Complement factor C1s IC50 from mixture screen
Complement factor C1s (EC 18.104.22.168) 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 ..
BioActive Compounds: 23
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 22.214.171.124) 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.
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).
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).
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.
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
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).
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]
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)
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.
Please direct correspondence to Andrew Napper (firstname.lastname@example.org).
* Activity Concentration. ** Test Concentration.
Data Table (Concise)