Synchronous spectrofluorimetry and chemometric modeling: A synergistic approach for analyzing simeprevir and daclatasvir, with application to pharmacokinetics evaluation.

Simeprevir and daclatasvir represent a cornerstone in the management of Hepatitis C Virus infection, a global health concern that affects millions of people worldwide. In this study, we propose a synergistic approach combining synchronous spectrofluorimetry and chemometric modeling i.e. Partial Least Squares (PLS-1) for the analysis of simeprevir and daclatasvir in different matrices. Moreover, the study employs firefly algorithms to further optimize the chemometric models via selecting the most informative features thus improving the accuracy and robustness of the calibration models. The firefly algorithm was able to reduce the number of selected wavelengths to 47-44% for simeprevir and daclatasvir, respectively offering a fast and sensitive technique for the determination of simeprevir and daclatasvir. Validation results underscore the models' effectiveness, as evidenced by recovery rates close to 100% with relative root mean square error of prediction (RRMSEP) of 2.253 and 2.1381 for simeprevir and daclatasvir, respectively. Moreover, the proposed models have been applied to determine the pharmacokinetics of simeprevir and daclatasvir, providing valuable insights into their distribution and elimination patterns. Overall, the study demonstrates the effectiveness of synchronous spectrofluorimetry coupled with multivariate calibration optimized by firefly algorithms in accurately determining and quantifying simeprevir and daclatasvir in HCV antiviral treatment, offering potential applications in pharmaceutical formulation analysis and pharmacokinetic studies for these drugs.

Simeprevir restores the anti-Staphylococcus activity of polymyxins.

Methicillin-resistant Staphylococcus aureus (MRSA) infection poses a severe threat to global public health due to its high mortality. Currently, polymyxins are mainly used for the treatment of Gram-negative bacterial-related infection, while exhibiting limited antibacterial activities against Staphylococcus aureus (S. aureus). However, the combination of antibiotics with antibiotic adjuvants is a feasible strategy for the hard-treated infection and toxicity reducing. We will investigate the antibacterial activity of simeprevir (SIM), which treated for genotype 1 and 4 chronic hepatitis C, combined with polymyxins against MRSA through high-throughput screening technology. In our study, the synergistic antibacterial effect of SIM and polymyxins against S. aureus in vitro was found by checkerboard assay and time-growth curve. The cytotoxicity of SIM combined with polymyxin B sulfate [PB(S)] or polymyxin E (PE) in vitro was evaluated using CCK-8, human RBC hemolysis and scratch assays. In addition, we investigated the eradication of biofilm formation of S. aureus by biofilm inhibition assay and the killing of persister cells. Moreover, we evaluated the therapeutic effect and in vivo toxicity of the combination against MRSA in murine subcutaneous abscess model. Furthermore, it was preliminarily found that SIM significantly enhanced the destruction of MRSA membrane by SYTOX Green and DISC3(5) probes. In summary, these results reveal that the therapy of SIM combined with polymyxins (especially PE) is promising for the treatment of MRSA infection.

Inhibitory effects of simeprevir on Staphylococcusepidermidis and itsbiofilm in vitro. / è¥¿å’ªåŒ¹éŸ¦å¯¹è¡¨çš®è‘¡è„çƒèŒåŠå ¶ç”Ÿç‰©è†œçš„ä½“å¤–æŠ‘åˆ¶ä½œç”¨.

OBJECTIVES: Staphylococcus epidermidis (S. epidermidis) is a Gram-positive opportunistic pathogen that often causes hospital infections. With the abuse of antibiotics, the resistance of S. epidermidis gradually increases, and drug repurposing has become a research hotspot in the treating of refractory drug-resistant bacterial infections. This study aims to study the antimicrobial and antibiofilm effects of simeprevir, an antiviral hepatitis drug, on S. epidermidis in vitro. METHODS: The micro-dilution assay was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of simeprevir against S. epidermidis. Crystal violet staining assay was used to detect the biofilm inhibitory effect of simeprevir. The antimicrobial activity of simeprevir against S. epidermidis and its biofilm were explored by SYTO9/PI fluorescent staining. The combined effect between simeprevir and gentamycin was assessed by checkerboard assay and was confirmed by time-inhibition assay. RESULTS: Simeprevir showed significant antimicrobial effects against S. epidermidis type strains and clinical isolates with the MIC and MBC at 2-16 µg/mL and 4-32 µg/mL, respectively. The antimicrobial effects of simeprevir were confirmed by SYTO9/PI staining. Simeprevir at MIC could significantly inhibit and break the biofilm on cover slides. Similarly, simeprevir also significantly inhibit the biofilm formation on the surface of urine catheters either in TSB [from (0.700±0.020) to (0.050±0.004)] (t=54.03, P<0.001), or horse serum [from (1.00±0.02) to (0.13±0.01)] (t=82.78, P<0.001). Synergistic antimicrobial effect was found between simeprevir and gentamycin against S. epidermidis with the fractional inhibitory concentration index of 0.5. CONCLUSIONS: Simeprevir shows antimicrobial effect and anti-biofilm activities against S. epidermidis.

Mathematical processing of absorption as green smart spectrophotometric methods for concurrent assay of hepatitis C antiviral drugs, Sofosbuvir and Simeprevir: application to combined pharmaceutical dosage forms and evaluation of the method greenness.

The present work was developed to create three rapid, simple, eco-friendly, cheap spectrophotometric methods for concurrent assay of Sofosbuvir (SOF) and Simeprevir (SMV) in their pure, laboratory prepared mixture and pharmaceutical dosage form with high degree of accuracy and precision. Three methods were developed including iso-absorptive point, ratio subtraction and dual wavelength. The linear range of the proposed methods was 3.0-50.0 and 2.0-50.0 µg mL-1 for SMV and SOF, respectively. The proposed methods were validated according to ICH guidelines in terms of linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed approach is highly simple and the procedure is environmentally green making it suitable for the drug analysis in routine works.

Comparative Real Life Egyptian Experience of the Combination of Sofosbuvir Plus Daclatasvir or Simeprevir for 12 Weeks in Naïve Cirrhotic Patients Infected with HCV Genotype 4.

BACKGROUND: Chronic infection with HCV is progressive worldwide health problem and the core reason for liver cirrhosis, portal hypertension, or hepatocellular carcinoma. HCV-G4 represents the most common threat to transplantation of the liver in Egypt. New interferon-free regimens have been started consuming direct-acting antiviral oral tablets for HCV cure. OBJECTIVES: In the current study, comparing the safety and efficacy of DAAs combination regimens including sofosbuvir with daclatasvir or sofosbuvir with simeprevir plus ribavirin for naïve cirrhotic Egyptian patients infected with HCV-G4 was our main goal. METHODS: We recruited 150 naïve cirrhotic HCV patients from the Tropical patients' clinic at Fayoum General Hospital. They were classified randomly into two groups, group one (n=75 patients) were administrated Sofosbuvir plus simeprevir (400 mg and 150 mg once daily respectively ) for twelve weeks, and group two (n=75 patients) were administrated Sofosbuvir plus Daclatasvir (400 mg and 60 mg once daily respectively) with ribavirin (1-1.2 gm daily weight-based) for twelve weeks. Clinical follow-up, laboratory investigations, and viral PCR were measured to detect treatment efficacy, safety, and any adverse events. RESULTS: Sustained virological response rates (SVR12) were 92%and 90.7% in the first and second groups, respectively. The major unfavorable events were fatigue, arthralgia, and weight loss without statistically meaningful differences between study groups. However, anemia and headache were significantly widespread in the second group (P=0.0161 and 0.0495, respectively). We observed four patients with photosensitivity in group I and not observed in the second group. CONCLUSION: The current study revealed that DAAs are safe and effective in the cure of naïve cirrhotic patients chronically infected by HCV-G4 with better results in those treated with sofosbuvir plus simeprevir regimen.

Elucidating the role of N440K mutation in SARS-CoV-2 spike - ACE-2 binding affinity and COVID-19 severity by virtual screening, molecular docking and dynamics approach.

COVID-19 has become a public health concern around the world. The frequency of N440K variant was higher during the second wave in South India. The mutation was observed in the Receptor Binding Domain region (RBD) of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike (S) protein. The binding affinity of SARS-CoV-2-Angiotensin-Converting Enzyme-2 (ACE-2) plays a major role in the transmission and severity of the disease. To understand the binding affinity of the wild and mutant SARS-CoV-2 S with ACE2, molecular modeling studies were carried out. We discovered that the wild SARS-CoV-2 S RBD-ACE-2 complex has a high binding affinity and stability than that of the mutant. The N440K strain escapes from antibody neutralization, which might increase reinfection and decrease vaccine efficiency. To find a potential inhibitor against mutant N440K SARS-CoV-2, a virtual screening process was carried out and found ZINC169293961, ZINC409421825 and ZINC22060839 as the best binding energy compounds. Communicated by Ramaswamy H. Sarma.

Successful treatment of hepatitis C genotype 4 using sofosbuvir, daclatasvir, simeprevir and ribavirin in Egyptian patients with direct-acting antiviral agent treatment failure.

Introduction: In chronic hepatitis C virus (HCV) patients in whom prior direct-acting antiviral agent (DAA) treatment had failed, outcomes after retreatment are optimal. Combination of sofosbuvir (SOF), daclatasvir (DCV), simeprevir (SIM), and ribavirin (RBV) in treatment experienced patients is recommended in current guidelines despite insufficient data. Our aim is to determine the efficacy and safety of SOF, DCV, SIM plus RBV in HCV infected patients who failed prior DAA treatment. Material and methods: One hundred and seventeen patients who failed to respond to SOF containing regimens were randomized according to previous response to therapy to non-responders and relapsers. Duration of therapy depends on fibrosis stages. SOF, DCV, SIM and weight based RBV 12 weeks for F1 and F2 (group I) and 24 weeks for F3 and F4 (group II). Results: In the non-responder group, a sustained virologic response (SVR) occurred in 100% in group I (F1 and F2) and 97% in group II (F3 and F4). Relapse was 3% in group II (F3 and F4). No patients from either group had breakthrough or non-response. In relapsers SVR was 100% in group I (F1 and F2) and 96% in group II (F3 and F4). Breakthrough, relapse and non-response were 2%, 4%, 2% respectively only in group II (F3 and F4). Conclusions: Combining multiple DAAs with different viral targets may be effective treatment protocol in previous non-responders and relapsers with short durations of treatment.

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.

Application of different spectrophotometric methods for quantitative analysis of direct acting antiviral drugs simeprevir and sofosbuvir.

Simeprevir and sofosbuvir are direct-acting antiviral drugs approved for the treatment of chronic HCV infection. Reports demonstrate the similarities between HCV and SARS-CoV-2 in terms of structure and replication mechanism. Therefore, it is suggested that a combination of simeprevir and sofosbuvir may be considered for COVID-19 patients. To date, no spectrophotometric methods have been published for quantitative analysis of simeprevir and sofosbuvir in combination. In this work, two simple spectrophotometric methods allowed quantitative analysis of the studied drugs in the mixed form. The zero-order direct method allowed quantitative analysis of simeprevir at 333 nm, with sofosbuvir showing zero absorbance values. The dual wavelength method allowed quantitative analysis of sofosbuvir by measuring the difference in absorbance values at 259.40 and 276 nm, where the difference in absorbance values of simeprevir was zero. With the applied methods, the investigated drugs in the mixtures and tablets prepared in the laboratory were successfully analyzed quantitatively with acceptable results.

Effects of simeprevir on the replication of SARS-CoV-2 in vitro and in transgenic hACE2 mice.

In a bid to contain the current COVID-19 (coronavirus disease 2019) pandemic, various countermeasures have been applied. To date, however, there is a lack of an effective drug for the treatment of COVID-19. Through molecular modelling studies, simeprevir, a protease inhibitor approved for the management of hepatitis C virus infection, has been predicted as a potential antiviral against SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causative agent of COVID-19. Here we assessed the efficacy of simeprevir against SARS-CoV-2 both in vitro in Vero E6 cells and in vivo in a human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model. The results showed that simeprevir could inhibit SARS-CoV-2 replication in Vero E6 cells with a half-maximal effective concentration (EC50) of 1.41 ± 0.12 µM. In a transgenic hACE2 mouse model of SARS-CoV-2 infection, intraperitoneal administration of simeprevir at 10 mg/kg/day for 3 consecutive days failed to suppress viral replication. These findings collectively imply that simeprevir does not inhibit SARS-CoV-2 in vivo and therefore do not support its application as a treatment against COVID-19 at a dosage of 10 mg/kg/day.

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.

Sofosbuvir-Velpatasvir (Epclusa)-Associated Photosensitivity in a Hepatitis C Patient: Case Report and Review of Photosensitivity to Hepatitis C Antiviral Agents.

Hepatitis C is a ribonucleic acid (RNA) virus, and its presence in individuals was previously considered to be a chronic condition. However, recent therapeutic advances with virus-directed treatment have resulted in a significant increase in cure rates as demonstrated by an absence of virus on repeat testing. Currently, several individual and combination antiviral therapies are available for the treatment of hepatitis C virus infection. However, each of the hepatitis C antiviral agents is associated with potential adverse skin effects whose incidence varies depending on the agent used for treatment. The cutaneous reactions, including photosensitivity, usually resolve once the antiviral treatment is completed. In this report, we discuss the case of a chronic hepatitis C patient who developed sofosbuvir-velpatasvir (Epclusa)-associated toxicity, while receiving the antiviral therapy. The 57-year-old man developed cutaneous phototoxicity when he started treatment with the drug. The adverse skin reaction promptly resolved once the treatment was completed. Clinicians who manage patients being treated with antiviral agents for hepatitis C infection should consider counseling these individuals regarding photoprotection including avoidance of sun exposure, daily sunscreen use, and wearing photoprotective clothing.

In vitro susceptibility of human Blastocystis subtypes to simeprevir.

INTRODUCTION AND AIM: Blastocystis is a common enteric parasite, having a worldwide distribution. Many antimicrobial agents are effective against it, yet side effects and drug resistance have been reported. Thus, ongoing trials are being conducted for exploring anti-Blastocystis alternatives. Proteases are attractive anti-protozoal drug targets, having documented roles in Blastocystis. Serine proteases are present in both hepatitis C virus and Blastocystis. Since drug repositioning is quite trendy, the in vitro efficacy of simeprevir (SMV), an anti-hepatitis serine protease inhibitor, against Blastocystis was investigated in the current study. METHODS: Stool samples were collected from patients, Alexandria, Egypt. Concentrated stools were screened using direct smears, trichrome, and modified Ziehl-Neelsen stains to exclude parasitic co-infections. Positive stool isolates were cultivated, molecularly subtyped for assessing the efficacy of three SMV doses (100,150, and 200 µg/ml) along 72 hours (h), on the most common subtype, through monitoring parasite growth, viability, re-culture, and also via ultrastructure verification. The most efficient dose and duration were later tested on other subtypes. RESULTS: Results revealed that Blastocystis was detected in 54.17% of examined samples. Molecularly, ST3 predominated (62%), followed by ST1 (8.6%) and ST2 (3.4%). Ascending concentrations of SMV progressively inhibited growth, viability, and re-culture of treated Blastocystis, with a non-statistically significant difference when compared to the therapeutic control metronidazole (MTZ). The most efficient dose and duration against ST3 was 150 µg/ml for 72 h. This dose inhibited the growth of ST3, ST1, and ST2 with percentages of 95.19%, 94.83%, and 94.74%, successively and viability with percentages of 98.30%, 98.09%, and 97.96%, successively. This dose abolished Blastocystis upon re-culturing. Ultra-structurally, SMV induced rupture of Blastocystis cell membrane leading to necrotic death, versus the reported apoptotic death caused by MTZ. In conclusion, 150 µg/ml SMV for 72 h proved its efficacy against ST1, ST2, and ST3 Blastocystis, thus sparing the need for pre-treatment molecular subtyping in developing countries.

Repurposing of FDA-approved drugs as inhibitors of sterol C-24 methyltransferase of Leishmania donovani to fight against leishmaniasis.

Leishmaniasis is a vector-borne disease caused by around 20 species of Leishmania. The main clinical forms of leishmaniasis are cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). VL is caused by Leishmania infantum in Central and South America, Mediterranean Basin, Middle East, and by L. donovani in Asia and Africa. Sterol C-24 methyltransferase (LdSMT) of L. donovani is a transferase enzyme of the sterol biosynthesis pathway. This pathway is one of the major targets for drug developments in Leishmania. Due to insufficient evidence about the exact function of SMT inside the cell and the uniqueness of the SMT enzyme in the Leishmania parasites made it a significant target for an effective drug development approach. We performed virtual screening of the Food and Drug Administration (FDA)-approved drug library against LdSMT and found simeprevir, an antiviral drug on top in the binding score. It showed a significant binding affinity with LdSMT. The binding was supported by hydrogen bonds and several other interactions. Simeprevir inhibited L. donovani growth of promastigotes with 50% inhibitory concentration (IC50 ) of 51.49 ± 5.87 µM. Further studies showed that simeprevir induced ROS generation in 44.7% of parasites at 125-µM concentration. Here, we for the first time reported simeprevir as an antileishmanial lead molecule using a drug repurposing approach.

Non-alcoholic fatty liver disease is a risk factor for occurrence of hepatocellular carcinoma after sustained virologic response in chronic hepatitis C patients: A prospective four-years follow-up study.

BACKGROUND AND AIM: The incidence of hepatocellular carcinoma (HCC) decreases significantly in chronic hepatitis C (CHC) patients with sustained virologic response (SVR) after pegylated-interferon plus ribavirin (PR) or direct-acting antiviral (DAAs) therapy. We follow-up a single cohort of CHC patients to identify risk factors associated with HCC development post-SVR. METHOD: CHC patients with SVR in Beijing/Hong Kong were followed up at 12-24 weekly intervals with surveillance for HCC by ultrasonography and alpha-fetoprotein (AFP). Multivariate Cox proportional hazards regression analysis was used to explore factors associated with HCC occurrence. RESULTS: Between October 2015 and May 2017, SVR was observed in 519 and 817 CHC patients after DAAs and PR therapy respectively. After a median post -SVR follow-up of 48 months, HCC developed in 54 (4.4%) SVR subjects. By adjusted Cox analysis, older age (≥55 years) [HR 2.4, 95% CI (1.3-4.3)], non-alcoholic fatty liver diseases [HR 2.4, 95%CI (1.3-4.2), higher AFP level (≥20 ng/ml) [HR 3.4, 95%CI (2.0-5.8)], higher liver stiffness measurement (≥14.6 kPa) [HR 4.2, 95%CI (2.3-7.6)], diabetes mellitus [HR 4.2, 95%CI (2.4-7.4)] at pre-treatment were associated with HCC occurrence. HCC patients in the DAAs induced SVR group had a higher prevalence of NAFLD as compared with those in the PR induced SVR group, 62% (18/29) vs 28% (7/25), p = 0.026. A nomogram formulated with the above six independent variables had a Concordance-Index of 0.835 (95% CI 0.783-0.866). CONCLUSION: Underlying NAFLD is associated with increased incidence of HCC in chronic HCV patients post-SVR, particularly in those treated with DAA.

Drug repurposing for identification of potential inhibitors against SARS-CoV-2 spike receptor-binding domain: An in silico approach.

BACKGROUND & OBJECTIVES: The world is currently under the threat of coronavirus disease 2019 (COVID-19) infection, caused by SARS-CoV-2. The objective of the present investigation was to repurpose the drugs with potential antiviral activity against receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein among 56 commercially available drugs. Therefore, an integrative computational approach, using molecular docking, quantum chemical calculation and molecular dynamics, was performed to unzip the effective drug-target interactions between RBD and 56 commercially available drugs. METHODS: The present in silico approach was based on information of drugs and experimentally derived crystal structure of RBD of SARS-CoV-2 S protein. Molecular docking analysis was performed for RBD against all 56 reported drugs using AutoDock 4.2 tool to screen the drugs with better potential antiviral activity which were further analysed by other computational tools for repurposing potential drug or drugs for COVID-19 therapeutics. RESULTS: Drugs such as chalcone, grazoprevir, enzaplatovir, dolutegravir, daclatasvir, tideglusib, presatovir, remdesivir and simeprevir were predicted to be potentially effective antiviral drugs against RBD and could have good COVID-19 therapeutic efficacy. Simeprevir displayed the highest binding affinity and reactivity against RBD with the values of -8.52 kcal/mol (binding energy) and 9.254 kcal/mol (band energy gap) among all the 56 drugs under investigation. INTERPRETATION & CONCLUSIONS: In the current investigation, simeprevir was identified as the potential antiviral drug based on the in silico findings in comparison to remdesivir, favipiravir and other 53 drugs. Further, laboratory and clinical investigations are needed to be carried out which will aid in the development of quick therapeutics designed for COVID-19.

Bio-activation of simeprevir in liver microsomes and characterization of its glutathione conjugates by liquid chromatography coupled to ultrahigh-resolution quadrupole time-of-flight mass spectrometry.

Liquid chromatography coupled to a triple quadrupole and, alternatively, to an ultrahigh-resolution quadrupole time-of-flight (UHR-QqTOF) mass spectrometers was used to collect qualitative and quantitative information from incubations of the anti-hepatitis C drug simeprevir with human and rat liver microsomes, respectively, supplemented with NADPH and glutathione. For this, different chromatographic methods using two different chromatographic columns, Kinetex® 2.6 µm C18 (50 × 3 mm) and Atlantis T3 (100 Å, 3 µm, 4.6 mm × 150 mm), have been employed. For determination and structural characterization of the reactive metabolites, we used information obtained from high-resolution mass spectrometry, namely accurate mass data to calculate the elemental composition, accurate MS/MS fragmentation patterns for confirmation of structural proposals, and the high mass spectral resolution to eliminate false-positive peaks. In this study, the use of high-resolution mass spectrometry (HR-MS) enabled the identification of 19 simeprevir metabolites generated by O- respectively N-demethylation, oxidation, dehydrogenation, hydrolysis, and formation of glutathione conjugates. The in silico study provides insights into the sites of simeprevir most amenable to reactions involving cytochrome P450. The developed methods have been successfully applied to analyze simeprevir and its metabolites simultaneously; based on this data, potential metabolic pathways of simeprevir are discussed. In general, the obtained results demonstrate that simeprevir is susceptible to form reactive simeprevir-glutathione adducts and cyclopropansulfonamide, which may explain the implication of simeprevir in idiosyncratic adverse drug reactions (IADRs) or hepatotoxicity.

Remission of long-term hepatic and renal disease induced by HCV after direct-acting antivirals therapy / Remissão de doença hepática e renal a longo prazo induzida pelo HCV após terapia antiviral de ação direta

Abstract In addition to liver disease, the hepatitis C virus (HCV) has been associated with autoimmune phenomena, such as mixed cryoglobulin and glomerulonephritis (GN). Until recently, both chronic hepatitis and HCV extra-hepatic manifestations were treated with peg-interferon plus ribavirin, however these drugs presented low efficacy and induced severe side effects. Nowadays, the HCV chronic hepatitis has been treated with direct acting antivirals (DAA), but studies on the DAA therapy for HCV-associated glomerulonephritis are scarce. Here, we describe two cases of HCV-associated glomerulonephritis that were treated with DAAs. In these two cases, previously experienced to peg-interferon plus ribavirin, the sofosbuvir plus simeprevir therapy was effective, without significant side effects, and interrupted the evolution of at least 20 years of both hepatic and renal diseases. These cases join the seven previously described cases that were treated with this DAAs association.

Resumo Além da doença hepática, o vírus da hepatite C (HCV) tem sido associado a fenômenos autoimunes, como crioglobulinemia mista (CM) e glomerulonefrite (GN). Até recentemente, a hepatite crônica e as manifestações extra-hepáticas do HCV eram tratadas com peg-interferon com ribavirina; no entanto, essas drogas apresentavam baixa eficácia e induziam efeitos colaterais graves. Atualmente, a hepatite crônica por HCV tem sido tratada com antivirais de ação direta (AAD), mas estudos sobre a terapia com AAD para glomerulonefrite associada ao HCV são escassos. Aqui, descrevemos dois casos de glomerulonefrite associada ao HCV que foram tratados com AAD. Nestes dois casos, previamente tratados com peg-interferon e ribavirina, a terapia com sofosbuvir com simeprevir foi eficaz, sem efeitos colaterais significativos, e interrompeu a evolução de pelo menos 20 anos de doenças hepáticas e renais. Esses casos se juntam aos sete casos descritos anteriormente que foram tratados com essa associação de AAD.

Pharmacoinformatics and molecular dynamics simulation studies reveal potential covalent and FDA-approved inhibitors of SARS-CoV-2 main protease 3CLpro.

The SARS-CoV-2 was confirmed to cause the global pandemic of coronavirus disease 2019 (COVID-19). The 3-chymotrypsin-like protease (3CLpro), an essential enzyme for viral replication, is a valid target to combat SARS-CoV and MERS-CoV. In this work, we present a structure-based study to identify potential covalent inhibitors containing a variety of chemical warheads. The targeted Asinex Focused Covalent (AFCL) library was screened based on different reaction types and potential covalent inhibitors were identified. In addition, we screened FDA-approved protease inhibitors to find candidates to be repurposed against SARS-CoV-2 3CLpro. A number of compounds with significant covalent docking scores were identified. These compounds were able to establish a covalent bond (C-S) with the reactive thiol group of Cys145 and to form favorable interactions with residues lining the substrate-binding site. Moreover, paritaprevir and simeprevir from FDA-approved protease inhibitors were identified as potential inhibitors of SARS-CoV-2 3CLpro. The mechanism and dynamic stability of binding between the identified compounds and SARS-CoV-2 3CLpro were characterized by molecular dynamics (MD) simulations. The identified compounds are potential inhibitors worthy of further development as COVID-19 drugs. Importantly, the identified FDA-approved anti-hepatitis-C virus (HCV) drugs paritaprevir and simeprevir could be ready for clinical trials to treat infected patients and help curb COVID-19. Communicated by Ramaswamy H. Sarma.

Computational investigation of possible inhibitors of the winged-helix domain of MUS81.

The methyl methanesulfonate and ultraviolet sensitive 81 (MUS81) is a structure-specific endonuclease that is highly conserved in eukaryotes and essential for homologous recombination repair. The winged-helix domain at the N-terminus of MUS81 (wMUS81) can bind DNA substrates and regulate the endonuclease activity. The repression of MUS81 activity could enhance the sensitivity to antitumor compounds of different tumour cells. Thus, MUS81 is a potential therapeutic target in cancer therapy. However, specific inhibitors of MUS81 have remained elusive. Here, for the first time, we attempt to discover the compounds disrupting the wMUS81 activity. The binding affinity of available drugs to wMUS81 was first estimated by molecular docking. pKa values were taken into consideration to eliminate unlikely protonation states of the ligands. Top-lead compounds were then estimated the binding affinity using the fast pulling ligand simulations. Finally, the free energy perturbation method accurately defined the absolute binding free energy of the top four ligands, revealing the most potential inhibitors of wMUS81 including simeprevir and nilotinib. Binding of simeprevir destabilizes the ß-hairpin region of wMUS81, likely disturbing the wMUS81 function. The van der Waals free binding energy majorly modulates the ligand-binding mechanism. The two conserved residues Leu189 and Arg196 are likely important in monitoring the interacting process of simeprevir to wMUS81.