Complement factor C1s
Complement factor C1s (EC 220.127.116.11) 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: 102
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Scott L. Diamond, University of Pennsylvania
MLSCN Grant: X01-MH076406-01
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 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 reported here 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 and active compounds were confirmed by single compound IC50 determination.
This assay is a part of the Molecular Library Screening Center Network (MLSCN).
Rationale for mixture screening
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 serine and cysteine 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 serine and cysteine 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).
This compound pooling strategy has been validated against the cysteine protease cathepsin B. Comparison of the results of screening 64,000 compounds from the MLSCN library as mixtures (Pubchem Assay ID 488) and as single compounds (Pubchem Assay ID 453), revealed the same profile of active hits in both cases. All but one of the active compounds discovered by single-compound screening was also identified by mixture screening.
200 plates containing 64,000 compounds from the MLSCN library were arranged in two 10 x 10 grids of 100 plates each. Sets of ten plates were pooled as follows to give 20 mixture plates per 100 single compound plates:
Plate 1_2_3_4_5_6_7_8_9_10 pooled to mixture plate 1-10
Plate 11_12_13_14_15_16_17_18_19_20 pooled to mixture plate 11-20
Plate 21_22_23_24_25_26_27_28_29_30 pooled to mixture plate 21-30
Plate 31_32_33_34_35_36_37_38_39_40 pooled to mixture plate 31-40
Plate 41_42_43_44_45_46_47_48_49_50 pooled to mixture plate 41-50
Plate 51_52_53_54_55_56_57_58_59_60 pooled to mixture plate 51-60
Plate 61_62_63_64_65_66_67_68_69_70 pooled to mixture plate 61-70
Plate 71_72_73_74_75_76_77_78_79_80 pooled to mixture plate 71-80
Plate 81_82_83_84_85_86_87_88_89_90 pooled to mixture plate 81-90
Plate 91_92_93_94_95_96_97_98_99_100 pooled to mixture plate 91-100
Plates were also pooled 'vertically':
Plates 1, 11, 21, 31, 41, 51, 61, 71, 81, 91 pooled to mixture plate 1-91
Plates 2, 12, 22, 32, 42, 52, 62, 72, 82, 92 pooled to mixture plate 2-92
And so on until mixture plate 10-100.
For example, the single compound in well A3 in Plate 1 is mixed with 9 compounds in well A3 in mixture plate 1-10, and also with 9 different compounds in well A3 in mixture plate 1-91.
The concentration of each mixture was 2.5 mM in DMSO (250 uM per compound). Pintool transfer into the HTS assay gave a final mixture concentration of 50 uM in 2% DMSO (5 uM per compound).
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. HTS was performed using 50 uM compound mixture (5 uM each of 10 compounds). Hits were confirmed on single compounds by IC50 determination as described below.
1.Fill low-volume plate with 4 uL water using Multidrop-micro
2.Add 5 uL assay buffer to columns 1 and 23 using Multidrop-384
3.Add 200 nL of compound mixture (2.5 mM in DMSO) using Evolution pintool
4.Add 1 uL of Boc-Leu-Gly-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
5.Add 5 uL enzyme (0.04 mg/mL in assay buffer) using Multidrop-384
6.Incubate for 2.5 hr at room temperature
7.Read fluorescence (excitation 355, emission 460) on Envision reader
Mixture percent inhibition---Data were analyzed in IDBS ActivityBase. Each HTS plate contained compound mixtures (50 uM in 2% DMSO) in columns 3-22, controls (enzyme, no compound mixture) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. HTS percent inhibition was calculated for each compound mixture 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:
% Inhibition = 100*(1-((signal-blank mean)/(control mean-blank mean)))
Assignment of percent inhibition to individual compounds---Percent inhibition results from the mixture HTS were retrieved in SARgen (IDBS) together with the identity of the 10 individual compounds within each mixture. As each compound was present in two mixtures the data were rearranged using a custom Excel macro to align both percent inhibition values associated with each compound. Compounds that gave >20% inhibition in both mixture locations were selected as hits and will be retested individually in dose-response.
Activity scoring is complicated by the fact that the two percent inhibition values associated with each compound in fact represent two different mixtures of 10 compounds. Thus simple averaging of the two values is not meaningful. Instead each compound was assigned a percent inhibition score based on the lower of the two percent inhibition values. This system appropriately scores hits showing activity in both mixtures, but avoids assignment of an erroneously high score to inactives that shared one location with an active compound.
The activity score reported here is based on percent inhibition observed in the primary HTS (see above):
Percent inhibition scores were calculated from the lower of the two percent inhibition values associated with each compound as follows:
(1) For percent inhibition between 0 and 100, score = percent inhibition
(2) For negative percent inhibition, score = 0
Compounds that gave percent inhibition >20 in both locations in the primary HTS were judged to be hits and these 102 compounds will be put through follow-up IC50 testing.
Activity outcome is reported as follows:
(1) Percent inhibition > 20 in both locations = active
(2) Percent inhibition < 20 in one or both locations = inactive
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 were submitted by Nuzhat Motlekar and Andrew Napper, 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 customised Excel macro for data analysis.
Please direct correspondence to Andrew Napper (firstname.lastname@example.org).
Data Table (Concise)