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BioAssay: AID 1627

Cathepsin L dose-response testing in the presence of cysteine

Human liver cathepsin L (EC 3.4.22.15) is a lysosomal cysteine protease. Recent interest in cathepsin L has been generated by research showing that proteolysis by this enzyme is required for the entry and replication of the SARS and Ebola viruses in human cells. Thus cathepsin L inhibitors have potential as novel anti-viral agents. ..more
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 Tested Compounds
 Tested Compounds
All(241)
 
 
Active(49)
 
 
Inactive(185)
 
 
Inconclusive(7)
 
 
 Tested Substances
 Tested Substances
All(241)
 
 
Active(49)
 
 
Inactive(185)
 
 
Inconclusive(7)
 
 
 Related BioAssays
 Related BioAssays
AID: 1627
Data Source: PCMD (CATL DOSE-RESPONSE CYS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-03-26

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 49
Depositor Specified Assays
AIDNameTypeComment
460Cathepsin Lscreening
825Cathepsin L dose-response confirmationconfirmatory
Description:
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 L (EC 3.4.22.15) is a lysosomal cysteine protease. Recent interest in cathepsin L has been generated by research showing that proteolysis by this enzyme is required for the entry and replication of the SARS and Ebola viruses in human cells. Thus cathepsin L inhibitors have potential as novel anti-viral agents.

Cathepsin L inhibitors may also be active against Plasmodium falciparum, the parasite responsible for human malaria. Plasmodium contains cathepsin L-like cysteine proteases known as falcipains that appear to promote virulence of the parasite through haemoglobin digestion and erythrocyte invasion.

A high-throughput screen for cathepsin L 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 and dose-response confirmation have been reported previously (AIDs 460 and 825). Confirmed actives from dose-response testing were retested using cysteine in the assay buffer instead of DTT. This allows the exclusion of DTT-reactive artifacts that inactivate the enzyme in the presence of DTT but are inactive in the presence of cysteine. Results of this dose-response confirmation are reported here.
Protocol
Materials

Human liver cathepsin L was purchased from Calbiochem (Cat #219402). Substrate Z-Phe-Arg-AMC was from Sigma-Aldrich. Assay buffer consisted of 20 mM sodium acetate, pH 5.5, containing 1 mM EDTA, and 5 mM cysteine. Low-volume 384-well black plates were from Corning (Item #3676).

Assay

Cathepsin L (8.7 ng/mL) was incubated with Z-Phe-Arg-AMC substrate (1 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.

IC50 protocol

1. Serial dilute compounds at 50x concentration in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM)
2. Fill Corning low-volume 384-well black 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 (10 uM in 5x assay buffer) using Multidrop-micro
6. Add 5 uL enzyme (17.4 ng/mL in assay buffer) using Multidrop-384
7. Incubate for 1 hr at room temperature
8. Read fluorescence (excitation 360, emission 460) on Envision reader

Data analysis

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; maximum percent activity and minimum percent activity fixed at 100 and 0, respectively; Hill slope limited to > -0.1.)
Comment
Activity scoring

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 >45% 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]

Active compound scores = 56-5
Inactive compound scores = 4-0

Activity Outcome

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

Contributors

This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were done by Parag Shah, and data were submitted by Andrew Napper, all of the University of Pennsylvania.

Please address correspondence to Andrew Napper (napper@seas.upenn.edu).
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierString
2IC50 mean*FloatμM
3QualifierString
4IC50 standard deviationFloatμM
5Number of IC50 determinationsInteger
6QualifierString
7IC50 #1FloatμM
8IC50 #1 Hill slopeFloat
9IC50 #1 R-squaredFloat
10IC50 #1 min concentrationFloatμM
11IC50 #1 percent activity at min concentrationFloat%
12IC50 #1 max concentrationFloatμM
13IC50 #1 percent activity at max concentrationFloat%
14IC50 #1 signal at 0.00152 microM (0.00152μM**)Float
15IC50 #1 signal at 0.00305 microM (0.00305μM**)Float
16IC50 #1 signal at 0.00610 microM (0.0061μM**)Float
17IC50 #1 signal at 0.01221 microM (0.01221μM**)Float
18IC50 #1 signal at 0.02441 microM (0.02441μM**)Float
19IC50 #1 signal at 0.04883 microM (0.04883μM**)Float
20IC50 #1 signal at 0.09766 microM (0.09766μM**)Float
21IC50 #1 signal at 0.19531 microM (0.19531μM**)Float
22IC50 #1 signal at 0.39063 microM (0.39063μM**)Float
23IC50 #1 signal at 0.78125 microM (0.78125μM**)Float
24IC50 #1 signal at 1.5625 microM (1.5625μM**)Float
25IC50 #1 signal at 3.125 microM (3.125μM**)Float
26IC50 #1 signal at 6.25 microM (6.25μM**)Float
27IC50 #1 signal at 12.5 microM (12.5μM**)Float
28IC50 #1 signal at 25 microM (25μM**)Float
29IC50 #1 signal at 50 microM (50μM**)Float
30IC50 #1 control meanFloat
31IC50 #1 control standard deviationFloat
32IC50 #1 number of control wellsInteger
33IC50 #1 control percent CVFloat%
34IC50 #1 blank meanFloat
35IC50 #1 blank standard deviationFloat
36IC50 #1 number of blank wellsInteger
37IC50 #1 blank percent CVFloat%
38IC50 #1 signal-background ratioFloat
39IC50 #1 plate Z-factorFloat
40QualifierString
41IC50 #2FloatμM
42IC50 #2 Hill slopeFloat
43IC50 #2 R-squaredFloat
44IC50 #2 min concentrationFloatμM
45C50 #2 percent activity at min concentrationFloat%
46IC50 #2 max concentrationFloatμM
47IC50 #2 percent activity at max concentrationFloat%
48IC50 #2 signal at 0.00152 microM (0.00152μM**)Float
49IC50 #2 signal at 0.00305microM (0.00305μM**)Float
50IC50 #2 signal at 0.00610 microM (0.0061μM**)Float
51IC50 #2 signal at 0.01221 microM (0.01221μM**)Float
52IC50 #2 signal at 0.02441 microM (0.02441μM**)Float
53IC50 #2 signal at 0.04883 microM (0.04883μM**)Float
54IC50 #2 signal at 0.09766 microM (0.09766μM**)Float
55IC50 #2 signal at 0.19531microM (0.19531μM**)Float
56IC50 #2 signal at 0.39063 microM (0.39063μM**)Float
57IC50 #2 signal at 0.78125 microM (0.78125μM**)Float
58IC50 #2 signal at 1.5625 microM (1.5625μM**)Float
59IC50 #2 signal at 3.125 microM (3.125μM**)Float
60IC50 #2 signal at 6.25 microM (6.25μM**)Float
61IC50 #2 signal at 12.5 microM (12.5μM**)Float
62IC50 #2 signal at 25 microM (25μM**)Float
63IC50 #2 signal at 50 microM (50μM**)Float
64IC50 #2 control meanFloat
65IC50 #2 control standard deviationFloat
66IC50 #2 number of control wellsInteger
67IC50 #2 control percent CVFloat%
68IC50 #2 blank meanFloat
69IC50 #2 blank standard deviationFloat
70IC50 #2 number of blank wellsInteger
71IC50 #2 blank percent CVFloat%
72IC50 #2 signal-background ratioFloat
73IC50 #2 plate Z-factorFloat
74QualifierString
75IC50 #3FloatμM
76IC50 #3 Hill slopeFloat
77IC50 #3 R-squaredFloat
78IC50 #3 min concentrationFloatμM
79IC50 #3 percent activity at min concentrationFloat%
80IC50 #3 max concentrationFloatμM
81IC50 #3 percent activity at max concentrationFloat%
82IC50 #3 signal at 0.00152 microM (0.00152μM**)Float
83IC50 #3 signal at 0.00305 microM (0.00305μM**)Float
84IC50 #3 signal at 0.00610 microM (0.0061μM**)Float
85IC50 #3 signal at 0.01221 microM (0.01221μM**)Float
86IC50 #3 signal at 0.02441 microM (0.02441μM**)Float
87IC50 #3 signal at 0.04883 microM (0.04883μM**)Float
88IC50 #3 signal at 0.09766 microM (0.09766μM**)Float
89IC50 #3 signal at 0.19531 microM (0.19531μM**)Float
90IC50 #3 signal at 0.39063 microM (0.39063μM**)Float
91IC50 #3 signal at 0.78125 microM (0.78125μM**)Float
92IC50 #3 signal at 1.5625 microM (1.5625μM**)Float
93IC50 #3 signal at 3.125 microM (3.125μM**)Float
94IC50 #3 signal at 6.25 microM (6.25μM**)Float
95IC50 #3 signal at 12.5 microM (12.5μM**)Float
96IC50 #3 signal at 25 microM (25μM**)Float
97IC50 #3 signal at 50 microM (50μM**)Float
98IC50 #3 control meanFloat
99IC50 #3 control standard deviationFloat
100IC50 #3 number of control wellsInteger
101IC50 #3 control percent CVFloat%
102IC50 #3 blank meanFloat
103IC50 #3 blank standard deviationFloat
104IC50 #3 number of blank wellsInteger
105IC50 #3 blank percent CVFloat%
106IC50 #3 signal-background ratioFloat
107IC50 #3 plate Z-factorFloat

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Classification
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