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There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally1-4. Efforts have focused on readily synthesizable molecules, inevitably leaving many chemotypes unexplored. Here we investigate structure-based docking of a bespoke virtual library of tetrahydropyridines-a scaffold that is poorly sampled by a general billion-molecule virtual library but is well suited to many aminergic G-protein-coupled receptors. Using three inputs, each with diverse available derivatives, a one pot C-H alkenylation, electrocyclization and reduction provides the tetrahydropyridine core with up to six sites of derivatization5-7. Docking a virtual library of 75 million tetrahydropyridines against a model of the serotonin 5-HT2A receptor (5-HT2AR) led to the synthesis and testing of 17 initial molecules. Four of these molecules had low-micromolar activities against either the 5-HT2A or the 5-HT2B receptors. Structure-based optimization led to the 5-HT2AR agonists (R)-69 and (R)-70, with half-maximal effective concentration values of 41 nM and 110 nM, respectively, and unusual signalling kinetics that differ from psychedelic 5-HT2AR agonists. Cryo-electron microscopy structural analysis confirmed the predicted binding mode to 5-HT2AR. The favourable physical properties of these new agonists conferred high brain permeability, enabling mouse behavioural assays. Notably, neither had psychedelic activity, in contrast to classic 5-HT2AR agonists, whereas both had potent antidepressant activity in mouse models and had the same efficacy as antidepressants such as fluoxetine at as low as 1/40th of the dose. Prospects for using bespoke virtual libraries to sample pharmacologically relevant chemical space will be considered.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca.sup.2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders.sup.1. CaSR is a family C G-protein-coupled receptor.sup.2 that functions as an obligate homodimer, with each protomer composed of a Ca.sup.2+-binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca.sup.2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor. Cryo-EM structures of human calcium-sensing receptor reveal intrinsic asymmetry in the receptor homodimer upon activation that is stabilized by calcimimetic drugs adopting distinct poses in the two protomers, priming one protomer for G-protein coupling., Author(s): Yang Gao [sup.1] [sup.2] , Michael J. Robertson [sup.1] [sup.2] , Sabrina N. Rahman [sup.4] , Alpay B. Seven [sup.1] [sup.2] , Chensong Zhang [sup.1] [sup.2] , Justin G. [...]

Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism.sup.1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric G.sub.i. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6-TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound G.sub.i can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6-TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation. Cryo-electron microscopy structures show that metabotropic glutamate receptor 2 forms a dimer to which only one G protein is coupled, revealing the basis for asymmetric signal transduction., Author(s): Alpay B. Seven [sup.1] , Ximena Barros-Álvarez [sup.1] , Marine de Lapeyrière [sup.2] , Makaía M. Papasergi-Scott [sup.1] , Michael J. Robertson [sup.1] , Chensong Zhang [sup.1] , Robert [...]

Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways.sup.1,2. Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human [beta]-arrestin 1 ([beta]arr1([DELTA]CT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with [beta]arr1([DELTA]CT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions. A cryo-electron microscopy structure of the neurotensin receptor 1 in complex with [beta]-arrestin 1 is reported., Author(s): Weijiao Huang [sup.1] , Matthieu Masureel [sup.1] , Qianhui Qu [sup.1] [sup.2] , John Janetzko [sup.1] , Asuka Inoue [sup.3] , Hideaki E. Kato [sup.1] [sup.6] , Michael J. [...]

After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit [beta]-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis.sup.1. Additionally, [beta]-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins.sup.2. In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of [beta]-arrestin with GPCRs are much less understood. Here we present a cryo-electron microscopy structure of [beta]-arrestin 1 ([beta]arr1) in complex with M2 muscarinic receptor (M2R) reconstituted in lipid nanodiscs. The M2R-[beta]arr1 complex displays a multimodal network of flexible interactions, including binding of the N domain of [beta]arr1 to phosphorylated receptor residues and insertion of the finger loop of [beta]arr1 into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric G.sub.o protein complex.sup.3. Moreover, the cryo-electron microscopy map reveals that the C-edge of [beta]arr1 engages the lipid bilayer. Through atomistic simulations and biophysical, biochemical and cellular assays, we show that the C-edge is critical for stable complex formation, [beta]arr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest that the cooperative interactions of [beta]-arrestin with both the receptor and the phospholipid bilayer contribute to its functional versatility. A cryo-electron microscopy structure of [beta]-arrestin 1 in complex with the M2 muscarinic receptor reconstituted in lipid nanodiscs is reported., Author(s): Dean P. Staus [sup.1] [sup.2] , Hongli Hu [sup.3] [sup.8] , Michael J. Robertson [sup.3] [sup.4] , Alissa L. W. Kleinhenz [sup.1] [sup.2] [sup.9] , Laura M. Wingler [sup.1] [...]

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein–lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR–G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid l-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands. Cryo-electron microscopy structures of the human calcium-sensing receptor in complex with Gi and Gq proteins reveal how this receptor activates distinct G protein subtypes and how its function is modulated by a variety of ligands. [ABSTRACT FROM AUTHOR]

Evolution has crafted thousands of enzymes that are efficient catalysts for a plethora of reactions. Human attempts at enzyme design trail far behind, but may benefit from exploiting evolutionary tactics. ©2007 Nature Publishing Group.

Evolution has crafted thousands of enzymes that are efficient catalysts for a plethora of reactions. Human attempts at enzyme design trail far behind, but may benefit from exploiting evolutionary tactics. ©2007 Nature Publishing Group.

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains--the 7-transmembrane domains--in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.The activation mechanism of metabotropic glutamate receptor subtype 5, a member of the family C G-protein-coupled receptors, is characterized by a combination of cryo-electron microscopy, crystallography and signalling studies., Author(s): Antoine Koehl [sup.1] , Hongli Hu [sup.1] [sup.2] , Dan Feng [sup.3] , Bingfa Sun [sup.3] , Yan Zhang [sup.1] [sup.2] , Michael J. Robertson [sup.1] [sup.2] , Matthew [...]

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment—one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.Bitopic functionalized ligands based on fentanyl can target the sodium ion-binding site of the mu-opioid receptor and selectively modulate downstream signalling pathways, potentially leading to safer analgesics. [ABSTRACT FROM AUTHOR]

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally1–4. Efforts have focused on readily synthesizable molecules, inevitably leaving many chemotypes unexplored. Here we investigate structure-based docking of a bespoke virtual library of tetrahydropyridines—a scaffold that is poorly sampled by a general billion-molecule virtual library but is well suited to many aminergic G-protein-coupled receptors. Using three inputs, each with diverse available derivatives, a one pot C–H alkenylation, electrocyclization and reduction provides the tetrahydropyridine core with up to six sites of derivatization5–7. Docking a virtual library of 75 million tetrahydropyridines against a model of the serotonin 5-HT2A receptor (5-HT2AR) led to the synthesis and testing of 17 initial molecules. Four of these molecules had low-micromolar activities against either the 5-HT2A or the 5-HT2B receptors. Structure-based optimization led to the 5-HT2AR agonists (R)-69 and (R)-70, with half-maximal effective concentration values of 41 nM and 110 nM, respectively, and unusual signalling kinetics that differ from psychedelic 5-HT2AR agonists. Cryo-electron microscopy structural analysis confirmed the predicted binding mode to 5-HT2AR. The favourable physical properties of these new agonists conferred high brain permeability, enabling mouse behavioural assays. Notably, neither had psychedelic activity, in contrast to classic 5-HT2AR agonists, whereas both had potent antidepressant activity in mouse models and had the same efficacy as antidepressants such as fluoxetine at as low as 1/40th of the dose. Prospects for using bespoke virtual libraries to sample pharmacologically relevant chemical space will be considered.New agonists of the serotonin 5-HT2A receptor (5-HT2AR) confer high brain permeability and antidepressant activity and—in contrast to classic 5-HT2AR agonists—lack psychedelic activity. [ABSTRACT FROM AUTHOR]

Glucose is a primary energy source in living cells. The discovery in 1960s that a sodium gradient powers the active uptake of glucose in the intestine1 heralded the concept of a secondary active transporter that can catalyse the movement of a substrate against an electrochemical gradient by harnessing energy from another coupled substrate. Subsequently, coupled Na+/glucose transport was found to be mediated by sodium–glucose cotransporters2,3 (SGLTs). SGLTs are responsible for active glucose and galactose absorption in the intestine and for glucose reabsorption in the kidney4, and are targeted by multiple drugs to treat diabetes5. Several members within the SGLT family transport key metabolites other than glucose2. Here we report cryo-electron microscopy structures of the prototypic human SGLT1 and a related monocarboxylate transporter SMCT1 from the same family. The structures, together with molecular dynamics simulations and functional studies, define the architecture of SGLTs, uncover the mechanism of substrate binding and selectivity, and shed light on water permeability of SGLT1. These results provide insights into the multifaceted functions of SGLTs.Cryo-electron microscopy structures of the sodium–glucose cotransporter SGLT1 and a related transporter SMCT1 define the architecture of this protein family and provide insights into substrate binding and transport function. [ABSTRACT FROM AUTHOR]

The surname of author Toon Laeremans was misspelled 'Laermans'. This error has been corrected online., Author(s): Antoine Koehl [sup.1] , Hongli Hu [sup.1] [sup.2] , Dan Feng [sup.3] , Bingfa Sun [sup.3] , Yan Zhang [sup.1] [sup.2] , Michael J. Robertson [sup.1] [sup.2] , Matthew [...]

Unrau and Bartel have described the isolation of RNA molecules that are able to catalyze the synthesis of the nucleotide 4-thiouridine from 4-thiouracil and activated ribose. The ribozyme evolves to form the glycosidic bond to join a sugar to a base, to produce a nucleotide. The discovery fills an important gape in a generalized scheme for the evolution of the RNA world.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor 1 (GPCR) that has a central role in regulating systemic calcium homeostasis 2,3 . Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional G i versus G q proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both G i and G q drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective G i and G q coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally 1-4 . Efforts have focused on readily synthesizable molecules, inevitably leaving many chemotypes unexplored. Here we investigate structure-based docking of a bespoke virtual library of tetrahydropyridines-a scaffold that is poorly sampled by a general billion-molecule virtual library but is well suited to many aminergic G-protein-coupled receptors. Using three inputs, each with diverse available derivatives, a one pot C-H alkenylation, electrocyclization and reduction provides the tetrahydropyridine core with up to six sites of derivatization 5-7 . Docking a virtual library of 75 million tetrahydropyridines against a model of the serotonin 5-HT 2A receptor (5-HT 2A R) led to the synthesis and testing of 17 initial molecules. Four of these molecules had low-micromolar activities against either the 5-HT 2A or the 5-HT 2B receptors. Structure-based optimization led to the 5-HT 2A R agonists (R)-69 and (R)-70, with half-maximal effective concentration values of 41 nM and 110 nM, respectively, and unusual signalling kinetics that differ from psychedelic 5-HT 2A R agonists. Cryo-electron microscopy structural analysis confirmed the predicted binding mode to 5-HT 2A R. The favourable physical properties of these new agonists conferred high brain permeability, enabling mouse behavioural assays. Notably, neither had psychedelic activity, in contrast to classic 5-HT 2A R agonists, whereas both had potent antidepressant activity in mouse models and had the same efficacy as antidepressants such as fluoxetine at as low as 1/40th of the dose. Prospects for using bespoke virtual libraries to sample pharmacologically relevant chemical space will be considered., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Stimulation of the metabotropic GABA B receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology 1 . GABA B belongs to family C of the G-protein-coupled receptors, which operate as dimers to transform synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins 2,3 . However, GABA B is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits 4,5 . Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABA B receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABA B has an essential role in relaying structural transitions by ordering the linker that connects the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of each of the subunits of GABA B caps and interacts with the hydrophilic head of a phospholipid that occupies the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G proteins. These results provide a starting framework through which to decipher the mechanistic modes of signal transduction mediated by GABA B dimers, and have important implications for rational drug design that targets these receptors.