Three-Dimensional Reconstruction of the Hepatitis C Virus Envelope Glycoprotein E1E2 Heterodimer by Electron Microscopic Analysis.

Despite the development of highly effective hepatitis C virus (HCV) treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins, E1 and E2, during the entry process, with E2 binding to cell receptors and E1 mediating endosomal fusion. The structure of E1E2 has only been partially resolved by X-ray crystallography of the core domain of E2 protein (E2c) and its complex with various neutralizing antibodies. Structural understanding of the E1E2 heterodimer in its native form can advance the design of candidates for HCV vaccine development. Here, we analyze the structure of the recombinant HCV E1E2 heterodimer with the aid of well-defined monoclonal anti-E1 and E2 antibodies, as well as a small-molecule chlorcyclizine-diazirine-biotin that can target and cross-link the putative E1 fusion domain. Three-dimensional (3D) models were generated after extensive 2D classification analysis with negative-stain single-particle data sets. We modeled the available crystal structures of the E2c and Fabs into 3D volumes of E1E2-Fab complexes based on the shape and dimension of the domain density. The E1E2 heterodimer exists in monomeric form and consists of a main globular body, presumably depicting the E1 and E2 stem/transmembrane domain, and a protruding structure representing the E2c region, based on anti-E2 Fab binding. At low resolution, a model generated from negative-stain analysis revealed the unique binding and orientation of individual or double Fabs onto the E1 and E2 components of the complex. Cryo-electron microscopy (cryo-EM) of the double Fab complexes resulted in a refined structural model of the E1E2 heterodimer, presented here. IMPORTANCE Recombinant HCV E1E2 heterodimer is being developed as a vaccine candidate. Using electron microscopy, we demonstrated unique features of E1E2 in complex with various neutralizing antibodies and small molecule inhibitors that are important to understanding its antigenicity and induction of immune response.

Ledipasvir/Sofosbuvir Is Effective as Sole Treatment of Porphyria Cutanea Tarda with Chronic Hepatitis C.

BACKGROUND AND AIMS: Chronic hepatitis C [CHC] is a risk factor for porphyria cutanea tarda [PCT]. To assess whether ledipasvir/sofosbuvir is effective for treating both PCT and CHC, we treated patients with CHC + PCT solely with ledipasvir/sofosbuvir and followed them for at least 1 year to assess cure of CHC and remission of PCT. METHODS: Between September 2017 and May 2020, 15 of 23 screened PCT + CHC patients were eligible and enrolled. All were treated with ledipasvir/sofosbuvir at recommended doses and durations, according to their stage of liver disease. We measured plasma and urinary porphyrins at baseline and monthly for the first 12 months and at 16, 20, and 24 mos. We measured serum HCV RNA at baseline, 8-12, and 20-24 mos. Cure of HCV was defined as no detectable serum HCV RNA ≥ 12 weeks after the end of treatment (EOT). Remission of PCT was defined clinically as no new blisters or bullae and biochemically as urinary uro- plus hepta-carboxyl porphyrins ≤ 100 mcg/g creatinine. RESULTS: All 15 patients, 13 of whom were men, were infected with HCV genotype 1. 2/15 withdrew or were lost to follow-up. Of the remaining 13, 12 achieved cure of CHC; 1 had complete virological response, followed by relapse of HCV after ledipasvir/sofosbuvir but was subsequently cured by treatment with sofosbuvir/velpatasvir. Of the 12 cured of CHC, all achieved sustained clinical remission of PCT. CONCLUSIONS: Ledipasvir/sofosbuvir [and likely other direct-acting antivirals] is an effective treatment for HCV in the presence of PCT and leads to clinical remission of PCT without additional phlebotomy or low-dose hydroxychloroquine treatment. TRIAL REGISTRATION: ClinicalTrials.gov NCT03118674.

Idiosyncratic drug-induced liver injury caused by givosiran in a patient with acute intermittent porphyria.

A 39-year-old woman with biochemically and clinically active acute intermittent porphyria (AIP) developed moderately severe liver injury after receiving her second dose of givosiran. Serologic evaluation ruled out hepatitis caused by viral, autoimmune, or other metabolic etiologies. The updated Roussel Uclaf Causality Assessment Method (RUCAM) score was 8 and the Revised Electronic Causality Assessment Method (RECAM) score for givosiran was 9. Results of liver tests returned to normal after givosiran was discontinued, and she has not received any more givosiran.

Recurrent symptoms of acute intermittent porphyria after biochemical normalization with givosiran-An ongoing clinical conundrum.

A 47-year-old woman with acute intermittent porphyria (AIP) has had recurring symptoms after achieving biochemical normalization of her urinary 5-aminolevulinic acid (ALA), porphobilinogen (PBG), and total porphyrins with givosiran. She has had normal liver tests, mildly decreased renal function, and sustained normal urinary ALA, PBG, and porphyrins with no rebound in her laboratory test results throughout treatment. She continues to tolerate monthly givosiran injections with no adverse effects, but she still experiences what she believes are acute porphyric attacks every 1-2 months.

Elagolix is porphyrogenic and may induce porphyric attacks in patients with the acute hepatic porphyrias.

Elagolix is an FDA-approved treatment for moderate-to-severe pain associated with endometriosis but has been associated with increased acute porphyric attacks in women with the acute hepatic porphyrias (AHPs). A fluorescence-based screening assay for drug porphyrogenicity in LMH cells indicates that elagolix is porphyrogenic; thus, elagolix should be avoided or used with caution in patients with the AHPs.

Eslicarbazepine acetate is porphyrogenic and should be used with caution in patients with the acute hepatic porphyrias.

Eslicarbazepine acetate, a third-generation antiepileptic drug (AED), has shown improved clinical response and safety in comparison to older generation AEDs for patients with partial-onset seizures. It is currently not known whether eslicarbazepine acetate is safe to use in patients with the acute hepatic porphyrias (AHPs) since a few first-generation AEDs, such as phenobarbital and carbamazepine, are known porphyrogenic agents. In this study, we used a recently published in vitro fluorescence-based screening assay to screen for porphyrogenicity in various agents. The assay confirmed that among the tested compounds used, allyl isopropyl acetamide, carbamazepine, eslicarbazepine acetate, and phenobarbital were porphyrogenic. Thus, eslicarbazepine acetate should be avoided if possible in patients with the AHPs, but if initiated, patients should be closely monitored and the drug should be discontinued if a porphyric exacerbation occurs.

Assessment of porphyrogenicity of drugs and chemicals in selected hepatic cell culture models through a fluorescence-based screening assay.

Compounds that induce 5-aminolevulinic acid [ALA] synthase-1 and/or cytochromes P-450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias [AHPs]. Currently, there is no simple, robust model used to assess and predict the porphyrogenicity of drugs and chemicals. Our aim was to develop a fluorescence-based in vitro assay for this purpose. We studied four different hepatic cell culture models: HepG2 cells, LMH cells, 3D HepG2 organoids, and 3D organoids of primary liver cells from people without known disease [normal human controls]. We took advantage of the fluorescent properties of protoporphyrin IX [PP], the last intermediate of the heme biosynthesis pathway, performing fluorescence spectrometry to measure the intensity of fluorescence emitted by these cells treated with selected compounds of importance to patients with AHPs. Among the four cell culture models, the LMH cells produced the highest fluorescence readings, suggesting that these cells retain more robust heme biosynthesis enzymes or that the other cell models may have lost their inducibility of ALA synthase-1 [ALAS-1]. Allyl isopropyl acetamide [AIA], a known potent porphyrogen and inducer of ALAS-1, was used as a positive control to help predict porphyrogenicity for tested compounds. Among the tested compounds (acetaminophen, acetylsalicylic acid, ß-estradiol, hydroxychloroquine sulfate, alpha-methyldopa, D (-) norgestrel, phenobarbital, phenytoin, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan), concentrations greater than 0.314 mM for norgestrel, phenobarbital, phenytoin, and sodium valproate produced fluorescence readings higher than the reading produced by the positive AIA control. Porphyrin accumulation was also measured by HPLC to confirm the validity of the assay. We conclude that LMH cell cultures in multi-well plates are an inexpensive, robust, and simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.

Spotlight on Givosiran as a Treatment Option for Adults with Acute Hepatic Porphyria: Design, Development, and Place in Therapy.

Small interfering ribonucleic acids [siRNAs] are short ribonucleic acid (RNA) fragments cleaved from double-stranded RNA molecules that target and bind to specific sequences on messenger RNA (mRNA), leading to their destruction. Therefore, the siRNA down-regulates the formation of selected mRNAs and their protein products. Givosiran is one such siRNA that uses this mechanism to treat acute hepatic porphyrias. Acute hepatic porphyrias are a group of rare, inherited metabolic disorders, characterized by acute potentially life-threatening attacks as well as chronic symptoms with a negative impact on quality of life. It has four types, each associated with distinct enzyme defects in the heme biosynthesis pathway in the liver. By targeting the expression of hepatic 5-aminolevulinic acid [ALA] synthase-1 [ALAS1], givosiran can down-regulate levels of toxic metabolites, leading to biochemical and clinical improvement. Givosiran selectively targets hepatocytes due to its linkage to N-acetylgalactosamine (GalNac) leading to its selective uptake via asialoglycoprotein receptors (ASGPR). We provide an up-to-date literature review regarding givosiran in the context of a clinical overview of the porphyrias, an overview of siRNAs for therapy of human disorders, the design and development of givosiran, key clinical trial results of givosiran for prevention of acute porphyric attacks, emerging concerns regarding chronic use of givosiran, and the overall management of acute hepatic porphyrias. These insights are important not only for the management of acute hepatic porphyrias but also for the emerging field of siRNAs and their role in novel therapies for various diseases.

A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells.

The CRISPR-Cas9 system has emerged as a powerful and efficient tool for genome editing. An important drawback of the CRISPR-Cas9 system is the constitutive endonuclease activity when Cas9 endonuclease and its sgRNA are co-expressed. This constitutive activity results in undesirable off-target effects that hinder studies using the system, such as probing gene functions or its therapeutic use in humans. Here, we describe a convenient method that allows temporal and tight control of CRISPR-Cas9 activity by combining transcriptional regulation of Cas9 expression and protein stability control of Cas9 in human stem cells. To achieve this dual control, we combined the doxycycline-inducible system for transcriptional regulation and FKBP12-derived destabilizing domain fused to Cas9 for protein stability regulation. We showed that approximately 5%-10% of Cas9 expression was observed when only one of the two controls was applied. By combining two systems, we markedly lowered the baseline Cas9 expression and limited the exposure time of Cas9 endonuclease in the cell, resulting in little or no undesirable on- or off-target effects. We anticipate that this dual conditional CRISPR-Cas9 system can serve as a valuable tool for systematic characterization and identification of genes for various pathological processes.

Chlorcyclizine Inhibits Viral Fusion of Hepatitis C Virus Entry by Directly Targeting HCV Envelope Glycoprotein 1.

Chlorcyclizine (CCZ) is a potent hepatitis C virus (HCV) entry inhibitor, but its molecular mechanism is unknown. Here, we show that CCZ directly targets the fusion peptide of HCV E1 and interferes with the fusion process. Generation of CCZ resistance-associated substitutions of HCV in vitro revealed six missense mutations in the HCV E1 protein, five being in the putative fusion peptide. A viral fusion assay demonstrated that CCZ blocked HCV entry at the membrane fusion step and that the mutant viruses acquired resistance to CCZ's action in blocking membrane fusion. UV cross-linking of photoactivatable CCZ-diazirine-biotin in both HCV-infected cells and recombinant HCV E1/E2 protein demonstrated direct binding to HCV E1 glycoprotein. Mass spectrometry analysis revealed that CCZ cross-linked to an E1 sequence adjacent to the putative fusion peptide. Docking simulations demonstrate a putative binding model, wherein CCZ binds to a hydrophobic pocket of HCV E1 and forms extensive interactions with the fusion peptide.

Fluoxazolevir inhibits hepatitis C virus infection in humanized chimeric mice by blocking viral membrane fusion.

Fluoxazolevir is an aryloxazole-based entry inhibitor of hepatitis C virus (HCV). We show that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 to prevent fusion. Nine of ten fluoxazolevir resistance-associated substitutions are in envelope protein 1, and four are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A 4-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotypes 1b, 2a or 3 resulted in a 2-log reduction in viraemia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3 log of viraemia but is associated with the emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment.