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1.
Nat Commun ; 14(1): 5938, 2023 09 23.
Article in English | MEDLINE | ID: mdl-37741852

ABSTRACT

GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, the lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study GPR61 structure and function. Here, we report a structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its allosteric binding site and mode of action. These findings offer mechanistic insights into an orphan GPCR while providing both a structural framework and tool compound to support further studies of GPR61 function and modulation.


Subject(s)
Drug Inverse Agonism , GTP-Binding Proteins , Receptors, G-Protein-Coupled , Allosteric Site , Appetite , Binding Sites , GTP-Binding Proteins/metabolism , Humans , Receptors, G-Protein-Coupled/agonists
2.
J Med Chem ; 66(1): 460-472, 2023 01 12.
Article in English | MEDLINE | ID: mdl-36562986

ABSTRACT

A series of small-molecule YEATS4 binders have been discovered as part of an ongoing research effort to generate high-quality probe molecules for emerging and/or challenging epigenetic targets. Analogues such as 4d and 4e demonstrate excellent potency and selectivity for YEATS4 binding versus YEATS1,2,3 and exhibit good physical properties and in vitro safety profiles. A new X-ray crystal structure confirms direct binding of this chemical series to YEATS4 at the lysine acetylation recognition site of the YEATS domain. Multiple analogues engage YEATS4 with nanomolar potency in a whole-cell nanoluciferase bioluminescent resonance energy transfer assay. Rodent pharmacokinetic studies demonstrate the competency of several analogues as in vivo-capable binders.


Subject(s)
Gene Expression Regulation , Protein Processing, Post-Translational , Protein Domains , Acetylation , Epigenesis, Genetic
3.
Nat Struct Mol Biol ; 30(1): 22-30, 2023 01.
Article in English | MEDLINE | ID: mdl-36522428

ABSTRACT

Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.


Subject(s)
Liver , Mitochondrial Membranes , Humans , Liver/metabolism , Mitochondrial Membranes/metabolism , Glycerol-3-Phosphate O-Acyltransferase/chemistry , Glycerol-3-Phosphate O-Acyltransferase/metabolism , Amino Acid Sequence
4.
J Biol Chem ; 298(6): 101972, 2022 06.
Article in English | MEDLINE | ID: mdl-35461811

ABSTRACT

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (ß, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, ß, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and ß Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.


Subject(s)
COVID-19 Drug Treatment , COVID-19 , Lactams/chemistry , SARS-CoV-2 , Viral Protease Inhibitors/chemistry , COVID-19/virology , Coronavirus 3C Proteases , Cysteine Endopeptidases/metabolism , Humans , Leucine , Nitriles , Pandemics , Proline , SARS-CoV-2/drug effects , Viral Proteins/metabolism
5.
Nat Commun ; 11(1): 1598, 2020 03 27.
Article in English | MEDLINE | ID: mdl-32221310

ABSTRACT

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.


Subject(s)
Antibodies/pharmacology , Cryoelectron Microscopy/methods , Membrane Proteins/chemistry , Antibodies/chemistry , Cell Membrane/metabolism , Cell Surface Display Techniques , Crystallography, X-Ray , Humans , Membrane Proteins/drug effects , Membrane Proteins/metabolism , Models, Molecular , Polymers , Propylamines , Protein Binding , Protein Conformation
6.
Biophys J ; 117(11): 2228-2239, 2019 12 03.
Article in English | MEDLINE | ID: mdl-31703801

ABSTRACT

Although the three-dimensional structures of G-protein coupled receptors (GPCRs), the largest superfamily of drug targets, have enabled structure-based drug design, there are no structures available for 87% of GPCRs. This is due to the stiff challenge in purifying the inherently flexible GPCRs. Identifying thermostabilized mutant GPCRs via systematic alanine scanning mutations has been a successful strategy in stabilizing GPCRs, but it remains a daunting task for each GPCR. We developed a computational method that combines sequence-, structure-, and dynamics-based molecular properties of GPCRs that recapitulate GPCR stability, with four different machine learning methods to predict thermostable mutations ahead of experiments. This method has been trained on thermostability data for 1231 mutants, the largest publicly available data set. A blind prediction for thermostable mutations of the complement factor C5a receptor 1 retrieved 36% of the thermostable mutants in the top 50 prioritized mutants compared to 3% in the first 50 attempts using systematic alanine scanning.


Subject(s)
Molecular Dynamics Simulation , Mutation , Receptor, Anaphylatoxin C5a/chemistry , Sequence Analysis/methods , Alanine/chemistry , Alanine/genetics , Amino Acid Substitution , HEK293 Cells , Humans , Machine Learning , Protein Domains , Protein Stability , Receptor, Anaphylatoxin C5a/genetics
7.
J Med Chem ; 61(23): 10415-10439, 2018 12 13.
Article in English | MEDLINE | ID: mdl-30130103

ABSTRACT

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.


Subject(s)
Drug Design , Drug Inverse Agonism , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Pyridines/administration & dosage , Pyridines/pharmacology , Administration, Oral , Animals , Biological Availability , Drug Evaluation, Preclinical , Humans , Mice , Pyridines/pharmacokinetics , Th17 Cells/drug effects , Th17 Cells/metabolism
9.
J Med Chem ; 60(23): 9860-9873, 2017 12 14.
Article in English | MEDLINE | ID: mdl-29148769

ABSTRACT

Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates. The discovery and optimization of 3-substituted azetidine carbamate irreversible inhibitors of MAGL were aided by the generation of inhibitor-bound MAGL crystal structures. Compound 6, a highly efficient and selective MAGL inhibitor against recombinant enzyme and in a cellular context, was tested in vivo and shown to elevate central 2-AG levels at a 10 mg/kg dose.


Subject(s)
Azetidines/pharmacology , Carbamates/pharmacology , Enzyme Inhibitors/pharmacology , Monoacylglycerol Lipases/antagonists & inhibitors , Piperidines/pharmacology , Animals , Azetidines/chemistry , Azetidines/pharmacokinetics , Carbamates/chemistry , Carbamates/pharmacokinetics , Cell Line , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Humans , Mice, Inbred C57BL , Models, Molecular , Monoacylglycerol Lipases/metabolism , Piperidines/chemistry , Piperidines/pharmacokinetics , Recombinant Proteins/metabolism
10.
J Med Chem ; 60(13): 5673-5698, 2017 07 13.
Article in English | MEDLINE | ID: mdl-28574706

ABSTRACT

Phosphodiesterase 2A (PDE2A) inhibitors have been reported to demonstrate in vivo activity in preclinical models of cognition. To more fully explore the biology of PDE2A inhibition, we sought to identify potent PDE2A inhibitors with improved brain penetration as compared to current literature compounds. Applying estimated human dose calculations while simultaneously leveraging synthetically enabled chemistry and structure-based drug design has resulted in a highly potent, selective, brain penetrant compound 71 (PF-05085727) that effects in vivo biochemical changes commensurate with PDE2A inhibition along with behavioral and electrophysiological reversal of the effects of NMDA antagonists in rodents. This data supports the ability of PDE2A inhibitors to potentiate NMDA signaling and their further development for clinical cognition indications.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 2/antagonists & inhibitors , Drug Design , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacology , Animals , Brain/drug effects , Brain/metabolism , Crystallography, X-Ray , Cyclic Nucleotide Phosphodiesterases, Type 2/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 2/metabolism , Dogs , Haplorhini , Humans , Mice , Molecular Docking Simulation , Phosphodiesterase Inhibitors/administration & dosage , Phosphodiesterase Inhibitors/pharmacokinetics , Rats
11.
ACS Chem Biol ; 11(9): 2529-40, 2016 09 16.
Article in English | MEDLINE | ID: mdl-27391855

ABSTRACT

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.


Subject(s)
Hydrolases/metabolism , Lysophospholipase/antagonists & inhibitors , Serine/metabolism , Animals , Crystallization , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Humans , Lysophospholipase/chemistry
12.
Protein Sci ; 24(1): 20-6, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25287857

ABSTRACT

Undecaprenyl pyrophosphate synthase (UPPs) is an essential enzyme in a key bacterial cell wall synthesis pathway. It catalyzes the consecutive condensations of isopentenyl pyrophosphate (IPP) groups on to a trans-farnesyl pyrophosphate (FPP) to produce a C55 isoprenoid, undecaprenyl pyrophosphate (UPP). Here we report the discovery and co-crystal structures of a drug-like UPPs inhibitor in complex with Streptococcus pneumoniae UPPs, with and without substrate FPP, at resolutions of 2.2 and 2.1 Å, respectively. The UPPs inhibitor has a low molecular weight (355 Da), but displays potent inhibition of UPP synthesis in vitro (IC50 50 nM) that translates into excellent whole cell antimicrobial activity against pathogenic strains of Streptococcal species (MIC90 0.4 µg mL(-1) ). Interestingly, the inhibitor does not compete with the substrates but rather binds at a site adjacent to the FPP binding site and interacts with the tail of the substrate. Based on the structures, an allosteric inhibition mechanism of UPPs is proposed for this inhibitor. This inhibition mechanism is supported by biochemical and biophysical experiments, and provides a basis for the development of novel antibiotics targeting Streptococcus pneumoniae.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/enzymology , Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/metabolism , Allosteric Regulation/drug effects , Anti-Bacterial Agents/chemistry , Crystallography, X-Ray , Drug Discovery , Enzyme Inhibitors/chemistry , Humans , Molecular Docking Simulation , Pneumococcal Infections/drug therapy , Pneumococcal Infections/microbiology , Polyisoprenyl Phosphates/metabolism , Sesquiterpenes/metabolism , Streptococcus pneumoniae/chemistry , Streptococcus pneumoniae/metabolism , Transferases/chemistry , Transferases/metabolism
13.
J Med Chem ; 54(13): 4536-47, 2011 Jul 14.
Article in English | MEDLINE | ID: mdl-21650160

ABSTRACT

Utilizing structure-based virtual library design and scoring, a novel chimeric series of phosphodiesterase 10A (PDE10A) inhibitors was discovered by synergizing binding site interactions and ADME properties of two chemotypes. Virtual libraries were docked and scored for potential binding ability, followed by visual inspection to prioritize analogs for parallel and directed synthesis. The process yielded highly potent and selective compounds such as 16. New X-ray cocrystal structures enabled rational design of substituents that resulted in the successful optimization of physical properties to produce in vivo activity and to modulate microsomal clearance and permeability.


Subject(s)
Antipsychotic Agents/chemical synthesis , Phosphodiesterase Inhibitors/chemical synthesis , Phosphoric Diester Hydrolases/metabolism , Schizophrenia/drug therapy , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Antipsychotic Agents/pharmacokinetics , Antipsychotic Agents/pharmacology , Avoidance Learning/drug effects , Binding Sites , Blood-Brain Barrier/metabolism , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Crystallography, X-Ray , Cyclic GMP/metabolism , Databases, Factual , Drug Design , Humans , In Vitro Techniques , Mice , Mice, Knockout , Microsomes, Liver/metabolism , Models, Molecular , Permeability , Phosphodiesterase Inhibitors/pharmacokinetics , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/genetics , Protein Conformation , Structure-Activity Relationship
14.
Proc Natl Acad Sci U S A ; 106(43): 18225-30, 2009 Oct 27.
Article in English | MEDLINE | ID: mdl-19828435

ABSTRACT

We report the X-ray crystal structure of a phosphodiesterase (PDE) that includes both catalytic and regulatory domains. PDE2A (215-900) crystallized as a dimer in which each subunit had an extended organization of regulatory GAF-A and GAF-B and catalytic domains connected by long alpha-helices. The subunits cross at the GAF-B/catalytic domain linker, and each side of the dimer contains in series the GAF-A and GAF-B of one subunit and the catalytic domain of the other subunit. A dimer interface extends over the entire length of the molecule. The substrate binding pocket of each catalytic domain is occluded by the H-loop. We deduced from comparisons with structures of isolated, ligand-bound catalytic subunits that the H-loop swings out to allow substrate access. However, in dimeric PDE2A (215-900), the H-loops of the two catalytic subunits pack against each other at the dimer interface, necessitating movement of the catalytic subunits to allow for H-loop movement. Comparison of the unliganded GAF-B of PDE2A (215-900) with previous structures of isolated, cGMP-bound GAF domains indicates that cGMP binding induces a significant shift in the GAF-B/catalytic domain linker. We propose that cGMP binding to GAF-B causes movement, through this linker region, of the catalytic domains, such that the H-loops no longer pack at the dimer interface and are, instead, free to swing out to allow substrate access. This increase in substrate access is proposed as the basis for PDE2A activation by cGMP and may be a general mechanism for regulation of all PDEs.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 2/chemistry , Allosteric Regulation , Animals , Biocatalysis , Cell Line , Crystallography, X-Ray , Cyclic Nucleotide Phosphodiesterases, Type 2/metabolism , Enzyme Activation , Humans , Models, Molecular , Protein Multimerization , Protein Structure, Quaternary , Structural Homology, Protein
15.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 8): 875-9, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19622872

ABSTRACT

Bacterial persistence is the ability of individual cells to randomly enter a period of dormancy during which the cells are protected against antibiotics. In Escherichia coli, persistence is regulated by the activity of a protein kinase HipA and its DNA-binding partner HipB, which is a strong inhibitor of both HipA activity and hip operon transcription. The crystal structure of the HipBA complex was solved by application of the SAD technique to a mercury derivative. In this article, the fortuitous and interesting effect of mercury soaks on the native HipBA crystals is discussed as well as the intriguing tryptophan-binding pocket found on the HipA surface. A HipA-regulation model is also proposed that is consistent with the available structural and biochemical data.


Subject(s)
DNA-Binding Proteins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Protein Kinase Inhibitors/chemistry , Anti-Bacterial Agents/therapeutic use , Binding Sites , Crystallization , Crystallography, X-Ray , DNA-Binding Proteins/metabolism , Drug Resistance, Bacterial , Escherichia coli Infections/drug therapy , Escherichia coli Infections/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Genes, Switch/genetics , Humans , Mercury/metabolism , Operon , Protein Conformation , Protein Kinase Inhibitors/metabolism , Structure-Activity Relationship , Tryptophan/metabolism
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