A single site study to investigate the current prevalence of anti-hepatitis C virus antibody among substance use disorder patients in Hiroshima-Insufficient testing and diagnosis.

AIMS: Hepatitis C virus (HCV) infection among drug users presents an important public health problem; however, little recognition and few approaches to address this issue in Japan. This study was conducted to investigate the current disease status by assessing anti-HCV antibody (Ab) seroprevalence among people who inject drugs (PWIDs) and people who use drugs (PWUDs) in Hiroshima, Japan. METHODS: This study was a psychiatric single-site chart review in patients with drug abuse problems in the Hiroshima region. The primary outcome was anti-HCV Ab prevalence among PWIDs who underwent anti-HCV Ab testing. The secondary outcomes included the prevalence of anti-HCV Ab among PWUDs who underwent anti-HCV Ab testing and the proportion of patients who underwent anti-HCV Ab examination. RESULTS: A total of 222 PWUD patients were enrolled. Among these, 16 patients (7.2%) had records of injection drug use (PWIDs). Eleven (68.8%) of the 16 PWIDs received anti-HCV Ab tests, and 4 (36.4%, 4/11) were anti-HCV Ab-positive. Among 222 PWUDs, 126 (56.8%) patients received anti-HCV Ab tests, and 57 of these patients (45.2%, 57/126) were anti-HCV Ab-positive. CONCLUSION: The prevalence of anti-HCV Ab among PWIDs and PWUDs who visited the study site was higher than the general population, which was 2.2% among hospitalized patients between May 2018 and November 2019. Considering the World Health Organization's (WHO) elimination goal and recent advances in HCV treatment, patients with drug abuse experience should be encouraged to take HCV tests and consult hepatologists for further investigations and treatment if they are positive for anti-HCV Ab.

Chemical Synthesis of Interleukin-2 and Disulfide Stabilizing Analogues.

Chemical protein synthesis allows the construction of well-defined structural variations and facilitates the development of deeper understanding of protein structure-function relationships and new protein engineering strategies. Herein, we report the chemical synthesis of interleukin-2 (IL-2) variants on a multimilligram scale and the formation of non-natural disulfide mimetics that improve stability against reduction. The synthesis was accomplished by convergent KAHA ligations; the acidic conditions of KAHA ligation proved to be valuable for the solubilization of the hydrophobic segments of IL-2. The bioactivity of the synthetic IL-2 and its analogues were shown to be equipotent to recombinant IL-2 and exhibit improved stability against reducing agents.

Antibiotic Discovery with Synthetic Fermentation: Library Assembly, Phenotypic Screening, and Mechanism of Action of ß-Peptides Targeting Penicillin-Binding Proteins.

In analogy to biosynthetic pathways leading to bioactive natural products, synthetic fermentation generates mixtures of molecules from simple building blocks under aqueous, biocompatible conditions, allowing the resulting cultures to be directly screened for biological activity. In this work, a novel ß-peptide antibiotic was successfully identified using the synthetic fermentation platform. Phenotypic screening was carried out in an initially random fashion, allowing simple identification of active cultures. Subsequent deconvolution, focused screening, and structure-activity relationship studies led to the identification of a potent antimicrobial peptide, showing strong selectivity for our model system Bacillus subtilis over human HEK293 cells. To determine the antibacterial mechanism of action, a peptide probe bearing a photoaffinity tag was readily synthesized through the use of appropriate synthetic fermentation building blocks and utilized for target identification using a quantitative mass spectrometry-based strategy. The chemoproteomic approach led to the identification of a number of bacterial membrane proteins as prospective targets. These findings were validated through binding affinity studies with penicillin-binding protein 4 using microscale thermophoresis, with the bioactive peptide showing a dissociation constant ( Kd) in the nanomolar range. Through these efforts, we provide a proof of concept for the synthetic fermentation approach presented here as a new strategy for the phenotypic discovery of novel bioactive compounds.

Facile folding of insulin variants bearing a prosthetic C-peptide prepared by α-ketoacid-hydroxylamine (KAHA) ligation.

The chemical synthesis of insulin is an enduring challenge due to the hydrophobic peptide chains and construction of the correct intermolecular disulfide pattern. We report a new approach to the chemical synthesis of insulin using a short, traceless, prosthetic C-peptide that facilitates the formation of the correct disulfide pattern during folding and its removal by basic treatment. The linear precursor is assembled by an ester forming α-ketoacid-hydroxylamine (KAHA) ligation that provides access to the linear insulin precursors in good yield from two readily prepared segments. This convergent and flexible route provides access to various human, mouse, and guinea pig insulins containing a single homoserine mutation that shows no detrimental effect on the biological activities.

De Novo Macrocyclic Peptide Inhibitors of Hepatitis B Virus Cellular Entry.

Hepatitis B virus (HBV) constitutes a significant public health burden, and currently available treatment options are not generally curative, necessitating the development of new therapeutics. Here we have applied random non-standard peptide integrated discovery (RaPID) screening to identify small macrocyclic peptide inhibitors of HBV entry that target the cell-surface receptor for HBV, sodium taurocholate cotransporting polypeptide (NTCP). In addition to their anti-HBV activity, these molecules also inhibit cellular entry by the related hepatitis D virus (HDV), and are active against diverse strains of HBV (including clinically relevant nucleos(t)ide analog-resistant and vaccine escaping strains). Importantly, these macrocyclic peptides, in contrast to other NTCP-binding HBV entry inhibitors, exhibited no inhibition of NTCP-mediated bile acid uptake, making them appealing candidates for therapeutic development.

MA026, an anti-hepatitis C virus compound, opens tight junctions of the epithelial cell membrane.

MA026 is an antiviral natural compound against hepatitis C virus (HCV). It was recently reported that MA026 binds claudin-1 (CLDN1) and inhibits HCV infection. Although CLDN1 is an important component of tight junctions (TJ) in the epithelial cell layer, the effects of MA026 on the TJ barrier function remained to be revealed. Here we report that MA026 irreversibly opens the TJ. MA026 irreversibly increased FD4 permeability and decreased transepithelial electrical resistance (TER) for at least 5 h. Although MA026 increased Ca2+ influx in layered MDCKII cells, the Ca2+ influx was less than that of capsaicin, a reversible TJ opener. Moreover, MA026 did not induce the dephosphorylation of cofilin and reorganization of F-actin structure. Although the mechanism is left to be disclosed, these results suggest that MA026 is a novel irreversible TJ opener probably by targeting CLDN1.

Cyclosporin derivatives inhibit hepatitis B virus entry without interfering with NTCP transporter activity.

BACKGROUND & AIMS: The sodium taurocholate co-transporting polypeptide (NTCP) is the main target of most hepatitis B virus (HBV) specific entry inhibitors. Unfortunately, these agents also block NTCP transport of bile acids into hepatocytes, and thus have the potential to cause adverse effects. We aimed to identify small molecules that inhibit HBV entry while maintaining NTCP transporter function. METHODS: We characterized a series of cyclosporine (CsA) derivatives for their anti-HBV activity and NTCP binding specificity using HepG2 cells overexpressing NTCP and primary human hepatocytes. The four most potent derivatives were tested for their capacity to prevent HBV entry, but maintain NTCP transporter function. Their antiviral activity against different HBV genotypes was analysed. RESULTS: We identified several CsA derivatives that inhibited HBV infection with a sub-micromolar IC50. Among them, SCY446 and SCY450 showed low activity against calcineurin (CN) and cyclophilins (CyPs), two major CsA cellular targets. This suggested that instead, these compounds interacted directly with NTCP to inhibit viral attachment to host cells, and have no immunosuppressive function. Importantly, we found that SCY450 and SCY995 did not impair the NTCP-dependent uptake of bile acids, and inhibited multiple HBV genotypes including a clinically relevant nucleoside analog-resistant HBV isolate. CONCLUSIONS: This is the first example of small molecule selective inhibition of HBV entry with no decrease in NTCP transporter activity. It suggests that the anti-HBV activity can be functionally separated from bile acid transport. These broadly active anti-HBV molecules are potential candidates for developing new drugs with fewer adverse effects. LAY SUMMARY: In this study, we identified new compounds that selectively inhibited hepatitis B virus (HBV) entry, and did not impair bile acid uptake. Our evidence offers a new strategy for developing anti-HBV drugs with fewer side effects.

A Novel Tricyclic Polyketide, Vanitaracin A, Specifically Inhibits the Entry of Hepatitis B and D Viruses by Targeting Sodium Taurocholate Cotransporting Polypeptide.

UNLABELLED: Anti-hepatitis B virus (HBV) drugs are currently limited to nucleos(t)ide analogs (NAs) and interferons. A challenge of drug development is the identification of small molecules that suppress HBV infection from new chemical sources. Here, from a fungus-derived secondary metabolite library, we identify a structurally novel tricyclic polyketide, named vanitaracin A, which specifically inhibits HBV infection. Vanitaracin A inhibited the viral entry process with a submicromolar 50% inhibitory concentration (IC50) (IC50 = 0.61 ± 0.23 µM), without evident cytotoxicity (50% cytotoxic concentration of >256 µM; selectivity index value of >419) in primary human hepatocytes. Vanitaracin A did not affect the HBV replication process. This compound was found to directly interact with the HBV entry receptor sodium taurocholate cotransporting polypeptide (NTCP) and impaired its bile acid transport activity. Consistent with this NTCP targeting, antiviral activity of vanitaracin A was observed with hepatitis D virus (HDV) but not hepatitis C virus. Importantly, vanitaracin A inhibited infection by all HBV genotypes tested (genotypes A to D) and clinically relevant NA-resistant HBV isolate. Thus, we identified a fungal metabolite, vanitaracin A, which was a potent, well-tolerated, and broadly active inhibitor of HBV and HDV entry. This compound, or its related analogs, could be part of an antiviral strategy for preventing reinfection with HBV, including clinically relevant nucleos(t)ide analog-resistant virus. IMPORTANCE: For achieving better treatment and prevention of hepatitis B virus (HBV) infection, anti-HBV agents targeting a new molecule are in great demand. Although sodium taurocholate cotransporting polypeptide (NTCP) has recently been reported to be an essential host factor for HBV entry, there is a limited number of reports that identify new compounds targeting NTCP and inhibiting HBV entry. Here, from an uncharacterized chemical library, we isolated a structurally new compound, named vanitaracin A, which inhibited the process of entry of HBV and hepatitis D virus (HDV). This compound was suggested to directly interact with NTCP and inhibit its transporter activity. Importantly, vanitaracin A inhibited the entry of all HBV genotypes examined and of a clinically relevant nucleos(t)ide analog-resistant HBV isolate.

Total synthesis and anti-hepatitis C virus activity of MA026.

The first total synthesis of MA026 and the identification of its candidate target protein for anti-hepatitis C virus activity are presented. MA026, a novel lipocyclodepsipeptide isolated from the fermentation broth of Pseudomonas sp. RtIB026, consists of a cyclodepsipeptide, a chain peptide, and an N-terminal (R)-3-hydroxydecanoic acid. The first subunit, side chain 2, was prepared by coupling fatty acid moiety 4 with tripeptide 5. The key macrocyclization of the decadepsipeptide at L-Leu(10)-D-Gln(11) provided the second subunit, cyclodepsipeptide 3. Late-stage condensation of the two key subunits and final deprotection afforded MA026. This convergent, flexible, solution-phase synthesis will be invaluable in generating MA026 derivatives for future structure-activity relationship studies. An infectious hepatitis C virus (HCV) cell culture assay revealed that MA026 suppresses HCV infection into host hepatocytes by inhibiting the entry process in a dose-dependent manner. Phage display screening followed by surface plasmon resonance (SPR) binding analyses identified claudin-1, an HCV entry receptor, as a candidate target protein of MA026.

The antitumor agent doxorubicin binds to Fanconi anemia group F protein.

Doxorubicin, a commonly used cancer chemotherapy agent, elicits several potent biological effects, including synergistic-antitumor activity in combination with cisplatin. However, the mechanism of this synergism remains obscure. Here, we employed an improved T7 phage display screening method to identify Fanconi anemia group F protein (FANCF) as a doxorubicin-binding protein. The FANCF-doxorubicin interaction was confirmed by pull-down assay and SPR analysis. FANCF is a component of the Fanconi anemia complex, which monoubiquitinates D2 protein of Fanconi anemia group as a cellular response against DNA cross-linkers such as cisplatin. We observed that the monoubiquitination was inhibited by doxorubicin treatment.

Total synthesis of (+)-Sch 725680: inhibitor of mammalian A-, B-, and Y-family DNA polymerases.

The total synthesis of (+)-Sch 725680, a member of the hydrogenated azaphilone family, has been accomplished. The synthesis confirmed the absolute configuration and biological activities of the natural product. A key reaction to construct a hydrogenated azaphilone core skeleton is a Ti-mediated aldol reaction.

Exploration of the binding proteins of perfluorooctane sulfonate by a T7 phage display screen.

Perfluorooctane sulfonate (PFOS) is a pollutant widely found throughout nature and is toxic to animals. We created a PFOS analogue on a polyethylene glycol polyacrylamide copolymer and isolated peptides that preferentially bound the PFOS analogue using a T7 phage display system. Bioinformatic analysis using the FASTAskan program on the RELIC bioinformatics server showed several human proteins that likely bound PFOS. Among them, we confirmed binding between PFOS and a recombinant soluble form of monocyte differentiation antigen CD14 (sCD14) by a surface plasmon biosensor. Furthermore, PFOS inhibited TNF-α production induced by the sCD14 in mouse macrophage RAW264.7 cells.