Human liver cathepsin B (EC 126.96.36.199) 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
BioActive Compounds: 75
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 188.8.131.52) 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).
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).
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.
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 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)))
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.
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.
Please direct correspondence to Andrew Napper (firstname.lastname@example.org).
Data Table (Concise)