Docking for EP4R antagonists active against inflammatory pain.

The lipid prostaglandin E2 (PGE2) mediates inflammatory pain by activating G protein-coupled receptors, including the prostaglandin E2 receptor 4 (EP4R). Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce nociception by inhibiting prostaglandin synthesis, however, the disruption of upstream prostanoid biosynthesis can lead to pleiotropic effects including gastrointestinal bleeding and cardiac complications. In contrast, by acting downstream, EP4R antagonists may act specifically as anti-inflammatory agents and, to date, no selective EP4R antagonists have been approved for human use. In this work, seeking to diversify EP4R antagonist scaffolds, we computationally dock over 400 million compounds against an EP4R crystal structure and experimentally validate 71 highly ranked, de novo synthesized molecules. Further, we show how structure-based optimization of initial docking hits identifies a potent and selective antagonist with 16 nanomolar potency. Finally, we demonstrate favorable pharmacokinetics for the discovered compound as well as anti-allodynic and anti-inflammatory activity in several preclinical pain models in mice.

Engineered odorant receptors illuminate structural principles of odor discrimination.

A central challenge in olfaction is understanding how the olfactory system detects and distinguishes odorants with diverse physicochemical properties and molecular configurations. Vertebrate animals perceive odors via G protein-coupled odorant receptors (ORs). In humans, ~400 ORs enable the sense of smell. The OR family is composed of two major classes: Class I ORs are tuned to carboxylic acids while Class II ORs, representing the vast majority of the human repertoire, respond to a wide variety of odorants. How ORs recognize chemically diverse odorants remains poorly understood. A fundamental bottleneck is the inability to visualize odorant binding to ORs. Here, we uncover fundamental molecular properties of odorant-OR interactions by employing engineered ORs crafted using a consensus protein design strategy. Because such consensus ORs (consORs) are derived from the 17 major subfamilies of human ORs, they provide a template for modeling individual native ORs with high sequence and structural homology. The biochemical tractability of consORs enabled four cryoEM structures of distinct consORs with unique ligand recognition properties. The structure of a Class I consOR, consOR51, showed high structural similarity to the native human receptor OR51E2 and yielded a homology model of a related member of the human OR51 family with high predictive power. Structures of three Class II consORs revealed distinct modes of odorant-binding and activation mechanisms between Class I and Class II ORs. Thus, the structures of consORs lay the groundwork for understanding molecular recognition of odorants by the OR superfamily.

Structure-based discovery of conformationally selective inhibitors of the serotonin transporter.

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

Structural basis of odorant recognition by a human odorant receptor.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1-3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Autoantibody mimicry of hormone action at the thyrotropin receptor.

Thyroid hormones are vital in metabolism, growth and development1. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR)2. In patients with Graves' disease, autoantibodies that activate the TSHR pathologically increase thyroid hormone activity3. How autoantibodies mimic thyrotropin function remains unclear. Here we determined cryo-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. Thyrotropin selects an upright orientation of the extracellular domain owing to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody from a patient with Graves' disease selects a similar upright orientation of the extracellular domain. Reorientation of the extracellular domain transduces a conformational change in the seven-transmembrane-segment domain via a conserved hinge domain, a tethered peptide agonist and a phospholipid that binds within the seven-transmembrane-segment domain. Rotation of the TSHR extracellular domain relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to other G-protein-coupled receptors with large extracellular domains.

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation.

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms.

The serum level of iron in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing the internalization and degradation of ferroportin1. Aberrant ferroportin activity can lead to diseases of iron overload, such as haemochromatosis, or iron limitation anaemias2. Here we determine cryogenic electron microscopy structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with hepcidin and the iron mimetic cobalt. These structures and accompanying molecular dynamics simulations identify two metal-binding sites within the N and C domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy terminus of hepcidin directly contacts the divalent metal in the ferroportin C domain. Hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, in which only ferroportin molecules loaded with iron are targeted for degradation. More broadly, our structural and functional insights may enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.

Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter.

Neurotransmitter/sodium symporters (NSSs) are responsible for Na+-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K+ inhibits Na+-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K+-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni2+ bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K+-induced closure of the transporter to the outside, which was counteracted by Na+ and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K+-effect. The K+-effect depended on an intact Na1 site and mutating the Na2 site potentiated K+ binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K+ to regulate the LeuT transport cycle.

Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter.

Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na(+)-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na(+)-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.

Novel and high affinity fluorescent ligands for the serotonin transporter based on (s)-citalopram.

Novel rhodamine-labeled ligands, based on (S)-citalopram, were synthesized and evaluated for uptake inhibition at the human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) and for binding at SERT, in transiently transfected COS7 cells. Compound 14 demonstrated high affinity binding and selectivity for SERT (K i = 3 nM). Visualization of SERT, using confocal laser scanning microscopy, validated compound 14 as a novel tool for studying SERT expression and distribution in living cells.

A virtual high-throughput screening approach to the discovery of novel inhibitors of the bacterial leucine transporter, LeuT.

Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.