Cardiac human bitter taste receptors contain naturally occurring variants that alter function.

Bitter taste receptors (T2R) are a subfamily of G protein-coupled receptors that enable humans to detect aversive and toxic substances. The ability to discern bitter compounds varies between individuals and is attributed mainly to naturally occurring T2R polymorphisms. T2Rs are also expressed in numerous non-gustatory tissues, including the heart, indicating potential contributions to cardiovascular physiology. In this study. T2Rs that have previously been identified in human cardiac tissues (T2Rs - 10, 14, 30, 31, 46 and 50) and their naturally occurring polymorphisms were functionally characterised. The ligand-dependent signaling responses of some T2R variants were completely abolished (T2R30 Leu252 and T2R46 Met228), whereas other receptor variants had moderate changes in their maximal response, but not potency, relative to wild type. Using a cAMP fluorescent biosensor, we reveal the productive coupling of T2R14, but not the T2R14 Phe201 variant, to endogenous Gαi. Modeling revealed that these variants resulted in altered interactions that generally affected ligand binding (T2R30 Leu252) or Gα protein interactions (T2R46 Met228 and T2R14 Phe201), rather than receptor structural stability. Interestingly, this study is the first to show a difference in signaling for T2R50 Tyr203 (rs1376251) which has been associated with cardiovascular disease. The observation of naturally occurring functional variation in the T2Rs with the greatest expression in the heart is important, as their discovery should prove useful in deciphering the role of T2Rs within the cardiovascular system.

Inhibiting a promiscuous GPCR: iterative discovery of bitter taste receptor ligands.

The human GPCR family comprises circa 800 members, activated by hundreds of thousands of compounds. Bitter taste receptors, TAS2Rs, constitute a large and distinct subfamily, expressed orally and extra-orally and involved in physiological and pathological conditions. TAS2R14 is the most promiscuous member, with over 150 agonists and 3 antagonists known prior to this study. Due to the scarcity of inhibitors and to the importance of chemical probes for exploring TAS2R14 functions, we aimed to discover new ligands for this receptor, with emphasis on antagonists. To cope with the lack of experimental structure of the receptor, we used a mixed experimental/computational methodology which iteratively improved the performance of the predicted structure. The increasing number of active compounds, obtained here through experimental screening of FDA-approved drug library, and through chemically synthesized flufenamic acid derivatives, enabled the refinement of the binding pocket, which in turn improved the structure-based virtual screening reliability. This mixed approach led to the identification of 10 new antagonists and 200 new agonists of TAS2R14, illustrating the untapped potential of rigorous medicinal chemistry for TAS2Rs. 9% of the ~ 1800 pharmaceutical drugs here tested activate TAS2R14, nine of them at sub-micromolar concentrations. The iterative framework suggested residues involved in the activation process, is suitable for expanding bitter and bitter-masking chemical space, and is applicable to other promiscuous GPCRs lacking experimental structures.

Discovery of 2-Aminopyrimidines as Potent Agonists for the Bitter Taste Receptor TAS2R14.

The bitter taste receptor TAS2R14 is a G protein-coupled receptor that is found on the tongue as well as in the human airway smooth muscle and other extraoral tissues. Because its activation causes bronchodilatation, TAS2R14 is a potential target for the treatment of asthma or chronic obstructive pulmonary disease. Structural variations of flufenamic acid, a nonsteroidal anti-inflammatory drug, led us to 2-aminopyridines showing considerable efficacy and potency in an IP1accumulation assay. In combination with an exchange of the carboxylic moiety by a tetrazole unit, a set of promising new TAS2R14 agonists was developed. The most potent ligand 28.1 (EC50 = 72 nM) revealed a six-fold higher potency than flufenamic acid and a maximum efficacy of 129%. Besides its unprecedented TAS2R14 activation, 28.1 revealed marked selectivity over a panel of 24 non-bitter taste human G protein-coupled receptors.

Taste GPCRs and their ligands.

Taste GPCRs are expressed in taste buds on the tongue and play a key role in food choice and consumption. They are also expressed extra-orally, with various physiological roles that are currently under study. Unraveling the roles of these receptors relies on the knowledge of their ligands. Combining sensory, cell-based and computational approaches enabled the discovery of numerous agonists and several antagonists. Here we provide a short overview of taste receptor families, main recent methods for ligands discovery, and current sources of information about known ligands. The future directions that are likely to impact the taste GPCR field include focus on ligand interactions with naturally occurring polymorphisms, as well as harnessing the power of CryoEM and of multiple signaling readout techniques.

Structure reveals the activation mechanism of the MC4 receptor to initiate satiation signaling.

Obesity is a global epidemic that causes morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is at the crux of appetite, energy homeostasis, and body-weight control in the central nervous system and is a prime target for anti-obesity drugs. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human MC4R-Gs signaling complex bound to the agonist setmelanotide, a cyclic peptide recently approved for the treatment of obesity. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that initiates satiation signaling. In addition, our findings indicate that calcium (Ca2+) is required for agonist, but not antagonist, efficacy. These results fill a gap in the understanding of MC4R activation and could guide the design of future weight-management drugs.

Lowered sensitivity of bitter taste receptors to ß-glucosides in bamboo lemurs: an instance of parallel and adaptive functional decline in TAS2R16?

Bitter taste facilitates the detection of potentially harmful substances and is perceived via bitter taste receptors (TAS2Rs) expressed on the tongue and oral cavity in vertebrates. In primates, TAS2R16 specifically recognizes ß-glucosides, which are important in cyanogenic plants' use of cyanide as a feeding deterrent. In this study, we performed cell-based functional assays for investigating the sensitivity of TAS2R16 to ß-glucosides in three species of bamboo lemurs (Prolemur simus, Hapalemur aureus and H. griseus), which primarily consume high-cyanide bamboo. TAS2R16 receptors from bamboo lemurs had lower sensitivity to ß-glucosides, including cyanogenic glucosides, than that of the closely related ring-tailed lemur (Lemur catta). Ancestral reconstructions of TAS2R16 for the bamboo-lemur last common ancestor (LCA) and that of the Hapalemur LCA showed an intermediate sensitivity to ß-glucosides between that of the ring-tailed lemurs and bamboo lemurs. Mutagenetic analyses revealed that P. simus and H. griseus had separate species-specific substitutions that led to reduced sensitivity. These results indicate that low sensitivity to ß-glucosides at the cellular level-a potentially adaptive trait for feeding on cyanogenic bamboo-evolved independently after the Prolemur-Hapalemur split in each species.

Tracking COVID-19 using taste and smell loss Google searches is not a reliable strategy.

Web search tools are widely used by the general public to obtain health-related information, and analysis of search data is often suggested for public health monitoring. We analyzed popularity of searches related to smell loss and taste loss, recently listed as symptoms of COVID-19. Searches on sight loss and hearing loss, which are not considered as COVID-19 symptoms, were used as control. Google Trends results per region in Italy or state in the US were compared to COVID-19 incidence in the corresponding geographical areas. The COVID-19 incidence did not correlate with searches for non-symptoms, but in some weeks had high correlation with taste and smell loss searches, which also correlated with each other. Correlation of the sensory symptoms with new COVID-19 cases for each country as a whole was high at some time points, but decreased (Italy) or dramatically fluctuated over time (US). Smell loss searches correlated with the incidence of media reports in the US. Our results show that popularity of symptom searches is not reliable for pandemic monitoring. Awareness of this limitation is important during the COVID-19 pandemic, which continues to spread and to exhibit new clinical manifestations, and for potential future health threats.

Dual binding mode of "bitter sugars" to their human bitter taste receptor target.

The 25 human bitter taste receptors (hTAS2Rs) are responsible for detecting bitter molecules present in food, and they also play several physiological and pathological roles in extraoral compartments. Therefore, understanding their ligand specificity is important both for food research and for pharmacological applications. Here we provide a molecular insight into the exquisite molecular recognition of bitter ß-glycopyranosides by one of the members of this receptor subclass, hTAS2R16. Most of its agonists have in common the presence of a ß-glycopyranose unit along with an extremely structurally diverse aglycon moiety. This poses the question of how hTAS2R16 can recognize such a large number of "bitter sugars". By means of hybrid molecular mechanics/coarse grained molecular dynamics simulations, here we show that the three hTAS2R16 agonists salicin, arbutin and phenyl-ß-D-glucopyranoside interact with the receptor through a previously unrecognized dual binding mode. Such mechanism may offer a seamless way to fit different aglycons inside the binding cavity, while maintaining the sugar bound, similar to the strategy used by several carbohydrate-binding lectins. Our prediction is validated a posteriori by comparison with mutagenesis data and also rationalizes a wealth of structure-activity relationship data. Therefore, our findings not only provide a deeper molecular characterization of the binding determinants for the three ligands studied here, but also give insights applicable to other hTAS2R16 agonists. Together with our results for other hTAS2Rs, this study paves the way to improve our overall understanding of the structural determinants of ligand specificity in bitter taste receptors.

Agonist Binding to Chemosensory Receptors: A Systematic Bioinformatics Analysis.

Human G-protein coupled receptors (hGPCRs) constitute a large and highly pharmaceutically relevant membrane receptor superfamily. About half of the hGPCRs' family members are chemosensory receptors, involved in bitter taste and olfaction, along with a variety of other physiological processes. Hence these receptors constitute promising targets for pharmaceutical intervention. Molecular modeling has been so far the most important tool to get insights on agonist binding and receptor activation. Here we investigate both aspects by bioinformatics-based predictions across all bitter taste and odorant receptors for which site-directed mutagenesis data are available. First, we observe that state-of-the-art homology modeling combined with previously used docking procedures turned out to reproduce only a limited fraction of ligand/receptor interactions inferred by experiments. This is most probably caused by the low sequence identity with available structural templates, which limits the accuracy of the protein model and in particular of the side-chains' orientations. Methods which transcend the limited sampling of the conformational space of docking may improve the predictions. As an example corroborating this, we review here multi-scale simulations from our lab and show that, for the three complexes studied so far, they significantly enhance the predictive power of the computational approach. Second, our bioinformatics analysis provides support to previous claims that several residues, including those at positions 1.50, 2.50, and 7.52, are involved in receptor activation.