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BioAssay: AID 1633

Cathepsin L probe dose-response testing

Human liver cathepsin L (EC 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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AID: 1633
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-03-27

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
460Cathepsin LScreeningdepositor-specified cross reference
825Cathepsin L dose-response confirmationConfirmatorydepositor-specified cross reference
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 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 in the presence of DTT and cysteine have been reported previously (AIDs 460 and 825). A compound with picomolar activity against cathepsin L was isolated, resynthesized, and the kinetics of inhibition were determined [Shah PP, Myers MC, Beavers MP, Purvis JE, Jing H, Grieser HJ, Sharlow ER, Napper AD, Huryn DM, Cooperman BS, Smith AB, Diamond SL. Kinetic Characterization and Molecular Docking of a Novel, Potent, and Selective Slow-binding Inhibitor of Human Cathepsin L, Molecular Pharmacology, 74, 34-41 (2008); Myers MC, Shah PP, Beavers MP, Napper AD, Diamond SL, Smith AB, Huryn DM. #Design, synthesis, and evaluation of inhibitors of cathepsin L: Exploiting a unique thiocarbazate chemotype,Bioorganic and Medicinal Chemistry Letters, 18, 3646-3651 (2008)]. Activity of this novel chemical probe is reported here.

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).


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.)
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]

Activity Outcome

IC50 values were determined as described in protocol above.
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


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 (
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
2IC50 mean*FloatμM
4IC50 standard deviationFloatμM
5Number of IC50 determinationsInteger
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
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
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.
Additional Information
Grant Number: MH076406-01

Data Table (Concise)
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