Study: Gut microbe-generated phenylacetylglutamine is an endogenous allosteric modulator of β2-adrenergic receptors. Image Credit: Explode / Shutterstock.com
Recent clinical studies have suggested that phenylacetylglutamine (PAGln), a novel gut microbial metabolite, can mechanistically modulate patients' risk of developing cardiovascular disease (CVD) and heart failure (HF). In a recent study published in tjournal Nature Communications, researchers examine the mechanisms involved in the association between PAGln and adverse cardiovascular outcomes.
Exploring new ways to treat CVDs
Chronic non-communicable diseases are responsible for almost 75% of deaths worldwide. CVDs, HF, and their comorbidities comprise a significant proportion of human mortality, thus emphasizing the urgent need for improving current diagnostic and therapeutic capabilities.
As research advances, the relationship between diet, gut microbiota, and public health is becoming increasingly evident. A growing body of literature highlights the association between gut microbial assemblages and psychological outcomes, including obesity, diabetes, and CVD risk.
PAGln has been hypothesized in several studies to correlate with adverse cardiovascular events. In fact, some studies have even used circulating and fecal concentrations of PAGln as proxies for predicting future CVD risk.
Nevertheless, the associations and mechanisms involved in the relationships between PAGln and CVD remain unclear. Verifying and elucidating these mechanisms may allow for the development of novel therapeutics for the management and prevention of CVDs.
About the study
Previous studies have identified the adrenergic receptor (AR)-binding potential of PAGln, which suggests its potential role in allosteric modulation. ARs are host receptors involved in a wide range of critical metabolic, homeostatic, and cardiovascular functions across the heart, adipose tissue, neurons, and vasculature. The present study aims to verify this hypothesis and further identify the regulatory 'fine-tuning' pathways linking PAGln to CVD outcomes.
The human embryonic kidney 293 (HEK293) line was used to stably express a tetracycline transactivator (tTA)-dependent luciferase reporter (HTLA) line and their genetically modified derivatives. To evaluate the potential allosteric effects of PAGln, cells were exposed to increasing concentrations of isoproterenol and norepinephrine, both of which are β-agonists. Cyclic adenosine monophosphate (cAMP)-dose-response assays were used to evaluate the binding efficacies of native phenylalanine and PAGln to β1-HEK293, β2-HEK293, HTLA, and parental-HEK293.
To measure G protein-coupled receptor (GPCR) transduction regulation, β-arrestin2 recruitment assays using HTLA cells were conducted, and relative luminescence was measured. Subsequently, radiology and binding assays were conducted to further verify GPCR affinity and estimate its relative expression.
To non-invasively monitor the dynamic allosteric modulation of ARs following PAGln treatment, dynamic mass redistribution (DMR) studies on mutant β2-HEK293 cells were performed.
To confirm whether these in vitro biochemical assays translate to real-world outcomes, the researchers also performed cardiac muscle function tests on heart failure patients' left ventricular apical tissue and contractility tests on mouse cardiomyocytes.
Study findings
The cAMP assay revealed that PAGln increased the production of cAMP cells expressing β2AR receptors but not β1AR receptors. This agnostic effect was only observed during acute transient exposure of less than ten minutes.
To determine whether these interactions occur under normal physiological conditions, the β-arrestin2 recruitment assay was used to assess the effects of PAGln as a negative allosteric modulator (NAM) with or without prior exposure to β-agonists. Exposure to PAGln for 15 minutes or longer, followed by treatment with β-agonists for 10 minutes, led to similar results obtained during the cAMP assay, thus indicating that PAGln elicits NAM effects in β2AR- but not β1AR-expressing HEK293 cells.
Isometric muscle contraction assays were employed to elucidate whether PAGln impacts NAM's effects on human heart function. A significant shift in the PAGln dose-response curve was observed as compared to when PAGln was absent, thereby confirming a strong NAM effect. These findings were consistent with those observed in the murine ventricular cardiomyocyte experiments.
Conclusions
The present study presents the first evidence of a gut microbiome metabolite functioning as a NAM of a host GPCR, which suggests the substantial coevolution of the microbiome and its host.
The condition-dependent NAM effects of PAGln on human cardiovascular tissue were also observed, thus highlighting the metabolite as a partial agonist of β2AR. PAGln was further identified as an ago-allosteric modulator, a special class of allosteric modulators that independently function as agnostics but as allosteric modulators when co-incubated with other agnostics.
Historically, drug discovery efforts targeting GPCRs have focused on agonists and antagonists that bind to the orthosteric site of the receptor. But the pursuit of allosteric modulators has become important in recent years, as they have the potential to fine-tune cellular responses with greater selectivity among the subtypes of GPCRs in tissues where the endogenous agonist exerts its physiological effect."
Taken together, these findings identify PAGln as a potential target for future anti-CVD drug discovery and the gut microbiome as an exciting opportunity for bioprospecting against chronic diseases.
Journal reference:
- Saha, P.P., Gogonea, V., Sweet, W. et al. (2024). Gut microbe-generated phenylacetylglutamine is an endogenous allosteric modulator of β2-adrenergic receptors. Nature Communications 15; 6696. doi:10.1038/s41467-024-50855-3