Fluorescence and Resonance Energy Transfer Shine New Light on GPCR Function

Hoffmann, Carsten; Bünemann, Moritz

doi:10.1002/9780470627327.ch10

Cited by 2 publications

(3 citation statements)

References 77 publications

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“…Similarly, experimental approaches such as Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), and single-molecule detection fluorescence can be employed to validate potential allosteric compounds; however, their implementation for the hunt of novel intracellular allosteric modulators via high-throughput screening is technically challenging [16][17][18] . Identification and subsequent characterization of novel endogenous GPCR intracellular allosteric modulators harbor multiple challenges due to the lack of accurate topology information for multiple GPCRs 19 , methods required to detect them, large-scale screening to evaluate their modulatory function, astronomically larger chemical space among others 1,20 .…”

Section: Introductionmentioning

confidence: 99%

“…For example, classical computational SAR (structure-activity relationships) techniques demand a substantial amount of experimentally validated compounds for the designated cavity for predictive modeling, which, unfortunately, at least in the case of GPCRs, is the limiting factor (Basith et al , 2018; Gupta et al , 2021). Similarly, experimental approaches such as Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), and single-molecule detection fluorescence can be employed to validate potential allosteric compounds; however, their implementation for the hunt of novel intracellular allosteric modulators via high-throughput screening is technically challenging (Zhou et al , 2021; Hoffmann & Bünemann, 2010; Jaeger et al ). Identification and subsequent characterization of novel endogenous GPCR intracellular allosteric modulators harbor multiple challenges due to the lack of accurate topology information for multiple GPCRs (Congreve et al , 2020), methods required to detect them, large-scale screening to evaluate their modulatory function, astronomically larger chemical space among others (Topiol, 2018a; Yang et al , 2021).…”

Section: Introductionmentioning

confidence: 99%

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Endogenous Intracellular Metabolites Allosterically Modulate GPCR-Gα Interface

Mohanty

Mittal

Gaur

et al. 2023

Preprint

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Traditionally, the exogenous allosteric modulators of G protein-coupled receptors (GPCRs) have been extensively investigated due to their pharmacological significance. However, to date, only a handful of endogenous intracellular allosteric modulators are known, that too with inconclusive binding information and their associated phenotypes. This limitation primarily stems from the non-availability of robust computational techniques that entails unbiased cavity identification across GPCR protein topology, cavity-specific ligand design, their synthesis, and cross-verification. Here, we introduce Gcoupler, which leverages an integrative approach combining de novo ligand design, statistical methods, and Graph Neural Networks for rational prediction of high-affinity ligands. Gcoupler offers an efficient and comparatively faster route to explore endogenous allosteric sites of GPCRs, including the GPCR-Gα interface. We tested and validated the applicability of Gcoupler in decrypting the cellular metabolites that could intrinsically but directly modulate the Ste2 (GPCR)-mediated pheromone-induced cell death in yeast. Our rigorous interrogation using Gcoupler and experimental approaches, including yeast genetic screening, high-resolution metabolomics, and functional assays identified endogenous hydrophobic metabolites as intracellular allosteric inhibitors of Ste2p signaling. Elevated intracellular levels of these metabolites, either naturally, through genetic alterations, or exogenous treatment, rescue the pheromone-induced programmed cell death. Mechanistic insights revealed that metabolites harbor high-binding affinity to the conserved GPCR-Gα interface and trigger a cohesive response that potentially obstructs downstream signaling. Finally, by utilizing isoproterenol-induced, GPCR-mediated human and primary neonatal rat hypertrophy models, we observed that elevated metabolite levels attenuate hypertrophic response, reinforcing the functional and evolutionary relevance of this allosteric modulation mechanism. In summary, our study reports a robust computational method that uncovered a novel, evolutionary conserved, and metabolite-driven regulatory mechanism of GPCR signaling.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Endogenous Intracellular Metabolites Allosterically Modulate GPCR-Gα Interface

Mohanty

Mittal

Gaur

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…For example, classical computational SAR (structure-activity relationships) techniques demand a substantial amount of experimentally validated compounds for the designated cavity for predictive modeling, which, unfortunately, at least in the case of GPCRs, is the limiting factor 14,15 . Similarly, experimental approaches such as Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), and single-molecule detection uorescence can be employed to validate potential allosteric compounds; however, their implementation for the hunt of novel intracellular allosteric modulators via high-throughput screening is technically challenging [16][17][18] . Identi cation and subsequent characterization of novel endogenous GPCR intracellular allosteric modulators harbor multiple challenges due to the lack of accurate topology information for multiple GPCRs 19 , methods required to detect them, large-scale screening to evaluate their modulatory function, astronomically larger chemical space among others 1,20 .…”

Section: Introductionmentioning

confidence: 99%

Artificial Intelligence Uncovers Evolutionarily Conserved Intracellular Allosteric Modulators of GPCR-Gα Interface

Mohanty

Mittal

Gaur

et al. 2023

Preprint

View full text Add to dashboard Cite

Traditionally, the exogenous allosteric modulators of G protein-coupled receptors (GPCRs) have been extensively investigated due to their pharmacological significance. However, to date, only a handful of endogenous intracellular allosteric modulators are known, that too with inconclusive binding information and their associated phenotypes. This limitation primarily stems from the non-availability of robust computational techniques that entails unbiased cavity identification across GPCR protein topology, cavity-specific ligand design, their synthesis, and cross-validation. Here, we introduce Gcoupler, which leverages an integrative approach combining de novo ligand design, statistical methods, and Graph Neural Networks for rationally predicting high-affinity ligands. Gcoupler offers an efficient and comparatively faster route to explore endogenous allosteric sites of GPCRs, including the GPCR-Gα interface. We tested and validated the applicability of Gcoupler in decrypting the cellular metabolites that could intracellularly but directly modulate the Ste2 (GPCR)-mediated pheromone-induced cell death in yeast. Our rigorous interrogation using Gcoupler and experimental approaches, including yeast genetic screening, RNA Sequencing, high-resolution metabolomics, and functional assays, identified endogenous hydrophobic metabolites as intracellular allosteric inhibitors of Ste2p signaling. Elevated intracellular levels of these metabolites, either naturally, through genetic alterations, or exogenous treatment, rescue the pheromone-induced programmed cell death. Mechanistic insights revealed that metabolites harbor high-binding affinity to the conserved GPCR-Gα interface and trigger a cohesive response that potentially obstructs downstream signaling. Finally, by utilizing isoproterenol-induced, GPCR-mediated human and neonatal rat cardiac hypertrophy models, we observed that elevated metabolite levels attenuate hypertrophic response, reinforcing the functional and evolutionary relevance of this mechanism. In summary, our study reports a robust computational method that uncovered a novel, evolutionary conserved, and metabolite-driven regulatory mechanism of GPCR signaling.

show abstract

Fluorescence and Resonance Energy Transfer Shine New Light on GPCR Function

Cited by 2 publications

References 77 publications

Endogenous Intracellular Metabolites Allosterically Modulate GPCR-Gα Interface

Endogenous Intracellular Metabolites Allosterically Modulate GPCR-Gα Interface

Artificial Intelligence Uncovers Evolutionarily Conserved Intracellular Allosteric Modulators of GPCR-Gα Interface

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