Synthesis of the full-length hepatitis B virus core protein and its capsid formation.

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.

Establishment and clinical application of SARS-CoV-2 catch column.

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).

Role of a bacterial glycolipid in Sec-independent membrane protein insertion.

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.

Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration.

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.

Identification of U83836E as a γ-glutamylcyclotransferase inhibitor that suppresses MCF7 breast cancer xenograft growth.

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.

Late-Stage Solubilization of Poorly Soluble Peptides Using Hydrazide Chemistry.

A novel late-stage solubilization of peptides using hydrazides is described. A solubilizing tag was attached through a selective N-alkylation at a hydrazide moiety with the aid of a 2-picoline-borane complex in 50% acetic acid-hexafluoro-2-propanol. The tag, which tolerates ligation and desulfurization conditions, can be detached by a Cu-mediated selective oxidative hydrolysis of the N-alkyl hydrazide. This new method was validated through the synthesis of HIV-1 protease.

Epimerization-Free Preparation of C-Terminal Cys Peptide Acid by Fmoc SPPS Using Pseudoproline-Type Protecting Group.

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.

A trimethyllysine-containing trityl tag for solubilizing hydrophobic peptides.

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.

Solubilizing Trityl-Type Tag To Synthesize Asx/Glx-Containing Peptides.

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.

Development of Trityl Group Anchored Solubilizing Tags for Peptide and Protein Synthesis.

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.

The versatile use of solubilizing trityl tags for difficult peptide/protein synthesis.

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.

HAP-01, the first chromogenic substrate for Aspergillus oryzae acid protease.

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.

Ring-closing metathesis of unprotected peptides in water.

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.

Peptide self-cleavage at a canaline residue: application to a solubilizing tag system for native chemical ligation.

Canaline (Can) is a non-proteinogenic amino acid containing an aminooxy group in its side chain. Can-containing peptides can be synthesized by standard Fmoc SPPS using Fmoc-Can(2-Cl-Trt). Here, for the first time, a Can residue within a peptide sequence was found to spontaneously cleave the main chain amide bond under slightly acidic conditions (pH 4-5). Contrastingly, Can-containing peptides are completely stable under the acidic conditions for HPLC purification (pH ca. 2) and under the neutral conditions for native chemical ligation (NCL). Taking advantage of these unique pH-dependent properties of Can, a novel solubilizing tag system for NCL-mediated protein synthesis using (Lys/Arg)n-Can was developed.

Easy-to-Attach/Detach Solubilizing-Tag-Aided Chemical Synthesis of an Aggregative Capsid Protein.

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 hepatitis B virus capsid protein that is usually difficult to handle was synthesized successfully.

A Novel Prodrug of a γ-Glutamylcyclotransferase Inhibitor Suppresses Cancer Cell Proliferation in†vitro and Inhibits Tumor Growth in a Xenograft Mouse Model of Prostate Cancer.

γ-Glutamylcyclotransferase (GGCT) depletion inhibits cancer cell proliferation. However, whether the enzymatic activity of GGCT is critical for the regulation of cancer cell growth remains unclear. In this study, a novel diester-type cell-permeable prodrug, pro-GA, was developed based on the structure of N-glutaryl-l-alanine (GA), by structure optimization using temporary fluorophore-tagged prodrug candidates. The antiproliferative activity of pro-GA was demonstrated using GGCT-overexpressing NIH-3T3 cells and human cancer cells including MCF7, HL-60, and PC3 cells. By contrast, normal cells were not significantly affected by pro-GA treatment. Moreover, pro-GA administration exhibited anticancer effects in a xenograft model using immunocompromised mice inoculated with PC3 cells. These results indicate that the enzymatic activity of GGCT accelerates tumor growth and that GGCT inhibition is a promising therapeutic strategy for the treatment of GGCT-overexpressing tumors.

1,2,4-Triazole-aided native chemical ligation between peptide-N-acyl-N'-methyl-benzimidazolinone and cysteinyl peptide.

1,2,4-Triazole facilitated native chemical ligation (NCL) between peptide-MeNbz (MeNbz: N-acyl-N'-methyl-benzimidazolinone) and a cysteinyl peptide in the absence of thiol additives. The method enabled one-pot desulfurization and iodine oxidation after NCL. Additionally, the direct isolation of the target peptide from the NCL reaction mixture with an activated thiopropyl-Sepharose resin was achieved.

N-Sulfanylethylaminooxybutyramide (SEAoxy): A Crypto-Thioester Compatible with Fmoc Solid-Phase Peptide Synthesis.

An N-sulfanylethylaminooxybutyramide (SEAoxy) has been developed as a novel thioester equivalent for native chemical ligation. SEAoxy peptide was straightforwardly synthesized by conventional Fmoc solid-phase peptide synthesis without a problem. Moreover, SEAoxy peptide could be directly applied to native chemical ligation owing to the intramolecular N-to-S acyl shift that releases the peptide-thioester in situ. This methodology was successfully applied to the synthesis of two bioactive peptides.

Imidazole-Aided Native Chemical Ligation: Imidazole as a One-Pot Desulfurization-Amenable Non-Thiol-Type Alternative to 4-Mercaptophenylacetic Acid.

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.