A simeprevir-inducible molecular switch for the control of cell and gene therapies.

Chemical inducer of dimerization (CID) modules can be used effectively as molecular switches to control biological processes, and thus there is significant interest within the synthetic biology community in identifying novel CID systems. To date, CID modules have been used primarily in engineering cells for in vitro applications. To broaden their utility to the clinical setting, including the potential to control cell and gene therapies, the identification of novel CID modules should consider factors such as the safety and pharmacokinetic profile of the small molecule inducer, and the orthogonality and immunogenicity of the protein components. Here we describe a CID module based on the orally available, approved, small molecule simeprevir and its target, the NS3/4A protease from hepatitis C virus. We demonstrate the utility of this CID module as a molecular switch to control biological processes such as gene expression and apoptosis in vitro, and show that the CID system can be used to rapidly induce apoptosis in tumor cells in a xenograft mouse model, leading to complete tumor regression.

Anti-hepatitis C virus drug simeprevir: a promising antimicrobial agent against MRSA.

Staphylococcus aureus is a major human pathogen, and the appearance of methicillin-resistant S. aureus (MRSA) renders S. aureus infections more challenging to treat. Therefore, new antimicrobial drugs are urgently needed to combat MRSA infections. Drug repurposing is an effective and feasible strategy. Here, we reported that the clinically approved anti-hepatitis C virus drug simeprevir had strong antibacterial activity against MRSA, with a minimum inhibitory concentration of 2-8 µg/mL. Simeprevir did not easily induce in vitro resistance. In addition, simeprevir significantly prevented S. aureus biofilm formation. Furthermore, simeprevir displayed limited toxicity in in vitro and in vivo assays. Moreover, simeprevir showed synergistic antimicrobial effects against both type and clinical strains of S. aureus. Simeprevir combined with gentamicin effectively reduced the bacterial burden in an MRSA-infected subcutaneous abscess mouse model. Results from a series of experiments, including membrane permeability assay, membrane potential assay, intracellular ATP level assay, and electron microscope observation, demonstrated that the action of simeprevir may be by disrupting bacterial cell membranes. Collectively, these results demonstrated the potential of simeprevir as an antimicrobial agent for the treatment of MRSA infections. KEY POINTS: • Simeprevir showed strong antibacterial activity against MRSA. • The antibacterial mechanism of simeprevir was mediated by membrane disruption and intracellular ATP depletion. • In vitro and in vivo synergistic antimicrobial efficacy between simeprevir and gentamicin was found.

Repurposing simeprevir, calpain inhibitor IV and a cathepsin F inhibitor against SARS-CoV-2 and insights into their interactions with Mpro.

The world has come to a sudden halt due to the incessant spread of a viral pneumonia dubbed COVID-19 caused by the beta-coronavirus, SARS-CoV-2. The main protease of SARS-CoV-2 plays a key role in the replication and propagation of the virus in the host cells. Inhibiting the protease blocks the replication of the virus; therefore it is considered as an attractive therapeutic target. Here we describe the screening of the DrugBank database, a public repository for small molecule therapeutics, to identify approved or experimental phase drugs that can be repurposed against the main protease of SARS-CoV-2. The initial screening was performed on more than 13,000 drug entries in the target database using an energy optimised pharmacophore hypothesis AARRR. A sub-set of the molecules selected based on the fitness score was further screened using molecular docking by sequentially filtering the molecules through the high throughput virtual screening, extra precision and standard precision docking modalities. The best hits were subjected to binding free energy estimation using the MM-GBSA method. Approved drugs viz, Cobicistat, Larotrectinib and Simeprevir were identified as potential candidates for repurposing. Drugs in the discovery phase identified as inhibitors include the known cysteine protease inhibitors, Calpain inhibitor IV and an experimental cathepsin F inhibitor. In order to analyse the stability of the binding interactions, the known cysteine protease inhibitors viz, Simeprevir, calpain inhibitor IV and the cathepsin F inhibitor in complex Mpro were subjected to molecular dynamics simulations at 100 ns. Based on the results Simeprevir was found to be a strong inhibitor of SARS-CoV-2 Mpro.Communicated by Ramaswamy H. Sarma.

Components of the phosphatidylserine endoplasmic reticulum to plasma membrane transport mechanism as targets for KRAS inhibition in pancreatic cancer.

KRAS is mutated in 90% of human pancreatic ductal adenocarcinomas (PDACs). To function, KRAS must localize to the plasma membrane (PM) via a C-terminal membrane anchor that specifically engages phosphatidylserine (PtdSer). This anchor-binding specificity renders KRAS-PM localization and signaling capacity critically dependent on PM PtdSer content. We now show that the PtdSer lipid transport proteins, ORP5 and ORP8, which are essential for maintaining PM PtdSer levels and hence KRAS PM localization, are required for KRAS oncogenesis. Knockdown of either protein, separately or simultaneously, abrogated growth of KRAS-mutant but not KRAS-wild-type pancreatic cancer cell xenografts. ORP5 or ORP8 knockout also abrogated tumor growth in an immune-competent orthotopic pancreatic cancer mouse model. Analysis of human datasets revealed that all components of this PtdSer transport mechanism, including the PM-localized EFR3A-PI4KIIIα complex that generates phosphatidylinositol-4-phosphate (PI4P), and endoplasmic reticulum (ER)-localized SAC1 phosphatase that hydrolyzes counter transported PI4P, are significantly up-regulated in pancreatic tumors compared to normal tissue. Taken together, these results support targeting PI4KIIIα in KRAS-mutant cancers to deplete the PM-to-ER PI4P gradient, reducing PM PtdSer content. We therefore repurposed the US Food and Drug Administration-approved hepatitis C antiviral agent, simeprevir, as a PI4KIIIα inhibitor In a PDAC setting. Simeprevir potently mislocalized KRAS from the PM, reduced the clonogenic potential of pancreatic cancer cell lines in vitro, and abrogated the growth of KRAS-dependent tumors in vivo with enhanced efficacy when combined with MAPK and PI3K inhibitors. We conclude that the cellular ER-to-PM PtdSer transport mechanism is essential for KRAS PM localization and oncogenesis and is accessible to therapeutic intervention.

Anti-HCV and anti-malaria agent, potential candidates to repurpose for coronavirus infection: Virtual screening, molecular docking, and molecular dynamics simulation study.

AIMS: Coronavirus disease 2019 (COVID-19) has appeared in Wuhan, China but the fast transmission has led to its widespread prevalence in various countries, which has made it a global concern. Another concern is the lack of definitive treatment for this disease. The researchers tried different treatment options which are not specific. The current study aims to identify potential small molecule inhibitors against the main protease protein of SARS-CoV-2 by the computational approach. MAIN METHODS: In this study, a virtual screening procedure employing docking of the two different datasets from the ZINC database, including 1615 FDA approved drugs and 4266 world approved drugs were used to identify new potential small molecule inhibitors for the newly released crystal structure of main protease protein of SARS-CoV-2. In the following to validate the docking result, molecular dynamics simulations were applied on selected ligands to identify the behavior and stability of them in the binding pocket of the main protease in 150 nanoseconds (ns). Furthermore, binding energy using the MMPBSA approach was also calculated. KEY FINDINGS: The result indicates that simeprevir (Hepatitis C virus NS3/4A protease inhibitor) and pyronaridine (antimalarial agent) could fit well to the binding pocket of the main protease and because of some other beneficial features including broad-spectrum antiviral properties and ADME profile, they might be a promising drug candidate for repurposing to the treatment of COVID-19. SIGNIFICANCE: Simeprevir and pyronaridine were selected by the combination of virtual screening and molecular dynamics simulation approaches as a potential candidate for treatment of COVID-19.

Identification of potential inhibitors against SARS-CoV-2 by targeting proteins responsible for envelope formation and virion assembly using docking based virtual screening, and pharmacokinetics approaches.

WHO has declared the outbreak of COVID-19 as a public health emergency of international concern. The ever-growing new cases have called for an urgent emergency for specific anti-COVID-19 drugs. Three structural proteins (Membrane, Envelope and Nucleocapsid protein) play an essential role in the assembly and formation of the infectious virion particles. Thus, the present study was designed to identify potential drug candidates from the unique collection of 548 anti-viral compounds (natural and synthetic anti-viral), which target SARS-CoV-2 structural proteins. High-end molecular docking analysis was performed to characterize the binding affinity of the selected drugs-the ligand, with the SARS-CoV-2 structural proteins, while high-level Simulation studies analyzed the stability of drug-protein interactions. The present study identified rutin, a bioflavonoid and the antibiotic, doxycycline, as the most potent inhibitor of SARS-CoV-2 envelope protein. Caffeic acid and ferulic acid were found to inhibit SARS-CoV-2 membrane protein while the anti-viral agent's simeprevir and grazoprevir showed a high binding affinity for nucleocapsid protein. All these compounds not only showed excellent pharmacokinetic properties, absorption, metabolism, minimal toxicity and bioavailability but were also remain stabilized at the active site of proteins during the MD simulation. Thus, the identified lead compounds may act as potential molecules for the development of effective drugs against SARS-CoV-2 by inhibiting the envelope formation, virion assembly and viral pathogenesis.

A proof-of-concept study in HCV-infected Huh7.5 cells for shortening the duration of DAA-based triple treatment regimens.

With the development of more effective direct-acting antivirals (DAAs), dual- or triple-therapy regimens represent the major strategy used to cure chronic hepatitis C virus (HCV) infection. Thus, shorter treatment duration regimens with low burden, few adverse effects and good patient adherence are urgently needed. This study theoretically demonstrates a proof-of-concept approach for shortening therapy duration by examining HCV-infected Huh7.5 cells after treatment with a high or low fixed dose of three DAAs (simeprevir + daclatasvir + sofosbuvir) for 6-15 days. The results demonstrated that HCV-infected Huh7.5 cells achieved an ultrarapid virologic response with undetectable HCV RNA and protein and were cured after treatment with the triple-therapy regimen for 15 days. When the treatment duration was shortened, virologic relapse might occur after treatment with a low fixed dose of the three DAAs for 9 days and did occur after treatment with a low fixed dose for 6 days, although HCV was below detectable levels at the end of treatment. However, virologic relapse could be avoided with treatment of a high fixed dose of the three DAAs for 9 or 6 days. Although a virologic breakthrough occurred after an intermittent treatment regimen at the low fixed dose, the high fixed dose cured HCV-positive Huh7.5 cells with intermittent treatment. In conclusion, HCV is persistently present below detectable levels in HCV-infected Huh7.5 cells for a long time after treatment, and a shortened therapy duration is associated with an increased risk of virologic relapse, but virologic relapse or breakthrough might be avoided by treatment with a combination of more highly effective DAAs.

Effectiveness and tolerability of direct-acting antivirals for chronic hepatitis C patients in a Southern state of Brazil

ABSTRACT Background This study aimed to evaluate the clinical effectiveness in terms of sustained virological response and tolerability of available second generation direct-acting antivirals in Brazilian patients. Methods This was a retrospective observational study conducted in six centers in Southern Brazil. The sample comprised adult patients who were chronically infected with hepatitis C virus, regardless of virus genotype, fibrosis stage, or prior treatment. Statistical analysis was performed to compare the effectiveness among the treatments, and also to uncover the factors influencing the achievement of sustained virological response. Results A total of 296 patients were included in the study, with the majority receiving sofosbuvir with daclatasvir (59%) or sofosbuvir with simeprevir (26%). Overall sustained virological response rates were approximately 91.6%. For genotype 1, sofosbuvir with daclatasvir had an sustained virological response rate of approximately 95%, while the sustained virological response rate of sofosbuvir with simeprevir was 92%; this difference was statistically significant only for subtype 1b. The only treatment used for genotype 3 patients was sofosbuvir with daclatasvir, and lower rates of sustained virological response were observed for this group, compared to genotype 1 (84% versus 95%, p < 0.05). Apart from this difference between genotypes, and a difference between patients who achieved rapid virologic response compared with those who did not, there were no other statistically significant factors associated with sustained virological response. Conclusions The results point to the effectiveness of second-generation direct-acting antivirals in hepatitis C virus Brazilian patients, especially those with genotype 1. Furthermore, that patients with genotype 3 need more attention and adjustments in available treatment options.

Rapidity and Severity of Hemoglobin Decreasing Associated with Erythrocyte Inosine Triphosphatase Activity and ATP Concentration during Chronic Hepatitis C Treatment.

The purpose of this study was to evaluate the association between therapy-induced hemoglobin (Hb) decreasing rapidity and severity with erythrocyte inosine triphosphatase (ITPase) activity and ATP concentration in chronic hepatitis C patients receiving chronic hepatitis C (HCV) treatment. Forty-three Japanese patients were included in the study. Erythrocyte ITPase activity before therapy was determined by HPLC-UV. Erythrocyte ATP concentrations before and during therapy were determined by luciferase assay. Genotyping for ITPA 94C>A (rs1127354) and IVS2+21 A>C (rs7270101) was conducted using TaqMan probes. The median ITPase activity (µmol/h/g hemoglobin) of ITPA 94 CC, CA, and AA genotypes was 136.8 (range, 80.4-289.6), 41.1 (24.3-93.1), and 11.8, respectively. ITPase activity and Hb decreasing showed a significantly inverse relationship at therapeutic weeks 2, 4, and 6 (p<0.01). Erythrocyte ATP concentration was decreased by therapy, and Hb decreasing was significantly and inversely correlated with erythrocyte ATP concentration at week 4 and after week 8 (p<0.001 and 0.05, respectively). ATP concentration for patients with ITPA 94CA was significantly lower than ITPA 94CC at week 4 (p=0.045). We concluded that ITPase activity plays an important function and that ATP concentration changes due to therapy are related to the Hb decreasing mechanism in the early period of therapy with HCV treatment.

Development and Validation of Two Screening Assays for the Hepatitis C Virus NS3 Q80K Polymorphism Associated with Reduced Response to Combination Treatment Regimens Containing Simeprevir.

Persons with hepatitis C virus (HCV) genotype 1a (GT1a) infections harboring a baseline Q80K polymorphism in nonstructural protein 3 (NS3) have a reduced virologic response to simeprevir in combination with pegylated interferon-alfa and ribavirin. We aimed to develop, validate, and freely disseminate an NS3 clinical sequencing assay to detect the Q80K polymorphism and potentially other HCV NS3 drug resistance mutations. HCV RNA was extracted from frozen plasma using a NucliSENS easyMAG automated nucleic acid extractor, amplified by nested reverse transcription-PCR, and sequenced using Sanger and/or next-generation (MiSeq) methods. Sanger chromatograms were analyzed using in-house software (RECall), and nucleotide mixtures were called automatically. MiSeq reads were iteratively mapped to the H77 reference genome, and consensus NS3 sequences were generated with nucleotides present at >20% called as mixtures. The accuracy, precision, and sensitivity for detecting the Q80K polymorphism were assessed in 70 samples previously sequenced by an external laboratory. A comparison of the sequences generated by the Sanger and MiSeq methods with those determined by an external lab revealed >98.5% nucleotide sequence concordance and zero discordant calls of the Q80K polymorphism. The results were both highly repeatable and reproducible (>99.7% nucleotide concordance and 100% Q80K concordance). The limits of detection (>2 and ∼5 log10 IU/ml for the Sanger and MiSeq assays, respectively) are sufficiently low to allow genotyping in nearly all chronically infected treatment-naive persons. No systematic bias in the under- or overamplification of minority variants was observed. Coinfection with other viruses (e.g., HIV and hepatitis B virus [HBV]) did not affect the assay results. The two independent HCV NS3 sequencing assays with the automated analysis procedures described here are useful tools to screen for the Q80K polymorphism and other HCV protease inhibitor drug resistance mutations.