HypoRiPPAtlas as an Atlas of hypothetical natural products for mass spectrometry database search

Recent analyses of public microbial genomes have found over a million biosynthetic gene clusters, the natural products of the majority of which remain unknown. Additionally, GNPS harbors billions of mass spectra of natural products without known structures and biosynthetic genes. We bridge the gap between large-scale genome mining and mass spectral datasets for natural product discovery by developing HypoRiPPAtlas, an Atlas of hypothetical natural product structures, which is ready-to-use for in silico database search of tandem mass spectra. HypoRiPPAtlas is constructed by mining genomes using seq2ripp, a machine-learning tool for the prediction of ribosomally synthesized and post-translationally modified peptides (RiPPs). In HypoRiPPAtlas, we identify RiPPs in microbes and plants. HypoRiPPAtlas could be extended to other natural product classes in the future by implementing corresponding biosynthetic logic. This study paves the way for large-scale explorations of biosynthetic pathways and chemical structures of microbial and plant RiPP classes

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Structure-Based Design of a Chemical Probe Set for the 5-HT5A Serotonin Receptor.

The 5-HT5A receptor (5-HT5AR), for which no selective agonists and a few antagonists exist, remains the least understood serotonin receptor. A single commercial antagonist, SB-699551, has been widely used to investigate the 5-HT5AR function in neurological disorders, including pain, but this molecule has substantial liabilities as a chemical probe. Accordingly, we sought to develop an internally controlled probe set. Docking over 6 million molecules against a 5-HT5AR homology model identified 5 mid-μM ligands, one of which was optimized to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT5AR with a more restricted off-target profile and decreased assay liabilities versus SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678. Surprisingly, analogs of UCSF678 that lost the 5-HT5AR activity revealed that 5-HT5AR engagement is nonessential for alleviating pain, contrary to studies with less-selective ligands. UCSF678 and analogs constitute a selective probe set with which to study the function of the 5-HT5AR

Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and CH Functionalization

Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon–carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%

A novel ligand “deschloroclozapine” selectively visualizes and activates chemogenetic receptors in non-human primates

Objectives: Designer Receptors Exclusively Activated by Designer Drug (DREADDs) is one of the chemogenetic technologies that afford to selectively and remotely control the activity of neuronal population expressing “designer receptor” by systemic delivery of the biologically inert compound. Muscarinic-based designer receptors, hM3Dq (excitatory) and hM4Di (inhibitory), can be activated by clozapine-N-oxide (CNO), are most widely used. DREADDs can be applicable for larger and discontinuous brain tissues, which non-human primate studies demand. For the application of DREADD to monkey study, it is desirable to monitor the DREADD expression in vivo. In addition, CNO has modest brain permeability and can be metabolized to clozapine, which is also a potent DREADD agonist. Since clozapine possesses activity at sites for numerous endogenous receptors, the CNO administration could be associated with off-target actions. In this study, we demonstrated that a novel ligand, deschloroclozapine (DCZ), served a dual purpose in chemogenetics: (1) as a selective compound for visualization of DREADD expression in vivo by positron emission tomography (PET) imaging and (2) as a selective agonist for muscarinic-based DREADDs.Methods and Results: In vitro inhibition binding assay with 3H-QNB revealed that DCZ is a high DREADD selective ligand; it exhibited high affinity to hM3Dq and hM4Di (6.3 and 4.2 nM, respectively: comparable to clozapine and 100-fold stronger than CNO), while it had moderate or low affinities (>50 nM) for a large number of endogenous receptors. It was confirmed by 11C-DCZ with PET in monkeys; significant uptake of 11C-DCZ was specifically found in DREADD expressing regions, while uptakes in non-DREADD expression regions were small. Agonist efficacy of DCZ was examined by electrophysiological recording in a monkey received a hM3Dq-vector injection. After systemic DCZ administration, but not a vehicle, neuronal activity in the hM3Dq-positive area was rapidly and significantly increased, whereas the activity in the area outside hM3Dq-positive sites did not change. A pharmacokinetic study revealed that any major significant metabolites of DCZ were not detected in the plasma and CSF after DCZ administration.Conclusions: These results indicate that (1) 11C-DCZ is a suitable and sensitive PET ligand for visualization of DREADD expression and (2) DCZ is a metabolically stable, extremely potent, highly brain-penetrable, and selective agonist for DREADDs, the combination of which provides clear benefits for non-human primates chemogenetics and future therapeutic applications.BrainPET201

Structural basis of ligand recognition at the human MT1 melatonin receptor

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms by synchronization to environmental cues and is involved in diverse physiological processes such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function. Melatonin is formed in the pineal gland in a light-regulated manner by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness by activating two high-affinity G-protein-coupled receptors, type 1A (MT$_1$) and type 1B (MT$_2$). Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids, and is one of the most popular supplements in the United States. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT$_1$ in complex with four agonists: the insomnia drug ramelteon, two melatonin analogues, and the mixed melatonin–serotonin antidepressant agomelatine. The structure of MT$_2$ is described in an accompanying paper. Although the MT$_1$ and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT$_1$, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT$_1$ mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors

Deschloroclozapine, a potent and selective chemogenetic actuator enables rapid neuronal and behavioral modulations in mice and monkeys

The chemogenetic technology Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) affords remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities, and potential off-target effects of CNO represent areas for improvement. Here we provide a new high affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 μg/kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 μg/kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents the most potent, selective, metabolically stable and fast-acting DREADD agonist reported with utility in both mice and non-human primates for a variety of applications

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