AlphaFold2 structures guide prospective ligand discovery.

AlphaFold2 (AF2) models have had wide impact, but they have had mixed success in retrospective ligand recognition. We prospectively docked large libraries against unrefined AF2 models of the σ2 and 5-HT2A receptors, testing hundreds of new molecules and comparing results to docking against the experimental structures. Hit rates were high and similar for the experimental and the AF2 structures, as were affinities. The success of docking against the AF2 models was achieved despite differences in orthosteric residue conformations versus the experimental structures. Determination of the cryo-electron microscopy structure for one of the more potent 5HT2A ligands from the AF2 docking revealed residue accommodations that resembled the AF2 prediction. AF2 models may sample conformations that differ from experimental structures but remain low energy and relevant for ligand discovery, extending the domain of structure-based ligand discovery.

Constitutive activation mechanism of a class C GPCR.

Class C G-protein-coupled receptors (GPCRs) are activated through binding of agonists to the large extracellular domain (ECD) followed by rearrangement of the transmembrane domains (TMDs). GPR156, a class C orphan GPCR, is unique because it lacks an ECD and exhibits constitutive activity. Impaired GPR156-Gi signaling contributes to loss of hearing. Here we present the cryo-electron microscopy structures of human GPR156 in the Go-free and Go-coupled states. We found that an endogenous phospholipid molecule is located within each TMD of the GPR156 dimer. Asymmetric binding of Gα to the phospholipid-bound GPR156 dimer restructures the first and second intracellular loops and the carboxy-terminal part of the elongated transmembrane 7 (TM7) without altering dimer conformation. Our findings reveal that GPR156 is a transducer for phospholipid signaling. Constant binding of abundant phospholipid molecules and the G-protein-induced reshaping of the cytoplasmic face provide a basis for the constitutive activation of GPR156.

AlphaFold2 structures template ligand discovery.

AlphaFold2 (AF2) and RosettaFold have greatly expanded the number of structures available for structure-based ligand discovery, even though retrospective studies have cast doubt on their direct usefulness for that goal. Here, we tested unrefined AF2 models prospectively, comparing experimental hit-rates and affinities from large library docking against AF2 models vs the same screens targeting experimental structures of the same receptors. In retrospective docking screens against the σ2 and the 5-HT2A receptors, the AF2 structures struggled to recapitulate ligands that we had previously found docking against the receptors' experimental structures, consistent with published results. Prospective large library docking against the AF2 models, however, yielded similar hit rates for both receptors versus docking against experimentally-derived structures; hundreds of molecules were prioritized and tested against each model and each structure of each receptor. The success of the AF2 models was achieved despite differences in orthosteric pocket residue conformations for both targets versus the experimental structures. Intriguingly, against the 5-HT2A receptor the most potent, subtype-selective agonists were discovered via docking against the AF2 model, not the experimental structure. To understand this from a molecular perspective, a cryoEM structure was determined for one of the more potent and selective ligands to emerge from docking against the AF2 model of the 5-HT2A receptor. Our findings suggest that AF2 models may sample conformations that are relevant for ligand discovery, much extending the domain of applicability of structure-based ligand discovery.

Structural studies of serotonin receptor family.

Serotonin receptors, also known as 5-HT receptors, belong to the G protein-coupled receptors (GPCRs) superfamily. They mediate the effects of serotonin, a neurotransmitter that plays a key role in a wide range of functions including mood regulation, cognition and appetite. The functions of serotonin are mediated by a family of 5-HT receptors including 12 GPCRs belonging to six major families: 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6 and 5-HT7. Despite their distinct characteristics and functions, these receptors' subtypes share common structural features and signaling mechanisms. Understanding the structure, functions and pharmacology of the serotonin receptor family is essential for unraveling the complexities of serotonin signaling and developing targeted therapeutics for neuropsychiatric disorders. However, developing drugs that selectively target specific receptor subtypes is challenging due to the structural similarities in their orthosteric binding sites. This review focuses on the recent advancements in the structural studies of 5-HT receptors, highlighting the key structural features of each subtype and shedding light on their potential as targets for mental health and neurological disorders (such as depression, anxiety, schizophrenia, and migraine) drugs. [BMB Reports 2023; 56(10): 527-536].

Molecular mechanism of biased signaling at the kappa opioid receptor.

The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may be key to maintaining this benefit while minimizing side-effect liabilities. As for most G protein-coupled receptors (GPCRs), however, the molecular mechanisms of ligand-specific signaling at KOR have remained unclear. To better understand the molecular determinants of KOR signaling bias, we apply structure determination, atomic-level molecular dynamics (MD) simulations, and functional assays. We determine a crystal structure of KOR bound to the G protein-biased agonist nalfurafine, the first approved KOR-targeting drug. We also identify an arrestin-biased KOR agonist, WMS-X600. Using MD simulations of KOR bound to nalfurafine, WMS-X600, and a balanced agonist U50,488, we identify three active-state receptor conformations, including one that appears to favor arrestin signaling over G protein signaling and another that appears to favor G protein signaling over arrestin signaling. These results, combined with mutagenesis validation, provide a molecular explanation of how agonists achieve biased signaling at KOR.

Signaling snapshots of a serotonin receptor activated by the prototypical psychedelic LSD.

Serotonin (5-hydroxytryptamine [5-HT]) 5-HT2-family receptors represent essential targets for lysergic acid diethylamide (LSD) and all other psychedelic drugs. Although the primary psychedelic drug effects are mediated by the 5-HT2A serotonin receptor (HTR2A), the 5-HT2B serotonin receptor (HTR2B) has been used as a model receptor to study the activation mechanisms of psychedelic drugs due to its high expression and similarity to HTR2A. In this study, we determined the cryo-EM structures of LSD-bound HTR2B in the transducer-free, Gq-protein-coupled, and ß-arrestin-1-coupled states. These structures provide distinct signaling snapshots of LSD's action, ranging from the transducer-free, partially active state to the transducer-coupled, fully active states. Insights from this study will both provide comprehensive molecular insights into the signaling mechanisms of the prototypical psychedelic LSD and accelerate the discovery of novel psychedelic drugs.

Bespoke library docking for 5-HT2A receptor agonists with antidepressant activity.

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.

Inactive and active state structures template selective tools for the human 5-HT5A receptor.

Serotonin receptors are important targets for established therapeutics and drug development as they are expressed throughout the human body and play key roles in cell signaling. There are 12 serotonergic G protein-coupled receptor members encoded in the human genome, of which the 5-hydroxytryptamine (5-HT)5A receptor (5-HT5AR) is the least understood and lacks selective tool compounds. Here, we report four high-resolution (2.73-2.80 Å) structures of human 5-HT5ARs, including an inactive state structure bound to an antagonist AS2674723 by crystallization and active state structures bound to a partial agonist lisuride and two full agonists, 5-carboxamidotryptamine (5-CT) and methylergometrine, by cryo-EM. Leveraging the new structures, we developed a highly selective and potent antagonist for 5-HT5AR. Collectively, these findings both enhance our understanding of this enigmatic receptor and provide a roadmap for structure-based drug discovery for 5-HT5AR.

Structure, function and pharmacology of human itch GPCRs.

The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently1. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions2-5. MRGPRX2 couples to both Gi and Gq in mast cells6. Here we describe agonist-stabilized structures of MRGPRX2 coupled to Gi1 and Gq in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and Gq. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.

Structure of a Hallucinogen-Activated Gq-Coupled 5-HT2A Serotonin Receptor.

Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.

Nanobody-enabled monitoring of kappa opioid receptor states.

Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.

Crystal structure of GSK3ß in complex with the flavonoid, morin.

GSK3ß is a key kinase that plays a role in cellular signaling pathways. In Alzheimer's disease (AD), GSK3ß has been implicated in hyperphosphorylation of tau proteins in the neuron, which is a hallmark of AD. Morin, a flavonoid that is abundant in nature, was found as an inhibitor of GSK3ß that can reduce tau pathology in vivo and in vitro. In this study, we determined the crystal structure of GSK3ß in complex with morin. The structure revealed that morin inhibits GSK3ß by binding to the ATP binding pocket. Our findings augment the potential of morin as a functional food to help prevent AD, as well as to provide structural information to develop new therapeutics based on the morin skeleton.

A missense allele of KARRIKIN-INSENSITIVE2 impairs ligand-binding and downstream signaling in Arabidopsis thaliana.

A smoke-derived compound, karrikin (KAR), and an endogenous but as yet unidentified KARRIKIN INSENSITIVE2 (KAI2) ligand (KL) have been identified as chemical cues in higher plants that impact on multiple aspects of growth and development. Genetic screening of light-signaling mutants in Arabidopsis thaliana has identified a mutant designated as ply2 (pleiotropic long hypocotyl2) that has pleiotropic light-response defects. In this study, we used positional cloning to identify the molecular lesion of ply2 as a missense mutation of KAI2/HYPOSENSITIVE TO LIGHT, which causes a single amino acid substitution, Ala219Val. Physiological analysis and genetic epistasis analysis with the KL-signaling components MORE AXILLARY GROWTH2 (MAX2) and SUPPRESSOR OF MAX2 1 suggested that the pleiotropic phenotypes of the ply2 mutant can be ascribed to a defect in KL-signaling. Molecular and biochemical analyses revealed that the mutant KAI2ply2 protein is impaired in its ligand-binding activity. In support of this conclusion, X-ray crystallography studies suggested that the KAI2ply2 mutation not only results in a narrowed entrance gate for the ligand but also alters the structural flexibility of the helical lid domains. We discuss the structural implications of the Ala219 residue with regard to ligand-specific binding and signaling of KAI2, together with potential functions of KL-signaling in the context of the light-regulatory network in Arabidopsis thaliana.

Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation.

Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia.

Constitutive activation of T cells by γ2-herpesviral GPCR through the interaction with cellular CXCR4.

Members of the herpesviral family use multiple strategies to hijack infected host cells and exploit cellular signaling for their pathogenesis and latent infection. Among the most intriguing weapons in the arsenal of pathogenic herpesviruses are the constitutively active virally-encoded G protein-coupled receptors (vGPCRs). Even though vGPCRs contribute to viral pathogenesis such as immune evasion and proliferative disorders, the molecular details of how vGPCRs continuously activate cellular signaling are largely unknown. Here, we report that the vGPCR of Herpesvirus saimiri (HVS), an oncogenic γ2-herpesvirus, constitutively activates T cells via a heteromeric interaction with cellular CXCR4. Constitutive T cell activation also occurs with expression of the vGPCR of Kaposi's sarcoma-associated herpesvirus (KSHV), but not the vGPCR of Epstein-Barr virus. Expression of HVS vGPCR down-regulated the surface expression of CXCR4 but did not induce the degradation of the chemokine receptor, suggesting that vGPCR/CXCR4 signaling continues in cytosolic compartments. The physical association of vGPCR with CXCR4 was demonstrated by proximity ligation assay as well as immunoprecipitation. Interestingly, the constitutive activation of T cells by HVS vGPCR is independent of proximal T cell receptor (TCR) signaling molecules, such as TCRß, Lck, and ZAP70, whereas CXCR4 silencing by shRNA abolished T cell activation by vGPCRs of HVS and KSHV. Furthermore, previously identified inactive vGPCR mutants failed to interact with CXCR4. These findings on the positive cooperativity of vGPCR with cellular CXCR4 in T cell activation extend our current understanding of the molecular mechanisms of vGPCR function and highlight the importance of heteromerization for GPCR activity.

Crystal structure and functional characterization of a light-driven chloride pump having an NTQ motif.

A novel light-driven chloride-pumping rhodopsin (ClR) containing an 'NTQ motif' in its putative ion conduction pathway has been discovered and functionally characterized in a genomic analysis study of a marine bacterium. Here we report the crystal structure of ClR from the flavobacterium Nonlabens marinus S1-08(T) determined under two conditions at 2.0 and 1.56 Å resolutions. The structures reveal two chloride-binding sites, one around the protonated Schiff base and the other on a cytoplasmic loop. We identify a '3 omega motif' formed by three non-consecutive aromatic amino acids that is correlated with the B-C loop orientation. Detailed ClR structural analyses with functional studies in E. coli reveal the chloride ion transduction pathway. Our results help understand the molecular mechanism and physiological role of ClR and provide a structural basis for optogenetic applications.

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR.

Induction of endoplasmic reticulum (ER)-to-Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)-mediated, Golgi-independent unconventional cell-surface trafficking of the folding-deficient ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation-dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508-CFTR, such as induction of ER-to-Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C-terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation-mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation-inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER-retained ΔF508-CFTR and mediates the ER stress-induced unconventional secretion pathway.

GC1118, an Anti-EGFR Antibody with a Distinct Binding Epitope and Superior Inhibitory Activity against High-Affinity EGFR Ligands.

The EGFR-targeted monoclonal antibodies are a valid therapeutic strategy for patients with metastatic colorectal cancer (mCRC). However, only a small subset of mCRC patients has therapeutic benefits and there are high demands for EGFR therapeutics with a broader patient pool and more potent efficacy. In this study, we report GC1118 exhibiting a different character in terms of binding epitope, affinity, mode of action, and efficacy from other anti-EGFR antibodies. Structural analysis of the EGFR-GC1118 crystal complex revealed that GC1118 recognizes linear, discrete N-terminal epitopes of domain III of EGFR, critical for EGF binding but not overlapping with those of other EGFR-targeted antibodies. GC1118 exhibited superior inhibitory activity against high-affinity EGFR ligands in terms of EGFR binding, triggering EGFR signaling, and proliferation compared with cetuximab and panitumumab. EGFR signaling driven by low-affinity ligands, on the contrary, was well inhibited by all the antibodies tested. GC1118 demonstrated robust antitumor activity in tumor xenografts with elevated expression of high-affinity ligands in vivo, whereas cetuximab did not. Considering the significant role of high-affinity EGFR ligands in modulating tumor microenvironment and inducing resistance to various cancer therapeutics, our study suggests a potential therapeutic advantage of GC1118 in terms of efficacy and a range of benefited patient pool. Mol Cancer Ther; 15(2); 251-63. ©2015 AACR.

In Vitro and In Vivo Investigation of the Inhibition of Trypanosoma brucei Cell Growth by Lipophilic Bisphosphonates.

We report the results of a screen of a library of 925 potential prenyl synthase inhibitors against Trypanosoma brucei farnesyl diphosphate synthase (TbFPPS) and against T. brucei, the causative agent of human African trypanosomiasis. The most potent compounds were lipophilic analogs of the bone resorption drug zoledronate, some of which had submicromolar to low micromolar activity against bloodstream form T. brucei and selectivity indices of up to ∼ 300. We evaluated the effects of two such inhibitors on survival and parasitemia in a T. brucei mouse model of infection and found that survival increased by up to 16 days. We also investigated the binding of three lipophilic bisphosphonates to an expressed TbFPPS using crystallography and investigated the thermodynamics of binding using isothermal titration calorimetry.