Cathepsin S dose-response confirmation
Human cathepsin S (EC 184.108.40.206) is a lysosomal cysteine protease that is expressed in antigen-presenting cells, especially dendritic cells, B-cells and macrophages. Cathepsin S plays a key role in the processing of antigenic peptides for presentation by MHC Class II molecules on the surface of antigen-presenting cells. Thus inhibitors of cathepsin S may be immunomodulators effective in the treatment of autoimmune diseases. ..more
BioActive Compounds: 34
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 cathepsin S (EC 220.127.116.11) is a lysosomal cysteine protease that is expressed in antigen-presenting cells, especially dendritic cells, B-cells and macrophages. Cathepsin S plays a key role in the processing of antigenic peptides for presentation by MHC Class II molecules on the surface of antigen-presenting cells. Thus inhibitors of cathepsin S may be immunomodulators effective in the treatment of autoimmune diseases.
A high-throughput screen for cathepsin S 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 501). Compounds identified as hits in HTS of mixtures of 10 compounds per well were tested individually in dose-response using the AMC release assay. Results of this dose-response confirmation are reported here.
Recombinant human cathepsin S was purchased from Calbiochem (Cat #219343). Substrate Z-Phe-Arg-AMC was from Bachem (Cat #I-1160.0050). Assay buffer consisted of 50 mM sodium phosphate, pH 6.5, 5 mM EDTA, 5 mM DTT, and 0.01% Triton X-100. Low-volume 384-well black plates were from Corning (Item #3676).
Cathepsin S (0.04 ug/mL) was incubated with Z-Phe-Arg-AMC substrate (15 uM) in 10 uL of assay buffer (see above) for 1 hr at room temperature. Activity of single compounds identified from mixture HTS were confirmed by IC50 determination as described below.
1.Serial dilute single 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-Phe-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
6.Add 5 uL enzyme (0.08 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. IC50 plates 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 >30% inhibition in both locations 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]
Forty-six compounds that gave percent inhibition >30 in both locations in the primary HTS were judged to be hits and were selected for follow-up IC50 testing. An additional 83 compounds that gave percent inhibition >30 in only one location were also selected for IC50 testing (see below for analysis of screening results). 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
All compounds that inhibited cathepsin S 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 S 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
Analysis of screening results
Results of retesting the 46 compounds that gave percent inhibition >30 in both locations in the primary HTS (AID 501) were as follows:
Hits (>30% inhibition in both locations) = 46
Hits active in IC50 = 28 (61% retest rate)
Hits marginal in IC50 = 2 (IC50 >50uM, percent inhibition 30-50% at 50 uM)
Hits inactive, one mixture contained another compound confirmed active = 1
Hits inactive, no other active compounds present = 15 (33% false positive rate)
An additional 1213 compounds in AID 501 gave >30% inhibition in one location only. Further computational and experimental analyses were carried out to explore whether any of these compounds might in fact be active. In most cases (904 out of 1213 compounds), the percent inhibition >30 in one location only could be attributed to a different component of that mixture that had already been selected as a hit based on the selection criteria described above. The validity of this conclusion was confirmed by IC50 testing of a representative selection comprising 39 of these compounds. All 39 were inactive (IC50 >50 uM, percent inhibition at 50 uM <30%). Thus the 904 compounds sharing a mixture with an active compound are reported as inactive in AID 501.
For the remaining 309 compounds out of the 1213 that showed >30% inhibition at one location only in AID 501, activity could not be attributed to another active compound in the mixture. Of these 309 compounds, 22 showed >23% inhibition in the second location. (23% represents 3 standard deviations from the mean control averaged over the entire HTS run.) These 22 compounds were selected for dose-response testing. The results are included in the data table in this AID and are summarized below:
Compounds that showed >30% inhibition in one location & 27-30% in the second = 10
Active in IC50 = 6 (60% retest rate)
Marginal in IC50 = 0 (IC50 >50uM, percent inhibition 30-50% at 50 uM)
Inactive in IC50 = 4 (40% false positive rate)
Compounds that showed >30% inhibition in one location & 23-27% in the second = 12
Active in IC50 = 0 (0% retest rate)
Marginal in IC50 = 0 (IC50 >50uM, percent inhibition 30-50% at 50 uM)
Inactive in IC50 = 12 (100% false positive rate)
The progressively lower retest rate as the activity criteria were relaxed from >30/>30 (61% retest) to >30/27-30 (60% retest), and then to >30/23-27 (0% retest) suggested that further relaxation of the activity criteria was unlikely to reveal additional active compounds. This was confirmed by retesting a selection of 19 compounds that showed 40-90% inhibition in one location but <23% inhibition in the second location. None were active (IC50 >50 uM), although one compound showed marginal inhibition at 50 uM.
Finally, three compounds that showed a significant fluorescence enhancement in the second location (<-30% inhibition) were retested in case activity was masked by the presence of a highly fluorescent compound in the second location. None of the three compounds were active in IC50 testing.
Summary of retest results
>30% inhibition in both locations = 46 compounds, of which 28 active (IC50 <50 uM)
>30 inhibition in one location, 27-30% in second = 10 compounds, 6 active
>30% inhibition in one location, 23-27% in second = 12 compounds, none active
>30% inhibition in one location, <-30% in second = 3 compounds, none active
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by 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)