AID	Name	Type	Comment
581	Cathepsin G	screening

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

Cathepsin G (EC 3.4.21.20) is a chymotrypsin-like serine protease that is secreted from neutrophils. Disregulated cathepsin G activity is implicated in the progression of various chronic inflammatory diseases such as asthma and chronic pulmonary obstructive disease. Thus cathepsin G inhibitors represent useful probes to further elucidate the role of this enzyme in inflammation and may provide a starting point for the development of novel therapeutic agents.

A high-throughput screen for cathepsin G 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 have been reported previously (AID 581). Compounds identified as hits in HTS of mixtures of 10 compounds per well were tested individually in dose-response using the AMC release assay. Results of this dose-response confirmation are reported here.

Materials

Human neutrophil cathepsin G was purchased from Calbiochem (Cat #219373). Substrate Suc-Ala-Ala-Pro-Phe-AMC was from Bachem (Cat #I-1465.0050). Assay buffer consisted of 100 mM HEPES, pH 7.4, 0.5 M sodium chloride. Low-volume 384-well black plates were from Corning (Item #3676).

Assay

Cathepsin G (4.2 ug/mL) was incubated with Suc-Ala-Ala-Pro-Phe-AMC substrate (15 uM) in 10 uL of assay buffer (see above) for 3 hr at room temperature. Activity of single compounds identified from mixture HTS were confirmed by IC50 determination as described below.

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 Suc-Ala-Ala-Pro-Phe-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
6.Add 5 uL enzyme (8.4 ug/mL in assay buffer) using Multidrop-384
7.Incubate for 3 hr at room temperature
8.Read fluorescence (excitation 355, emission 460) on Envision reader

Data analysis

Data were analyzed in IDBS ActivityBase. 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).

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 >30% inhibition in both locations 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

Compounds that gave percent inhibition >20 in both locations in the primary HTS (AID 581) 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 all IC50 determinations = inactive
(3) IC50 <50 uM in one or more determinations & >50 uM in one or more = inconclusive

Analysis of screening results

Results of retesting the compounds that gave percent inhibition >20 in both locations in the primary HTS were as follows:

Hits (>20% inhibition in both locations) = 92
Hits active in IC50 = 68 (74% retest rate)
Hits marginal in IC50 = 6 (IC50 >50uM, percent inhibition 30-50% at 50 uM)

Contributors

This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Edinson Lucumi, and data were submitted by Andrew Napper, all of the University of Pennsylvania.

Our thanks go to Parag Shah and Bill Denney for enormous help in setting up the HTS lab and troubleshooting its operation.

Correspondence

Please direct correspondence to Andrew Napper (napper@seas.upenn.edu).