Human cathepsin S (EC 18.104.22.168) 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: 33
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 cathepsin S (EC 22.214.171.124) 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.
The high-throughput screen for cathepsin S 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. The MLSCN compound library was screened as mixtures of 10 compounds per well and active compounds were confirmed by single compound IC50 determination.
This assay is a part of the Molecular Library Screening Center Network (MLSCN).
Rationale for mixture screening
One of our goals at the Penn Center for Molecular Discovery (PCMD) is to develop capabilities for screening multiple members of target classes, for example cysteine and serine proteases. Many HTS labs focus effort on one target of interest within a class due to resource and time constraints. A few compounds are then tested for selectivity against additional target class members during the hit-to-lead process. Our goal is to test the entire MLSCN compound library against multiple cysteine and serine proteases to obtain a profile of activity against these enzymes classes. This profile may then be used to immediately identify selective compounds during subsequent screening of novel enzyme targets. It may also be possible to identify a subset of the library with an enhanced hit rate towards these enzyme families that might provide the basis for gene family screening.
One strategy that enables the rapid screening of multiple targets is to screen compound mixtures. We chose to evaluate an orthogonal pooling strategy that gives 10 compounds per well, as described in detail below. An advantage of this method is that each compound is in two plates, mixed with a different set of 9 other compounds at each location. This duplication improves the HTS statistics and greatly simplifies deconvolution of the results as activity in both wells containing a given compound immediately identifies that compound as a hit. This method has been used with some success in several pharmaceutical companies (see for example Devlin, J.J., et al. Drug Dev. Res. 35, 80-85, 1996).
This compound pooling strategy has been validated against the cysteine protease cathepsin B. Comparison of the results of screening 64,000 compounds from the MLSCN library as mixtures (Pubchem Assay ID 488) and as single compounds (Pubchem Assay ID 453), revealed the same profile of active hits in both cases. All but one of the active compounds discovered by single-compound screening was also identified by mixture screening.
200 plates containing 64,000 compounds from the MLSCN library were arranged in two 10 x 10 grids of 100 plates each. Sets of ten plates were pooled as follows to give 20 mixture plates per 100 single compound plates:
Plate 1_2_3_4_5_6_7_8_9_10 pooled to mixture plate 1-10
Plate 11_12_13_14_15_16_17_18_19_20 pooled to mixture plate 11-20
Plate 21_22_23_24_25_26_27_28_29_30 pooled to mixture plate 21-30
Plate 31_32_33_34_35_36_37_38_39_40 pooled to mixture plate 31-40
Plate 41_42_43_44_45_46_47_48_49_50 pooled to mixture plate 41-50
Plate 51_52_53_54_55_56_57_58_59_60 pooled to mixture plate 51-60
Plate 61_62_63_64_65_66_67_68_69_70 pooled to mixture plate 61-70
Plate 71_72_73_74_75_76_77_78_79_80 pooled to mixture plate 71-80
Plate 81_82_83_84_85_86_87_88_89_90 pooled to mixture plate 81-90
Plate 91_92_93_94_95_96_97_98_99_100 pooled to mixture plate 91-100
Plates were also pooled 'vertically':
Plates 1, 11, 21, 31, 41, 51, 61, 71, 81, 91 pooled to mixture plate 1-91
Plates 2, 12, 22, 32, 42, 52, 62, 72, 82, 92 pooled to mixture plate 2-92
And so on until mixture plate 10-100.
For example, the single compound in well A3 in Plate 1 is mixed with 9 compounds in well A3 in mixture plate 1-10, and also with 9 different compounds in well A3 in mixture plate 1-91.
The concentration of each mixture was 2.5 mM in DMSO (250 uM per compound). Pintool transfer into the HTS assay gave a final mixture concentration of 50 uM in 2% DMSO (5 uM per compound).
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. HTS was performed using 50 uM compound mixture (5 uM each of 10 compounds). Hits obtained will be confirmed on single compounds by IC50 determination in a dose-response mode.
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 mixture (2.5 mM in DMSO) using Evolution pintool
4.Add 1 uL of Z-Phe-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
5.Add 5 uL enzyme (0.08 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
Mixture percent inhibition---Data were analyzed in IDBS ActivityBase. Each HTS plate contained compound mixtures (50 uM in 2% DMSO) in columns 3-22, controls (enzyme, no compound mixture) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. HTS percent inhibition was calculated for each compound mixture 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)))
Assignment of percent inhibition to individual compounds---Mixture percent inhibition data were retrieved in IDBS SARgen, together with the 10 compounds contained in each mixture. The data were rearranged in Excel using VLookup functions such that each row contained a single compound and the two percent inhibition values corresponding to the location of the compound in the mixture plates.
Activity scoring is complicated by the fact that the two percent inhibition values associated with each compound in fact represent two different mixtures of 10 compounds. Thus simple averaging of the two values is not meaningful. Instead each compound was assigned a percent inhibition score based on the lower of the two percent inhibition values. This system appropriately scores hits showing activity in both mixtures, but avoids assignment of an erroneously high score to inactives that shared one location with an active compound.
Percent inhibition scores were calculated from the lower of the two percent inhibition values associated with each compound as follows:
(1) For positive percent inhibition, score = lower of the two percent inhibitions
(2) For negative percent inhibition, score = 0
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.
Activity outcome is reported as follows:
(1) Percent inhibition > 30 in both wells tested: Active
(2) Percent inhibition < 30 in one or both wells tested: Inactive
Summary of results
>30% inhibition in both locations = 33 compounds
<30% inhibition in one or both locations = 61996
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by 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)