AID	Name	Type	Comment
825	Cathepsin L dose-response confirmation	confirmatory
1627	Cathepsin L dose-response testing in the presence of cysteine	confirmatory
1633	Cathepsin L probe dose-response testing	confirmatory
1636	Cathepsin L probe #2 dose-response testing	confirmatory

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).

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)))

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).