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1.
Nat Commun ; 12(1): 815, 2021 02 05.
Article in English | MEDLINE | ID: mdl-33547286

ABSTRACT

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.


Subject(s)
Aminopyridines/chemistry , Azepines/chemistry , Orexin Receptor Antagonists/chemistry , Orexin Receptors/chemistry , Peptides/chemistry , Sleep Aids, Pharmaceutical/chemistry , Sulfonamides/chemistry , Triazoles/chemistry , Aminopyridines/metabolism , Azepines/metabolism , Binding Sites , Cloning, Molecular , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Humans , Molecular Dynamics Simulation , Orexin Receptor Antagonists/metabolism , Orexin Receptors/agonists , Orexin Receptors/metabolism , Peptides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sleep Aids, Pharmaceutical/metabolism , Sulfonamides/metabolism , Triazoles/metabolism
2.
Nat Struct Mol Biol ; 24(7): 570-577, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28581512

ABSTRACT

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.


Subject(s)
Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/chemistry , Allosteric Regulation , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Protein Binding , Protein Conformation
3.
Proc Natl Acad Sci U S A ; 114(3): E297-E306, 2017 01 17.
Article in English | MEDLINE | ID: mdl-28039433

ABSTRACT

Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of-but adjacent to-the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.


Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Receptor, trkA/antagonists & inhibitors , Receptor, trkA/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Drug Evaluation, Preclinical , Humans , Kinetics , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Models, Molecular , Protein Conformation , Protein Kinase Inhibitors/chemical synthesis , Receptor, trkA/genetics , Receptor, trkB/antagonists & inhibitors , Receptor, trkB/chemistry , Receptor, trkB/genetics , Receptor, trkC/antagonists & inhibitors , Receptor, trkC/chemistry , Receptor, trkC/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/drug effects , Recombinant Proteins/genetics , Structure-Activity Relationship , Surface Plasmon Resonance
4.
J Biol Chem ; 288(47): 34073-34080, 2013 Nov 22.
Article in English | MEDLINE | ID: mdl-24108127

ABSTRACT

The emergence of antibiotic-resistant strains of pathogenic bacteria is an increasing threat to global health that underscores an urgent need for an expanded antibacterial armamentarium. Gram-negative bacteria, such as Escherichia coli, have become increasingly important clinical pathogens with limited treatment options. This is due in part to their lipopolysaccharide (LPS) outer membrane components, which dually serve as endotoxins while also protecting Gram-negative bacteria from antibiotic entry. The LpxC enzyme catalyzes the committed step of LPS biosynthesis, making LpxC a promising target for new antibacterials. Here, we present the first structure of an LpxC enzyme in complex with the deacetylation reaction product, UDP-(3-O-(R-3-hydroxymyristoyl))-glucosamine. These studies provide valuable insight into recognition of substrates and products by LpxC and a platform for structure-guided drug discovery of broad spectrum Gram-negative antibiotics.


Subject(s)
Amidohydrolases/chemistry , Escherichia coli/enzymology , Myristic Acids/chemistry , Protons , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Amidohydrolases/metabolism , Crystallography, X-Ray , Lipopolysaccharides/biosynthesis , Lipopolysaccharides/chemistry , Myristic Acids/metabolism , Protein Structure, Tertiary , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/metabolism
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