RESUMO
Glycans decorate all cells and are critical mediators of cellular processes through recognition by glycan-binding proteins (GBPs). While targeting glycan-protein interactions has great therapeutic potential, these interactions are challenging to study as they are generally transient and exhibit low binding affinities. Glycan-based photo-crosslinkable probes have enabled covalent capture and identification of unknown GBP receptors and glycoconjugate ligands. Here, we review recent progress in photo-crosslinking approaches targeting glycan-mediated interactions. We discuss two prominent emerging strategies: 1) development of photo-crosslinkable oligosaccharide ligands to identify GBP receptors; and 2) cell-surface glyco-engineering to identify glycoconjugate ligands of GBPs. Overall, photoaffinity labeling affords valuable insights into complex glycan-protein networks and is poised to help elucidate the glycan-protein interactome, providing novel targets for therapeutic intervention.
Assuntos
Marcadores de Fotoafinidade , Polissacarídeos , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/metabolismo , Polissacarídeos/metabolismo , Polissacarídeos/química , Humanos , Ligação Proteica , Proteínas/metabolismo , Proteínas/química , Ligantes , Animais , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismoRESUMO
Lipid-protein interactions serve as the basis for many of the diverse roles of lipids. However, these noncovalent binding events are often weak, transient, or dependent upon environmental cues. Photoaffinity labeling can preserve these interactions under native conditions, enabling their biochemical profiling. Typically, photoaffinity labeling probes contain a diazirine photocrosslinker and a click chemistry handle for enrichment and downstream analysis. In this review, we summarize recent advances in the understanding the mechanisms of diazirine photocrosslinking, and we provide an overview of recent applications of photoaffinity labeling to reveal the interactions of diverse types of lipids with specific members of the proteome.
Assuntos
Diazometano , Marcadores de Fotoafinidade , Química Click , Lipídeos , Marcadores de Fotoafinidade/metabolismoRESUMO
Minimalist photo-reactive probes, which consist of a photo-reactive group and a tag for detection of target proteins, are useful tools in chemical biology. Although several diazirine-based and aryl azide-based minimalist probes are available, no keto-based minimalist probe has yet been reported. Here we describe minimalist probes based on a 2-thienyl-substituted α-ketoamide bearing an alkyne group on the thiophene ring. The 3-alkyne probe showed the highest photo-affinity labeling efficiency.
Assuntos
Azidas , Marcadores de Fotoafinidade , Marcadores de Afinidade , Alcinos , Marcadores de Fotoafinidade/metabolismo , ProteínasRESUMO
The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.
Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Proteômica , Trifosfato de Adenosina/metabolismo , Fosfatos de Dinucleosídeos/química , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , L-Lactato Desidrogenase/metabolismo , Fosfoglicerato Quinase/metabolismo , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/metabolismo , Ligação Proteica , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.
Assuntos
Colesterol/análogos & derivados , Diazometano/análogos & derivados , Canais Iônicos de Abertura Ativada por Ligante/química , Marcadores de Fotoafinidade/química , Alcinos/síntese química , Alcinos/química , Alcinos/metabolismo , Sítios de Ligação , Colesterol/síntese química , Colesterol/metabolismo , Cianobactérias/química , Diazometano/síntese química , Diazometano/metabolismo , Corantes Fluorescentes/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/metabolismo , Ligação ProteicaRESUMO
Baculiferins are a group of marine sponge-derived polycyclic alkaloids with anti-HIV (human immunodeficiency virus) activities. To identify additional baculiferin-based congeners for SAR analysis and to investigate the mode of action, a total of 18 new baculiferin-type derivatives were synthesized. The inhibitory activities of the congeners against the HIV-1 virus were evaluated in vitro, and the relevant SAR was discussed. Compound 18 exerted the most potent activity toward VSV-G-pseudotyped HIV-1 (IC50 of 3.44 µM) and HIV-1 strain SF33 (IC50 of 2.80 µM) in vitro. To identify the cellular targets, three photoaffinity baculiferin probes were simultaneously synthesized. Photoaffinity labeling experiments together with LC-MS/MS data identified aspartate-tRNA ligase (DARS) as a putative target protein of 18. The overexpression and knockdown of DARS in HEK293T cells provided additional data to demonstrate that DARS is a potential target protein in the regulation of HIV virus infection. The modes of antiviral baculiferins 13 and 18 binding to DARS were determined by a molecular docking simulation. Thus, baculiferin 18 is considered a promising lead as a new molecular target for the development of anti-HIV agents.
Assuntos
Alcaloides/farmacologia , Fármacos Anti-HIV/farmacologia , Aspartato-tRNA Ligase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , HIV-1/efeitos dos fármacos , Alcaloides/síntese química , Alcaloides/metabolismo , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/metabolismo , Aspartato-tRNA Ligase/química , Aspartato-tRNA Ligase/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/metabolismo , Marcadores de Fotoafinidade/farmacologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Settlement and metamorphosis of planktonic larvae into benthic adults are critical components of a diverse range of marine invertebrate-mediated processes such as the formation of mussel beds and coral reefs, the recruitment of marine shellfisheries, and the initiation of macrobiofouling. Although larval settlement and metamorphosis induced by natural chemical cues is widespread among marine invertebrates, the mechanisms of action remain poorly understood. Here, we identified that the molecular target of adenosine (an inducer of larval settlement and metamorphosis from conspecific adults in the invasive biofouling mussel Mytilopsis sallei) is adenosine kinase (ADK). The results of transcriptomic analyses, pharmacological assays, temporal and spatial gene expression analyses, and siRNA interference, suggest that ATP-dependent phosphorylation of adenosine catalyzed by ADK activates the downstream AMPK-FoxO signaling pathway, inducing larval settlement and metamorphosis in M. sallei. This study not only reveals the role of the ADK-AMPK-FoxO pathway in larval settlement and metamorphosis of marine invertebrates but it also deepens our understanding of the functions and evolution of adenosine signaling, a process that is widespread in biology and important in medicine.
Assuntos
Adenosina/análogos & derivados , Adenosina/farmacologia , Bivalves/efeitos dos fármacos , Larva/efeitos dos fármacos , Metamorfose Biológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Adenosina/metabolismo , Adenosina Quinase/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição Forkhead/metabolismo , Marcadores de Fotoafinidade/metabolismo , Marcadores de Fotoafinidade/farmacologia , Transcriptoma/efeitos dos fármacosRESUMO
Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice variants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate the analgesic action of novel opioid drugs such as 3'-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that IBNtxA is a potent analgesic effective in a spectrum of pain models but lacks many side-effects associated with traditional opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efficiently radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously, as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specific ligand-protein contacts and its signaling complexes.
Assuntos
Analgésicos Opioides/metabolismo , Azidas/metabolismo , Encéfalo/metabolismo , Naltrexona/análogos & derivados , Marcadores de Fotoafinidade/metabolismo , Receptores Opioides/metabolismo , Analgésicos Opioides/síntese química , Animais , Azidas/síntese química , Encéfalo/efeitos dos fármacos , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Naltrexona/síntese química , Naltrexona/metabolismo , Marcadores de Fotoafinidade/síntese química , Ligação Proteica/fisiologia , Ensaio Radioligante/métodosRESUMO
RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here, we present our efforts to develop such a platform using photoaffinity labeling. This technique, termed Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), enables the rapid identification of small molecule binding locations within their RNA targets and can provide information on ligand selectivity across multiple different RNAs. These data, when supplemented with small molecule SAR data and RNA probing data enable the construction of a computational model of the RNA-ligand structure, thereby enabling the rational design of novel RNA-targeted ligands.
Assuntos
Azidas/química , Diazometano/análogos & derivados , Marcadores de Fotoafinidade/química , RNA/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Azidas/metabolismo , Azidas/efeitos da radiação , Sítios de Ligação , Diazometano/metabolismo , Diazometano/efeitos da radiação , Ligantes , Simulação de Acoplamento Molecular , Marcadores de Fotoafinidade/metabolismo , Marcadores de Fotoafinidade/efeitos da radiação , Estudo de Prova de Conceito , RNA/química , Transcrição Reversa , Análise de Sequência de DNARESUMO
Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1ß3 and α1ß3γ2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the ß subunit M4 (ß3Pro-415, ß3Leu-417, and ß3Thr-418) and M3 (ß3Arg-309) helices located at the base of a pocket in the ß+-α- subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3ß-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAAR ß+-α- subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.
Assuntos
Receptores de GABA-A/metabolismo , Esteroides/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Modelos Moleculares , Marcadores de Fotoafinidade/análise , Marcadores de Fotoafinidade/metabolismo , Pregnanolona/análise , Pregnanolona/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-A/química , Esteroides/químicaRESUMO
Linking neural circuitry to behavior by mapping active neurons in vivo is a challenge. Both genetically encoded calcium indicators (GECIs) and intermediate early genes (IEGs) have been used to pinpoint active neurons during a stimulus or behavior but have drawbacks such as limiting the movement of the organism, requiring a priori knowledge of the active region or having poor temporal resolution. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) was engineered to overcome these spatial-temporal challenges. CaMPARI is a photoconvertible protein that only converts from green to red fluorescence in the presence of high calcium concentration and 405 nm light. This allows the experimenter to precisely mark active neurons within defined temporal windows. The photoconversion can then be quantified by taking the ratio of the red fluorescence to the green. CaMPARI promises the ability to trace active neurons during a specific stimulus; however, CaMPARI's uses in adult Drosophila have been limited to photoconversion during fly immobilization. Here, we demonstrate a method that allows photoconversion of multiple freely-moving intact adult flies during a stimulus. Flies were placed in a dish with filter paper wet with acetic acid (pH = 2) or neutralized acetic acid (pH = 7) and exposed to photoconvertible light (60 mW) for 30 min (500 ms on, 200 ms off). Immediately following photoconversion, whole flies were fixed and imaged by confocal microscopy. The red:green ratio was quantified for the DC4 glomerulus, a bundle of neurons expressing Ir64a, an ionotropic receptor that senses acids in the Drosophila antennal lobe. Flies exposed to acetic acid showed 1.3-fold greater photoconversion than flies exposed to neutralized acetic acid. This finding was recapitulated using a more physiological stimulus of apple cider vinegar. These results indicate that CaMPARI can be used to label neurons in intact, freely-moving adult flies and will be useful for identifying the circuitry underlying complex behaviors.
Assuntos
Cálcio/metabolismo , Corantes Fluorescentes/metabolismo , Locomoção/fisiologia , Neurônios/metabolismo , Fatores Etários , Animais , Animais Geneticamente Modificados , Cálcio/análise , Drosophila melanogaster , Corantes Fluorescentes/análise , Neurônios/química , Marcadores de Fotoafinidade/análise , Marcadores de Fotoafinidade/metabolismo , Coloração e Rotulagem/métodosRESUMO
The critical roles played by GABAA receptors as inhibitory regulators of excitation in the central nervous system has been known for many years. Aberrant GABAA receptor function and trafficking deficits have also been associated with several diseases including anxiety, depression, epilepsy, and insomnia. As a consequence, important drug groups such as the benzodiazepines, barbiturates, and many general anesthetics have become established as modulators of GABAA receptor activity. Nevertheless, there is much we do not understand about the roles and mechanisms of GABAA receptors at neural network and systems levels. It is therefore crucial to develop novel technologies and especially chemical entities that can interrogate GABAA receptor function in the nervous system. Here, we describe the chemistry and characterization of a novel set of 4-PIOL and 4-PHP analogues synthesized with the aim of developing a toolkit of drugs that can photoinactivate GABAA receptors. Most of these new analogues show higher affinities/potencies compared with the respective lead compounds. This is indicative of cavernous areas being present near their binding sites that can be potentially associated with novel receptor interactions. The 4-PHP azide-analogue, 2d, possesses particularly impressive nanomolar affinity/potency and is an effective UV-inducible photoinhibitor of GABAA receptors with considerable potential for photocontrol of GABAA receptor function in situ.
Assuntos
Isoxazóis/metabolismo , Marcadores de Fotoafinidade/metabolismo , Piperidinas/metabolismo , Receptores de GABA-A/metabolismo , Células HEK293 , Humanos , Isoxazóis/análise , Marcadores de Fotoafinidade/análise , Piperidinas/análise , Estrutura Secundária de Proteína , Receptores de GABA-A/análise , Receptores de GABA-A/químicaRESUMO
Agonists at the α2 adrenergic receptor produce sedation, increase focus, provide analgesia, and induce centrally mediated hypotension and bradycardia, yet neither their dynamic interactions with adrenergic receptors nor their modulation of neuronal circuit activity is completely understood. Photoaffinity ligands of α2 adrenergic agonists have the potential both to capture discrete moments of ligand-receptor interactions and to prolong naturalistic drug effects in discrete regions of tissue in vivo. We present here the synthesis and characterization of a novel α2 adrenergic agonist photolabel based on the imidazole medetomidine called azi-medetomidine. Azi-medetomidine shares protein association characteristics with its parent compound in experimental model systems and by molecular dynamics simulation of interactions with the α2A adrenergic receptor. Azi-medetomidine acts as an agonist at α2A adrenergic receptors, and produces hypnosis in Xenopus laevis tadpoles. Azi-medetomidine competes with the α2 agonist clonidine at α2A adrenergic receptors, which is potentiated by photolabeling, and azi-medetomidine labels moieties on the α2A adrenergic receptor as determined by mass spectrometry in a manner consistent with a simulated model. This novel α2 adrenergic agonist photolabel can serve as a powerful tool for in vitro and in vivo investigations of adrenergic signaling.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/síntese química , Agonistas de Receptores Adrenérgicos alfa 2/metabolismo , Medetomidina/síntese química , Medetomidina/metabolismo , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/metabolismo , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Humanos , Ligantes , Estrutura Secundária de Proteína , Receptores Adrenérgicos alfa 2/metabolismo , Xenopus laevisRESUMO
Crenolanib (CP-868,596), a potent inhibitor of FLT3 and PDGFRα/ß, is currently under phaseâ III clinical investigation for the treatment of acute myeloid leukemia. However, the protein targets of Crenolanib in cancer cells remain obscure, which results in difficulties in understanding the mechanism of actions and side effects. To alleviate this issue, in this study, a photoaffinity probe and two fluorescent probes were created based on Crenolanib, followed by competitive protein profiling and bioimaging studies, with the aim of characterizing the cellular targets. A series of unknown protein hits, such as MAPK1, SHMT2, SLC25A11, and HIGD1A, were successfully identified by means of pull-down/LC-MS/MS; these might provide valuable clues for understanding drug action and potential toxicities. Moreover, the fluorescent probes are suitable for imaging drug distribution at the single-cell level.
Assuntos
Benzimidazóis/farmacologia , Corantes Fluorescentes/farmacologia , Marcadores de Fotoafinidade/farmacologia , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Microscopia de Fluorescência/métodos , Simulação de Acoplamento Molecular , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/metabolismo , Piperidinas/síntese química , Piperidinas/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
A novel synthetic approach is used to prepare a diverse set of "first-in-class" dihydropyridine-based TGFß receptor degraders bearing photoaffinity labels. These probes serve as valuable tools to study TGFß receptor fates and dynamics - an important challenge in chemical biology.
Assuntos
Marcadores de Fotoafinidade/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Alcinos/química , Azirinas/química , Desenho de Fármacos , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Marcadores de Fotoafinidade/químicaRESUMO
Cholesterol is an essential component of cell membranes, and is required for mammalian pentameric ligand-gated ion channel (pLGIC) function. Computational studies suggest direct interactions between cholesterol and pLGICs but experimental evidence identifying specific binding sites is limited. In this study, we mapped cholesterol binding to Gloeobacter ligand-gated ion channel (GLIC), a model pLGIC chosen for its high level of expression, existing crystal structure, and previous use as a prototypic pLGIC. Using two cholesterol analogue photolabeling reagents with the photoreactive moiety on opposite ends of the sterol, we identified two cholesterol binding sites: an intersubunit site between TM3 and TM1 of adjacent subunits and an intrasubunit site between TM1 and TM4. In both the inter- and intrasubunit sites, cholesterol is oriented such that the 3OH group points toward the center of the transmembrane domains rather than toward either the cytosolic or extracellular surfaces. We then compared this binding to that of the cholesterol metabolite, allopregnanolone, a neurosteroid that allosterically modulates pLGICs. The same binding pockets were identified for allopregnanolone and cholesterol, but the binding orientation of the two ligands was markedly different, with the 3OH group of allopregnanolone pointing to the intra- and extracellular termini of the transmembrane domains rather than to their centers. We also found that cholesterol increases, whereas allopregnanolone decreases the thermal stability of GLIC. These data indicate that cholesterol and neurosteroids bind to common hydrophobic pockets in the model pLGIC, GLIC, but that their effects depend on the orientation and specific molecular interactions unique to each sterol.
Assuntos
Colesterol/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/fisiologia , Neurotransmissores/metabolismo , Sítios de Ligação/fisiologia , Membrana Celular/metabolismo , Colesterol/fisiologia , Cianobactérias/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Ligantes , Modelos Moleculares , Neurotransmissores/fisiologia , Marcadores de Fotoafinidade/metabolismo , Pregnanolona/metabolismo , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologiaRESUMO
AXL has been defined as a novel target for cancer therapeutics. However, only a few potent and selective inhibitors targeting AXL are available to date. Recently, our group has developed a lead compound, 9im, capable of displaying potent and specific inhibition of AXL. To further identify the cellular on/off targets, in this study, competitive affinity-based proteome profiling was carried out, leading to the discovery of several unknown cellular targets such as BCAP31, LPCAT3, POR, TM9SF3, SCCPDH and CANX. In addition, trans-cyclooctene (TCO) and acedan-containing probes were developed to image the binding between 9im and its target proteins inside live cells and tumor tissues. These probes would be useful tools in the detection of AXL in various biosystems.
Assuntos
Corantes Fluorescentes/metabolismo , Marcadores de Fotoafinidade/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Quinolonas/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Química Click , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/efeitos da radiação , Humanos , Masculino , Camundongos Endogâmicos ICR , Microscopia de Fluorescência , Simulação de Acoplamento Molecular , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/efeitos da radiação , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/efeitos da radiação , Proteoma/química , Proteínas Proto-Oncogênicas/química , Quinolonas/química , Quinolonas/farmacologia , Quinolonas/efeitos da radiação , Receptores Proteína Tirosina Quinases/química , Raios Ultravioleta , Receptor Tirosina Quinase AxlRESUMO
Nocistatin (NST) is a neuropeptide produced from the same precursor protein of opioid peptide nociceptin/orphanin FQ, and it is involved in a broad range of central functions including pain transmission in the nervous system. However, the composition and structure of the receptor(s) for NST remain unclear. Here, we developed NST photoaffinity probe to identify NST receptor. The NST photoaffinity probe contains an azide moiety for the tagging of the binding protein as well as biotin for protein detection. Intrathecal administration of a NST photoaffinity probe, biotin-(AC5)2-[Y6,azF14]bNST, inhibited the nociceptin/orphanin FQ-evoked tactile pain allodynia in a manner similar to that of NST. The biotin-(AC5)2-[Y6,azF14]bNST-binding proteins were primarily localized in the gray matter of the spinal cord. After photo-crosslinking of the protein complex with biotin-(AC5)2-[Y6,azF14]bNST, two dominant binding protein bands were observed at 58 and 64â¯kDa. Thus, biotin-(AC5)2-[Y6,azF14]bNST has pharmacological activity and is useful for characterizing the NST receptor.
Assuntos
Peptídeos Opioides/análise , Marcadores de Fotoafinidade/química , Medula Espinal/química , Animais , Biotinilação , Hiperalgesia/metabolismo , Masculino , Camundongos , Peptídeos Opioides/metabolismo , Marcadores de Fotoafinidade/metabolismo , Ligação Proteica , Medula Espinal/metabolismoRESUMO
Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding "G site" and a substrate binding "H site". Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the "G site", and (2) an ABP designed to mimic a substrate molecule and have "H site" activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and "G" and "H" site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.
Assuntos
Glutationa Transferase/metabolismo , Marcadores de Fotoafinidade/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Glutationa/metabolismo , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Fígado/enzimologia , Pulmão/enzimologia , Camundongos , Marcadores de Fotoafinidade/química , Ligação ProteicaRESUMO
Posttranslational modifications (PTMs) of lysine are crucial histone marks that regulate diverse biological processes. The functional roles and regulation mechanism of many newly identified lysine PTMs, however, remain yet to be understood. Here we report a photoaffinity crotonyl lysine (Kcr) analogue that can be genetically and site-specifically incorporated into histone proteins. This, in conjunction with the genetically encoded photo-lysine as a "control probe", enables the capture and identification of enzymatic machinery and/or effector proteins for histone lysine crotonylation.