Cathepsin B Inhibitor Series SAR Study
Human liver cathepsin B (EC 188.8.131.52) is a lysosomal cysteine protease. There has been a recent resurgence of interest in cathepsin B due to research showing that proteolysis by this enzyme is required for the entry and replication of the Ebola and SARS viruses in human cells. Thus cathepsin B inhibitors have potential as novel anti-viral agents. ..more
BioActive Compounds: 10
Human liver cathepsin B (EC 184.108.40.206) is a lysosomal cysteine protease. There has been a recent resurgence of interest in cathepsin B due to research showing that proteolysis by this enzyme is required for the entry and replication of the Ebola and SARS viruses in human cells. Thus cathepsin B inhibitors have potential as novel anti-viral agents.
Cathepsin B is also implicated in cancer progression. Upregulation and secretion of this enzyme occurs in many types of tumors and correlates positively with their invasive and metastatic capabilities. Cathepsin B facilitates tumor invasion by dissolving extracellular barriers. Inhibitors of cathepsin B thus have been studied as potential anti-cancer agents.
Two high-throughput screens against cathepsin B reported previously under AID 453 and AID 488 each independently revealed the same series of aminopyrazole inhibitors. Six compounds active in the original HTS were resynthesized, and 21 new compounds were made to explore SAR. As in the original HTS and IC50 follow-up, compounds were tested for inhibitory activity in an end-point assay that monitored the release of the fluorophore aminomethyl coumarin (AMC) upon enzymatic hydrolysis of an AMC-labeled dipeptide.
This assay is a part of the Molecular Library Screening Center Network (MLSCN).
Human liver cathepsin B was purchased from Calbiochem (Cat #219362). Substrate Z-Arg-Arg-AMC was from Bachem (Cat #I-1135.0050). Assay buffer consisted of 100 mM sodium-potassium phosphate, pH 6.8 (86 mM potassium phosphate, monobasic; 7 mm sodium phosphate, monobasic; 7 mm sodium phosphate, tribasic), 1 mM EDTA, and 2 mM DTT. Low-volume 384-well black plates were from Corning (Item #3676).
Cathepsin B (0.065 ug/mL) was incubated with Z-Arg-Arg-AMC substrate (15 uM) in 10 uL of assay buffer (see above) for 1 hr at room temperature. Inhibitor IC50 values were determined as described below.
1.Serial dilute 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 Z-Arg-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
6.Add 5 uL enzyme (0.13 ug/mL in assay buffer) using Multidrop-384
7.Incubate for 1 hr at room temperature
8.Read fluorescence (excitation 355, emission 460) on Envision reader
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 is based on the linear-log formula developed by Eduard Sergienko at the San Diego Center for Chemical Genomics. The combined activity score is based on triplicate IC50 determinations as follows:
Combined activity score = 1.667 x (IC50 score #1 + IC50 score #2 + IC50 score #3).
IC50 scores were calculated as follows:
(1) Score = 3.45 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 = [3.45 x (0-3)] + [(100-percent activity at max concentration)/3.4]
Compounds that gave three replicate IC50s <50 uM were judged to be active. These compounds giving three replicate IC50s >50 uM are reported as inactive. 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. One compound gave IC50s >50 uM but nevertheless showed >30% inhibition at 50 uM; this compound is reported as inconclusive.
Scott Diamond---PCMD director
PCMD Chemistry Core:
Amos B. Smith, III---Director
Donna Huryn---Associate Director
Michael Myers---Postdoctoral researcher
PCMD Assay Development/HTS Core:
Andrew Napper---Director of HTS
Nuzhat Motlekar---HTS Specialist
PCMD Informatics Core
Mary Pat Beavers---Director
This assay was submitted to the PCMD by Scott Diamond, compounds were synthesized by Michael Myers, IC50 determinations were conducted by Nuzhat Motlekar, 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.
Please direct correspondence to Andrew Napper (firstname.lastname@example.org).
* Activity Concentration. ** Test Concentration.
Data Table (Concise)