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1.
Nat Commun ; 12(1): 6685, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34795227

RESUMO

Phosphoproteomics integrating data-independent acquisition (DIA) enables deep phosphoproteome profiling with improved quantification reproducibility and accuracy compared to data-dependent acquisition (DDA)-based phosphoproteomics. DIA data mining heavily relies on a spectral library that in most cases is built on DDA analysis of the same sample. Construction of this project-specific DDA library impairs the analytical throughput, limits the proteome coverage, and increases the sample size for DIA phosphoproteomics. Herein we introduce a deep neural network, DeepPhospho, which conceptually differs from previous deep learning models to achieve accurate predictions of LC-MS/MS data for phosphopeptides. By leveraging in silico libraries generated by DeepPhospho, we establish a DIA workflow for phosphoproteome profiling which involves DIA data acquisition and data mining with DeepPhospho predicted libraries, thus circumventing the need of DDA library construction. Our DeepPhospho-empowered workflow substantially expands the phosphoproteome coverage while maintaining high quantification performance, which leads to the discovery of more signaling pathways and regulated kinases in an EGF signaling study than the DDA library-based approach. DeepPhospho is provided as a web server as well as an offline app to facilitate user access to model training, predictions and library generation.

2.
Sci Adv ; 7(30)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34290087

RESUMO

Transmembrane proteins play vital roles in mediating synaptic transmission, plasticity, and homeostasis in the brain. However, these proteins, especially the G protein-coupled receptors (GPCRs), are underrepresented in most large-scale proteomic surveys. Here, we present a new proteomic approach aided by deep learning models for comprehensive profiling of transmembrane protein families in multiple mouse brain regions. Our multiregional proteome profiling highlights the considerable discrepancy between messenger RNA and protein distribution, especially for region-enriched GPCRs, and predicts an endogenous GPCR interaction network in the brain. Furthermore, our new approach reveals the transmembrane proteome remodeling landscape in the brain of a mouse depression model, which led to the identification of two previously unknown GPCR regulators of depressive-like behaviors. Our study provides an enabling technology and rich data resource to expand the understanding of transmembrane proteome organization and dynamics in the brain and accelerate the discovery of potential therapeutic targets for depression treatment.

3.
Biomolecules ; 11(7)2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201418

RESUMO

Allosteric modulators have emerged with many potential pharmacological advantages as they do not compete the binding of agonist or antagonist to the orthosteric sites but ultimately affect downstream signaling. To identify allosteric modulators targeting an extra-helical binding site of the glucagon-like peptide-1 receptor (GLP-1R) within the membrane environment, the following two computational approaches were applied: structure-based virtual screening with consideration of lipid contacts and ligand-based virtual screening with the maintenance of specific allosteric pocket residue interactions. Verified by radiolabeled ligand binding and cAMP accumulation experiments, two negative allosteric modulators and seven positive allosteric modulators were discovered using structure-based and ligand-based virtual screening methods, respectively. The computational approach presented here could possibly be used to discover allosteric modulators of other G protein-coupled receptors.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Células CHO , Cricetinae , Cricetulus , Glucagon/administração & dosagem , Glucagon/química , Glucagon/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular/métodos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
4.
ACS Chem Biol ; 16(6): 991-1002, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34048655

RESUMO

Allosteric ligands provide new opportunities to modulate G protein-coupled receptor (GPCR) function and present therapeutic benefits over orthosteric molecules. Negative allosteric modulators (NAMs) can inhibit the activation of a receptor and downstream signal transduction. Screening NAMs for a GPCR target is particularly challenging because of the difficulty in distinguishing NAMs from antagonists bound to the orthosteric site as they both show inhibitory effects in receptor signaling assays. Here we report an affinity mass spectrometry (MS)-based approach tailored to screening potential NAMs of a GPCR target especially from fragment libraries. Compared to regular surface plasmon resonance or NMR-based methods for fragment screening, our approach features a reduction of the protein and compound consumption by 2-4 orders of magnitude and an increase in the data acquisition speed by 2-3 orders of magnitude. Our affinity MS-based fragment screening led to the identification of a new NAM of the adenosine A2A receptor (A2AAR) bearing an unprecedented azetidine moiety predicted to occupy the allosteric sodium binding site. Molecular dynamics simulations, ligand structure-activity relationship (SAR) studies, and in-solution NMR analyses further revealed the unique binding mode and antagonistic property of this compound that differs considerably from HMA (5-(N,N-hexamethylene)amiloride), a well-characterized NAM of A2AAR. Taken together, our work would facilitate fragment-based screening of allosteric modulators, as well as guide the design of novel NAMs acting at the sodium ion pocket of class A GPCRs.


Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Receptor A2A de Adenosina/metabolismo , Sódio/metabolismo , Agonistas do Receptor A2 de Adenosina/química , Antagonistas do Receptor A2 de Adenosina/química , Sítio Alostérico/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Descoberta de Drogas , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptor A2A de Adenosina/química
6.
Signal Transduct Target Ther ; 6(1): 7, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33414387

RESUMO

As one of the most successful therapeutic target families, G protein-coupled receptors (GPCRs) have experienced a transformation from random ligand screening to knowledge-driven drug design. We are eye-witnessing tremendous progresses made recently in the understanding of their structure-function relationships that facilitated drug development at an unprecedented pace. This article intends to provide a comprehensive overview of this important field to a broader readership that shares some common interests in drug discovery.

7.
ACS Chem Biol ; 15(12): 3275-3284, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33258587

RESUMO

The GPR52, a class A orphan G protein-coupled receptor (GPCR), is regarded as a promising therapeutic target for the treatment of Huntington's disease and multiple psychiatric disorders. Although the recently solved structure of GPR52 has revealed a binding mechanism likely shared by all reported agonists, the small molecule antagonist E7 cannot fit into this agonist-binding pocket, and its interaction mode with the receptor remains unknown. Here, we employed targeted proteomics and affinity mass spectrometry approaches to uncover a unique binding mode of E7 which acts as a covalent and allosteric ligand of GPR52. Among three Cys residues identified in this study to form covalent conjugates with E7, the intracellular C1564.40 makes the most significant contribution to the antagonism activity of E7. Discovery of this novel intracellular site for covalent attachment of an antagonist would facilitate the design of GPR52-selective negative allosteric modulators which could serve as potential therapeutics for treating Huntington's disease.


Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Regulação Alostérica , Humanos , Doença de Huntington/metabolismo , Ligantes , Receptores Acoplados a Proteínas G/metabolismo
8.
Nat Commun ; 11(1): 5699, 2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33177502

RESUMO

G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.


Assuntos
Encéfalo/citologia , Imagem Molecular/métodos , Neurônios/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Citoesqueleto/metabolismo , Feminino , Recuperação de Fluorescência Após Fotodegradação , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência/métodos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/análise , Receptor CB1 de Canabinoide/genética
11.
ACS Cent Sci ; 6(2): 213-225, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32123739

RESUMO

Subtype selectivity and functional bias are vital in current drug discovery for G protein-coupled receptors (GPCRs) as selective and biased ligands are expected to yield drug leads with optimal on-target benefits and minimal side-effects. However, structure-based design and medicinal chemistry exploration remain challenging in part because of highly conserved binding pockets within subfamilies. Herein, we present an affinity mass spectrometry approach for screening herbal extracts to identify active ligands of a GPCR, the 5-HT2C receptor. Using this method, we discovered a naturally occurring aporphine 1857 that displayed strong selectivity for activating 5-HT2C without activating the 5-HT2A or 5-HT2B receptors. Remarkably, this novel ligand exhibited exclusive bias toward G protein signaling for which key residues were identified, and it showed comparable in vivo efficacy for food intake suppression and weight loss as the antiobesity drug, lorcaserin. Our study establishes an efficient approach to discovering novel GPCR ligands by exploring the largely untapped chemical space of natural products.

12.
iScience ; 23(3): 100903, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32109675

RESUMO

Data-independent acquisition mass spectrometry (DIA-MS) is a powerful technique that enables relatively deep proteomic profiling with superior quantification reproducibility. DIA data mining predominantly relies on a spectral library of sufficient proteome coverage that, in most cases, is built on data-dependent acquisition-based analysis of the same sample. To expand the proteome coverage for a pre-determined protein family, we report herein on the construction of a hybrid spectral library that supplements a DIA experiment-derived library with a protein family-targeted virtual library predicted by deep learning. Leveraging this DIA hybrid library substantially deepens the coverage of three transmembrane protein families (G protein-coupled receptors, ion channels, and transporters) in mouse brain tissues with increases in protein identification of 37%-87% and peptide identification of 58%-161%. Moreover, of the 412 novel GPCR peptides exclusively identified with the DIA hybrid library strategy, 53.6% were validated as present in mouse brain tissues based on orthogonal experimental measurement.

13.
Anal Chim Acta ; 1102: 53-62, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32043996

RESUMO

Chemical cross-linking would conceivably cause structural disruption of a protein, but few cross-linkers have been fully evaluated in this aspect. Furthermore, integral membrane proteins may differ from soluble proteins in the selection of suitable cross-linkers, which has never been investigated. In this study, we systematically evaluated the impact of five conventional cross-linkers targeting Lys, Asp and Glu, and two Arg-reactive cross-linkers on the structural and functional integrity of two G protein-coupled receptors (GPCRs). Perturbation of the receptor structure and ligand-binding activity was observed, depending on the receptor and cross-linking conditions. In particular, our study demonstrated that the concentrations of PDH and KArGO need to be fine-tuned in order to minimize the structural and functional disturbance of specific GPCRs. A set of amenable cross-linkers was selected to acquire the most comprehensive cross-link maps for two GPCRs. Our in-depth cross-linking mass spectrometry (CXMS) analysis has revealed dynamic features of structural regions in GPCRs that are not observable in the crystal structures. Thus, CXMS analysis of GPCRs using the expanded toolkit would facilitate structural modeling of uncharacterized receptors and gain new insights into receptor-ligand interactions.


Assuntos
Reagentes para Ligações Cruzadas/química , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Receptores Adrenérgicos alfa 2/química , Cromatografia em Gel , Cromatografia Líquida , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica , Estabilidade Proteica , Receptores Adrenérgicos alfa 2/metabolismo , Espectrometria de Massas em Tandem/métodos
14.
ACS Chem Neurosci ; 11(4): 549-559, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31968160

RESUMO

The 5-HT2C receptor has emerged as a promising target in the treatment of a variety of central nervous system disorders. We have first identified aporphines as a new class of 5-HT2C receptor agonists. Structure-activity relationship results indicate that the aporphine core may be required for 5-HT2C receptor activity, and substitutions at its C1 position are important for 5-HT2C receptor activity. Our efforts to optimize our hit 15781 lead to the identification of the highly potent and selective 5-HT2C agonist 18b (MQ02-439) with an EC50 value of 104 nM and weak antagonism at the 5-HT2A and 5-HT2B receptors. The findings may serve as good starting points for the development of more potent and selective 5-HT2C agonists as valuable pharmacological tools or potential drug candidates.


Assuntos
Aporfinas/farmacologia , Receptor 5-HT2C de Serotonina/efeitos dos fármacos , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Ensaio Radioligante , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptor 5-HT2C de Serotonina/metabolismo , Relação Estrutura-Atividade
15.
Curr Opin Biotechnol ; 64: 24-31, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31606719

RESUMO

The recent rapid advance of systematic mapping of protein-metabolite interactions (PMIs) in both prokaryotic and eukaryotic cells has been catalyzed by development of innovative and effective proteomics or metabolomics strategies all based on large-scale mass spectrometry (MS) analysis of biomolecules. Both metabolite-centric and protein-centric approaches have been established to profile PMIs in the native cellular matrix treated by specific metabolites or proteins. Here we will review the development and application of versatile MS-based proteomics and metabolomics techniques for global PMI mapping in different species, which lead to the discovery of numerous uncharacterized PMIs that may reveal new interaction-derived functionality. We further discuss the strengths and limitations of different PMI mapping approaches as well as the key elements in MS quantification and data mining for reliable PMI identification.


Assuntos
Metabolômica , Proteômica , Mineração de Dados , Espectrometria de Massas
16.
Anal Chem ; 91(13): 8162-8169, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31094506

RESUMO

Affinity mass spectrometry (MS) enables rapid screening of compound mixtures for ligands bound to a specific protein target, yet its current throughput is limited to individually assay pools of 400-2000 compounds. Typical affinity MS screens implemented in pharmaceutical industry laboratories identify putative ligands based on qualitative analysis of compound binding to the target whereas no quantitative information is acquired to discriminate high- and low-affinity ligands in the screening phase. Furthermore, these screens require purification of a stabilized form of the protein target, which poses a great challenge for membrane receptor targets. Here, we describe a new, potentially general affinity MS strategy that allows screening of 20,000 compounds in one pool for highly efficient ligand discovery toward a G protein-coupled receptor (GPCR) target. Quantitative measurement of compound binding to the receptor enables high-affinity ligand selection using both the purified receptor and receptor-embedded cell membranes. This high-throughput, label-free and quantitative affinity MS screen resulted in discovery of three new antagonists of the A2A adenosine receptor.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Antagonistas do Receptor A2 de Adenosina/análise , Membrana Celular/metabolismo , Humanos , Ligantes , Ligação Proteica , Receptores Acoplados a Proteínas G/antagonistas & inibidores
17.
Analyst ; 144(9): 2881-2890, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30788466

RESUMO

Although natural herbs have been a rich source of compounds for drug discovery, identification of bioactive components from natural herbs suffers from low efficiency and prohibitive cost of the conventional bioassay-based screening platforms. Here we develop a new strategy that integrates virtual screening, affinity mass spectrometry (MS) and targeted metabolomics for efficient discovery of herb-derived ligands towards a specific protein target site. Herb-based virtual screening conveniently selects herbs of potential bioactivity whereas affinity MS combined with targeted metabolomics readily screens candidate compounds in a high-throughput manner. This new integrated approach was benchmarked on screening chemical ligands that target the hydrophobic pocket of the nucleoprotein (NP) of Ebola viruses for which no small molecule ligands have been reported. Seven compounds identified by this approach from the crude extracts of three natural herbs were all validated to bind to the NP target in pure ligand binding assays. Among them, three compounds isolated from Piper nigrum (HJ-1, HJ-4 and HJ-6) strongly promoted the formation of large NP oligomers and reduced the protein thermal stability. In addition, cooperative binding between these chemical ligands and an endogenous peptide ligand was observed, and molecular docking was employed to propose a possible mechanism. Taken together, we established a platform integrating in silico and experimental screening approaches for efficient discovery of herb-derived bioactive ligands especially towards non-enzyme protein targets.


Assuntos
Produtos Biológicos/metabolismo , Espectrometria de Massas/métodos , Metabolômica/métodos , Nucleoproteínas/metabolismo , Extratos Vegetais/metabolismo , Proteínas do Core Viral/metabolismo , Sítios de Ligação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Descoberta de Drogas/métodos , Ebolavirus/química , Ligantes , Simulação de Acoplamento Molecular , Proteínas do Nucleocapsídeo , Nucleoproteínas/química , Ophiopogon/química , Piper nigrum/química , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Ligação Proteica , Salvia miltiorrhiza/química , Sementes/química , Proteínas do Core Viral/química
18.
Nat Chem Biol ; 15(2): 206, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30573766

RESUMO

In the version of this article originally published, the present address for Petr Popov was incorrectly listed as 'Koltech Institute of Science & Technology, Moscow, Russia'. The correct present address is 'Skolkovo Institute of Science and Technology, Moscow, Russia'. The error has been corrected in the HTML and PDF versions of the paper.

19.
Methods Mol Biol ; 1859: 171-184, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30421229

RESUMO

Quantification of targeted metabolites, especially trace metabolites and structural isomers, in complex biological materials is an ongoing challenge for metabolomics. In this chapter, we summarize high-resolution mass spectrometry-based approaches mainly used for targeted metabolite and metabolomics analysis, and then introduce an MS1/MS2-combined PRM workflow for quantification of central carbon metabolism intermediates, amino acids, and shikimate pathway-related metabolites. Major steps in the workflow, including cell culture, metabolite extraction, LC-MS analysis and data processing, are described. Furthermore, we adapt this new approach to a dynamic 13C-labeling experiment and demonstrate its unique advantage in capturing and correcting isotopomer labeling curves to facilitate nonstationary 13C-labeling metabolism analysis.


Assuntos
Isótopos de Carbono/química , Marcação por Isótopo/métodos , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Aminoácidos/metabolismo , Isótopos de Carbono/metabolismo , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/metabolismo , Marcação por Isótopo/instrumentação , Metabolômica/instrumentação , Ácido Chiquímico/metabolismo , Espectrometria de Massas em Tandem/instrumentação
20.
Methods Mol Biol ; 1859: 301-316, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30421238

RESUMO

Dynamic or isotopically nonstationary 13C labeling experiments are a powerful tool not only for precise carbon flux quantification (e.g., metabolic flux analysis of photoautotrophic organisms) but also for the investigation of pathway bottlenecks, a cell's phenotype, and metabolite channeling. In general, isotopically nonstationary metabolic flux analysis requires three main components: (1) transient isotopic labeling experiments; (2) metabolite quenching and isotopomer analysis using LC-MS; (3) metabolic network construction and flux quantification. Labeling dynamics of key metabolites from 13C-pulse experiments allow flux estimation of key central pathways by solving ordinary differential equations to fit time-dependent isotopomer distribution data. Additionally, it is important to provide biomass requirements, carbon uptake rates, specific growth rates, and carbon excretion rates to properly and precisely balance the metabolic network. Labeling dynamics through cascade metabolites may also identify channeling phenomena in which metabolites are passed between enzymes without mixing with the bulk phase. In this chapter, we outline experimental protocols to probe metabolic pathways through dynamic labeling. We describe protocols for labeling experiments, metabolite quenching and extraction, LC-MS analysis, computational flux quantification, and metabolite channeling observations.


Assuntos
Marcação por Isótopo/métodos , Análise do Fluxo Metabólico/métodos , Metabolômica/métodos , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/química , Escherichia coli/metabolismo , Marcação por Isótopo/instrumentação , Análise do Fluxo Metabólico/instrumentação , Redes e Vias Metabólicas , Metaboloma , Metabolômica/instrumentação , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
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