Small molecule compound ZYZ-384 targets SMYD3 to alleviate aging

Abstract

SMYD3 is a chromatin modifier that facilitates the trimethylation of histone 3 lysine 4 (H3K4) to induce diverse biological activities. We have provided a brief demonstration of the anti-aging effect of ZYZ-384, a newly developed inhibitor targeting SMYD3. In order to validate the anti-senescence effect of ZYZ-384, we utilized angiotensin II to induce senescence in two types of human endothelial cells (HMEC-1) and mouse endothelial cells (SVEC4-10), creating cellular models for senescence. Additionally, we employed D-galactose-induced subacute senescence animal models as well as natural senescence animal models. At the cellular level, we assessed proliferation capacity and intracellular markers associated with aging. Aging markers, SASP and differential metabolites were evaluated at an organismal level using animal models. Compared to senescent cells or animals, ZYZ-384 application significantly inhibited levels of aging markers in both senescent cell and senescent animal models while promoting cell proliferation. Furthermore, it suppressed expression of SMYD3 and H3K4me3 along with over expression of HSP 90 and NF-κB. Our study demonstrates that ZYZ-384 is an effective inhibitor targeting SMYD3 which can effectively delay aging.

Keywords: Cell proliferation; Chromatin modifier; D-galactose; Senescence.

Fig. 1

The effects of ZYZ-384 on the inhibition and growth rate of human HMEC-1 or mouse SVEC4-10 cells. The proliferation activity of cells was detected by CCK-8 assay. Cells were exposed to Ang II at a concentration of 100 nmol/L or gradient concentrations of ZYZ-384, incubated for 48 h, and then analyzed for their proliferation or inhibitory effects. (A) Effect of ZYZ-384 on the viability of human HMEC-1 cells. (B) Effect of ZYZ-384 on the viability of mouse SVEC4-10 cells. (C) The stimulating proliferation effect of ZYZ-384 on human HMEC-1 cells. (D) The stimulating proliferation effect of ZYZ-384 on mouse SVEC4-10 cells. (E) SA-β-Gal staining. Aged cells exhibit a blue response. (^#P < 0.05 vs. Ctrl group, ^{# #}P < 0.01 vs. Ctrl group; ^*P < 0.05 vs. Ang II group, ^{* *}P < 0.01 vs. Ang II group).

Fig. 2

The proliferation effect of ZYZ-384 on human HMEC-1 or mouse SVEC4-10 cells is evidenced by the presence of bright green fluorescence. (A) (C) The impact of ZYZ-384 on the proliferation of human HMEC-1 cells and their fluorescence activity. (B) (D) The impact of ZYZ-384 on the proliferation of mouse SVEC4-10 cells and their fluorescence activity. (^#P < 0.05 vs. Ctrl group, ^{# #}P < 0.01 vs. Ctrl group; ^*P < 0.05 vs. Ang II group, ^{* *}P < 0.01 vs. Ang II group).

Fig. 3

(A-D) The impact of ZYZ-384 on the expression levels of aging markers p21, p16, and p53 in human HMEC-1 cells, as well as the fluorescence activity of the cells. (^#P < 0.05 vs. Ctrl group, ^{# #}P < 0.01 vs. Ctrl group; ^*P < 0.05 vs. Ang II group, ^{* *}P < 0.01 vs. Ang II group).

Fig. 4

(A-D) The effect of ZYZ-384 on the expression levels of aging markers p21, p16, and p53 in mouse SVEC4-10 cells, as well as the fluorescence activity of the cells. (^{# #}P < 0.01 vs. Ctrl group; ^*P < 0.05 vs. Ang II group, ^{* *}P < 0.01 vs. Ang II group).

Fig. 5

Hematoxylin-eosin staining images of a subacute aging mouse model formed by subcutaneous injection of D-galactose are shown from left to right, depicting the brain, kidney, spleen, liver, and heart.

Fig. 6

Effect of ZYZ-384 on the organ aging marker p16 in subacute senescence animal model. (A) Immunohistochemical staining images. (B) IHC score. The score was based on the intensity of staining and the proportion of positive cells. (^#P < 0.05 vs. Ctrl group, ^{# #}P < 0.01 vs. Ctrl group; ^*P < 0.05 vs. Model group, ^{* *}P < 0.01 vs. Model group).

Fig. 7

Effect of ZYZ-384 on the organ aging marker p21 in subacute senescence animal model. (A) Immunohistochemical staining images. (B) IHC score. (^{# #}P < 0.01 vs. Ctrl group; ^*P < 0.05 vs. Model group, ^{* *}P < 0.01 vs. Model group).

Fig. 8

Effect of ZYZ-384 on the organ aging marker Rb in subacute senescence animal model. (A) Immunohistochemical staining images. (B) IHC score. (^#P < 0.05 vs. Ctrl group, ^{# #}P < 0.01 vs. Ctrl group; ^*P < 0.05 vs. Model group, ^{* *}P < 0.01 vs. Model group).

Fig. 9

(A) (B) PCA analysis of the positive and negative ion models in the samples shows good dispersion with no overlap, indicating reliable data. (C) Venn diagram. (D) Heat map showing potential biomarker levels in different groups. Red indicates up-regulation, green indicates down-regulation. (E) (F) KEGG enrichment analysis in different groups. A represents ZYZ-384.

Fig. 10

Effects of different concentrations of ZYZ-384 on SOD, MDA, IL-1β, IL-6, MCP-1, ALT, AST, GLOB, CREA, CK, BUN, Cat, TC and TG in plasma of a D-galactose induced subacute aging animal model. (^{# #}P < 0.01 vs. Ctrl group; ^*P < 0.05 vs. Model group, ^{* *}P < 0.01 vs. Model group).

Fig. 11

Effect of ZYZ-384 on ethology and morphology of natural aging model. (A) The ethology results, from left to right, include the rotating rod test, running experiment, and Barnes Maze experiment. (B) The SA-β-Gal staining of frozen sections of hippocampus, kidney, spleen, liver and heart. (^*P < 0.05 vs. Model group; ^#P < 0.05 vs. ZYZ-384 H group; ^△P < 0.01 vs. siRNA SMYD3 group).

Fig. 12

Effects of ZYZ-384 on SMYD3 protein in animal models of natural aging. (A) Immunohistochemical staining images. (B) IHC score. (^{* *}P < 0.01 vs. Model group; ^#P < 0.05 vs. ZYZ-384 H group; ^{# #}P < 0.01 vs. ZYZ-384 H group; ^△P < 0.05 vs. siRNA SMYD3 group; ^{△ △}P < 0.01 vs. siRNA SMYD3 group).

Fig. 13

Effects of ZYZ-384 on H3K4me3 protein in animal models of natural aging. (A) Immunohistochemical staining images. (B) IHC score. (^{* *}P < 0.01 vs. Model group; ^#P < 0.05 vs. ZYZ-384 H group; ^{# #}P < 0.01 vs. ZYZ-384 H group; ^△P < 0.05 vs. siRNA SMYD3 group; ^{△ △}P < 0.01 vs. siRNA SMYD3 group).

Fig. 14

Effect of ZYZ-384 on Hsp90 in animal models of natural aging. (A) Immunohistochemical staining images. (B) IHC score. (^{* *}P < 0.01 vs. Model group; ^#P < 0.05 vs. ZYZ-384 H group; ^{# #}P < 0.01 vs. ZYZ-384 H group; ^∆P < 0.05 vs. siRNA SMYD3 group; ^∆∆P < 0.01 vs. siRNA SMYD3 group).

Fig. 15

Effect of ZYZ-384 on NF-κB p65 in animal models of natural aging. (A) Immunohistochemical staining images. (B) IHC score. (^{* *}P < 0.01 vs. Model group; ^{# #}P < 0.01 vs. ZYZ-384 H group; ^∆∆P < 0.01 vs. siRNA SMYD3 group).