2012

Tanshinones as selective and slow-binding inhibitors for SARS-CoV cysteine proteases

Bioorganic & Medicinal Chemistry
Volume 20, Issue 19, 1 October 2012, Pages 5928-5935

Ji-Young Park, Jang Hoon Kim, Young Min Kim, Hyung Jae Jeong, Dae Wook Kim, Ki Hun Park, Hyung-Jun Kwon, Su-Jin Park, Woo Song Lee, Young Bae Ryu

Abstract

In the search for anti-SARS-CoV, tanshinones derived from Salvia miltiorrhiza were found to be specific and selective inhibitors for the SARS-CoV 3CLpro and PLpro, viral cysteine proteases. A literature search for studies involving the seven isolated tanshinone hits showed that at present, none have been identified as coronaviral protease inhibitors. We have identified that all of the isolated tanshinones are good inhibitors of both cysteine proteases. However, their activity was slightly affected by subtle changes in structure and targeting enzymes. All isolated compounds (17) act as time dependent inhibitors of PLpro, but no improved inhibition was observed following preincubation with the 3CLpro. In a detail kinetic mechanism study, all of the tanshinones except rosmariquinone (7) were identified as noncompetitive enzyme isomerization inhibitors. However, rosmariquinone (7) showed a different kinetic mechanism through mixed-type simple reversible slow-binding inhibition. Furthermore, tanshinone I (5) exhibited the most potent nanomolar level inhibitory activity toward deubiquitinating (IC50 = 0.7 μM). Additionally, the inhibition is selective because these compounds do not exert significant inhibitory effects against other proteases including chymotrysin, papain, and HIV protease. These findings provide potential inhibitors for SARS-CoV viral infection and replication.

Keywords

Tanshinone, SARS-CoV, 3CLpro, PLpro, Slow-binding inhibitor


Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13

Bioorganic & Medicinal Chemistry Letters
Volume 22, Issue 12, 15 June 2012, Pages 4049-4054

Mi-SunYu, June Leeb, Jin Moo Lee, Younggyu KimcYoung-WonChin, Jun-GooJeee, Young-Sam Keum, Yong-Joo Jeong

Abstract

Severe acute respiratory syndrome (SARS) is an infectious disease with a strong potential for transmission upon close personal contact and is caused by the SARS-coronavirus (CoV). However, there are no natural or synthetic compounds currently available that can inhibit SARS-CoV. We examined the inhibitory effects of 64 purified natural compounds against the activity of SARS helicase, nsP13, and the hepatitis C virus (HCV) helicase, NS3h, by conducting fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. While none of the compounds, examined in our study inhibited the DNA unwinding activity or ATPase activity of human HCV helicase protein, we found that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity, but not the unwinding activity, nsP13. In addition, we observed that myricetin and scutellarein did not exhibit cytotoxicity against normal breast epithelial MCF10A cells. Our study demonstrates for the first time that selected naturally-occurring flavonoids, including myricetin and scultellarein might serve as SARS-CoV chemical inhibitors.

Keywords

Myricetin, Scutellarein, SARS-coronavirus (SARS-CoV) helicase

Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506

Virus Research
Volume 165, Issue 1, April 2012, Pages 112-117

Javier Carbajo- Lozoya, Marcel A. Müller, Stephan Kallies, Volker Thiel, Christian Drosten, Albrechtvon Brunn

Abstract

Recent research has shown that Coronavirus (CoV) replication depends on active immunophilin pathways. Here we demonstrate that the drug FK506 (Tacrolimus) inhibited strongly the growth of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E at low, non-cytotoxic concentrations in cell culture. As shown by plaque titration, qPCR, Luciferase- and green fluorescent protein (GFP) reporter gene expression, replication was diminished by several orders of magnitude. Knockdown of the cellular FK506-binding proteins FKBP1A and FKBP1B in CaCo2 cells prevented replication of HCoV-NL63, suggesting the requirement of these members of the immunophilin family for virus growth.

Keywords

SARS-CoV, HCoV-NL63, HCoV-229E, FK506, Tacrolimus, Immunophilins, FKBP1A (FKBP12), FKBP1B (FKBP12.6), Inhibition of viral replication


Diagnostics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid antigen using chicken immunoglobulin Y

Poultry Science
Volume 91, Issue 3, 1 March 2012, Pages 636-642

A .Palaniyappan, D. Das, S. Kammila, M.R. Suresh, H.H. Sunwoo

Abstract

The goal of this study was to develop a quantitative detection system for severe acute respiratory syndrome-associated coronavirus (SARS-CoV), targeting the nucleocapsid protein (NP), to determine the presence and degree of infection in suspected individuals. Because the NP is the viral protein shed during infection and its template mRNA is the most abundant subgenomic RNA, it is a suitable candidate for developing antibodies for diagnostic applications. In this study, we have prepared full-length SARS-CoV NP expressed in Escherichia coli and purified. Full-length NP was used for the preparation of mouse monoclonal antibody and chicken polyclonal IgY antibodies for the development of heterosandwich ELISA for early diagnostics of SARS-suspected individuals. The sensitivity of the developed heterosandwich ELISA can detect the viral antigen at 18.5 pg/mL of recombinant NP. This study describes ultrasensitive ELISA using 19B6 monoclonal antibody as the capture antibody and IgY as the detecting antibody against the most abundant SARS-CoV NP antigens. One of the most important findings was the use of inexpensive polyclonal IgY antibody to increase the sensitivity of the detection system for SARS-CoV at the picogram level. Furthermore, the immunoassay of SARS-CoV NP antigen developed could be an effective and sensitive method of diagnosing SARS-suspected individuals during a future SARS-CoV outbreak.

Key words

SARS-CoV, nucleocapsid protein, 19B6 monoclonal antibody, immunoglobulin Y, heterosandwich ELISA



Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506

Virus Research
Volume 165, Issue 1, April 2012, Pages 112-117

Javier Carbajo-Lozoya, Marcel A.Müller, Stephan Kallies, Volker Thiel, Christian Drosten, Albrechtvon Brunn

Abstract

Recent research has shown that Coronavirus (CoV) replication depends on active immunophilin pathways. Here we demonstrate that the drug FK506 (Tacrolimus) inhibited strongly the growth of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E at low, non-cytotoxic concentrations in cell culture. As shown by plaque titration, qPCR, Luciferase- and green fluorescent protein (GFP) reporter gene expression, replication was diminished by several orders of magnitude. Knockdown of the cellular FK506-binding proteins FKBP1A and FKBP1B in CaCo2 cells prevented replication of HCoV-NL63, suggesting the requirement of these members of the immunophilin family for virus growth.

Keywords

SARS-CoV, HCoV-NL63, HCoV-229E, FK506, Tacrolimus, Immunophilins, FKBP1A (FKBP12), FKBP1B, (FKBP12.6), Inhibition of viral replication

A hexapeptide of the receptor-binding domain of SARS corona virus spike protein blocks viral entry into host cells via the human receptor ACE2

Antiviral Research
Volume 94, Issue 3, June 2012, Pages 288-296

Anna-WinonaStruck, Marco Axmanna, Susanne Pfefferle, ChristianDrosten, Bernd Meyera

Abstract

In vitro infection of Vero E6 cells by SARS coronavirus (SARS-CoV) is blocked by hexapeptide Tyr-Lys-Tyr-Arg-Tyr-Leu. The peptide also inhibits proliferation of coronavirus NL63. On human cells both viruses utilize angiotensin-converting enzyme 2 (ACE2) as entry receptor. Blocking the viral entry is specific as alpha virus Sindbis shows no reduction in infectivity. Peptide 438YKYRYL443 is part of the receptor-binding domain (RBD) of the spike protein of SARS-CoV. Peptide libraries were screened by surface plasmon resonance (SPR) to identify RBD binding epitopes. 438YKYRYL443 carries the dominant binding epitope and binds to ACE2 with KD = 46 μM. The binding mode was further characterized by saturation transfer difference (STD) NMR spectroscopy and molecular dynamic simulations. Based on this information the peptide can be used as lead structure to design potential entry inhibitors against SARS-CoV and related viruses.

Keywords

SARS coronavirus, Spike protein, ACE2, Entry inhibitor, Virus proliferation assay, SPR screening

Diagnostics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid antigen using chicken immunoglobulin Y

Poultry Science
Volume 91, Issue 3, 1 March 2012, Pages 636-642

A. Palaniyappan, D.Das, S.Kammila, M.R.Suresh, H.H.Sunwoo

Abstract

The goal of this study was to develop a quantitative detection system for severe acute respiratory syndrome-associated coronavirus (SARS-CoV), targeting the nucleocapsid protein (NP), to determine the presence and degree of infection in suspected individuals. Because the NP is the viral protein shed during infection and its template mRNA is the most abundant subgenomic RNA, it is a suitable candidate for developing antibodies for diagnostic applications. In this study, we have prepared full-length SARS-CoV NP expressed in Escherichia coli and purified. Full-length NP was used for the preparation of mouse monoclonal antibody and chicken polyclonal IgY antibodies for the development of heterosandwich ELISA for early diagnostics of SARS-suspected individuals. The sensitivity of the developed heterosandwich ELISA can detect the viral antigen at 18.5 pg/mL of recombinant NP. This study describes ultrasensitive ELISA using 19B6 monoclonal antibody as the capture antibody and IgY as the detecting antibody against the most abundant SARS-CoV NP antigens. One of the most important findings was the use of inexpensive polyclonal IgY antibody to increase the sensitivity of the detection system for SARS-CoV at the picogram level. Furthermore, the immunoassay of SARS-CoV NP antigen developed could be an effective and sensitive method of diagnosing SARS-suspected individuals during a future SARS-CoV outbreak.

Key words

SARS-CoV nucleocapsid protein, 19B6 monoclonal antibody, immunoglobulin Y, heterosandwich ELISA