Virus Serine Protease Research Articles

Dual function of Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 μM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.

Novel 1,2,4-triazolethiol–thiophen Hybrids: Facile Synthesis, Characterization, ADMET Prediction and Molecular Docking

In the present contribution, novel 1,2,4-triazolethiol–thiophene hybrids, namely 4-ethyl-5-(thiophen-2-yl)-4H-1,2,4-triazole-3-thiol (1) and 4-phenyl-5-(thiophen-2-yl)-4H-1,2,4-triazole-3-thiol (2), which were readily fabricated from addition of isothiocyanatoethane or isothiocyanatobenzene, respectively, to thiophene-2-carbohydrazide followed by addition a KOH solution to provoke the cyclization to the 1,2,4-triazole ring. The formation of compounds 1 and 2 was firmly confirmed by the means of elemental analysis, IR, 1H and 13C{1H} NMR spectroscopy. The DFT-based computations in gas phase were additionally applied to shed light on the structure and electronic features of the title compounds. Theoretical calculations revealed that for both molecules their corresponding thione derivatives, namely 4-ethyl-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1') and 4-phenyl-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2'), are 15.00 and 11.96 kcal/mol, respectively, more energetically favorable in gas phase. However, a comparison of the experimental and calculated IR and NMR spectra testify to the thiol tautomers of compounds 1 and 2 for both compounds in solid state and in DMSO-d 6. The chemical activity of 1 and 2 was estimated by reactivity descriptors and MEP surface. ADMET properties of the reported compounds were predicted in silico using online services. Potential inhibition of a series of the tick-borne encephalitis (TBE) proteins by compounds 1 and 2 was studied using molecular docking, which, in turn, allowed to reveal the ligand efficiency scores for the resulting protein–ligand complexes. It was established that compound 1 exhibits the best activity against the tick-borne encephalitis virus Serine protease NS3, while compound 2 is preferable for the RNA-stimulated ATPase activity of tick-borne encephalitis virus helicase.

N-sulfonyl peptide-hybrids as a new class of dengue virus protease inhibitors.

Dengue virus (DENV) from the Flaviviridae family causes an epidemic disease that seriously threatens human life. The viral serine protease NS2B-NS3 is a promising target for drug development against DENV and other flaviviruses. We here report the design, synthesis, and in-vitro characterization of potent peptidic inhibitors of DENV protease with a sulfonyl moiety as N-terminal cap, thereby creating sulfonamide-peptide hybrids. The in-vitro target affinities of some synthesized compounds were in the nanomolar range, with the most promising derivative reaching a Ki value of 78nM against DENV-2 protease. The synthesized compounds did not have relevant off-target activity nor cytotoxicity. The metabolic stability of compounds against rat liver microsomes and pancreatic enzymes was remarkable. In general, the integration of sulfonamide moieties at the N-terminus of peptidic inhibitors proved to be a promising and attractive strategy for further drug development against DENV infections.

Penicillin Derivatives Inhibit the SARS-CoV-2 Main Protease by Reaction with Its Nucleophilic Cysteine.

The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mpro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that β-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl–enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by β-lactams and related acylating agents.

SAR evolution towards potent C-terminal carboxamide peptide inhibitors of Zika virus NS2B-NS3 protease

Zika virus (ZIKV) is a member of the Flaviviridae family that can cause neurological disorders and congenital malformations. The NS2B-NS3 viral serine protease is an attractive target for the development of new antiviral agents against ZIKV. We report here a SAR study on a series of substrate-like linear tripeptides that inhibit in a non-covalent manner the NS2B-NS3 protease. Optimization of the residues at positions P1, P2, P3 and of the N-terminal and C-terminal portions of the tripeptide allowed the identification of inhibitors with sub-micromolar potency with phenylglycine as arginine-mimicking group and benzylamide as C-terminal fragment. Further SAR exploration and application of these structural changes to a series of peptides having a 4-substituted phenylglycine residue at the P1 position led to potent compounds showing double digit nanomolar inhibition of the Zika protease (IC50 = 30 nM) with high selectivity against trypsin-like proteases and the proteases of other flavivirus, such as Dengue 2 virus (DEN2V) and West Nile virus (WNV).

Doxycycline Interferes with Zika Virus Serine Protease and Inhibits Virus Replication in Human Skin Fibroblasts.

Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (−7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection.

Metabolite profiling and in vitro evaluation of Lepisanthes fruticosa fruit pulp extract as inhibitor against dengue and West Nile virus NS2B-NS3 proteases

Background: Dengue virus serotype 2 (DENV2) and West Nile virus (WNV) fevers are mosquito-borne diseases with no effective treatment at present. In recent years, the development of plant-based antivirals targeting the viral NS2B-NS3 serine proteases has been the main focus as the synthetic antivirals available are not specific and less safe. Objectives: To evaluate the inhibitory activity of Lepisanthes fruticosa pulp extract against NS2B-NS3 proteases from DENV2 and WNV and identify the metabolites from this fruit extract. Materials and Methods: In vitro DENV2 and WNV NS2B-NS3 proteases assays were carried out using the methanolic extract of L. fruticosa pulp. Liquid chromatography-electron spray ionization-mass spectrometry/mass spectrometry (LC-ESI-MS/MS) and gas chromatography-mass spectrometry/mass spectrometry (GC-MS/MS) were performed to determine the metabolites present in this fruit species extract. Results: L. fruticosa extract exhibited inhibitory activity toward DENV2 and WNV NS2B-NS3 proteases with 50% inhibitory concentration value of 1.733 ± 0.195 and 9.245 ± 0.938 mg/mL, respectively. LC-ESI-MS/MS of L. fruticosa extract identified epigallocatechin-catechin, epigallocatechin, epicatechin, catechin, cyanidin rutinoside, procyanidin trimer, rutin, myricetin rhamnohexoside, luteolin glucoside and its derivative which were from the flavonoid group. In addition, GC-MS/MS identified fatty acids and sterols. Conclusion: The inhibitory activity of L. fruticosa pulp extract toward NS2B-NS3 proteases from DENV2 and WNV suggests this fruit species as a potential source for the development of antiviral. Metabolites from the groups of flavonols, flavones, and sterols identified in L. fruticosa pulp may contribute to the inhibitory properties of L. fruticosa.

Beyond Basicity: Discovery of Nonbasic DENV-2 Protease Inhibitors with Potent Activity in Cell Culture.

The viral serine protease NS2B-NS3 is one of the promising targets for drug discovery against dengue virus and other flaviviruses. The molecular recognition preferences of the protease favor basic, positively charged moieties as substrates and inhibitors, which leads to pharmacokinetic liabilities and off-target interactions with host proteases such as thrombin. We here present the results of efforts that were aimed specifically at the discovery and development of noncharged, small-molecular inhibitors of the flaviviral proteases. A key factor in the discovery of these compounds was a cellular reporter gene assay for the dengue protease, the DENV2proHeLa system. Extensive structure-activity relationship explorations resulted in novel benzamide derivatives with submicromolar activities in viral replication assays (EC50 0.24 μM), selectivity against off-target proteases, and negligible cytotoxicity. This structural class has increased drug-likeness compared to most of the previously published active-site-directed flaviviral protease inhibitors and includes promising candidates for further preclinical development.

DockCoV2: a drug database against SARS-CoV-2

The current state of the COVID-19 pandemic is a global health crisis. To fight the novel coronavirus, one of the best-known ways is to block enzymes essential for virus replication. Currently, we know that the SARS-CoV-2 virus encodes about 29 proteins such as spike protein, 3C-like protease (3CLpro), RNA-dependent RNA polymerase (RdRp), Papain-like protease (PLpro), and nucleocapsid (N) protein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) for viral entry and transmembrane serine protease family member II (TMPRSS2) for spike protein priming. Thus in order to speed up the discovery of potential drugs, we develop DockCoV2, a drug database for SARS-CoV-2. DockCoV2 focuses on predicting the binding affinity of FDA-approved and Taiwan National Health Insurance (NHI) drugs with the seven proteins mentioned above. This database contains a total of 3,109 drugs. DockCoV2 is easy to use and search against, is well cross-linked to external databases, and provides the state-of-the-art prediction results in one site. Users can download their drug-protein docking data of interest and examine additional drug-related information on DockCoV2. Furthermore, DockCoV2 provides experimental information to help users understand which drugs have already been reported to be effective against MERS or SARS-CoV. DockCoV2 is available at https://covirus.cc/drugs/.

Identification of Transcription Factors Regulating SARS-CoV-2 Entry Genes in the Intestine.

Gastrointestinal symptoms of coronavirus disease 2019 (COVID-19), including diarrhea, nausea, and vomiting, are more common than previously thought. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) likely causes these symptoms by infecting the epithelial cells lining the gastrointestinal tract,1 with angiotensin I converting enzyme 2 (ACE2) functioning as the viral receptor2 and transmembrane serine protease 2 (TMPRSS2) functioning in viral spike protein priming.3 Du et al4 recently reported that ACE2 not only is expressed in lung alveolar type II (AT2) cells but also highly expressed in absorptive enterocytes.

A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays.

Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available. One of the promising targets for drug discovery against dengue and other flaviviruses is the viral serine protease NS2B-NS3. We present the design, synthesis, and in vitro and cellular characterization of a novel chemotype of potent small-molecule non-peptidic dengue protease inhibitors derived from 4-benzyloxyphenylglycine. A newly developed luciferase-based DENV-2 protease reporter system in HeLa cells (DENV2proHeLa) was employed to determine the activity of the compounds in a cellular environment. Specificity and selectivity of the DENV2proHeLa system were confirmed by viral titer reduction assays. The compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays, are selective against other serine proteases, and do not show relevant cytotoxicity. An extensive structure-activity relationship study provides a perspective for further drug development against flaviviral infections.

The Nuclear Pore Complex: A Target for NS3 Protease of Dengue and Zika Viruses.

During flavivirus infection, some viral proteins move to the nucleus and cellular components are relocated from the nucleus to the cytoplasm. Thus, the integrity of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC), was evaluated during infection with dengue virus (DENV) and Zika virus (ZIKV). We found that while during DENV infection the integrity and distribution of at least three nucleoporins (Nup), Nup153, Nup98, and Nup62 were altered, during ZIKV infection, the integrity of TPR, Nup153, and Nup98 were modified. In this work, several lines of evidence indicate that the viral serine protease NS2B3 is involved in Nups cleavage. First, the serine protease inhibitors, TLCK and Leupeptin, prevented Nup98 and Nup62 cleavage. Second, the transfection of DENV and ZIKV NS2B3 protease was sufficient to inhibit the nuclear ring recognition detected in mock-infected cells with the Mab414 antibody. Third, the mutant but not the active (WT) protease was unable to cleave Nups in transfected cells. Thus, here we describe for the first time that the NS3 protein from flavivirus plays novel functions hijacking the nuclear pore complex, the main controller of the nuclear-cytoplasmic transport.

Anti-dengue infectivity evaluation of bioflavonoid from Azadirachta indica by dengue virus serine protease inhibition

Dengue virus (DENV) serine protease enzyme, i.e. NS2B-NS3pro (non-structural protein 2B-non-structural protein 3) has been approved as prime drug target for the drug discovery against dengue infection, because of its essential role in viral replication. This study demonstrates the potential of bioflavonoids from Azadirachta indica against dengue infection using computational and experimental approach. Initially, 49 bioflavonoids reported in Azadirachta indica were collected and virtually screened on the catalytic triad of DENV protease, results in the identification of kaempferol-3-O-rutinoside (−9.555 kcal/mol), rutin (−9.324 kcal/mol), hyperoside (−7.879 kcal/mol), and epicatechin (−7.622 kcal/mol) as potent viral protease inhibitors against reference compound quercetin (−6.94 kcal/mol). Subsequently, these docked complexes were analyzed for the stability via molecular dynamics simulations and free binding energy calculations, suggested the considerable stability of selected bioflavonoids with viral protease. Additionally, density functional theory and ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) analysis indicated the least chemical reactivity and considerable medicinal properties, respectively for the screened bioflavonoids by comparison to quercetin. Accordingly, kaempferol 3-O-β-rutinoside and epicatechin were evaluated at various concentrations for cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) and in vitro antiviral activity (focus forming unit assay) against DENV-2 strain. The antiviral assay showed dose dependent inhibition of DENV-2 infectivity by the selected compounds while maximum 77.7% and 66.2% viral inhibition were recorded for 100 µM kaempferol 3-O-β-rutinoside and 1000 µM epicatechin, respectively without significant cell toxicity. These results suggested the potential of bioflavonoids from Azadirachta indica in the development of effective drug against dengue infection. Communicated by Ramaswamy H. Sarma

The identification of active compounds in Ganoderma lucidum var. antler extract inhibiting dengue virus serine protease and its computational studies

The number of global dengue incidences is alarmingly high in recent years. The global distribution of four dengue serotypes has also added economic burden in the dengue-endemic countries. To discover the potent dengue virus inhibitors in the antler form of Ganoderma lucidum (Lingzhi or Reishi), the water extraction of normal G. lucidum and its antler form were conducted and the chemical compounds were identified by LC-MS. Six distinct chemical compounds identified in high abundance were hesperetin, thymidine, lucidenic acid, 11-aminoundecanoic acid, 5-carboxyvanillic acid and ganocin B. The water extracts of G. lucidum in its antler form inhibited the DENV2 NS2B-NS3 protease activity at 84.6 ± 0.7%, higher than the normal G. lucidum. Then, molecular docking was performed on the homology model built from an in-house sequence. Docking simulation results showed that hesperetin and ganocin B were the best leads to bind at the catalytic triad of DENV2 NS2B-NS3pro via hydrogen bonding, van der Waals and pi-pi interactions. Extensive overlapping of HOMO-LUMO orbitals at the ringed regions of hesperetin helped to facilitate the entry of ligand to the catalytic triad cleft. LC-MS, molecular docking and density functional theory analyses confirmed that hesperetin was the strongest inhibitor against NS2B-NS3 protease. Communicated by Ramaswamy H. Sarma

A comparison of machine learning algorithms for the prediction of Hepatitis C NS3 protease cleavage sites

Abstract Hepatitis is a global disease that is on the rise and is currently the cause of more deaths than the human immunodeficiency virus each year. As a result, there is an increasing need for antivirals. Previously, effective antivirals have been found in the form of substrate-mimetic antiviral protease inhibitors. The application of machine learning has been used to predict cleavage patterns of viral proteases to provide information for future drug design. This study has successfully applied and compared several machine learning algorithms to hepatitis C viral NS3 serine protease cleavage data. Results have found that differences in sequence-extraction methods can outweigh differences in algorithm choice. Models produced from pseudo-coded datasets all performed with high accuracy and outperformed models created with orthogonal-coded datasets. However, no single pseudo-model performed significantly better than any other. Evaluation of performance measures also show that the correct choice of model scoring system is essential for unbiased model assessment.

A Comparison of Machine Learning Algorithms for the Prediction of Hepatitis C NS3 Protease Cleavage Sites

Hepatitis is a global disease that is on the rise and is currently the cause of more deaths than the human immunodeficiency virus each year. As a result, there is an increasing need for antivirals. Previously, effective anti-virals have been found in the form of substrate-mimetic antiviral protease inhibitors. The application of machine learning has been used to predict cleavage patterns of viral proteases to provide information for future drug design. This study has successfully applied and compared several machine learning algorithms to hepatitis C viral NS3 serine protease cleavage data. Results have found that differences in sequence-extraction methods can outweigh differences in algorithm choice. Models produced from pseudo-coded data sets all performed with high accuracy and outperformed models created with orthogonal-coded data sets. However, no single pseudo-model performed significantly better than any other. Evaluation of performance measures also show that the correct choice of model scoring system is essential for unbiased model assessment.

Phosphonate inhibitors of West Nile virus NS2B/NS3 protease

West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)P(OPh)2 displaying Ki and k2/Ki values of 0.4 µM and 28 265 M−1s−1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT protease.

Isosorbide-based peptidomimetics as inhibitors of hepatitis C virus serine protease

Hepatitis C infection is a cause of chronic liver diseases such as cirrhosis and carcinoma. The current therapy for hepatitis C has limited efficacy and low tolerance. The HCV encodes a serine protease which is critical for viral replication, and few protease inhibitors are currently on the market. In this paper, we describe the synthesis and screening of novel isosorbide-based peptidomimetic inhibitors, in which the compounds 1d, 1e, and 1i showed significant inhibition of the protease activity in vitro at 100µM. The compound 1e also showed dose-response (IC50=36±3µM) and inhibited the protease mutants D168A and V170A at 100µM, indicating it as a promising inhibitor of the HCV NS3/4A protease. Our molecular modeling studies suggest that the activity of 1e is associated with a change in the interactions of S2 and S4 subsites, since that the increased flexibility favors a decrease in activity against D168A, whereas the appearance of a hydrophobic cavity in the S4 subsite increase the inhibition against V170A strain.

Unraveling the structural basis of grazoprevir potency against clinically relevant substitutions in hepatitis C virus NS3/4A protease from genotype 1a

Grazoprevir is a potent pan-genotype and macrocyclic inhibitor of hepatitis C virus (HCV) NS3/4A protease and was developed for treating chronic HCV infection. In HCV genotype (GT) 1a, grazoprevir maintains potent activity against a majority of NS3 resistance-associated amino acid substitutions, including the highly prevalent and naturally occurring Q80K polymorphism that impacts simeprevir, another NS3/4A protease inhibitor. The basis for an unexpected difference in the clinical impact of some NS3 substitutions was investigated. Phenotypic analysis of resistance-associated substitutions identified in NS3 from GT1a-infected patients who failed therapy with grazoprevir (in combination with elbasvir, an inhibitor of HCV NS5A protein) showed that positions 56, 156, and 168 in NS3 were most impactful because they diminished protein-inhibitor interactions. Although an amino acid substitution from aspartic acid to alanine at position 168 (D168A) reduced the potency of grazoprevir, its combination with R155K unexpectedly nullified this effect. Molecular dynamics and free-energy surface studies indicated that Asp-168 is important in anchoring Arg-155 for ligand binding but is not critical for Lys-155 because of the inherent flexibility of its side chain. Moreover, modeling studies supported a strong direct cation-heterocycle interaction between the Lys-155 side chain of the double substitution, R155K/D168A, and the lone pair on the quinoxaline in grazoprevir. This unique interaction provides a structural basis for grazoprevir's higher potency than simeprevir, an inhibitor to which the double substitution confers a significant reduction in potency. Our findings are consistent with the detection of R155K/D168A in NS3 from virologic failures treated with simeprevir but not grazoprevir.

Why Should We Target Viral Serine Proteases When Developing Antiviral agents?

Why Should We Target Viral Serine Proteases When Developing Antiviral agents?