AID	Name	Type	Comment
460	Cathepsin L	screening
1627	Cathepsin L dose-response testing in the presence of cysteine	confirmatory
1633	Cathepsin L probe dose-response testing	confirmatory
1636	Cathepsin L probe #2 dose-response testing	confirmatory

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 3.4.22.15) 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 have been reported previously (AID 460). Actives from the HTS were tested in dose-response using the AMC release assay. Results of this dose-response confirmation are reported here.

Materials

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 DTT. Low-volume 384-well black plates were from Corning (Item #3676).

Assay

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

Compounds that gave percent inhibition >45 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 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 compounds that gave percent inhibition >45 in the primary HTS were as follows:

Hits (>45% inhibition) = 102
Hits active in IC50 = 49 (48% retest rate)

Contributors

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 Parag Shah (ppshah@seas.upenn.edu).