Aim: Discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors using a structure-based drug discovery strategy. Materials & methods: Virtual screening employing covalent and noncovalent docking was performed to discover Mpro inhibitors, which were subsequently evaluated in biochemical and cellular assays. Results: 91 virtual hits were selected for biochemical assays, and four were confirmed as reversible inhibitors of SARS CoV-2 Mpro with IC50 values of 0.4-3 μM. They were also shown to inhibit SARS-CoV-1 Mpro and human cathepsin L. Molecular dynamics simulations indicated the stability of the Mpro inhibitor complexes and the interaction of ligands at the subsites. Conclusion: This approach led to the discovery of novel thiosemicarbazones as potent SARS-CoV-2 Mpro inhibitors.

• MeSH
• Antiviral Agents pharmacology   chemistry   MeSH
• COVID-19 * MeSH
• Protease Inhibitors pharmacology   chemistry   MeSH
• Humans MeSH
• SARS-CoV-2 MeSH
• Molecular Docking Simulation MeSH
• Thiosemicarbazones * pharmacology   MeSH
• Viral Nonstructural Proteins MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Adenosine Triphosphatases * metabolism   MeSH
• RNA, Double-Stranded metabolism   MeSH
• Phosphates metabolism   MeSH
• DNA, Single-Stranded * metabolism   MeSH
• Humans MeSH
• RNA Helicases * metabolism   MeSH
• Viral Nonstructural Proteins * metabolism   MeSH
• Encephalitis Viruses, Tick-Borne * enzymology   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Zika virus (ZIKV) has been characterized as one of many potential pathogens and placed under future epidemic outbreaks by the WHO. However, a lack of potential therapeutics can result in an uncontrolled pandemic as with other human pandemic viruses. Therefore, prioritized effective therapeutics development has been recommended against ZIKV. In this context, the present study adopted a strategy to explore the lead compounds from Azadirachta indica against ZIKV via concurrent inhibition of the NS2B-NS3 protease (ZIKVpro) and NS5 RNA dependent RNA polymerase (ZIKVRdRp) proteins using molecular simulations. Initially, structure-based virtual screening of 44 bioflavonoids reported in Azadirachta indica against the crystal structures of targeted ZIKV proteins resulted in the identification of the top four common bioflavonoids, viz. Rutin, Nicotiflorin, Isoquercitrin, and Hyperoside. These compounds showed substantial docking energy (-7.9 to -11.01 kcal/mol) and intermolecular interactions with essential residues of ZIKVpro (B:His51, B:Asp75, and B:Ser135) and ZIKVRdRp (Asp540, Ile799, and Asp665) by comparison to the reference compounds, O7N inhibitor (ZIKVpro) and Sofosbuvir inhibitor (ZIKVRdRp). Besides, long interval molecular dynamics simulation (500 ns) on the selected docked poses reveals stability of the respective docked poses contributed by intermolecular hydrogen bonds and hydrophobic interactions. The predicted complex stability was further supported by calculated end-point binding free energy using molecular mechanics generalized born surface area (MM/GBSA) method. Consequently, the identified common bioflavonoids are recommended as promising therapeutic inhibitors of ZIKVpro and ZIKVRdRp against ZIKV for further experimental assessment.

• MeSH
• Antiviral Agents chemistry   MeSH
• Azadirachta * chemistry   MeSH
• Flavonoids chemistry   MeSH
• Zika Virus Infection * drug therapy   MeSH
• Protease Inhibitors chemistry   MeSH
• Humans MeSH
• Lead pharmacology   MeSH
• Peptide Hydrolases pharmacology   MeSH
• RNA-Dependent RNA Polymerase MeSH
• Molecular Docking Simulation MeSH
• Viral Nonstructural Proteins metabolism   MeSH
• Zika Virus * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Drug repurposing requires a limited resource, cost-effective and faster method to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, this in silico studies attempts to identify the drug-likeness properties of ravidasvir, an II/III phase clinical trial chronic hepatitis C drug against 3-Chymotrypsin-like protease (3CLpro) of SARS-CoV-2 to combat the ongoing coronavirus disease 2019 (COVID-19) pandemic. This protease is predominantly involved in virus replication cycle; hence it is considered as a potent drug target. The molecular docking results showed that ravidasvir was found to be potent inhibitors of 3CLpro with scoring function based binding energy is -26.7 kJ/mol. Further dynamic behaviour of apo form and complex form of ravidasvir with 3CLpro were studied using molecular dynamics (MD) simulations over 500 ns each, total 2 μs time scale. The motion of the protein was studied using principal component analysis of the MD simulation trajectories. The binding free energy calculated using MM/PBSA method from the MD simulation trajectory was -190.3 ± 70.2 kJ/mol and -106.0 ± 26.7 kJ/mol for GROMOS96 54A7 and AMBER99SB-ILDN force field, respectively. This in silico studies suggesting ravidasvir might be a potential lead molecule against SARS-CoV-2 for further optimization and drug development to combat the life-threatening COVID-19 pandemic.Communicated by Ramaswamy H. Sarma.

• MeSH
• Adipates MeSH
• Benzimidazoles MeSH
• COVID-19 * MeSH
• Cysteine Endopeptidases chemistry   MeSH
• COVID-19 Drug Treatment MeSH
• Protease Inhibitors chemistry   MeSH
• Coronavirus 3C Proteases MeSH
• Humans MeSH
• Pandemics MeSH
• SARS-CoV-2 * MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Succinates MeSH
• Valine analogs & derivatives   MeSH
• Viral Nonstructural Proteins chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

• MeSH
• Antiviral Agents pharmacology   MeSH
• COVID-19 MeSH
• COVID-19 Drug Treatment MeSH
• Fluorescent Dyes chemistry   MeSH
• Protease Inhibitors pharmacology   MeSH
• Coronavirus Papain-Like Proteases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Drug Evaluation, Preclinical MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• RNA Helicases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• RNA Viruses enzymology   MeSH
• High-Throughput Screening Assays methods   MeSH
• SARS-CoV-2 enzymology   MeSH
• Serine Endopeptidases chemistry   genetics   metabolism   MeSH
• Viral Nonstructural Proteins antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Encephalitis Viruses, Tick-Borne enzymology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND & AIMS: Eight-week glecaprevir/pibrentasvir leads to high rates of sustained virological response at post-treatment week 12 (SVR12) across HCV genotypes (GT) 1-6 in treatment-naïve patients without cirrhosis. We evaluated glecaprevir/pibrentasvir once daily for 8 weeks in treatment-naïve patients with compensated cirrhosis. METHODS: EXPEDITION-8 was a single-arm, multicenter, phase IIIb trial. The primary and key secondary efficacy analyses were to compare the lower bound of the 95% CI of the SVR12 rate in i) patients with GT1,2,4-6 in the per protocol (PP) population, ii) patients with GT1,2,4-6 in the intention-to-treat (ITT) population, iii) patients with GT1-6 in the PP population, and iv) patients with GT1-6 in the ITT population, to pre-defined efficacy thresholds based on historical SVR12 rates for 12 weeks of glecaprevir/pibrentasvir in the same populations. Safety was also assessed. RESULTS: A total of 343 patients were enrolled. Most patients were male (63%), white (83%), and had GT1 (67%). The SVR12 rate in patients with GT1-6 was 99.7% (n/N = 334/335; 95%CI 98.3-99.9) in the PP population and 97.7% (n/N = 335/343; 95% CI 96.1-99.3) in the ITT population. All primary and key secondary efficacy analyses were achieved. One patient (GT3a) experienced relapse (0.3%) at post-treatment week 4. Common adverse events (≥5%) were fatigue (9%), pruritus (8%), headache (8%), and nausea (6%). Serious adverse events (none related) occurred in 2% of patients. No adverse event led to study drug discontinuation. Clinically significant laboratory abnormalities were infrequent. CONCLUSIONS: Eight-week glecaprevir/pibrentasvir was well tolerated and led to a similarly high SVR12 rate as the 12-week regimen in treatment-naïve patients with chronic HCV GT1-6 infection and compensated cirrhosis. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03089944. LAY SUMMARY: This study was the first to evaluate an 8-week direct-acting antiviral (DAA) regimen active against all major types of hepatitis C virus (HCV) in untreated patients with compensated cirrhosis. High virological cure rates were achieved with glecaprevir/pibrentasvir across HCV genotypes 1-6, and these high cure rates did not depend on any patient or viral characteristics present before treatment. This may simplify care and allow non-specialist healthcare professionals to treat these patients, contributing to global efforts to eliminate HCV.

• MeSH
• Antiviral Agents administration & dosage   adverse effects   MeSH
• Benzimidazoles administration & dosage   adverse effects   MeSH
• Quinoxalines administration & dosage   adverse effects   MeSH
• Hepatitis C, Chronic blood   complications   drug therapy   virology   MeSH
• Cyclopropanes administration & dosage   adverse effects   MeSH
• Drug Combinations MeSH
• Genotype * MeSH
• Hepacivirus enzymology   genetics   MeSH
• Liver Cirrhosis complications   drug therapy   MeSH
• Aminoisobutyric Acids administration & dosage   adverse effects   MeSH
• Leucine administration & dosage   adverse effects   analogs & derivatives   MeSH
• Middle Aged MeSH
• Humans MeSH
• Lactams, Macrocyclic administration & dosage   adverse effects   MeSH
• Polymorphism, Genetic MeSH
• Proline administration & dosage   adverse effects   analogs & derivatives   MeSH
• Pyrrolidines administration & dosage   adverse effects   MeSH
• RNA, Viral blood   genetics   MeSH
• Aged MeSH
• Sustained Virologic Response MeSH
• Sulfonamides administration & dosage   adverse effects   MeSH
• Viral Nonstructural Proteins genetics   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Clinical Trial, Phase III MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH

Many picornaviruses hijack the Golgi resident Acyl-coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4-kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4-phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small-angle X-ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.

• MeSH
• Acyl Coenzyme A chemistry   metabolism   MeSH
• Adaptor Proteins, Signal Transducing chemistry   metabolism   MeSH
• Humans MeSH
• Membrane Proteins chemistry   metabolism   MeSH
• Picornaviridae chemistry   metabolism   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Genotype 1b (GT1b) is the most common subtype of the hepatitis C virus (HCV). We present an integrated analysis of 1070 participants with HCV GT1b infection from 30 countries who received elbasvir/grazoprevir for 12 weeks. METHODS: This is a retrospective analysis of data from participants with chronic HCV GT1b infection enrolled in 11 phase II/III clinical trials. All participants received elbasvir 50 mg plus grazoprevir 100 mg once daily for 12 weeks. The primary end point of all studies was sustained virologic response 12 weeks after completion of therapy (SVR12, HCV RNA < 15 IU/ml). RESULTS: SVR12 was 97.2% (1040/1070). Of the 30 participants who failed to attain SVR12, 15 relapsed and 15 had nonvirologic failure. Among participant subgroups, SVR12 was high in those with compensated cirrhosis (188/189, 99.5%), HIV co-infection (51/54, 94.4%), and baseline viral load > 800,000 IU/ml (705/728, 96.8%). Resistance-associated substitutions (RASs) at NS5A positions 28, 30, 31, or 93 were present in 21.6% of participants at baseline. SVR12 was 99.6% (820/823) in participants without baseline NS5A RASs and 94.7% (215/227) in those with baseline NS5A RASs. Serious adverse events occurred in 3.2% (34/1070) of participants, nine of which occurred after study medication was completed. CONCLUSIONS: Elbasvir/grazoprevir for 12 weeks represents an effective treatment option for participants with HCV GT1b infection. SVR12 was high in all participant subgroups, including those with compensated cirrhosis, HIV co-infection, and high baseline viral load. CLINICALTRIALS. GOV IDENTIFIERS: The trials discussed in this paper were registered with Clinicaltrial.gov as the following: NCT02092350 (C-SURFER), NCT02105662 (C-EDGE Co-Infection), NCT02105467 (C-EDGE treatment-naive), NCT02105701 (C-EDGE treatment-experienced), NCT01717326 (C-WORTHy), NCT02251990 (C-CORAL), NCT02105688 (C-EDGE COSTAR), NCT02252016 (C-EDGE IBLD), NCT02115321 (C-SALT), NCT02203149 (Japan phase 2/3 study), NCT02358044 (C-EDGE Head-2-Head).

• MeSH
• Antiviral Agents adverse effects   therapeutic use   MeSH
• Benzofurans adverse effects   therapeutic use   MeSH
• Quinoxalines adverse effects   therapeutic use   MeSH
• Hepatitis C, Chronic complications   drug therapy   virology   MeSH
• Adult MeSH
• Genotype MeSH
• Hepacivirus genetics   MeSH
• HIV Infections complications   MeSH
• Imidazoles adverse effects   therapeutic use   MeSH
• Liver Cirrhosis physiopathology   virology   MeSH
• Clinical Trials, Phase II as Topic MeSH
• Clinical Trials, Phase III as Topic MeSH
• Coinfection complications   MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Retrospective Studies MeSH
• Aged MeSH
• Sustained Virologic Response MeSH
• Drug Resistance, Viral genetics   MeSH
• Viral Load MeSH
• Viral Nonstructural Proteins genetics   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Léčba chronické hepatitidy typu C kombinací přímo působících perorálních virostatik (directly acting antivirals, DAA) má vysokou účinnost (až 100 %), minimum kontraindikací a mimořádně příznivý bezpečnostní profil. Fixní kombinace elbasviru s grazoprevirem (Zepatier) představuje nově dostupnou, vysoce účinnou variantu této léčby určenou pro pacienty infikované virem hepatitidy typu C (HCV) genotypu 1 nebo 4. Elbasvir (EBR) je inhibitor NS5A (nestrukturálního proteinu 5A) druhé vlny 1. generace, grazoprevir (GZR) proteázový inhibitor 2. generace. Léčivý přípravek Zepatier obsahuje 50 mg EBR a 100 mg GZR. Standardní dávkování představuje jedna tableta denně.

Chronic hepatitis C therapy using directly acting antivirals (DAA) has high efficacy (up to 100%), minimum contraindications plus an extraordinarily favorable safety profile. Fixed combination of elbasvir and grazoprevir represents a newly available, highly effective variant of this therapy, indicated for patients infected with hepatitis C virus (HCV), genotypes 1 or 4. Elbasvir (EBR) is a NS5A inhibitor of the second wave of the 1st generation; grazoprevir (GZR) is a protease inhibitor of the 2nd generation. The preparation Zepatier includes 50 mg of EBR and 100 mg of GZR. The standard dose is one tablet daily.

• Keywords
• elbasvir, grazoprevir,
• MeSH
• Antiviral Agents pharmacology   metabolism   therapeutic use   MeSH
• Benzofurans pharmacology   metabolism   therapeutic use   MeSH
• Quinoxalines pharmacology   metabolism   therapeutic use   MeSH
• Hepatitis C, Chronic * drug therapy   MeSH
• Drug Combinations MeSH
• Imidazoles pharmacology   metabolism   therapeutic use   MeSH
• Protease Inhibitors pharmacology   metabolism   therapeutic use   MeSH
• Clinical Studies as Topic MeSH
• Quantitative Structure-Activity Relationship MeSH
• Humans MeSH
• Drug-Related Side Effects and Adverse Reactions MeSH
• Viral Nonstructural Proteins pharmacology   metabolism   therapeutic use   MeSH
• Check Tag
• Humans MeSH

Serological diagnosis of Zika virus is challenging due to high cross-reactivity of Zika virus with other flavivirus antibodies. Recently, a Zika NS1-based enzyme-linked immunosorbent assay (ELISA) was developed and shown to be highly specific for Zika antibody detection; however, sensitivity was evaluated for only a small number of confirmed Zika-infected patients. In this study, we measured the sensitivity and kinetics of Zika IgM and IgG antibodies using the Zika NS1-based ELISA in 105 samples from 63 returning travelers infected with Zika virus (proven by PCR or neutralization assay) from Israel, Czech Republic, Italy, Belgium, Germany, and Chile. Zika virus IgM was detected from 2 to 42 days post-symptom onset (PSO) with an overall sensitivity of 79% in the first month and 68% until 2 months PSO, while IgG antibodies were detected from 5 days to 3 years PSO with 79% sensitivity. Interestingly, significant differences in IgM sensitivity and IgM detection period were observed between Israeli and European/Chilean Zika-infected travelers, adding to the complexity of Zika infection diagnosis and suggesting that other diagnostic methods should be complemented to reduce false-negative results.

• MeSH
• Time Factors MeSH
• Travel MeSH
• Child MeSH
• Adult MeSH
• Enzyme-Linked Immunosorbent Assay methods   MeSH
• Communicable Diseases, Imported diagnosis   MeSH
• Immunoglobulin G blood   MeSH
• Immunoglobulin M blood   MeSH
• Zika Virus Infection diagnosis   MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Child, Preschool MeSH
• Antibodies, Viral blood   MeSH
• Aged MeSH
• Sensitivity and Specificity MeSH
• Serologic Tests methods   MeSH
• Antibody Formation MeSH
• Viral Nonstructural Proteins immunology   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Geographicals
• Chile MeSH
• Europe MeSH