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BioAssay: AID 577

HTS to identify Inhibitors of West Nile Virus NS2bNS3 Proteinase

The HTS assay to identify Inhibitors of West Nile Virus NS2bNS3 Proteinase was proposed by Dr Alex Strongin of the Burnham Institute XO1-MH077601, and was developed and screened at the University of Pittsburgh Molecular Library Screening Center part of the Molecular Library Screening Center Network (MLSCN). ..more
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 Tested Compounds
 Tested Compounds
All(65217)
 
 
Active(119)
 
 
Inactive(65099)
 
 
 Tested Substances
 Tested Substances
All(65239)
 
 
Active(119)
 
 
Inactive(65120)
 
 
AID: 577
Data Source: University of Pittsburgh Molecular Library Screening Center (In vitro West Nile Virus NS2bNS3 Proteinase HTS)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2007-01-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 119
Related Experiments
AIDNameTypeComment
653West Nile Virus NS2bNS3 Proteinase Inhibitor Dose Response Confirmation.Confirmatorydepositor-specified cross reference
Description:
The HTS assay to identify Inhibitors of West Nile Virus NS2bNS3 Proteinase was proposed by Dr Alex Strongin of the Burnham Institute XO1-MH077601, and was developed and screened at the University of Pittsburgh Molecular Library Screening Center part of the Molecular Library Screening Center Network (MLSCN).

Extracted from the XO1-MH077601 Proposal submitted by Dr. Alex Strongin, Burnham Institute:

West Nile virus, a member of the Flaviviridae family, was first isolated in 1937 in the West Nile district of Uganda. West Nile virus is transmitted to animals including humans, through mosquito bites. Mosquitoes become infected when they feed on infected birds. In 2003, West Nile virus was detected in as many as 46 of the United States. The virus infected as many as 10,000 people and was the cause of approximately 300 deaths. The data for 2004 are even more troubling because the virus has spread and intensified throughout the US. West Nile virus is also a potential bioterrorism weapon. West Nile virus, Dengue and Yellow Fever flaviviruses are Priority Pathogens according to the classification issued by the NIAID.

West Nile virus, as other, similar flaviviruses, has an icosahedral core (30- to 35-nm in size) composed of multiple copies of a 12-kDa capsid protein. The capsid encloses a single-stranded RNA with a single reading frame encoding a polypeptide precursor of approximately 3,400 amino acid residues (Fig. 1). There are three structural proteins (C, prM and E) and seven non-structural proteins encoded by the West Nile virus genome (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). Both furin of the host and the virus-encoded NS3 serine proteinase are required to process the polyprotein precursor into the individual functional proteins.

The full-length NS3 peptide sequence in West Nile and Dengue viruses represents a multifunctional protein. The N-terminal 184 amino acid-long fragment of NS3 represents the NS3 proteinase. The C-terminal portion of the NS3 protein encodes a nucleotide triphosphatase, an RNA triphosphatase and a helicase. The NS3 proteinase is required for the maturation of the virus. The NS3 proteinase is responsible for cleaving the NS2a/NS2b, NS2b/NS3, NS3/NS4a and NS4b/NS5 junction regions. This proteinase is also responsible for the cleavage at the C-terminal region of the C protein. As is the case with a number of flaviviruses, the NS2b protein, that is located in the polypeptide precursor upstream of the NS3 proteinase, functions as a cofactor and promotes the proteolytic activity of the NS3 enzyme. The cofactor activity of the 40 amino acid long central portion of the NS2b is roughly equivalent to that of the entire NS2b sequence. Most importantly, inactivating mutations of the NS3 cleavage sites in the polyprotein precursor abolish virus infectivity. We hypothesize that the processing NS3 proteinase, which is an essential component of the virus life cycle, is the most promising drug target for anti-flaviviral inhibitors, from which novel, anti-viral therapies will emerge.

Currently, there are millions of cases of flaviviridae infections, especially Dengue throughout the world. West Nile virus is ranked as a Category B Priority Pathogen. In addition, West Nile virus is an emerging natural viral pathogen in the US. We believe that targeting the individual, unique NS3 processing protease, which is critical for the maturation of the viral proteins, will be the most successful drug strategy to block the flaviviral infection.

The primary objective of the HTS described here is to identify small molecule inhibitors that will inactivate the flaviviral NS3 serine proteinase. A homogenous, mix-and-measure, fluorescence peptide cleavage assay was proposed as the primary screening assay format. The cDNA fragment of the West Nile and Dengue genome encoding the NS2b-NS3 proteinase were cloned from cDNA fragments provided by Drs. Richard Kinney, CDC, Fort Collins, CO, and Michael Diamond, Washington University, St. Louis, MO. The wild-type NS2b-NS3 proteinase construct was expressed in E. coli and pilot-scale quantities of the homogeneous material were purified by Dr. Strongin and his colleagues at the Burnham Institute. Autolysis of the NS2b-NS3 precursor was used to generate the soluble, mature and homogenous NS3 proteinase. The cleavage assay employs the proteolytic enzyme, purified NS3 proteinase of West Nile virus provided by Dr Strongin of the Burnham Institute, and a commercially available fluorescence substrate Pyr-RTKR-AMC.
Protocol
NS2bNS3 HTS Assay Protocol.

Aprotinin, Sigma, A-4529.
Brij-35 solution, 30%, Sigma, B4184-1L
Dimethylsulfoxide, (DMSO) Sigma, 154938.
Glycerol, Sigma, G5516-1L.
Pyr-RTKR-AMC Substrate, BaChem I-1650.
Tris Base, Molecular Biology Grade, EMD Biosciences, 648310.

Consumables
384-well black low-volume assay plate, Greiner 784076.
384-well low profile pyramid bottom reservoir, Innovative Microplate S30022
PK384 Robotic Tip series, 30uL Axygen, PK-384-R


Preparation of Stock Solutions:

Aprotinin, 1 mM: To make 1 mM Aprotinin dissolve 6.51 mg Aprotinin in 1 mL 20% glycerol. Aliquot and store at -20#C.

Aprotinin Inhibitor Solution: Prepare Fresh Daily
100 nM: Dilute .5 uL 1 mM Aprotinin and 150uL DMSO in 4.85 mL 20% glycerol.
3uM : Dilute 3uL 1mM Aprotinin and 30uL DMSO in 967uL 20% glycerol.
4uM : Dilute 4uL 1mM Aprotinin and 30uL DMSO in 967uL 20% glycerol.

10X Assay Buffer: 100 mM Tris, pH8.0, 0.05% Brij-35:
100 mL 1 M Tris, pH 8.0
1.67 mL 30% Brij-35
898.3 ml Milli-Q H2O
Filter-sterilize

1X Assay Buffer:
1 mL 10X Assay Buffer
2 mL glycerol
7 mL Milli-Q H2O

DMSO, 3%:
Dilute DMSO to 3% in 20% glycerol.

Pyr-RTKR-AMC, 1 mM:
To make 1 mM Pyr-TRTK-AMC, dissolve 25 mg in 30 mL 20% glycerol.
Aliquot and store at -20#C.
Dilute 1mM stock to 180uM. Prepare fresh daily.

Glycerol, 20%:
Make a 20% solution of glycerol by mixing 200 mL glycerol with 800 mL Milli-Q H2O.

Tris, 1 M, pH 8.0:
Make a 1 M stock by dissolving 121 g Tris with 800 mL Milli-Q H2O.
Adjust pH to 8.0 with HCl.
Adjust final volume to 1 L.

NS2b-NS3 Stock:
1 mg/mL stock in 1X Assay Buffer: Stored in -20#C freezer.

NS2b-NS3 Enzyme solution; 2ug/mL NS2b/NS3, 10 mM Tris, pH 8.0, 0.005% Brij-35:
Thaw enzyme on ice and dilute 1 mg/ml NS2b-NS3 to 2ug/mL in 3X Assay Buffer, 20% glycerol. Prepare fresh daily.

3.0 mL 10X Assay Buffer
2.0 mL glycerol
20 uL 1 mg/ml NS2b-NS3
4.98 mL Milli-Q H2O


Pyr-RTKR-AMC Substrate 180uM:
Dilute 180uL 1 mM Pyr-RTKR-AMC in 820uL 20% glycerol.

Maximum Stimulation Control
5uL/ well 3% DMSO, 20% glycerol
5uL/well 2ug/ml NS2b-NS3
5uL/well 180uM Pyr-RTKR-AMC

Minimum Stimulation Control
5uL/ well 3uM Aprotinin Inhibitor Solution
5uL/well 2ug/ml NS2b-NS3
5uL/well 180uM Pyr-RTKR-AMC

50% Stimulation Control
5uL/ well 100 nM Aprotinin Inhibitor Solution
5uL/well 2ug/ml NS2b-NS3
5uL/well 180uM Pyr-RTKR-AMC

HTS Protocol:

1) Dilute compound plates in 20 % gluycerol. 10 to 100 uM final, 1% DMSO.
2) Transfer 5 uL of diluted compounds and plate controls in 20 % glycerol to wells of 384-well black low-volume 384-well assay plate, Greiner # 784076.
3) Transfer 5 uL/well 2 ug/ml NS2b-NS3 enzyme solution (3X assay buffer) to all wells.
4) Transfer 5 uL/well of 180 uM Pyr-RTKR-AMC substrate to all wells.
5) Centrifuge plate at 800xg for 30 secs-1 min.
6) Incubate assay plate at ambient temperature with shaking for 90 min. Do not stack plates!
7) Stop assay by transfer of 5 uL/well of 4 #M Aprotinin to assay plate.
8) Read fluorescence intensity at Ex 360 nm, Em 460 nm on the M5 plate reader.
Comment
The West Nile Virus NS2bNS3 HTS conducted at the PMLSC utilized a Z score statistical scoring method to identify active compounds (Brideau et al, 2003 J. Biomolecular Screening 8(6): p634-637.

The Z score for a compound is computed on a plate-by-plate basis. The Z score for the raw fluorescent intensity value Xi is defined as Zi = (Xi-Xm)/Sm, where Xm is the mean of all the raw fluorescent intensity values of the compounds on a plate (n=320), and Sm is the standard deviation of all these values. A cut off Z score of -3 was selected as the active criterion for the West Nile Virus NS2bNS3 HTS.

West Nile Virus NS2bNS3 Activity scoring rules:

PUBCHEM_ACTIVITY_OUTCOME

1 - Substance is considered inactive when Z-score is > -3
2 - Substance is considered active when Z-score is 3 - Substance activity outcome is inconclusive

PUBCHEM_ACTIVITY_SCORE

0-40 scoring range is reserved for primary HTS data
a) if Z-score is b) if Z-score is > -3 the score is 10


Definition of Hit Criteria: It is anticipated that all HTS actives will be confirmed in 10-pt IC50 dose response curves. Actives confirmed as dose dependent inhibitors of the West Nile Virus NS2bNS3 proteinase will be further evaluated and should exhibit:
No quenching of AMC fluorescence.
IC50 < 10 #M
Structural confirmation by LCMS.

Secondary assay testing paradigm:
Confirmed dose dependent NS2b-NS3 inhibitors will be screened for selectivity against Dengue Virus NS2b-NS3 proteinase (to be provided by Dr. Strongin). The assay provider will also profile confirmed inhibitors against host serine proteases including; furin, plasmin, urokinase, and trypsin. The assay provider plans to follow up interesting hits utilizing a variety of methodologies; iterative enzymatic tests, rational structure based design, computer-assisted docking, NMR ligand binding studies, and a variety of cell based assays including compound cytotoxicity evaluations, West Nile Virus subgenomic replicon and reporter gene assays, CytoPathic effect inhibition assays, and reduction in viral titer assays.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1HTS FI Ex360_ Em465Raw fluorescent intensity value at excitation 360 nm and emission 465 nm measured on the M5 microtiter plate readerFloat
2Z-scoreZ-score computed on a plate by plate basis, where the Z-zcore for the raw value of Xi is: Zi=(Xi-Xm)/SmFloat
3Plate Mean FIMean of the raw fluorescent intensity values of all the compounds on a plate (n=320)at excitation 360 nm and emission 465 nm measured on the M5 microtiter plate readerFloat
4Plate STD Dev FIStandard deviation from the Mean of the raw fluorescent intensity values of all the compounds on a plate (n=320)at excitation 360 nm and emission 465 nm measured on the M5 microtiter plate readerFloat
5HTS Assay DateDate the HTS assay was runString

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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