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BioAssay: AID 787

Complement factor C1s IC50 from mixture screen

Complement factor C1s (EC 3.4.21.42) is a trypsin-like serine protease that is activated in one of the first steps in the classical complement cascade. Despite the essential role for the complement cascade in immune defense, unregulated activation leading to acute inflammation and tissue damage has been implicated in many disease states. Under normal conditions the activity of C1s is modulated by more ..
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 Tested Compounds
 Tested Compounds
All(184)
 
 
Active(23)
 
 
Inactive(160)
 
 
Inconclusive(1)
 
 
 Tested Substances
 Tested Substances
All(184)
 
 
Active(23)
 
 
Inactive(160)
 
 
Inconclusive(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 787
Data Source: PCMD (C1s IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-08-16
Modify Date: 2008-10-07

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: complement component 1, s subcomponent [Homo sapiens]
Description ..   
Protein Family: Tryp_SPc

Gene:C1S     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 23
Related Experiments
AIDNameTypeComment
538Complement factor C1sScreeningdepositor-specified cross reference: Complement factor C1s mixture screen
829Complement C1s ELISAConfirmatorydepositor-specified cross reference
Description:
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

Complement factor C1s (EC 3.4.21.42) is a trypsin-like serine protease that is activated in one of the first steps in the classical complement cascade. Despite the essential role for the complement cascade in immune defense, unregulated activation leading to acute inflammation and tissue damage has been implicated in many disease states. Under normal conditions the activity of C1s is modulated by its endogenous inhibitor, C1 esterase inhibitor. Pathological conditions lead to excessive activation of C1s; thus a small molecule inhibitor would be useful in the treatment of ischemia-reperfusion injury and other complement-mediated diseases.

Assay

The high-throughput screen for complement factor C1s inhibitors has been reported earlier (AID 538). The assay consisted of an end-point assay monitoring the release of the fluorophore aminomethyl coumarin (AMC) upon enzymatic hydrolysis of an AMC-labeled tripeptide. The MLSCN compound library was screened as mixtures of 10 compounds per well. Active compounds were confirmed by single compound IC50 determination. The IC50 is reported in this submission.

This assay is a part of the Molecular Library Screening Center Network (MLSCN).
Protocol
Materials
Activated human complement factor C1s was purchased from Calbiochem (Cat #204879). Substrate Boc-Leu-Gly-Arg-AMC was from Bachem (Cat #I-1105.0050). Assay buffer consisted of 50 mM HEPES, pH 7.5, 0.2 M sodium chloride, and 0.2% polyethylene glycol (PEG). Low-volume 384-well black plates were from Corning (Item #3676).
Assay
Complement factor C1s (0.02 mg/mL) was incubated with Boc-Leu-Gly-Arg-AMC substrate (15 uM) in 10 uL of assay buffer (see above) for 2.5 hr at room temperature. IC50 was performed using a serial dilution of compounds from 50 uM to 75 nM. All IC50 determinations were done in triplicate.
IC50 protocol
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 Boc-Leu-Gly-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
6.Add 5 uL enzyme (0.04 mg/mL in assay buffer) using Multidrop-384
7.Incubate for 2.5 hr at room temperature
8.Read fluorescence (excitation 355, emission 460) on Envision reader
Data analysis
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).
Comment
Activity scoring
The activity score reported here is based on follow-up IC50 testing on compounds that showed >20% inhibition in the primary HTS:
IC50 score = sum (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
Compounds that gave percent inhibition >20 in the primary HTS were judged to be hits and these compounds were selected for follow-up IC50 testing. 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 2 or more IC50 determinations = inactive
Analysis of screening results
Results of retesting the compounds that gave percent inhibition >20 in the primary HTS were as follows:
Hits from primary screen (>20% inhibition) = 184
Hits active in IC50 = 23 (12 % retest rate)
Contributors
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Nuzhat Motlekar and Chun-Hao Chiu, and data was submitted by Nuzhat Motlekar, all of the University of Pennsylvania.
We would like to thank Dr. Mandar Ghatnekar and Rajaram Gurumurthi (Infosys Technologies Ltd.) for providing us with a customized tool for data analysis.
Correspondence
Please direct 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
3IC50 standard deviationFloatμM
4number of IC50 determinationsInteger
5QualifierString
6IC50 # 1FloatμM
7IC50 # 1 Hill slopeFloat
8IC50 # 1 R-squaredFloat
9IC50 # 1 min concentrationFloatμM
10IC50 # 1 percent activity at min concentrationFloat%
11IC50 # 1 max concentrationFloatμM
12IC50 # 1 percent activity at max concentrationFloat%
13IC50 # 1 FU at 0.00152 microM (0.00152μM**)Float
14IC50 # 1 FU at 0.00305 microM (0.00305μM**)Float
15IC50 # 1 FU at 0.00610 microM (0.00161μM**)Float
16IC50 # 1 FU at 0.01221 microM (0.01221μM**)Float
17IC50 # 1 FU at 0.02441 microM (0.02441μM**)Float
18IC50 # 1 FU at 0.04883 microM (0.04883μM**)Float
19IC50 # 1 FU at 0.09766 microM (0.09766μM**)Float
20IC50 # 1 FU at 0.19531 microM (0.19531μM**)Float
21IC50 # 1 FU at 0.39063 microM (0.39063μM**)Float
22IC50 # 1 FU at 0.78125 microM (0.78125μM**)Float
23IC50 # 1 FU at 1.5625 microM (1.5625μM**)Float
24IC50 # 1 FU at 3.125 microM (3.125μM**)Float
25IC50 # 1 FU at 6.25 microM (6.25μM**)Float
26IC50 # 1 FU at 12.5 microM (12.5μM**)Float
27IC50 # 1 FU at 25 microM (25μM**)Float
28IC50 # 1 FU at 50 microM (50μM**)Float
29IC50 # 1 control meanFloat
30IC50 # 1 control standard deviationFloat
31IC50 # 1 number of control wellsInteger
32IC50 # 1 control percent CVFloat%
33IC50 # 1 blank meanFloat
34IC50 # 1 blank standard deviationFloat
35IC50 # 1 number of blank wellsInteger
36IC50 # 1 blank percent CVFloat%
37IC50 # 1 signal-background ratioFloatratio
38IC50 # 1 plate Z-factorFloat
39QualifierString
40IC50 # 2FloatμM
41IC50 # 2 Hill slopeFloat
42IC50 # 2 R-squaredFloat
43IC50 # 2 min concentrationFloatμM
44IC50 # 2 percent activity at min concentrationFloat%
45IC50 # 2 max concentrationFloatμM
46IC50 # 2 percent activity at max concentrationFloat%
47IC50 # 2 FU at 0.00152 microM (0.00152μM**)Float
48IC50 # 2 FU at 0.00305 microM (0.00305μM**)Float
49IC50 # 2 FU at 0.00610 microM (0.0061μM**)Float
50IC50 # 2 FU at 0.01221 microM (0.01221μM**)Float
51IC50 # 2 FU at 0.02441 microM (0.02441μM**)Float
52IC50 # 2 FU at 0.04883 microM (0.04883μM**)Float
53IC50 # 2 FU at 0.09766 microM (0.09766μM**)Float
54IC50 # 2 FU at 0.19531 microM (0.19531μM**)Float
55IC50 # 2 FU at 0.39063 microM (0.39063μM**)Float
56IC50 # 2 FU at 0.78125 microM (0.78125μM**)Float
57IC50 # 2 FU at 1.5625 microM (1.5625μM**)Float
58IC50 # 2 FU at 3.125 microM (3.125μM**)Float
59IC50 # 2 FU at 6.25 microM (6.25μM**)Float
60IC50 # 2 FU at 12.5 microM (12.5μM**)Float
61IC50 # 2 FU at 25 microM (25μM**)Float
62IC50 # 2 FU at 50 microM (50μM**)Float
63IC50 # 2 control meanFloat
64IC50 # 2 control standard deviationFloat
65IC50 # 2 number of control wellsInteger
66IC50 # 2 control percent CVFloat%
67IC50 # 2 blank meanFloat
68IC50 # 2 blank standard deviationFloat
69IC50 # 2 number of blank wellsInteger
70IC50 # 2 blank percent CVFloat%
71IC50 # 2 signal-background ratioFloatratio
72IC50 # 2 plate Z-factorFloat
73QualifierString
74IC50 # 3 FloatμM
75IC50 # 3 Hill slopeFloat
76IC50 # 3 R-squaredFloat
77IC50 # 3 min concentrationFloatμM
78IC50 # 3 percent activity at min concentrationFloat%
79IC50 # 3 max concentrationFloatμM
80IC50 # 3 percent activity at max concentrationFloat%
81IC50 # 3 FU at 0.00152 microM (0.00152μM**)Float
82IC50 # 3 FU at 0.00305 microM (0.00305μM**)Float
83IC50 # 3 FU at 0.00610 microM (0.0061μM**)Float
84IC50 # 3 FU at 0.01221 microM (0.01221μM**)Float
85IC50 # 3 FU at 0.02441 microM (0.02441μM**)Float
86IC50 # 3 FU at 0.04883 microM (0.04883μM**)Float
87IC50 # 3 FU at 0.09766 microM (0.09766μM**)Float
88IC50 # 3 FU at 0.19531 microM (0.19531μM**)Float
89IC50 # 3 FU at 0.39063 microM (0.39063μM**)Float
90IC50 # 3 FU at 0.78125 microM (0.78125μM**)Float
91IC50 # 3 FU at 1.5625 microM (1.5625μM**)Float
92IC50 # 3 FU at 3.125 microM (3.125μM**)Float
93IC50 # 3 FU at 6.25 microM (6.25μM**)Float
94IC50 # 3 FU at 12.5 microM (12.5μM**)Float
95IC50 # 3 FU at 25 microM (25μM**)Float
96IC50 # 3 FU at 50 microM (50μM**)Float
97IC50 # 3 control meanFloat
98IC50 # 3 control standard deviationFloat
99IC50 # 3 number of control wellsInteger
100IC50 # 3 control percent CVFloat%
101IC50 # 3 blank meanFloat
102IC50 # 3 blank standard deviationFloat
103IC50 # 3 number of blank wellsInteger
104IC50 # 3 blank percent CVFloat%
105IC50 # 3 signal-background ratioFloatratio
106IC50 # 3 plate Z-factorFloat

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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