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1.
Nat Commun ; 11(1): 4370, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873792

RESUMO

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sítio Alostérico/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/genética , Simulação de Acoplamento Molecular , Mutação , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/ultraestrutura , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
2.
PLoS One ; 15(7): e0224952, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32692785

RESUMO

Tauopathies are a class of neurodegenerative disorders characterized by abnormal deposition of post-translationally modified tau protein in the human brain. Tauopathies are associated with Alzheimer's disease (AD), chronic traumatic encephalopathy (CTE), and other diseases. Hyperphosphorylation increases tau tendency to aggregate and form neurofibrillary tangles (NFT), a pathological hallmark of AD. In this study, okadaic acid (OA, 100 nM), a protein phosphatase 1/2A inhibitor, was treated for 24h in mouse neuroblastoma (N2a) and differentiated rat primary neuronal cortical cell cultures (CTX) to induce tau-hyperphosphorylation and oligomerization as a cell-based tauopathy model. Following the treatments, the effectiveness of different kinase inhibitors was assessed using the tauopathy-relevant tau antibodies through tau-immunoblotting, including the sites: pSer202/pThr205 (AT8), pThr181 (AT270), pSer202 (CP13), pSer396/pSer404 (PHF-1), and pThr231 (RZ3). OA-treated samples induced tau phosphorylation and oligomerization at all tested epitopes, forming a monomeric band (46-67 kDa) and oligomeric bands (170 kDa and 240 kDa). We found that TBB (a casein kinase II inhibitor), AR and LiCl (GSK-3 inhibitors), cyclosporin A (calcineurin inhibitor), and Saracatinib (Fyn kinase inhibitor) caused robust inhibition of OA-induced monomeric and oligomeric p-tau in both N2a and CTX culture. Additionally, a cyclin-dependent kinase 5 inhibitor (Roscovitine) and a calcium chelator (EGTA) showed contrasting results between the two neuronal cultures. This study provides a comprehensive view of potential drug candidates (TBB, CsA, AR, and Saracatinib), and their efficacy against tau hyperphosphorylation and oligomerization processes. These findings warrant further experimentation, possibly including animal models of tauopathies, which may provide a putative Neurotherapy for AD, CTE, and other forms of tauopathy-induced neurodegenerative diseases.


Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Ácido Okadáico/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Animais , Linhagem Celular , Ciclosporina/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Cloreto de Lítio/farmacologia , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ratos , Tauopatias/metabolismo , Tauopatias/patologia , Triazóis/farmacologia
3.
Proc Natl Acad Sci U S A ; 117(28): 16313-16323, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601209

RESUMO

Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.


Assuntos
Peroxirredoxinas/química , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
4.
Nat Chem Biol ; 16(7): 766-775, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483376

RESUMO

Cell surfaces are glycosylated in various ways with high heterogeneity, which usually leads to ambiguous conclusions about glycan-involved biological functions. Here, we describe a two-step chemoenzymatic approach for N-glycan-subtype-selective editing on the surface of living cells that consists of a first 'delete' step to remove heterogeneous N-glycoforms of a certain subclass and a second 'insert' step to assemble a well-defined N-glycan back onto the pretreated glyco-sites. Such glyco-edited cells, carrying more homogeneous oligosaccharide structures, could enable precise understanding of carbohydrate-mediated functions. In particular, N-glycan-subtype-selective remodeling and imaging with different monosaccharide motifs at the non-reducing end were successfully achieved. Using a combination of the expression system of the Lec4 CHO cell line and this two-step glycan-editing approach, opioid receptor delta 1 (OPRD1) was investigated to correlate its glycostructures with the biological functions of receptor dimerization, agonist-induced signaling and internalization.


Assuntos
Membrana Celular/química , Células Epiteliais/química , Glicoconjugados/química , Oligossacarídeos/química , Receptores Opioides delta/química , Animais , Células CHO , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetulus , Encefalina Leucina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica , Glicoconjugados/metabolismo , Glicosilação , Células HEK293 , Humanos , Camundongos , Oligossacarídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Transgenes
5.
Nature ; 584(7820): 298-303, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32555460

RESUMO

Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis1. GABAB belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits2. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABAB. Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.


Assuntos
Microscopia Crioeletrônica , Receptores de GABA-B/química , Receptores de GABA-B/ultraestrutura , Regulação Alostérica/efeitos dos fármacos , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Sítios de Ligação/efeitos dos fármacos , Agonistas dos Receptores de GABA-B/química , Agonistas dos Receptores de GABA-B/metabolismo , Agonistas dos Receptores de GABA-B/farmacologia , Humanos , Modelos Moleculares , Domínios Proteicos/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Receptores de GABA-B/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
6.
Biochem Biophys Res Commun ; 528(1): 35-38, 2020 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-32451080

RESUMO

The recent release of COVID-19 spike glycoprotein allows detailed analysis of the structural features that are required for stabilizing the infective form of its quaternary assembly. Trying to disassemble the trimeric structure of COVID-19 spike glycoprotein, we analyzed single protomer surfaces searching for concave moieties that are located at the three protomer-protomer interfaces. The presence of some druggable pockets at these interfaces suggested that some of the available drugs in Drug Bank could destabilize the quaternary spike glycoprotein formation by binding to these pockets, therefore interfering with COVID-19 life cycle. The approach we propose here can be an additional strategy to fight against the deadly virus. Ligands of COVID-19 spike glycoprotein that we have predicted in the present computational investigation, might be the basis for new experimental studies in vitro and in vivo.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Pneumonia Viral/tratamento farmacológico , Multimerização Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Antivirais/uso terapêutico , Betacoronavirus/química , Betacoronavirus/fisiologia , Sítios de Ligação , Infecções por Coronavirus/epidemiologia , Ligantes , Modelos Moleculares , Pandemias , Pneumonia Viral/epidemiologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico
7.
ACS Comb Sci ; 22(6): 297-305, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32402186

RESUMO

A new coronavirus (CoV) caused a pandemic named COVID-19, which has become a global health care emergency in the present time. The virus is referred to as SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) and has a genome similar (∼82%) to that of the previously known SARS-CoV (SARS coronavirus). An attractive therapeutic target for CoVs is the main protease (Mpro) or 3-chymotrypsin-like cysteine protease (3CLpro), as this enzyme plays a key role in polyprotein processing and is active in a dimeric form. Further, Mpro is highly conserved among various CoVs, and a mutation in Mpro is often lethal to the virus. Thus, drugs targeting the Mpro enzyme significantly reduce the risk of mutation-mediated drug resistance and display broad-spectrum antiviral activity. The combinatorial design of peptide-based inhibitors targeting the dimerization of SARS-CoV Mpro represents a potential therapeutic strategy. In this regard, we have compiled the literature reports highlighting the effect of mutations and N-terminal deletion of residues of SARS-CoV Mpro on its dimerization and, thus, catalytic activity. We believe that the present review will stimulate research in this less explored yet quite significant area. The effect of the COVID-19 epidemic and the possibility of future CoV outbreaks strongly emphasize the urgent need for the design and development of potent antiviral agents against CoV infections.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/metabolismo , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Descoberta de Drogas , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Mutação/efeitos dos fármacos , Pandemias , Peptídeos/farmacologia , Pneumonia Viral/virologia , Conformação Proteica/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
8.
Nat Commun ; 11(1): 2444, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415068

RESUMO

The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Imagem com Lapso de Tempo
9.
Biochem Biophys Res Commun ; 528(1): 35-38, 2020 07 12.
Artigo em Inglês | MEDLINE | ID: covidwho-388951

RESUMO

The recent release of COVID-19 spike glycoprotein allows detailed analysis of the structural features that are required for stabilizing the infective form of its quaternary assembly. Trying to disassemble the trimeric structure of COVID-19 spike glycoprotein, we analyzed single protomer surfaces searching for concave moieties that are located at the three protomer-protomer interfaces. The presence of some druggable pockets at these interfaces suggested that some of the available drugs in Drug Bank could destabilize the quaternary spike glycoprotein formation by binding to these pockets, therefore interfering with COVID-19 life cycle. The approach we propose here can be an additional strategy to fight against the deadly virus. Ligands of COVID-19 spike glycoprotein that we have predicted in the present computational investigation, might be the basis for new experimental studies in vitro and in vivo.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Pneumonia Viral/tratamento farmacológico , Multimerização Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Antivirais/uso terapêutico , Betacoronavirus/química , Betacoronavirus/fisiologia , Sítios de Ligação , Infecções por Coronavirus/epidemiologia , Ligantes , Modelos Moleculares , Pandemias , Pneumonia Viral/epidemiologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico
10.
J Med Chem ; 63(10): 5274-5286, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32364733

RESUMO

The cyclic dimeric peptide 1229U91 (GR231118) has an unusual structure and displays potent, insurmountable antagonism of the Y1 receptor. To probe the structural basis for this activity, we have prepared ring size variants and heterodimeric compounds, identifying the specific residues underpinning the mechanism of 1229U91 binding. The homodimeric structure was shown to be dispensible, with analogues lacking key pharmacophoric residues in one dimer arm retaining high antagonist affinity. Compounds 11d-h also showed enhanced Y1R selectivity over Y4R compared to 1229U91.


Assuntos
Neuropeptídeos/química , Neuropeptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Neuropeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/fisiologia , Receptores de Neuropeptídeo Y/antagonistas & inibidores
11.
ACS Comb Sci ; 22(6): 297-305, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: covidwho-247796

RESUMO

A new coronavirus (CoV) caused a pandemic named COVID-19, which has become a global health care emergency in the present time. The virus is referred to as SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) and has a genome similar (∼82%) to that of the previously known SARS-CoV (SARS coronavirus). An attractive therapeutic target for CoVs is the main protease (Mpro) or 3-chymotrypsin-like cysteine protease (3CLpro), as this enzyme plays a key role in polyprotein processing and is active in a dimeric form. Further, Mpro is highly conserved among various CoVs, and a mutation in Mpro is often lethal to the virus. Thus, drugs targeting the Mpro enzyme significantly reduce the risk of mutation-mediated drug resistance and display broad-spectrum antiviral activity. The combinatorial design of peptide-based inhibitors targeting the dimerization of SARS-CoV Mpro represents a potential therapeutic strategy. In this regard, we have compiled the literature reports highlighting the effect of mutations and N-terminal deletion of residues of SARS-CoV Mpro on its dimerization and, thus, catalytic activity. We believe that the present review will stimulate research in this less explored yet quite significant area. The effect of the COVID-19 epidemic and the possibility of future CoV outbreaks strongly emphasize the urgent need for the design and development of potent antiviral agents against CoV infections.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/metabolismo , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Descoberta de Drogas , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Mutação/efeitos dos fármacos , Pandemias , Peptídeos/farmacologia , Pneumonia Viral/virologia , Conformação Proteica/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
12.
Mol Pharmacol ; 97(5): 336-350, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32111699

RESUMO

Positive allosteric modulators (PAMs) of AMPA receptors boost cognitive performance in preclinical and clinical studies. Their therapeutic window is narrow, however, and clinical application will likely only occur if greater discrimination in activity is achieved. Toward that end, we compared the modulatory activity of two PAMs recently considered as clinical candidates, LY451395 (mibampator) and PF-04958242/BIIB104, on recombinant and native AMPA receptors (AMPARs). We found that the principle molecular determinant that shaped modulatory activity of both PAMs on deactivation (recombinant) and decay (synaptic) of AMPARs was the auxiliary protein incorporated into the receptor complexes. AMPARs containing the stargazin/γ2 transmembrane AMPAR regulatory protein (TARP) were slowed to a >10-fold degree by both PAMs as compared with those incorporating γ8 TARP. Neither subunit composition nor flip/flop splice variation had substantive effect. Similarly, stargazin/γ2-containing mossy fiber EPSCs in cerebellar granule neurons were slowed to a ∼5-fold greater degree than EPSCs in hippocampal CA1 pyramidal cell neurons, which express the γ8 TARP. LY451395 exhibited greater efficacy than BIIB104 at both synapses. These studies provide insight into the receptor constituents that determine efficacy of sulfonamide PAMs. We conclude that compounds that discriminate between AMPARs complexed with distinct TARPs, and particularly those with lower stargazin/γ2 efficacy such as BIIB104, could act as viable procognitive therapeutics. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of AMPA receptors enhance cognitive function in a variety of preclinical models. A clearer understanding of the critical determinants of PAM activity could yield critical insight into pathways to maximize their therapeutic index. Here we show that auxiliary proteins for AMPARs play a major, but thus far underappreciated, role in shaping recombinant and neuronal AMPAR modulation by two clinical candidate PAMs. These data will inform both clinical outcomes as well as future rational development of new modulators.


Assuntos
Proteínas de Membrana/metabolismo , Receptores de AMPA/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Compostos de Bifenilo/farmacologia , Cerebelo/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Resultado do Tratamento , Triazóis/farmacologia
13.
Cancer Sci ; 111(5): 1774-1784, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32112605

RESUMO

The signal transducer and activator of transcription 3 (STAT3) signaling pathway is a key mediator of cancer cell proliferation, survival and invasion. Aberrant STAT3 has been demonstrated in various malignant cancers. YHO-1701 is a novel quinolinecarboxamide derivative generated from STX-0119. Here, we examined the effect of YHO-1701 on STAT3 and evaluated antitumor activity of YHO-1701 as a single agent and in combination. YHO-1701 inhibited STAT3-SH2 binding to phospho-Tyr peptide selectively and more potently than STX-0119 in biochemical assays. Molecular docking studies with STAT3 suggested more stable interaction of YHO-1701 with the SH2 domain. YHO-1701 exhibited approximately 10-fold stronger activity than STX-0119 in abrogating the STAT3 signaling pathway of human oral cancer cell line SAS. YHO-1701 also blocked multi-step events by inhibiting STAT3 dimerization and suppressed STAT3 promoter activity. As expected, YHO-1701 exerted strong antiproliferative activity against human cancer cell lines addicted to STAT3 signaling. Orally administered YHO-1701 showed statistically significant antitumor effects with long exposure to high levels of YHO-1701 at tumor sites in SAS xenograft models. Moreover, combination regimen with sorafenib led to significantly stronger antitumor activity. In addition, the suppression level of survivin (a downstream target) was superior for the combination as compared with monotherapy groups within tumor tissues. Thus, YHO-1701 had a favorable specificity for STAT3 and pharmacokinetics after oral treatment; it also contributed to the enhanced antitumor activity of sorafenib. The evidence presented here provides justification using for this approach in future clinical settings.


Assuntos
Antineoplásicos/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimioterapia Combinada , Humanos , Interleucina-6/sangue , Camundongos , Simulação de Acoplamento Molecular , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Oxidiazóis/química , Oxidiazóis/farmacologia , Multimerização Proteica/efeitos dos fármacos , Quinolinas/química , Quinolinas/farmacologia , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Survivina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Domínios de Homologia de src
14.
J Med Chem ; 63(7): 3596-3609, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32159953

RESUMO

Opioid receptors (ORs) are among the best-studied G protein-coupled receptors due to their involvement in neurological disorders and important role in pain treatment. Contrary to the classical monomeric model, indirect evidence suggests that ORs might form dimers, which could be endowed with a distinct pharmacological profile, and, thus, be targeted to develop innovative pharmacological therapies. However, direct evidence for the spontaneous formation of OR dimers in living cells under physiological conditions is missing. Despite a growing interest in the κ opioid receptor (KOR), KOR-selective fluorescent probes are particularly scarce in the literature. Herein, we present the first set of fluorescent KOR-selective probes with antagonistic properties. Two of these were employed in single-molecule microscopy (SMM) experiments to investigate KOR homodimerization, localization, and trafficking. Our findings indicate that most KORs labeled with the new fluorescent probes are present as apparently freely diffusing monomers on the surface of a simple cell model.


Assuntos
Corantes Fluorescentes/farmacologia , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Multimerização Proteica/efeitos dos fármacos , Receptores Opioides kappa/antagonistas & inibidores , Animais , Células CHO , Cricetulus , Corantes Fluorescentes/síntese química , Células HEK293 , Humanos , Ligantes , Naltrexona/síntese química , Receptores Opioides kappa/metabolismo , Imagem Individual de Molécula
15.
Proc Natl Acad Sci U S A ; 117(12): 6521-6530, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32156734

RESUMO

Necroptosis is a regulated necrotic cell death pathway involved in development and disease. Its signaling cascade results in the formation of disulfide bond-dependent amyloid-like polymers of mixed lineage kinase domain-like protein (MLKL), which mediate proinflammatory cell membrane disruption. We screened compound libraries provided by the National Cancer Institute and identified a small-molecule inhibitor of necroptosis named necroptosis-blocking compound 1 (NBC1). Biotin-labeled NBC1 specifically conjugates to heat shock protein Hsp70. NBC1 and PES-Cl, a known Hsp70 substrate-binding inhibitor, block the formation of MLKL polymers, but not MLKL tetramers in necroptosis-induced cells. In vitro, recombinant Hsp70 interacts with the N-terminal domain (NTD) of MLKL and promotes NTD polymerization, which has been shown to mediate the cell killing activity. Furthermore, the substrate-binding domain (SBD) of Hsp70 is sufficient to promote MLKL polymerization. NBC1 covalently conjugates cysteine 574 and cysteine 603 of the SBD to block its function. In addition, an SBD mutant with both cysteines mutated to serines loses its ability to promote MLKL polymerization. Interestingly, knockdown of Hsp70 in cells leads to MLKL destabilization, suggesting that MLKL might also be a client protein of Hsp70. In summary, using NBC1, an inhibitor of necroptosis, we identified Hsp70 as a molecular chaperone performing dual functions in necroptosis. It stabilizes MLKL protein under normal condition and promotes MLKL polymerization through its substrate-binding domain during necroptosis.


Assuntos
Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Necroptose/efeitos dos fármacos , Proteínas Quinases/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Células HT29 , Humanos , Estrutura Molecular , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/genética , Multimerização Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
16.
J Med Chem ; 63(6): 3205-3214, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32124607

RESUMO

Transthyretin (TTR) modulates the deposition, processing, and toxicity of Abeta (Aß) peptides. We have shown that this effect is enhanced in mice by treatment with small molecules such as iododiflunisal (IDIF, 4), a good TTR stabilizer. Here, we describe the thermodynamics of the formation of binary and ternary complexes among TTR, Aß(1-42) peptide, and TTR stabilizers using isothermal titration calorimetry (ITC). A TTR/Aß(1-42) (1:1) complex with a dissociation constant of Kd = 0.94 µM is formed; with IDIF (4), this constant improves up to Kd = 0.32 µM, indicating the presence of a ternary complex TTR/IDIF/Aß(1-42). However, with the drugs diflunisal (1) or Tafamidis (2), an analogous chaperoning effect could not be observed. Similar phenomena could be recorded with the shorter peptide Aß(12-28) (7). We propose the design of a simple assay system for the search of other chaperones that behave like IDIF and may become potential candidate drugs for Alzheimer's disease (AD).


Assuntos
Peptídeos beta-Amiloides/metabolismo , Benzoxazóis/metabolismo , Diflunisal/análogos & derivados , Diflunisal/metabolismo , Fragmentos de Peptídeos/metabolismo , Pré-Albumina/metabolismo , Multimerização Proteica/efeitos dos fármacos , Bioensaio/métodos , Calorimetria/métodos , Humanos , Termodinâmica
17.
PLoS One ; 15(2): e0223870, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32092083

RESUMO

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized in detail before. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues, we were not able to detect either oxidase or monooxygenase activity when L-lactate was used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.


Assuntos
Mononucleotídeo de Flavina/farmacologia , Oxigenases de Função Mista/metabolismo , Pediococcus acidilactici/enzimologia , Multimerização Proteica/efeitos dos fármacos , Catálise , Domínio Catalítico , Cristalografia por Raios X , Ácido Láctico/metabolismo , Pediococcus acidilactici/metabolismo , Proteínas Recombinantes
18.
Nat Commun ; 11(1): 941, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071304

RESUMO

Oxidative stress is a major pathogenic mechanism in Parkinson's disease (PD). As an important cellular antioxidant, glutathione (GSH) balances the production and incorporation of free radicals to protect neurons from oxidative damage. GSH level is decreased in the brains of PD patients. Hence, clarifying the molecular mechanism of GSH deficiency may help deepen our knowledge of PD pathogenesis. Here we report that the astrocytic dopamine D2 receptor (DRD2) regulates GSH synthesis via PKM2-mediated Nrf2 transactivation. In addition we find that pyridoxine can dimerize PKM2 to promote GSH biosynthesis. Further experiments show that pyridoxine supplementation increases the resistance of nigral dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in wild-type mice as well as in astrocytic Drd2 conditional knockout mice. We conclude that dimerizing PKM2 may be a potential target for PD treatment.


Assuntos
Glutationa/biossíntese , Intoxicação por MPTP/patologia , Fator 2 Relacionado a NF-E2/genética , Fármacos Neuroprotetores/administração & dosagem , Piruvato Quinase/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Astrócitos , Técnicas de Observação do Comportamento , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Dopamina/metabolismo , Neurônios Dopaminérgicos , Intoxicação por MPTP/diagnóstico , Intoxicação por MPTP/tratamento farmacológico , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Multimerização Proteica/efeitos dos fármacos , Piridoxina/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Receptores de Dopamina D2/genética , Substância Negra/citologia , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Ativação Transcricional
19.
J Med Chem ; 63(6): 3131-3141, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32105468

RESUMO

Structure-based stabilization of protein-protein interactions (PPIs) is a promising strategy for drug discovery. However, this approach has mainly focused on the stabilization of native PPIs, and non-native PPIs have received little consideration. Here, we identified a non-native interaction interface on the three-dimensional dimeric structure of the N-terminal domain of the MERS-CoV nucleocapsid protein (MERS-CoV N-NTD). The interface formed a conserved hydrophobic cavity suitable for targeted drug screening. By considering the hydrophobic complementarity during the virtual screening step, we identified 5-benzyloxygramine as a new N protein PPI orthosteric stabilizer that exhibits both antiviral and N-NTD protein-stabilizing activities. X-ray crystallography and small-angle X-ray scattering showed that 5-benzyloxygramine stabilizes the N-NTD dimers through simultaneous hydrophobic interactions with both partners, resulting in abnormal N protein oligomerization that was further confirmed in the cell. This unique approach based on the identification and stabilization of non-native PPIs of N protein could be applied toward drug discovery against CoV diseases.


Assuntos
Alcaloides/farmacologia , Antivirais/farmacologia , Indóis/farmacologia , Proteínas do Nucleocapsídeo/metabolismo , Multimerização Proteica/efeitos dos fármacos , Alcaloides/química , Alcaloides/metabolismo , Sequência de Aminoácidos , Animais , Antivirais/química , Antivirais/metabolismo , Chlorocebus aethiops , Cristalografia por Raios X , Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Indóis/química , Indóis/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Simulação de Acoplamento Molecular , Proteínas do Nucleocapsídeo/química , Ligação Proteica , Domínios Proteicos , Alinhamento de Sequência , Células Vero
20.
Inorg Chem ; 59(4): 2527-2535, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32027132

RESUMO

Protein aggregation has attracted substantial interest because of its role in causing many serious illnesses, such as neurodegenerative diseases and type II diabetes. Recent studies have shown that protein aggregation can be prevented by forming metal ion complexes with a target protein, which affects their conformation in solution and their physical properties, such as aggregation. Thus, understanding the interactions between aggregating molecules and bioactive metal ions such as Cu2+ is beneficial for new drug discovery. Pramlintide, a synthetic peptide drug, and its natural counterpart rat amylin are known to be resistant to aggregation because of the presence of proline residues, which are usually ß-sheet "breakers" within their amino acid sequence. Here, we investigate the Cu2+ coordination properties of pramlintide and rat amylin using nuclear magnetic resonance, circular dichroism, electron paramagnetic resonance, ultraviolet-visible spectroscopy, potentiometry, and mass spectrometry. We test the influence of Cu2+ on the aggregation properties of these amylin analogues with thioflavin T assays. We find that both peptides form stable complexes with Cu2+ with similar affinities at a 1:1 ratio. The N-termini of both peptides are involved in Cu2+ binding; His18 imidazole is an equally attractive binding site in the case of pramlintide. Our results show that Cu2+ ions influence the aggregation of pramlintide, but not that of rat amylin.


Assuntos
Cobre/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Complexos de Coordenação/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Ratos
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