RESUMO
Chronic infection with hepatitis B virus (HBV) is a major cause of cirrhosis and liver cancer. Capsid assembly modulators can induce error-prone assembly of HBV core proteins to prevent the formation of infectious virions, representing promising candidates for treating chronic HBV infections. To explore novel capsid assembly modulators from unexplored mirror-image libraries of natural products, we have investigated the synthetic process of the HBV core protein for preparing the mirror-image target protein. In this report, the chemical synthesis of full-length HBV core protein (Cp183) containing an arginine-rich nucleic acid-binding domain at the C-terminus is presented. Sequential ligations using four peptide segments enabled the synthesis of Cp183 via convergent and C-to-N direction approaches. After refolding under appropriate conditions, followed by the addition of nucleic acid, the synthetic Cp183 assembled into capsid-like particles.
Assuntos
Hepatite B , Ácidos Nucleicos , Humanos , Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírus da Hepatite B , Hepatite B/metabolismo , Proteínas do Core Viral/análise , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo , Replicação Viral , Antivirais/metabolismoRESUMO
BACKGROUND: A certain number of patients with coronavirus disease 2019 (COVID-19), particularly those who test positive for SARS-CoV-2 in the serum, are hospitalized. Further, some even die. We examined the effect of blood adsorption therapy using columns that can eliminate SARS-CoV-2 on the improvement of the prognosis of severe COVID-19 patients. METHODS: This study enrolled seven patients receiving mechanical ventilation. The patients received viral adsorption therapy using SARS-catch column for 3 days. The SARS-catch column was developed by immobilizing a specific peptide, designed based on the sequence of human angiotensin-converting enzyme 2 (hACE2), to an endotoxin adsorption column (PMX). In total, eight types of SARS-CoV-2-catch (SCC) candidate peptides were developed. Then, a clinical study on the effects of blood adsorption therapy using the SARS-catch column in patients with severe COVID-19 was performed, and the data in the present study were compared with historical data of severe COVID-19 patients. RESULTS: Among all SCC candidate peptides, SCC-4N had the best adsorption activity against SARS-CoV-2. The SARS-catch column using SCC-4N removed 65% more SARS-CoV-2 than PMX. Compared with historical data, the weaning time from mechanical ventilation was faster in the present study. In addition, the rate of negative blood viral load in the present study was higher than that in the historical data. CONCLUSION: The timely treatment with virus adsorption therapy may eliminate serum SARS-CoV-2 and improve the prognosis of patients with severe COVID-19. However, large-scale studies must be performed in the future to further assess the finding of this study (jRCTs052200134).
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , PeptídeosRESUMO
γ-Glutamylcyclotransferase (GGCT) is involved in glutathione homeostasis, in which it catalyzes the reaction that generates 5-oxoproline and free amino acids from γ-glutamyl peptides. Increasing evidence shows that GGCT has oncogenic functions and is overexpressed in various cancer tissues, and that inhibition of GGCT activity exerts anticancer effects in vitro and in vivo. Here, we demonstrate that U83836E ((2R)-2-[[4-(2,6-dipyrrolidin-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-3,4-dihydro-2,5,7,8,-tetramethyl-2H-1-benzopyran-6-ol, dihydrochloride), a lazaroid that inhibits lipid peroxidation, inhibits GGCT enzymatic activity. U83836E was identified from a high-throughput screen of low molecular weight compounds using a fluorochrome-conjugated GGCT probe. We directly quantified that U83836E specifically inhibited GGCT by measuring the product of a fluorochrome-conjugated GGCT substrate assay, and showed that U83836E inhibited GGCT activity in extracts of NIH3T3 cells overexpressing GGCT. Moreover, U83836E significantly inhibited tumor growth in a xenograft model that used immunodeficient mice orthotopically inoculated with MCF7 human breast cancer cells. These results indicate that U83836E may be a useful GGCT inhibitor for the development of potential cancer therapeutics.
Assuntos
Neoplasias da Mama/patologia , Cromanos/farmacologia , Inibidores Enzimáticos/farmacologia , Piperazinas/farmacologia , gama-Glutamilciclotransferase/antagonistas & inibidores , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos SCID , Células NIH 3T3 , Ensaios Antitumorais Modelo de Xenoenxerto , gama-Glutamilciclotransferase/metabolismoRESUMO
Preparation of C-terminal Cys-containing peptide acid by Fmoc solid-phase peptide synthesis (SPPS) is difficult due to base-mediated epimerization at Cys. In this paper, use of a C-terminal pseudoproline structure and Trt(2-Cl) resin achieved epimerization-free direct preparation of the C-terminal Cys-containing peptide acid by Fmoc SPPS. Additionally, the C-terminal Cys(ΨDmp,Hpro)-containing protected peptide segment was applied to an epimerization-free segment condensation reaction.
RESUMO
A novel solubilizing tag system for Asp/Asn/Glu/Gln-containing peptides is described. In this method, an Asp/Glu[Dbz-Cys-NH2 ]-containing peptide (Dbz: 3,4-diaminobenzoic acid) is first synthesized through fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis. The solubilizing moiety containing an oligo-Lys group is then attached to the peptide in hexafluoroisopropanol through a trityl anchor to afford a hydrophilic tagged peptide. To detach the solubilizing tag, the Dbz moiety of the tagged peptide is activated with NaNO2 , and the Asp/Asn/Glu/Gln-containing peptide is obtained through hydrolysis or ammonolysis. This synthetic approach proved to be compatible with native chemical ligation, and amyloid ß-protein 1-42 was successfully synthesized by the solubilizing-tag-aided native chemical ligation-desulfurization method.
Assuntos
Aminoácidos/química , Peptídeos/síntese química , Estrutura Molecular , Peptídeos/química , SolubilidadeRESUMO
Recently, solubilizing tag methods (Trt-K and Trt-R method) were developed for the challenging synthesis of peptides/proteins by means of native chemical ligation. In this system, the solubilizing tag can be attached to the Cys side chain by simply mixing the tag-introducing reagent under acidic conditions. The tagged peptides/proteins exhibited high water solubility thanks to the introduction of redundant oligo-Lys/Arg. In the final reaction, the tag can be quickly and cleanly detached by a standard deprotection reaction with trifluoroacetic acid. Herein, the development and application of these methods are described.
Assuntos
Cisteína/química , Peptídeos/síntese química , Proteínas/síntese química , Estrutura Molecular , Peptídeos/química , Proteínas/química , SolubilidadeRESUMO
Hydrophobic membrane peptides/proteins having low water solubility are often difficult to prepare. To overcome this issue, temporal introduction of solubilizing tags has been demonstrated to be beneficial. Following our recent work on the solubilization of a difficult target by using a hydrophilic oligo-Lys tag bearing a trityl linker (Trt-K method), this paper describes a comparative study of the solubilizing abilities of several peptidic trityl tags containing Lys, Arg, Glu, Asn, Nε-tri-Me-Lys or Cys-sulfonate using two hydrophobic model peptides. Among the tags evaluated, that containing Nε-tri-Me-Lys exhibits superior solubilizing ability.
Assuntos
Lisina/análogos & derivados , Peptídeos/síntese química , Sequência de Aminoácidos , Aminoácidos/química , Interações Hidrofóbicas e Hidrofílicas , Lisina/química , Solubilidade , Relação Estrutura-Atividade , Ácidos Sulfônicos/química , ÁguaRESUMO
Solubilizing trityl tags (Trt-oligoLys/Arg) proved applicable to metal-free radical-triggered desulfurization and an Ag-mediated thioester method. Additionally, using the solubilizing trityl tag strategy, synthesis of the influenza BM2 proton channel, which previously required organic solvent-aided native chemical ligation (NCL) and desulfurization due to its low solubility, was achieved without using organic solvents.
Assuntos
Técnicas de Química Sintética/métodos , Compostos de Tritil/química , Proteínas Virais/síntese química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Leucina/química , Solubilidade , Enxofre/química , Proteínas Virais/químicaRESUMO
The first chromogenic substrate for Aspergillus oryzae acid protease, 'HAP-01', was successfully developed after evaluating the substrate specificity of the enzyme. Furthermore, with HAP-01, digestion-triggered chromophore release was employed as a novel chromogenic technique.
Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Nitrofenóis/química , Peptídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Piridinas , Pirimidinas , Especificidade por SubstratoRESUMO
Ring-closing metathesis (RCM) is an attractive reaction for the preparation of artificially designed peptides. Until now, RCM has been used for fully or partially protected peptides. Herein, the first RCM of unprotected peptides in water was achieved using a water-soluble Ru catalyst.
RESUMO
A solubilizing Trt-K10 tag was developed for the effective chemical preparation of peptides/proteins with low solubility. The Trt-K10 tag comprises a hydrophilic oligo-Lys sequence and a trityl anchor, and can be selectively introduced to a side chain thiol of Cys of deprotected peptides/proteins with a trityl alcohol-type introducing reagent Trt(OH)-K10 under acidic conditions. Significantly, the ligation product in the reaction mixture of a thiol-additive-free native chemical ligation can be modified directly in a one-pot manner to facilitate the isolation of the product by high-performance liquid chromatography. Finally, the Trt-K10 tag can be readily removed with a standard trifluoroacetic acid cocktail. Using this easy-to-attach/detach tag-aided method, a hepatitisâ B virus capsid protein that is usually difficult to handle was synthesized successfully.
Assuntos
Proteínas do Capsídeo/síntese química , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Cisteína/química , Vírus da Hepatite B/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Polilisina/química , Solubilidade , Compostos de Sulfidrila/químicaRESUMO
We report a novel strategy for native chemical ligation (NCL). Alanines not located at a ligation site are temporarily replaced with cysteines, and this enables efficient thiol-additive-free NCL, with subsequent desulfurization to regenerate the target peptide. We synthesized stresscopin-related peptide and neuroendocrine regulatory peptide-2 (NERP-2) by this method. We confirmed that both conventional alkyl thioester and thioester-equivalent N-acyl-N'-methyl-benzimidazolinone (MeNbz) can be adopted as thioester components for thiol-additive-free NCL of multi-Cys-containing peptides.
Assuntos
Alanina/química , Cisteína/química , Peptídeos/química , Compostos de Sulfidrila/química , Sequência de Aminoácidos , Benzimidazóis/química , Hormônio Liberador da Corticotropina/química , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida , Urocortinas/químicaRESUMO
Various bioactive proteins have been synthesized by native chemical ligation (NCL) and its combination with subsequent desulfurization (e.g., conversion from Cys to Ala). In NCL, excess 4-mercaptophenylacetic acid (MPAA) is generally added to facilitate the reaction. However, co-elution of MPAA with the ligation product during preparative high-performance liquid chromatography sometimes reduces its usefulness. In addition, contamination of MPAA disturbs subsequent desulfurization. Here, we report for the first time that imidazole can be adopted as an alternative to MPAA in NCL using a peptide-alkylthioester. The efficiency of the imidazole-aided NCL (Im-NCL) is similar to that of traditional MPAA-aided NCL. As model cases, we successfully synthesized adiponectin(19-107) and [Ser(PO3 H2 )65 ]-ubiquitin using Im-NCL with a one-pot desulfurization.
RESUMO
N(α) -Trifluoroacetyl-Cys-Leu-NH2 (TfaC-Leu-NH2 ) was incorporated into thioesters through its side-chain thiol group to develop a more reactive peptide-thioester than the commonly used peptide-3-mercaptopropionic acid (MPA)-thioester. The TfaC-thioester could be readily synthesized by solid-phase peptide synthesis (SPPS) with Boc chemistry using in situ neutralization protocols in sufficient yield without any side reaction associated with the use of TfaC. This thioester proved to display a much higher reactivity in the thiol-free native chemical ligation (NCL) reaction than the MPA-thioester and to be comparable to the thioarylester, such as the 4-mercaptophenylacetic acid (MPAA)-thioester, in terms of the ligation rate. We were able to demonstrate the usefulness of the TfaC-thioester by using it to synthesize neuromedin S via a one-pot sequential NCL approach followed by desulfurization. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 503-511, 2016.
Assuntos
Neuropeptídeos/química , Neuropeptídeos/síntese química , Compostos de Sulfidrila/químicaRESUMO
Cancer-related γ-glutamyl cyclotransferase (GGCT) specifically converts γ-glutamyl amino acids (γ-Glu-Xaa) into pyroglutamate and the corresponding amino acids (Xaa). Here we report a novel GGCT fluorogenic probe "LISA-101" containing a masked O-acylated fluorophore "resorufin" on the side chain of the P amino acid (Xaa). Upon GGCT treatment, the P amino acid was liberated and spontaneously released the intact fluorophore. Thus, the fluorescence was regained. LISA-101 will expand the strategies for cancer studies.
Assuntos
Anticorpos Monoclonais , Corantes Fluorescentes , Neoplasias/diagnóstico , gama-Glutamilciclotransferase , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Células MCF-7 , Estrutura Molecular , gama-Glutamilciclotransferase/síntese química , gama-Glutamilciclotransferase/metabolismoRESUMO
Light it up: human chromosome 7 ORF 24, a tumor-related protein, has been identified as a γ-glutamyl cyclotransferase (GGCT) in the glutathione homeostasis cycle. The singular substrate preference of the enzyme has hampered chemical probe development, and no fluorogenic probe has been reported. Here we report the first fluorogenic dipeptide probe, LISA-4, which should contribute toward further understanding of GGCT.
Assuntos
Corantes Fluorescentes/metabolismo , Nitrogênio/metabolismo , Oxigênio/metabolismo , gama-Glutamilciclotransferase/metabolismo , Sítios de Ligação , Biocatálise , Cromossomos Humanos Par 7 , Dipeptídeos/química , Dipeptídeos/metabolismo , Corantes Fluorescentes/química , Humanos , Simulação de Acoplamento Molecular , Nitrogênio/química , Fases de Leitura Aberta , Oxigênio/química , Estrutura Terciária de Proteína , gama-Glutamilciclotransferase/genéticaRESUMO
N-sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl(3)- or SOCl(2)-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.
Assuntos
Aminoácidos/química , Anilidas/síntese química , Fluorenos/química , Peptídeos/síntese química , Compostos de Enxofre/síntese química , Acilação , Ésteres , Cinética , Fosfatos/química , Técnicas de Síntese em Fase Sólida , Compostos de Sulfidrila/químicaAssuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Fluorescência , Corantes Fluorescentes/farmacologia , gama-Glutamilciclotransferase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Camundongos , Estrutura Molecular , Células NIH 3T3 , gama-Glutamilciclotransferase/metabolismoRESUMO
Non-proteinaceous components in membranes regulate membrane protein insertion cooperatively with proteinaceous translocons. An endogenous glycolipid in the Escherichia coli membrane called membrane protein integrase (MPIase) is one such component. Here, we focused on the Sec translocon-independent pathway and examined the mechanisms of MPIase-facilitated protein insertion using physicochemical techniques. We determined the membrane insertion efficiency of a small hydrophobic protein using solid-state nuclear magnetic resonance, which showed good agreement with that determined by the insertion assay using an in vitro translation system. The observed insertion efficiency was strongly correlated with membrane physicochemical properties measured using fluorescence techniques. Diacylglycerol, a trace component of E. coli membrane, reduced the acyl chain mobility in the core region and inhibited the insertion, whereas MPIase restored them. We observed the electrostatic intermolecular interactions between MPIase and the side chain of basic amino acids in the protein, suggesting that the negatively charged pyrophosphate of MPIase attracts the positively charged residues of a protein near the membrane surface, which triggers the insertion. Thus, this study demonstrated the ingenious approach of MPIase to support membrane insertion of proteins by using its unique molecular structure in various ways.
Assuntos
Proteínas de Escherichia coli , Proteínas de Membrana , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicolipídeos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Canais de Translocação SEC/metabolismoRESUMO
Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.