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BioAssay: AID 453

Cathepsin B

Human liver cathepsin B (EC 3.4.22.1) is a lysosomal cysteine protease. There has been a recent resurgence of interest in cathepsin B due to research showing that proteolysis by this enzyme is required for the entry and replication of the Ebola and SARS viruses in human cells. Thus cathepsin B inhibitors have potential as novel anti-viral agents. ..more
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 Tested Compounds
 Tested Compounds
All(63332)
 
 
Active(75)
 
 
Inactive(63257)
 
 
 Tested Substances
 Tested Substances
All(63332)
 
 
Active(75)
 
 
Inactive(63257)
 
 
 Related BioAssays
 Related BioAssays
AID: 453
Data Source: PCMD (CATB)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-08-08
Modify Date: 2008-10-07

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 75
Related Experiments
AIDNameTypeComment
488Cathepsin B compound mixture screeningScreeningdepositor-specified cross reference
501Cathepsin SScreeningdepositor-specified cross reference
538Complement factor C1sScreeningdepositor-specified cross reference
581Cathepsin GScreeningdepositor-specified cross reference
820Cathepsin B dose-response confirmationConfirmatorydepositor-specified cross reference
830Cathepsin B mixture HTS dose-response confirmationConfirmatorydepositor-specified cross reference
Description:
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Dr. Scott L. Diamond, University of Pennsylvania
MLSCN Grant: X01-MH076406-01

Human liver cathepsin B (EC 3.4.22.1) is a lysosomal cysteine protease. There has been a recent resurgence of interest in cathepsin B due to research showing that proteolysis by this enzyme is required for the entry and replication of the Ebola and SARS viruses in human cells. Thus cathepsin B inhibitors have potential as novel anti-viral agents.

Cathepsin B is also implicated in cancer progression. Upregulation and secretion of this enzyme occurs in many types of tumors and correlates positively with their invasive and metastatic capabilities. Cathepsin B facilitates tumor invasion by dissolving extracellular barriers. Inhibitors of cathepsin B thus have been studied as potential anti-cancer agents.

The high-throughput screen for cathepsin B inhibitors reported here consisted of an end-point assay monitoring the release of the fluorophore aminomethyl coumarin (AMC) upon enzymatic hydrolysis of an AMC-labeled dipeptide.

This assay is a part of the Molecular Library Screening Center Network (MLSCN).
Protocol
Materials

Human liver cathepsin B was purchased from Calbiochem (Cat #219362). Substrate Z-Arg-Arg-AMC was from Bachem (Cat #I-1135.0050). Assay buffer consisted of 100 mM sodium-potassium phosphate, pH 6.8 (86 mM potassium phosphate, monobasic; 7 mm sodium phosphate, monobasic; 7 mm sodium phosphate, tribasic), 1 mM EDTA, and 2 mM DTT. Low-volume 384-well black plates were from Corning (Item #3676).

Assay

Cathepsin B (0.065 ug/mL) was incubated with Z-Arg-Arg-AMC substrate (15 uM) in 10 uL of assay buffer (see above) for 1 hr at room temperature. HTS was performed using 10 uM compound.

HTS protocol

1.Fill low-volume plate with 4 uL water using Multidrop-micro
2.Add 5 uL assay buffer to columns 1 and 23 using Multidrop-384
3.Add 200 nL of compound (0.5 mM in DMSO) using Evolution pintool
4.Add 1 uL of Z-Arg-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
5.Add 5 uL enzyme (0.13 ug/mL in assay buffer) using Multidrop-384
6.Incubate for 1 hr at room temperature
7.Read fluorescence (excitation 355, emission 460) on Envision reader


Data analysis

Data were analyzed in IDBS ActivityBase. Each HTS plate contained compounds (10 uM in 2% DMSO) in columns 3-22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. HTS percent inhibition was calculated for each compound from the signal in fluorescence units (FU) and the mean of the plate controls and the mean of the plate blanks using the following equation:

% Inhibition = 100*(1-((signal-blank mean)/(control mean-blank mean)))
Comment
Activity scoring

For positive percent inhibition, score = percent inhibition

For negative percent inhibition, score = 0

(Note that compounds that fluoresce at excitation 355/emission 460 nm give a signal higher than the plate control that corresponds to a substantial negative percent inhibition)

Results of screening the MLSCN library of compounds against Cathepsin B were as follows:

Compounds that gave percent inhibition >20 at 10 uM concentration (Hits): 75
Compounds that gave percent inhibition <20 at 10 uM concentration (Inactive): 63257

The hits will be retested in a dose-response assay for confirmation of activity.

Contributors

This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Andrew Napper and Nuzhat Motlekar, and data were submitted by Nuzhat Motlekar and Andrew Napper, all of the University of Pennsylvania.

Our thanks go to Parag Shah and Bill Denney for enormous help in setting up the HTS lab and troubleshooting its operation.

Correspondence

Please direct correspondence to Andrew Napper (napper@seas.upenn.edu).
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Percent inhibitionFloat%
2Percent inhibition signal (FU)Float
3Percent inhibition control mean (FU)Float
4Percent inhibition control standard deviation (FU)Float
5Percent inhibition number of control wellsInteger
6Percent inhibition control percent CVFloat%
7Percent inhibition blank mean (FU)Float
8Percent inhibition blank standard deviation (FU)Float
9Percent inhibition number of blank wellsInteger
10Percent inhibition blank percent CVFloat%
11Percent inhibition signal-to-background ratioFloat
12Percent inhibition plate Z-factorFloat

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