change in the gene expression of the indicated gene groups in MTZ, 8-OAc, or Rote treated condition as compared with DMSO control. Each dot represents a single gene and lines are median and interquartile range

Fig. 5

Lifespan extension of MTZ, 8-OAc, and Rote utilize different mechanisms. (A) Survival of wild-type N2 animals grown on atfs-1 RNAi on DMSO control or 5 μM MTZ, 8-OAc, or Rote from day 1 of adulthood. (B) Survival of wild-type N2 animals grown on hsf-1 RNAi on DMSO control or 5 μM MTZ, 8-OAc, or Rote from day 1 of adulthood. (C) Survival of wild-type N2 animals grown on daf-16 RNAi on DMSO control or 5 μM MTZ, 8-OAc, or Rote from day 1 of adulthood. (D) Survival of wild-type N2 animals grown on NAC-supplemented plates containing DMSO control or 5 μM MTZ, 8-OAc, or Rote from day 1 of adulthood. (E) Graphical description of MTZ and 8-OAc as novel compounds used to inhibit complex I and promote mitohormesis, potentially through the activation of ATFS-1 and HSF1