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BioAssay: AID 460

Cathepsin L

Human liver cathepsin L (EC 3.4.22.15) is a lysosomal cysteine protease. Recent interest in cathepsin L has been generated by research showing that proteolysis by this enzyme is required for the entry and replication of the SARS and Ebola viruses in human cells. Thus cathepsin L inhibitors have potential as novel anti-viral agents. ..more
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 Tested Compounds
 Tested Compounds
All(57816)
 
 
Active(100)
 
 
Inactive(57716)
 
 
 Tested Substances
 Tested Substances
All(57821)
 
 
Active(100)
 
 
Inactive(57721)
 
 
 Related BioAssays
 Related BioAssays
AID: 460
Data Source: PCMD (CATL)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-09-08
Modify Date: 2008-10-07

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 100
Depositor Specified Assays
AIDNameTypeComment
825Cathepsin L dose-response confirmationconfirmatory
1627Cathepsin L dose-response testing in the presence of cysteineconfirmatory
1633Cathepsin L probe dose-response testingconfirmatory
1636Cathepsin L probe #2 dose-response testingconfirmatory
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 L (EC 3.4.22.15) is a lysosomal cysteine protease. Recent interest in cathepsin L has been generated by research showing that proteolysis by this enzyme is required for the entry and replication of the SARS and Ebola viruses in human cells. Thus cathepsin L inhibitors have potential as novel anti-viral agents.

Cathepsin L inhibitors may also be active against Plasmodium falciparum, the parasite responsible for human malaria. Plasmodium contains cathepsin L-like cysteine proteases known as falcipains that appear to promote virulence of the parasite through haemoglobin digestion and erythrocyte invasion.

The high-throughput screen for cathepsin L 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 Libraries Screening Center Network (MLSCN).
Protocol
Materials

Human liver cathepsin L was purchased from Calbiochem (Cat #219402). Substrate Z-Phe-Arg-AMC was from Sigma-Aldrich. Assay buffer consisted of 20 mM sodium acetate, pH 5.5, containing 1 mM EDTA, and 5 mM DTT. Low-volume 384-well black plates were from Corning (Item #3676).

Assay

Cathepsin L (8.7 ng/mL) was incubated with Z-Phe-Arg-AMC substrate (1 uM) in 10 uL of assay buffer (see above) for 1 hr at room temperature. HTS was performed using 10 uM compound as described below.

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-Phe-Arg-AMC substrate (10 uM in 5x assay buffer) using Multidrop-micro
5. Add 5 uL enzyme (17.4 ng/mL in assay buffer) using Multidrop-384
6. Incubate for 1 hr at room temperature
7. Read fluorescence (excitation 360, 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)

Activity Outcome

Activity outcome is reported as follows:

(1) percent inhibition > 45 = active
(2) perecent inhibition < 45 = inactive

Analysis of screening results

Results of testing compounds the MLSCN library of compounds against Cathepsin L are as follows:

Hits (>45% inhibition): 100 compounds
Inactives (<45% inhibition): 57721 compounds

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 done by Parag Shah, and data were submitted by Nuzhat Motlekar and Andrew Napper, all of the University of Pennsylvania.

Please address correspondence to Parag Shah (ppshah@seas.upenn.edu).
Result Definitions
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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)
Classification
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