Cathepsin B mixture HTS dose-response confirmation
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 more ..
BioActive Compounds: 40
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
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).
Validation of the compound pooling strategy was provided by screening the MLSCN library against cathepsin B (EC 188.8.131.52), a human lysosomal cysteine protease. 64,000 compounds were screened in a single three-hour HTS run; the pooling strategy and the primary HTS results have been reported in AID 488. Actives from the HTS were tested in dose-response, and results of this dose-response confirmation are reported here. We have previously screened cathepsin B by single compound HTS (Pubchem Assay IDs 453 and 820), allowing us to compare the hit profile obtained by single and mixture screening. The most important consideration was to ensure that all of the active compounds discovered by single-compound screening were also identified by mixture screening.
The dose-response confirmation reported here employed the same assay as used in the high-throughput screen (AID 488), namely an end-point assay monitoring the release of the fluorophore aminomethyl coumarin (AMC) upon enzymatic hydrolysis of an AMC-labeled dipeptide.
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. 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-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. 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 >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 both locations in the primary HTS were selected for follow-up IC50 testing. Additional compounds that had shown activity in prior single compound screening (AIDs 453 and 820) were also tested to allow comparison of mixture and single-compound HTS.
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 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
Comparison between mixture screening and single compound HTS
Results of screening the same set of 64,000 compounds as mixtures of 10 and as single compounds were analyzed and compared as follows:
Summary of mixture results:
Hits (>20% inhibition in both locations) = 38
Hits active in IC50 = 30 (79% retest rate)
Hits marginal in IC50 = 3 (IC50 >50uM, percent inhibition 30-50% at 50 uM)
Hits inactive, one mixture contained another compound confirmed active = 4
Hits inactive, no other active compounds present = 1 (2.6% false positive rate)
Summary of single compound results (from Assay IDs 453 and 820):
Hits (>20% inhibition) = 73
Hits active in IC50 = 35 (48% retest rate)
Hits marginal in IC50 = 16 (IC50 >50uM, percent inhibition 30-50% at 50 uM)
Hits inactive = 22 (30% false positive rate)
[Note: IC50 results described as #marginal# above are listed as inactive in PubChem data table.]
The above retest statistics show that the mixture screening gave a much higher retest rate than the single compound screening (79% vs. 48%) and a >10-fold lower false positive rate (2.6% vs. 30%). This reflects the statistical value of screening each mixture component in duplicate.
The most important factor to consider in validating the mixture screening is the false negative rate. (i.e. Are there any confirmed active compounds from the single compound screening that were missed in the mixture HTS?). Excluding three compounds identified by mixture screening that were not hits in the single compound screening (<20% inhibition), there were 27 confirmed active hits shared between both single compound and mixture HTS. Thus the mixture screening missed 8 of the 35 confirmed actives from the single compound screening. However, this analysis looks significantly different if the DTT-reactive artifacts are excluded (see above for discussion of DTT-reactive artifacts).
Overlap of single compound and mixture actives (all compounds included):
Total single compound HTS hits active in IC50 testing = 35
Number of single compound IC50 actives that were hits in mixture HTS = 27 (77%)
Number of single compound IC50 actives missed in mixture HTS = 8 (23%)
Overlap of single compound and mixture actives (DTT-reactive artifacts excluded):
Total single compound HTS hits active in IC50 testing = 20
Number of single compound IC50 actives that were hits in mixture HTS = 19 (95%)
Number of single compound IC50 actives missed in mixture HTS = 1 (5%)
Mixture screening identified all but one (95%) of the true actives from the single compound screening, as well as three compounds not found in the single compound HTS. The fact that many of the DTT-reactive artifacts were not hits in the mixture screening suggests that other mixture components attenuate either the reduction of DTT-reactive compounds or the inactivation of the enzyme by the reactive oxygen species generated by the DTT-compound redox chemistry. Thus screening of cathepsin B against compound mixtures appeared to protect the enzyme from artifactual inactivation.
Screening of the MLSCN compound library as mixtures of 10 against cathepsin B identified 19 active compounds. These actives include all but one of the compounds previously identified by single compound screening against cathepsin B (Assay IDs 453 and 820). Thus mixture screening represents a viable alternative to single compound screening of proteases. Based on this validation the PCMD plans to profile additional cysteine and serine proteases using the mixture protocol described here.
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Nuzhat Motlekar and Andrew Napper, 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)