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Dengue virus type 2 NS3 protease and NS2B-NS3 protease precursor induce apoptosis

Shafee N, AbuBakar S

J Gen Virol, 2003 Aug;84(Pt 8):2191-2195.
PMID: 12867651 DOI: 10.1099/vir.0.19022-0

Apoptosis was detected in Vero cell cultures expressing transfected dengue virus type 2 (DENV-2) genes. Approximately 17.5 and 51.5 % of cells expressing NS3 serine protease and NS2B-NS3(185) serine protease precursor protein [NS2B-NS3(185)(pro)] genes, respectively, were apoptotic. The percentage of apoptotic cells was significantly higher in cell cultures expressing NS2B-NS3(185)(pro). NS2B-NS3(185)(pro) was detected as NS2B-NS3(185)(pro)-EGFP fusion protein in cytoplasmic vesicular structures in the apoptotic cells. Site-directed mutagenesis which replaced His(51) with Ala within the protease catalytic triad significantly reduced the ability of the expressed NS3 and NS2B-NS3(185)(pro) to induce apoptosis. Results from the present study showed that DENV-2-encoded NS3 serine protease induces apoptosis, which is enhanced in cells expressing its precursor, NS2B-NS3(185)(pro). These findings suggest the importance of NS2B as a cofactor to NS3 protease-induced apoptosis.

Matched MeSH terms: Serine Endopeptidases
Fulltext Pharmacophore modelling of vanillin derivatives, favipiravir, chloroquine, hydroxychloroquine, monolaurin and tetrodotoxin as MPro inhibitors of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)

Law WY, Asaruddin MR, Bhawani SA, Mohamad S

BMC Res Notes, 2020 Nov 11;13(1):527.
PMID: 33176880 DOI: 10.1186/s13104-020-05379-6

OBJECTIVES: The aim of this study was to use Ligand-based pharmacophore modelling approach for four established antiviral drugs, namely remdesivir, lopinavir, ritonavir and hydroxychloroquine for COVID-19 inhibitors as training sets. In this study Twenty vanillin derivatives together with monolaurin and tetrodotoxin were used as test sets to evaluate as potential SARS-CoV-2 inhibitors. The Structure-based pharmacophore modelling approach was also performed using 5RE6, 5REX and 5RFZ in order to analyse the binding site and ligand-protein complex interactions.
RESULTS: The pharmacophore modelling mode of 5RE6 displayed two Hydrogen Bond Acceptors (HBA) and one Hydrophobic (HY) interaction. Besides, the pharmacophore model of 5REX showed two HBA and two HY interactions. Finally, the pharmacophore model of 5RFZ showed three HBA and one HY interaction. Based on ligand-based approach, 20 Schiff-based vanillin derivatives, showed strong MPro inhibition activity. This was due to their good alignment and common features to PDB-5RE6. Similarly, monolaurin and tetrodotoxin displayed some significant activity against SARS-CoV-2. From structure-based approach, vanillin derivatives (1) to (12) displayed some potent MPro inhibition against SARS-CoV-2. Favipiravir, chloroquine and hydroxychloroquine also showed some significant MPro inhibition.

Matched MeSH terms: Cysteine Endopeptidases
Fulltext Study the antiviral activity of some derivatives of tetracycline and non-steroid anti inflammatory drugs towards dengue virus

Rothan HA, Buckle MJ, Ammar YA, Mohammadjavad P, Shatrah O, Noorsaadah AR, et al.

Trop Biomed, 2013 Dec;30(4):681-90.
PMID: 24522138

Various clinical symptoms are caused by dengue virus ranging from mild fever to severe hemorrhagic fever while there is no successful anti-dengue therapeutics available. Among different strategies towards identifying and developing anti-dengue therapeutics, testing anti-dengue properties of known drugs could represent an efficient strategy for which information of its medical approval, toxicity and side effects is readily available. In this study, we evaluated the antiviral activity of some medical compounds towards dengue NS2B-NS3 protease (DENV2 NS2B-NS3pro) as a target to inhibit dengue virus replication. Mefenamic acid, a non-steroid anti inflammatory drug and doxycycline, a derivative antibiotic of tetracycline both showed significant inhibition potential against DENV2 NS2B-NS3pro Ki values 32 ± 2 μM and 55 ± 5 μM respectively. The effective cytotoxic concentrations of 50% (CC50) against Vero cells were evaluated for mefenamic acid (150 ± 5 μM) and doxycycline (125 ± 4 μM). Concentrations lower than CC50 were used to test the inhibition potential of these compounds against DENV2 replication in Vero cells. The results showed significant reduction in viral load after applying mefenamic acid and doxycyline in concentration dependent manner. Mefenamic acid reduced viral RNA at EC50 of 32 ± 4 μM whilst doxycycline EC50 was 40 ± 3 μM. Mefenamic acid showed higher selectivity against dengue virus replication in vitro compared to doxycycline. These findings underline the need for further experimental and clinical studies on these drugs utilizing its anti-dengue and anti-inflammatory activities to attenuate the clinical symptoms of dengue infection.

Matched MeSH terms: Serine Endopeptidases
Fulltext Antiviral cationic peptides as a strategy for innovation in global health therapeutics for dengue virus: high yield production of the biologically active recombinant plectasin peptide

Rothan HA, Mohamed Z, Suhaeb AM, Rahman NA, Yusof R

OMICS, 2013 Nov;17(11):560-7.
PMID: 24044366 DOI: 10.1089/omi.2013.0056

Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.

Matched MeSH terms: Serine Endopeptidases/metabolism
Fulltext Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication

Rothan HA, Bahrani H, Mohamed Z, Abd Rahman N, Yusof R

PLoS One, 2014;9(4):e94561.
PMID: 24722532 DOI: 10.1371/journal.pone.0094561

Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.

Matched MeSH terms: Serine Endopeptidases/metabolism
Fulltext A quaternary ammonium silane antimicrobial triggers bacterial membrane and biofilm destruction

Daood U, Matinlinna JP, Pichika MR, Mak KK, Nagendrababu V, Fawzy AS

Sci Rep, 2020 07 03;10(1):10970.
PMID: 32620785 DOI: 10.1038/s41598-020-67616-z

To study the antimicrobial effects of quaternary ammonium silane (QAS) exposure on Streptococcus mutans and Lactobacillus acidophilus bacterial biofilms at different concentrations. Streptococcus mutans and Lactobacillus acidophilus biofilms were cultured on dentine disks, and incubated for bacterial adhesion for 3-days. Disks were treated with disinfectant (experimental QAS or control) and returned to culture for four days. Small-molecule drug discovery-suite was used to analyze QAS/Sortase-A active site. Cleavage of a synthetic fluorescent peptide substrate, was used to analyze inhibition of Sortase-A. Raman spectroscopy was performed and biofilms stained for confocal laser scanning microscopy (CLSM). Dentine disks that contained treated dual-species biofilms were examined using scanning electron microscopy (SEM). Analysis of DAPI within biofilms was performed using CLSM. Fatty acids in bacterial membranes were assessed with succinic-dehydrogenase assay along with time-kill assay. Sortase-A protein underwent conformational change due to QAS molecule during simulation, showing fluctuating alpha and beta strands. Spectroscopy revealed low carbohydrate intensities in 1% and 2% QAS. SEM images demonstrated absence of bacterial colonies after treatment. DAPI staining decreased with 1% QAS (p

Matched MeSH terms: Cysteine Endopeptidases