Identification of Potential Human Protein Targets for Soybean Isoflavones

Soy isoflavones have been reported as endocrine disruptors due to their ability to modulate the activity of estrogen receptors (ERs) in mammals; however, its ability to modulate other metabolic pathways is not entirely clear, which makes it necessary to identify new pharmacological targets that interact with these compounds present in soybean. In this work, a virtual screening was executed to identify potential targets of nine soy isoflavones, employing human proteins target from PharmMapper. The best 25 fit scores were selected and prepared for AutoDock Vina docking protocols. The results suggest that equol, daidzein and biochanin A, have the potential to interact with targets such as phenylethanolamine N-methyltransferase, sex hormone-binding globulin and vitamin D3 receptor, respectively. The validations of docking protocols showed good pose reproducibility (root-mean-square deviation (RMSD) ranged 0.001-3.854 Å) and a modest correlation between binding affinities and agonist concentration, AC50 (correlation coefficient (R) = 0.643, p < 0.001). Protein interaction network revealed that predicted targets for soy isoflavones are involved in different pathways, including neurotransmission, metabolism, and cancer remarking the need of a better understanding of the effects of these compounds on human health.

A novel luminescence-based β-arrestin membrane recruitment assay for unmodified GPCRs

G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, G protein-independent signaling via the arrestin pathway has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either the G protein or arrestin-signaling pathways. Currently available screening techniques that measure arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure arrestin recruitment to any unmodified receptor.NanoLuc, an engineered luciferase from ophlorus gracilirostris (deep sea shrimp), is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. Here, we have identified a novel split site and have fused the N-terminal fragment to a membrane tether and the C-terminal fragment to the N-terminus of either β-arrestin 1 or 2. Upon receptor activation, arrestin is recruited to the plasma membrane in an agonist concentration-dependent manner and the two NanoLuc fragments complement to reconstitute functional luciferase, which allows quantification of recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification. The split NanoLuc arrestin recruitment assay has promise as a new generic assay for measuring arrestin recruitment to diverse GPCR types in heterologous or native cells.

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on time-resolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.

GLP-1-Based Strategies: A Physiological Analysis of Differential Mode of Action

DPP4 inhibitors and GLP-1 receptor agonists used in incretin-based strategies treat Type 2 diabetes with different modes of action. The pharmacological blood GLP-1R agonist concentration targets pancreatic and some extrapancreatic GLP-1R, whereas DPP4i favors the physiological activation of the gut-brain-periphery axis that could allow clinicians to adapt the management of Type 2 diabetes, according to the patient's pathophysiological characteristics.

Understanding the Effect of Different Assay Formats on Agonist Parameters: A Study Using the µ-Opioid Receptor

The correct interpretation of data is fundamental to the study of G-protein–coupled receptor pharmacology. Often, new assay technologies are assimilated into the drug discovery environment without full consideration of the data generated. In this study, the authors look at µ-opioid receptor agonists in three different assays: (1) [35S]GTPγS binding, (2) inhibition of forskolin-stimulated cAMP production, and (3) β-arrestin recruitment. Agonist-concentration effect curves were performed before and after treatment with the irreversible antagonist β-funaltrexamine, and where appropriate, these data were fitted to the operational model of agonism. The Z′ value was highest in the β-arrestin assay, followed by the [35S]GTPγS and cAMP assays. The cAMP data fitted well to the operational model, as did the [35S]GTPγS data, but the [35S]GTPγS assay led to an apparent overestimation of KA values. However, in the β-arrestin assay, data did not fit the operational model, as treatment with β-funaltrexamine reduced the Emax proportionally to receptor number, with no change in EC50. In addition, the EC50 values generated correlated well with affinity values. In conclusion, the β-arrestin recruitment assay does not fit with traditional pharmacological theory but is of great utility as the EC50 value generated is a good approximation of affinity.