MRGPRX4 mediates phospho-drug-associated pruritus in a humanized mouse model.

The phosphate modification of drugs is a common chemical strategy to increase solubility and allow for parenteral administration. Unfortunately, phosphate modifications often elicit treatment- or dose-limiting pruritus through an unknown mechanism. Using unbiased high-throughput drug screens, we identified the Mas-related G protein-coupled receptor X4 (MRGPRX4), a primate-specific, sensory neuron receptor previously implicated in itch, as a potential target for phosphate-modified compounds. Using both Gq-mediated calcium mobilization and G protein-independent GPCR assays, we found that phosphate-modified compounds potently activate MRGPRX4. Furthermore, a humanized mouse model expressing MRGPRX4 in sensory neurons exhibited robust phosphomonoester prodrug-evoked itch. To characterize and confirm this interaction, we further determined the structure of MRGPRX4 in complex with a phosphate-modified drug through single-particle cryo-electron microscopy (cryo-EM) and identified critical amino acid residues responsible for the binding of the phosphate group. Together, these findings explain how phosphorylated drugs can elicit treatment-limiting itch and identify MRGPRX4 as a potential therapeutic target to suppress itch and to guide future drug design.

Mach's principle and Mach's hypotheses.

We argue that the fundamental assertion underlying Mach's critique of Newton's first law is that inertial motion is not motion in the absence of causes; rather, it is motion whose cause lies in some homogeneous aspect of the environment. We distinguish this formal requirement (Mach's principle) from two hypotheses which Mach considers concerning the origin of inertia: that the distant stars play (1) a merely "collateral" or (2) a "fundamental" role in the causal determination of inertial motion. In his later writings, Mach deliberately avoids referring to the concept of causation, and indeed, this has made the interpretation of Mach's principle a subject of widespread controversy. However, in his earlier writings, the substance of Mach's critique is less ambiguously expressed. Therefore, close attention is given to Mach's early writings and the evolution of his thought. Various accounts in the secondary literature on Mach's principle, in particular those of Norton and DiSalle, are assessed on this basis. We end with a defence of the Machian status and legitimacy of the early Einstein's research program.

Silane Effects on Adhesion Enhancement of 2K Polyurethane Adhesives.

With excellent properties such as great flexibility, outstanding chemical resistance, and superb mechanical strength, two-part polyurethane (2K PU) adhesives have been widely applied in many applications, including those in transportation and construction. Despite the extensive use, their adhesion to nonpolar polymer substrates still needs to be improved and has been widely studied. The incorporation of silane molecules and the use of plasma treatment on substrate surfaces are two popular methods to increase the adhesion of 2K PU adhesives, but their detailed adhesion enhancement mechanisms are still largely unknown. In this research, sum frequency generation (SFG) vibrational spectroscopy was used to probe the influence of added or coated silanes on the interfacial structure at the buried polypropylene (PP)/2K PU adhesive interface in situ. How plasma treatment on PP could improve adhesion was also investigated. To achieve maximum adhesion, two methods to involve silanes were studied. In the first method, silanes were directly mixed with the 2K PU adhesive before use. In the second method, silane molecules were spin-coated onto the PP substrate before the PU adhesive applied. It was found that the first method could not improve the 2K PU adhesion to PP, while the second method could substantially enhance such adhesion. SFG studies demonstrated that with the second method silane molecules chemically reacted at the interface to connect PP and 2K PU adhesive to improve the adhesion. With the first method, silane molecules could not effectively diffuse to the interface to enhance adhesion. In this research, plasma treatment was also found to be a useful method to improve the adhesion of the 2K PU adhesive to nonpolar polymer materials.

Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor.

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-ß-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-ß-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-ß-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.

Ligand and G-protein selectivity in the κ-opioid receptor.

The κ-opioid receptor (KOR) represents a highly desirable therapeutic target for treating not only pain but also addiction and affective disorders1. However, the development of KOR analgesics has been hindered by the associated hallucinogenic side effects2. The initiation of KOR signalling requires the Gi/o-family proteins including the conventional (Gi1, Gi2, Gi3, GoA and GoB) and nonconventional (Gz and Gg) subtypes. How hallucinogens exert their actions through KOR and how KOR determines G-protein subtype selectivity are not well understood. Here we determined the active-state structures of KOR in a complex with multiple G-protein heterotrimers-Gi1, GoA, Gz and Gg-using cryo-electron microscopy. The KOR-G-protein complexes are bound to hallucinogenic salvinorins or highly selective KOR agonists. Comparisons of these structures reveal molecular determinants critical for KOR-G-protein interactions as well as key elements governing Gi/o-family subtype selectivity and KOR ligand selectivity. Furthermore, the four G-protein subtypes display an intrinsically different binding affinity and allosteric activity on agonist binding at KOR. These results provide insights into the actions of opioids and G-protein-coupling specificity at KOR and establish a foundation to examine the therapeutic potential of pathway-selective agonists of KOR.

Neurotensin Receptor Allosterism Revealed in Complex with a Biased Allosteric Modulator.

The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward ß-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here, we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and GoA with and without the brain-penetrant small-molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gα subunit selective and positive allosteric modulation and agonism for ß-arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1.

Molecular Behavior of 1K Polyurethane Adhesive at Buried Interfaces: Plasma Treatment, Annealing, and Adhesion.

One-part (1K) polyurethane (PU) adhesive has excellent bulk strength and environmental resistance. It is therefore widely used in many fields, such as construction, transportation, and flexible lamination. However, when contacting non-polar polymer materials, the poor adhesion of 1K PU adhesive may not be able to support its outdoor applications. To solve this problem, plasma treatment of the non-polar polymer surface has been utilized to improve adhesion between the polymer and 1K PU adhesive. The detailed mechanisms of adhesion enhancement of the 1K PU adhesive caused by plasma treatment on polymer substrates have not been studied extensively because adhesion is a property of buried interfaces which are difficult to probe. In this study, sum frequency generation (SFG) vibrational spectroscopy was used to investigate the buried PU/polypropylene (PP) interfaces in situ nondestructively. Fourier-transform infrared spectroscopy, the X-ray diffraction technique, and adhesion tests were used as supplemental methods to SFG in the study. The 1K PU adhesive is a moisture-curing adhesive and usually needs several days to be fully cured. Here, time-dependent SFG experiments were conducted to monitor the molecular behaviors at the buried 1K PU adhesive/PP interfaces during the curing process. It was found that the PU adhesives underwent rearrangement during the curing process with functional groups gradually becoming ordered at the interface. Stronger adhesion between the plasma-treated PP substrate and the 1K PU adhesive was observed, which was achieved by the interfacial chemical reactions and a more rigid interface. Annealing the samples increased the reaction speed and enhanced the bulk PU strength with higher crystallinity. In this research, molecular mechanisms of adhesion enhancement of the 1K PU adhesive caused by the plasma treatment on PP and by annealing the PU/PP samples were elucidated.

Ligand recognition and allosteric modulation of the human MRGPRX1 receptor.

The human MAS-related G protein-coupled receptor X1 (MRGPRX1) is preferentially expressed in the small-diameter primary sensory neurons and involved in the mediation of nociception and pruritus. Central activation of MRGPRX1 by the endogenous opioid peptide fragment BAM8-22 and its positive allosteric modulator ML382 has been shown to effectively inhibit persistent pain, making MRGPRX1 a promising target for non-opioid pain treatment. However, the activation mechanism of MRGPRX1 is still largely unknown. Here we report three high-resolution cryogenic electron microscopy structures of MRGPRX1-Gαq in complex with BAM8-22 alone, with BAM8-22 and ML382 simultaneously as well as with a synthetic agonist compound-16. These structures reveal the agonist binding mode for MRGPRX1 and illuminate the structural requirements for positive allosteric modulation. Collectively, our findings provide a molecular understanding of the activation and allosteric modulation of the MRGPRX1 receptor, which could facilitate the structure-based design of non-opioid pain-relieving drugs.

Molecular basis for selective activation of DREADD-based chemogenetics.

Designer receptors exclusively activated by designer drugs (DREADDs) represent a powerful chemogenetic technology for the remote control of neuronal activity and cellular signalling1-4. The muscarinic receptor-based DREADDs are the most widely used chemogenetic tools in neuroscience research. The Gq-coupled DREADD (hM3Dq) is used to enhance neuronal activity, whereas the Gi/o-coupled DREADD (hM4Di) is utilized to inhibit neuronal activity5. Here we report four DREADD-related cryogenic electron microscopy high-resolution structures: a hM3Dq-miniGq complex and a hM4Di-miniGo complex bound to deschloroclozapine; a hM3Dq-miniGq complex bound to clozapine-N-oxide; and a hM3R-miniGq complex bound to iperoxo. Complemented with mutagenesis, functional and computational simulation data, our structures reveal key details of the recognition of DREADD chemogenetic actuators and the molecular basis for activation. These findings should accelerate the structure-guided discovery of next-generation chemogenetic tools.

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.

Conformational Variability in Ground-State CFTR Lipoprotein Particle Cryo-EM Ensembles.

Cystic fibrosis transmembrane regulator (CFTR) is a dynamic membrane protein belonging to the ABC transporter family. It is unusual within this family as it is an ion channel, as opposed to a transporter. Activation of CFTR requires ATP and phosphorylation by PKA, and dysregulation of CFTR mediated salt and water homeostasis can lead to cystic fibrosis. Recent advancements in structural biological methods have led to more than 10 published CFTR structures, and, so far, all of these structures of CFTR, determined by cryo-EM, have been limited to detergent-purified protein preparations. To visualize CFTR in an environment that more closely represents its native membranous environment, we utilized two different lipoprotein particle encapsulation techniques: one in which the ion channel is first purified and then reconstituted using the membrane scaffolding protein Saposin A and another that uses the solubilizing polymer Sokalan CP9 (DIBMA) to extract CFTR directly from membranes. Structures derived from these types of preparations may better correlate to their function, for instance, the single-channel measurements from membrane vesicles.

Molecular insights into the regulation of constitutive activity by RNA editing of 5HT2C serotonin receptors.

RNA editing is a process by which post-transcriptional changes of mRNA nucleotides alter protein function through modification of the amino acid content. The 5HT2C serotonin receptor, which undergoes 32 distinct RNA-editing events leading to 24 protein isoforms, is a notable example of this process. These 5HT2C isoforms display differences in constitutive activity, agonist/inverse agonist potencies, and efficacies. To elucidate the molecular mechanisms responsible for these effects of RNA editing, we present four active-state 5HT2C-transducer-coupled structures of three representative isoforms (INI, VGV, and VSV) with the selective drug lorcaserin (Belviq) and the classic psychedelic psilocin. We also provide a comprehensive analysis of agonist activation and constitutive activity across all 24 protein isoforms. Collectively, these findings reveal a unique hydrogen-bonding network located on intracellular loop 2 that is subject to RNA editing, which differentially affects GPCR constitutive and agonist signaling activities.

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.

High-speed high-resolution data collection on a 200 keV cryo-TEM.

Limitations to successful single-particle cryo-electron microscopy (cryo-EM) projects include stable sample generation, production of quality cryo-EM grids with randomly oriented particles embedded in thin vitreous ice and access to microscope time. To address the limitation of microscope time, methodologies to more efficiently collect data on a 200 keV Talos Arctica cryo-transmission electron microscope at speeds as fast as 720 movies per hour (∼17 000 per day) were tested. In this study, key parameters were explored to increase data collection speed including: (1) using the beam-image shift method to acquire multiple images per stage position, (2) employing UltrAufoil TEM grids with R0.6/1 hole spacing, (3) collecting hardware-binned data and (4) adjusting the image shift delay factor in SerialEM. Here, eight EM maps of mouse apoferritin at 1.8-1.9 Šresolution were obtained in the analysis with data collection times for each dataset ranging from 56 min to 2 h. An EM map of mouse apoferritin at 1.78 Šwas obtained from an overnight data collection at a speed of 500 movies per hour and subgroup analysis performed, with no significant variation observed in data quality by image shift distance and image shift delay. The findings and operating procedures detailed herein allow for rapid turnover of single-particle cryo-EM structure determination.

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.

Functional integrity of membrane protein rhodopsin solubilized by styrene-maleic acid copolymer.

Membrane proteins often require solubilization to study their structure or define the mechanisms underlying their function. In this study, the functional properties of the membrane protein rhodopsin in its native lipid environment were investigated after being solubilized with styrene-maleic acid (SMA) copolymer. The static absorption spectra of rhodopsin before and after the addition of SMA were recorded at room temperature to quantify the amount of membrane protein solubilized. The samples were then photobleached to analyze the functionality of rhodopsin upon solubilization. Samples with low or high SMA/rhodopsin ratios were compared to find a threshold in which the maximal amount of active rhodopsin was solubilized from membrane suspensions. Interestingly, whereas the highest SMA/rhodopsin ratios yielded the most solubilized rhodopsin, the rhodopsin produced under these conditions could not reach the active (Meta II) state upon photoactivation. The results confirm that SMA is a useful tool for membrane protein research, but SMA added in excess can interfere with the dynamics of protein activation.

The 3-year outcomes of corneal tattooing for severely disfigured corneas.

BACKGROUND: To evaluate the efficacy of corneal tattooing for various clinical applications. METHODS: The medical charts of 62 eyes of 62 patients who underwent corneal tattooing between March 2016 and August 2017 were retrospectively reviewed. The causes of opacity and various methods of corneal tattooing were analyzed. RESULTS: Among our 62 patients, 38 were males and 24 were females. Average age was 48.47 ± 15.30 (range, 12-74) years old. The mean follow-up period was 40.19 ± 2.34 (range, 36-43) months. The most common causes of corneal opacity were ocular trauma (33 eyes, 53.2%), prior retinal surgery (9 eyes, 14.5%), congenital etiologies (8 eyes, 12.9%) and measles (5 eyes, 8.0%). The most common associated ocular findings were strabismus (23 eyes, 37.0%), phthisis bulbi (17 eyes, 27.4%) and band keratopathy (13 eyes, 20.9%). Cosmetic outcomes improved without serious complications in all cases. CONCLUSIONS: Corneal tattooing is a viable option with an expanding set of indications, such as discolored previous corneal tattoos, white pupil due to inoperable cataract with clear cornea and dense corneal opacities in blind eyes. Elective corneal tattooing seems to be a viable and convenient method to improve cosmesis with minimal complications and high patient satisfaction.

Longterm, Real-world Safety of Adalimumab in Rheumatoid Arthritis: Analysis of a Prospective US-based Registry.

OBJECTIVE: To assess longterm safety in a US cohort of patients with rheumatoid arthritis (RA) treated with adalimumab (ADA) in real-world clinical care settings. METHODS: This observational study analyzed the longterm incidence of safety outcomes among patients with RA initiating ADA, using data from the Corrona RA registry. Patients were adults (≥ 18 yrs) who initiated ADA treatment between January 2008 and June 2017, and who had at least 1 followup visit. RESULTS: In total, 2798 ADA initiators were available for analysis, with a mean age of 54.5 years, 77% female, and mean disease duration of 8.3 years. Nearly half (48%) were biologic-naive, and 9% were using prednisone ≥ 10 mg at ADA initiation. The incidence rates per 100 person-years for serious infections, congestive heart failure requiring hospitalization, malignancy (excluding nonmelanoma skin cancer), and all-cause mortality were 1.86, 0.15, 0.64, and 0.33, respectively. The incidence of serious infections was higher in the first year of therapy (3.44, 95% CI 2.45-4.84) than in subsequent years, while other measured adverse effects did not vary substantially by duration of exposure. The median time to ADA discontinuation was 11 months, while the median time to first serious infection among those experiencing a serious infection event was 12 months. CONCLUSION: Analysis of longterm data from this prospective real-world registry demonstrated a safety profile consistent with previous studies in patients with RA. This analysis did not identify any new safety signals associated with ADA treatment and provides guidance for physicians prescribing ADA for extended periods.

Octahedron-based Projections as Intermediate Representations for Computer Imaging: TOAST, TEA, and More.

This paper defines and discusses a set of rectangular all-sky projections that have no singular points, notably the Tesselated Octahedral Adaptive Spherical Transformation (or TOAST) developed initially for the WorldWide Telescope. These have proven to be useful as intermediate representations for imaging data where the application transforms dynamically from a standardized internal format to a specific format (projection, scaling, orientation, etc.) requested by the user. TOAST is strongly related to the Hierarchical Triangular Mesh pixelization and is particularly well adapted to situations where one wishes to traverse a hierarchy of images increasing in resolution. Because it can be recursively computed using a very simple algorithm it is particularly adaptable to use with graphical processing units.

Cryo-EM Visualization of an Active High Open Probability CFTR Anion Channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic fibrosis, is a unique member of the large family of ATP-binding cassette transport proteins. Regulation of CFTR channel activity is stringently controlled by phosphorylation and nucleotide binding. Structural changes that underlie transitions between active and inactive functional states are not yet fully understood. Indeed the first 3D structures of dephosphorylated, ATP-free, and phosphorylated ATP-bound states were only recently reported. Here we have determined the structure of inactive and active states of a thermally stabilized CFTR, the latter with a very high channel open probability, confirmed after reconstitution into proteoliposomes. These structures, obtained at nominal resolution of 4.3 and 6.6 Å, reveal a unique repositioning of the transmembrane helices and regulatory domain density that provide insights into the structural transition between active and inactive functional states of CFTR. Moreover, we observe an extracellular vestibule that may provide anion access to the pore due to the conformation of transmembrane helices 7 and 8 that differs from the previous orthologue CFTR structures. In conclusion, our work contributes detailed structural information on an active, open state of the CFTR anion channel.