Cathepsin B dose-response confirmation
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: 37
Screening Center: Penn Center for Molecular Discovery
Center Affiliation: University of Pennsylvania
Network: Molecular Library Screening Center Network (MLSCN)
Assay Provider: Scott Diamond, University of Pennsylvania
Grant number: 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.
A high-throughput screen for cathepsin B inhibitors was designed as an end-point assay monitoring the release of the fluorophore aminomethyl coumarin (AMC) upon enzymatic hydrolysis of an AMC-labeled dipeptide. Primary HTS results have been reported previously (AID 453). Actives from the HTS were tested in dose-response using the AMC release assay. Results of this dose-response confirmation are reported here.
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 actives were confirmed by IC50 determination as described below.
1.Serial dilute compounds at 50x concentration in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM)
2.Fill low-volume plate with 4 uL water using Multidrop-micro
3.Add 5 uL assay buffer to columns 1 and 23 using Multidrop-384
4.Add 200 nL of compound (in DMSO from step 1) using Evolution pintool
5.Add 1 uL of Z-Arg-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
6.Add 5 uL enzyme (0.13 ug/mL in assay buffer) using Multidrop-384
7.Incubate for 1 hr at room temperature
8.Read fluorescence (excitation 355, emission 460) on Envision reader
Data were analyzed in IDBS ActivityBase. Each IC50 plate contained compounds in columns 3-22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. Each column 3-22 contained 16 two-fold dilutions of a single compound, ranging in concentration from 50 uM to 1.5 nM. Percent activity was calculated for each dilution of 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:
% Activity = 100*((signal-blank mean)/(control mean-blank mean))
Dose response curves of percent activity were fit using XLfit equation 205 (four parameter logistic fit with maximum percent activity and minimum percent activity fixed at 100 and 0, respectively).
Activity scoring is based on the linear-log formula developed by Eduard Sergienko at the San Diego Center for Chemical Genomics. The activity score reported here is calculated from the results of follow-up IC50 testing on compounds that showed >20% inhibition in the primary HTS:
Activity score = IC50 score #1 + IC50 score #2 + IC50 score #3.
IC50 scores were calculated as follows:
(1) Score = 5.75 x (pIC50-3), where pIC50 = -log(10) of IC50 in mol/L
(2) For IC50 >50 uM (zero in IC50 column), score was calculated from percent activity at maximum concentration tested in assay (50 uM):
Score = [5.75 x (0-3)] + [(100-percent activity at max concentration)/1.75]
Compounds that gave percent inhibition >20 in the primary HTS were judged to be hits and these compounds were selected for follow-up IC50 testing. IC50 values were determined as described in protocol above. The percent activity at the maximum concentration is reported and can be used to estimate the potency of compounds for which the IC50 values were >50 uM.
Activity outcome is reported as follows:
(1) IC50 <50 uM in all three IC50 determinations = active
(2) IC50 >50 uM in all IC50 determinations = inactive
(3) IC50 <50 uM in one or more determinations & >50 uM in one or more = inconclusive
Analysis of screening results
Results of retesting compounds that gave percent inhibition >20 in the primary HTS were as follows:
Hits (>20% inhibition in both locations) = 76
Hits active in IC50 = 36 (47% retest rate)
All compounds that inhibited cathepsin B under the conditions described above were also tested in the presence of cysteine in the assay buffer in place of DTT. It has been reported previously (Smith G.K., et al. Arch. Biochem. Biopys. 399, 195-205, 2002) that redox-sensitive compounds can be reduced by DTT to produce reactive oxygen species such as hydrogen peroxide. Under these conditions enzymes that contain an active-site cysteine are inactivated by thiol oxidation. There are numerous examples of such compounds that react with DTT and thus cause enzyme inactivation but show no activity in the presence of cysteine.
Compounds that inhibited cathepsin B in the presence of DTT but not cysteine were judged to be artifacts. These compounds are reported as active, but are flagged as artifacts in the assaydata_comment field. To retrieve the 'artifact' notation alongside the activity score the data must be retrieved as follows:
(1) Next to 'Test Results' click on the 'Select' box (not 'show')
(2) Under 'Select Bioassay Results', click on the plus-sign icon next to 'Contributed Cross References'
(3) Next to 'Comment', click the check box
(4) Next to 'Test Results', click on the 'Show' box
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 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).
* Activity Concentration. ** Test Concentration.
Data Table (Concise)