A novel SUCNR1 inhibitor alleviates dysbiosis through inhibition of host responses without direct interaction with host microbiota

Abstract

Type 2 diabetes (T2D) is a chronic metabolic disorder in which insulin resistance and impaired insulin secretion result in altered metabolite balance, specifically elevated levels of circulating glucose and succinate, which increases the risk of many pathologies, including periodontitis. Succinate, a tricarboxylic acid (TCA) cycle intermediate, can be produced and metabolized by both host cells and host microbiota, where elevated levels serve as an inflammation and pathogen threat signal through activating the succinate G protein-coupled receptor, SUCNR1. Modulating succinate-induced SUCNR1 signaling remains a promising therapeutic approach for pathologies resulting in elevated levels of succinate, such as T2D and periodontitis. Here, we demonstrate hyperglycemia and elevated intracellular succinate in a T2D mouse model and determine gut microbiome composition. Drawing on previous work demonstrating the ability of a novel SUCNR1 antagonist, compound 7a, to block inflammation and alleviate dysbiosis in a mouse model, we examined if compound 7a has an impact on the growth and virulence gene expression of bacterial and fungal human microbiota in vitro, and if 7a could reduce bone loss in a periodontitis-induced mouse model. T2D mice harbored a significantly different gut microbiome, suggesting the altered metabolite profile of T2D causes shifts in host-microbial community structure, with enrichment in succinate producers and consumers and mucin-degrading bacteria. Bacterial and fungal cultures showed that 7a did not influence growth or virulence gene expression, suggesting the therapeutic effects of 7a are a direct result of 7a interacting with host cells and that alterations in microbial community structure are driven by reduced host SUCNR1 signaling. This work further suggests that targeting SUCNR1 signaling is a promising therapeutic approach in metabolic, inflammatory, or immune disorders with elevated succinate levels.

Keywords: SUCNR1 antagonist; antimicrobial; microbiome; periodontitis; succinate; type 2 diabetes.

Figure 1:. Elevated levels of succinate correlate with an altered microbiome in type 2 diabetes

Glucose tolerance test results showing blood glucose levels at time 0 (resting fasting glucose levels) and after glucose addition show significant differences in blood glucose levels between T2D (HG) and normal glycemic mice (NG) (A) (n=4-5 Student’s T-test, SEM). Intracellular gingival tissue succinate levels are significantly elevated in HG mice compared to NG (B) (n=4-5, Student’s T-test, SEM). PCoA analysis of Bray-Curtis showed that the gut microbiome of HG mice was significantly altered compared to the NG mice (C). Hierarchical clustering heatmap of the scaled relative abundance of the top 20 relatively abundant genera in the NG (n=5) and HG (n=8) samples indicated different genus-level bacterial profiles between groups (D). LEfSe analysis showed the effect size of each discriminative taxa (E). The genus Prevotellaceae UCG-001 was significantly enriched in the T2D (HG) mice with the highest LDA score (E). Evolutionary relationships between significantly differentially relatively abundant taxa show single genera driving significant differences at higher taxonomic levels and no strong phylum-level patterns of significance (F). Bar plots of the relative abundance of suspected succinate-producing bacterial genera, Bacteroides and Prevotellaceae UCG-001, showing enrichment in the T2D mice (HG) compared to the normal glycemic controls (NG) (G). *, p<0.05; **, p<0.01 ***P < 0.005.

Figure 2:. Blocking SUCNR1 significantly reduces alveolar bone loss in a mouse periodontitis model

Demonstration of the experimental design (created with BioRender.com) (A). The distance between the cementoenamel junction (CEJ) and alveolar bone (AB) crest is significantly reduced in mice treated with a topical SUCNR1 antagonist (i.e., 7a). A representative image of microcomputed tomography (μCT) of maxillae from each group (scale bar, 500 mm). (B) and quantitative μCT results are shown (C) (n=8, Student’s T-test, SEM,***p < 0.005).

Figure 3:. A novel SUCNR1 antagonist does not affect virulence factor gene expression

The compound 7a at 1 μM and 100 μM did not significantly impact virulence gene expression compared to a DMSO-vehicle equivalent of several human microbiota, as determined by relative expression levels of select genes. A) Porphyromonas gingivalis ATCC 33277, B) Fusobacterium nucleatum ATCC 10953, C) Escherichia coli ATCC 25922GFP, D) Candida albicans ATCC 18804. (n=3, One-way ANOVA, SEM).

Figure 3:. A novel SUCNR1 antagonist does not affect virulence factor gene expression

The compound 7a at 1 μM and 100 μM did not significantly impact virulence gene expression compared to a DMSO-vehicle equivalent of several human microbiota, as determined by relative expression levels of select genes. A) Porphyromonas gingivalis ATCC 33277, B) Fusobacterium nucleatum ATCC 10953, C) Escherichia coli ATCC 25922GFP, D) Candida albicans ATCC 18804. (n=3, One-way ANOVA, SEM).

Figure 4.. A novel SUCNR1 antagonist does not impact the growth of human microbiota

The compound 7a at 1 μM and 100 μM did not impact cell growth in liquid media compared to a DMSO-vehicle equivalent of several human microbiota, as determined by optical density measurements (OD 600) of cell cultures in exponential-phase growth (n=3, One-way ANOVA, SEM).