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1.
Cell ; 187(10): 2536-2556.e30, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38653237

RESUMO

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.


Assuntos
Cisteína , Neoplasias , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Cisteína/metabolismo , Cisteína/química , Ligantes , Melanoma/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Oxirredução , Transdução de Sinais , Fatores de Transcrição SOXE/química , Fatores de Transcrição SOXE/metabolismo
2.
J Am Chem Soc ; 137(35): 11461-75, 2015 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-26270632

RESUMO

Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.


Assuntos
Hidrolases/metabolismo , Espaço Intracelular/metabolismo , Imagem Molecular , Sondas Moleculares/metabolismo , Alcenos/química , Alcinos/química , Azidas/química , Compostos de Boro/química , Sobrevivência Celular , Reação de Cicloadição , Ciclo-Octanos/química , Fluoresceína/química , Corantes Fluorescentes/química , Células HeLa , Humanos , Hidrolases/química , Hidrolases/genética , Ligantes , Modelos Moleculares , Sondas Moleculares/química , Sondas Moleculares/genética , Conformação Proteica , Engenharia de Proteínas
3.
Bioorg Med Chem Lett ; 25(4): 908-13, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25582600

RESUMO

Herein we describe design strategies that led to the discovery of novel pyridopyrazine-1,6-dione γ-secretase modulators (GSMs) incorporating an indole motif as a heterocyclic replacement for a naphthyl moiety that was present in the original lead 9. Tactics involving parallel medicinal chemistry and in situ monomer synthesis to prepare focused libraries are discussed. Optimized indole GSM 29 exhibited good alignment of in vitro potency and physicochemical properties, and moderate reduction of brain Aß42 was achieved in a rat efficacy model when dosed orally at 30mg/kg. Labeling experiments using a clickable, indole-derived GSM photoaffinity probe demonstrated that this series binds to the presenilin N-terminal fragment (PS1-NTF) of the γ-secretase complex.


Assuntos
Secretases da Proteína Precursora do Amiloide/efeitos dos fármacos , Descoberta de Drogas , Indóis/farmacologia , Presenilinas/efeitos dos fármacos , Pirazinas/química , Animais , Indóis/química , Ratos
4.
J Biol Chem ; 288(14): 9710-9720, 2013 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-23396974

RESUMO

γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic ß-amyloid peptides (i.e. Aß42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aß42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilinas/química , Doença de Alzheimer/metabolismo , Proteínas Amiloidogênicas/química , Animais , Sítios de Ligação , Domínio Catalítico , Sistema Livre de Células , Células HeLa , Humanos , Concentração Inibidora 50 , Luz , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Marcadores de Fotoafinidade/farmacologia , Fotoquímica/métodos , Ligação Proteica , Proteolipídeos/metabolismo , Ratos , Ratos Sprague-Dawley
5.
bioRxiv ; 2023 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-37961514

RESUMO

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap , an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFκB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.

6.
J Med Chem ; 65(21): 14326-14336, 2022 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-36314537

RESUMO

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.


Assuntos
Artrite Reumatoide , Doenças Autoimunes , Lúpus Eritematoso Sistêmico , Humanos , Tirosina Quinase da Agamaglobulinemia , Inibidores de Proteínas Quinases/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Lúpus Eritematoso Sistêmico/tratamento farmacológico
7.
ACS Med Chem Lett ; 12(5): 782-790, 2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34055226

RESUMO

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that plays a critical role in the activation of B cells, macrophages, and osteoclasts. Given the key role of these cell types in the pathology of autoimmune disorders, BTK inhibitors have the potential to improve treatment outcomes in multiple diseases. Herein, we report the discovery and characterization of a novel potent and selective covalent 4-oxo-4,5-dihydro-3H-1-thia-3,5,8-triazaacenaphthylene-2-carboxamide BTK inhibitor chemotype. Compound 27 irreversibly inhibits BTK by targeting a noncatalytic cysteine residue (Cys481) for covalent bond formation. Compound 27 is characterized by selectivity for BTK, potent in vivo BTK occupancy that is sustained after it is cleared from systemic circulation, and dose-dependent efficacy at reducing joint inflammation in a rat collagen-induced arthritis model.

8.
Biochemistry ; 48(30): 7261-70, 2009 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-19527073

RESUMO

Fucose-alpha(1-2)-galactose [Fucalpha(1-2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, and memory. However, an understanding of their precise roles has been hampered by a lack of information regarding Fucalpha(1-2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative Fucalpha(1-2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that Fucalpha(1-2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a "glycocode" to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of Fucalpha(1-2)Gal sugars in the OB is regulated by the alpha(1-2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of Fucalpha(1-2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes.


Assuntos
Dissacarídeos/análise , Epitopos , Plasticidade Neuronal/fisiologia , Bulbo Olfatório , Proteoma , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Cromatografia de Afinidade , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lectinas/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bulbo Olfatório/química , Bulbo Olfatório/citologia , Bulbo Olfatório/fisiologia , Ratos , Distribuição Tecidual , Galactosídeo 2-alfa-L-Fucosiltransferase
9.
ACS Chem Neurosci ; 7(8): 1166-73, 2016 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-27253220

RESUMO

γ-Secretase is a multiprotein complex that catalyzes intramembrane proteolysis associated with Alzheimer's disease and cancer. Here, we have developed potent sulfonamide clickable photoaffinity probes that target γ-secretase in vitro and in cells by incorporating various photoreactive groups and walking the clickable alkyne handle to different positions around the molecule. We found that benzophenone is preferred over diazirine as a photoreactive group within the sulfonamide scaffold for labeling γ-secretase. Intriguingly, the placement of the alkyne at different positions has little effect on probe potency but has a significant impact on the efficiency of labeling of γ-secretase. Moreover, the optimized clickable photoprobe, 163-BP3, was utilized as a cellular probe to effectively assess the target engagement of inhibitors with γ-secretase in primary neuronal cells. In addition, biotinylated 163-BP3 probes were developed and used to capture the native γ-secretase complex in the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) solubilized state. Taken together, these next generation clickable and biotinylated sulfonamide probes offer new tools to study γ-secretase in biochemical and cellular systems. Finally, the data provide insights into structural features of the sulfonamide inhibitor binding site in relation to the active site and into the design of clickable photoaffinity probes.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Domínio Catalítico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Sulfonamidas/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Benzofenonas/metabolismo , Sítios de Ligação/efeitos dos fármacos , Biotinilação , Células Cultivadas , Córtex Cerebral/citologia , Ácidos Cólicos/metabolismo , Diazometano/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/metabolismo , Marcadores de Fotoafinidade , Presenilina-1/metabolismo , Especificidade por Substrato
11.
Proc Natl Acad Sci U S A ; 103(1): 21-6, 2006 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-16373512

RESUMO

Although fucose-alpha(1-2)-galactose [Fucalpha(1-2)Gal] carbohydrates have been implicated in cognitive processes such as long-term memory, the molecular mechanisms by which these sugars influence neuronal communication are not well understood. Here, we present molecular insights into the functions of Fucalpha(1-2)Gal sugars, demonstrating that they play a role in the regulation of synaptic proteins and neuronal morphology. We show that synapsins Ia and Ib, synapse-specific proteins involved in neurotransmitter release and synaptogenesis, are the major Fucalpha(1-2)Gal glycoproteins in mature cultured neurons and the adult rat hippocampus. Fucosylation has profound effects on the expression and turnover of synapsin in cells and protects synapsin from degradation by the calcium-activated protease calpain. Our studies suggest that defucosylation of synapsin has critical consequences for neuronal growth and morphology, leading to stunted neurite outgrowth and delayed synapse formation. We also demonstrate that Fucalpha(1-2)Gal carbohydrates are not limited to synapsin but are found on additional glycoproteins involved in modulating neuronal architecture. Together, our studies identify important roles for Fucalpha(1-2)Gal sugars in the regulation of neuronal proteins and morphological changes that may underlie synaptic plasticity.


Assuntos
Dissacarídeos/metabolismo , Regulação da Expressão Gênica , Hipocampo/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Sinapsinas/metabolismo , Animais , Calpaína/metabolismo , Fucose , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neurônios/citologia
12.
J Biol Chem ; 277(33): 29359-62, 2002 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-12087086

RESUMO

Adipocyte complement-related protein of 30 kDa (Acrp30)/adiponectin is an adipocyte-derived hormone that affects lipid and glucose metabolism in muscle and liver, but its physical and biochemical properties are poorly characterized. Here we have used several approaches to show that Acrp30 expressed in and purified from Escherichia coli and human embryonic kidney 293T cells forms trimers and hexamers; 293T cells also produce a higher molecular weight species. Similar Acrp30 oligomers were found in mouse serum as well as in 3T3-L1 adipocyte-conditioned medium, although in different proportions. In parallel, we assessed whether Acrp30 is a signaling molecule by searching for promoter or enhancer elements that respond to Acrp30 or its isolated trimeric globular C-terminal domain, gAcrp30. Acrp30 addition to C2C12 myocytes or myotubes led to activation of NF-kappa B transcription factor in a manner dependent upon phosphorylation and degradation of I kappa B-alpha. Importantly, only hexameric and larger isoforms of Acrp30 activated NF-kappa B; trimeric Acrp30 or gAcrp30 could not activate NF-kappa B. Our data indicate that oligomerization of Acrp30 is important for at least some of its biological activities, and changes in the relative abundance of each oligomeric isoform in plasma may regulate Acrp30 activity.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , NF-kappa B/metabolismo , Proteínas/fisiologia , Transdução de Sinais , Células 3T3 , Adiponectina , Sequência de Aminoácidos , Animais , Biopolímeros , Linhagem Celular , Cromatografia em Gel , Meios de Cultivo Condicionados , Humanos , Camundongos , Dados de Sequência Molecular
13.
Proc Natl Acad Sci U S A ; 99(25): 16309-13, 2002 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-12456889

RESUMO

gACRP30, the globular subunit of adipocyte complement-related protein of 30 kDa (ACRP30), improves insulin sensitivity and increases fatty acid oxidation. The mechanism by which gACRP30 exerts these effects is unknown. Here, we examined if gACRP30 activates AMP-activated protein kinase (AMPK), an enzyme that has been shown to increase muscle fatty acid oxidation and insulin sensitivity. Incubation of rat extensor digitorum longus (EDL), a predominantly fast twitch muscle, with gACRP30 (2.5 micro g/ml) for 30 min led to 2-fold increases in AMPK activity and phosphorylation of both AMPK on Thr-172 and acetyl CoA carboxylase (ACC) on Ser-79. Accordingly, concentration of malonyl CoA was diminished by 30%. In addition, gACRP30 caused a 1.5-fold increase in 2-deoxyglucose uptake. Similar changes in malonyl CoA and ACC were observed in soleus muscle incubated with gACRP30 (2.5 micro g/ml), although no significant changes in AMPK activity or 2-deoxyglucose uptake were detected. When EDL was incubated with full-length hexameric ACRP30 (10 micro g/ml), AMPK activity and ACC phosphorylation were not altered. Administration of gACRP30 (75 micro g) to C57 BL6J mice in vivo led to increased AMPK activity and ACC phosphorylation and decreased malonyl CoA concentration in gastrocnemius muscle within 15-30 min. Both in vivo and in vitro, activation of AMPK was the first effect of gACRP30 and was transient, whereas alterations in malonyl CoA and ACC occurred later and were more sustained. Thus, gACRP30 most likely exerts its actions on muscle fatty acid oxidation by inactivating ACC via activation of AMPK and perhaps other signal transduction proteins.


Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Ácidos Graxos/metabolismo , Glucose/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Quinases Ativadas por AMP , Acetil-CoA Carboxilase/metabolismo , Adiponectina , Animais , Transporte Biológico/efeitos dos fármacos , Desoxiglucose/metabolismo , Ativação Enzimática , Feminino , Resistência à Insulina , Masculino , Malonil Coenzima A/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Oxirredução , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas/química , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
14.
J Biol Chem ; 278(50): 50810-7, 2003 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-14522956

RESUMO

Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and- stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys22-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys22 with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-kappaB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-alpha at Thr172 and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Proteínas/química , Transdução de Sinais , Adiponectina , Alanina/química , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Cromatografia em Gel , Microscopia Crioeletrônica , Cisteína/química , Dimerização , Dissulfetos/química , Ditiotreitol/farmacologia , Genes Reporter , Humanos , Ligantes , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Músculos/metabolismo , Mutagênese Sítio-Dirigida , NF-kappa B/metabolismo , Conformação Proteica , Isoformas de Proteínas , Homologia de Sequência de Aminoácidos
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