Human liver cathepsin B (catalog number 219362) was purchased from Calbiochem (San Diego, CA). Substrate Z-Arg-Arg-AMC was from Bachem (King of Prussia, PA). Assay buffer components and AMC standard were purchased from Sigma-Aldrich (St. Louis, MO). Low-volume non–binding surface 384-well black plates used for the fluorescent assay were from Corning (Lowell, MA). Polypropylene V-bottom plates from Greiner Bio-One (Monroe, NC) were used for compound storage and dilution. Heat seals were applied using a PlateLoc®
heat sealer (Velocity 11, Menlo Park, CA), and polystyrene lids were from Nunc®
(Rochester, NY). The compounds tested here were supplied by BioFocus DPI (South San Francisco, CA) as part of the MLSCN.15
A library of 64,000 compounds from the Molecular Libraries Small Molecule repository was shipped on dry ice from BioFocus DPI as frozen 10 mM solutions in dimethyl sulfoxide (DMSO) in heat-sealed 384-well polypropylene plates. Prior to compound plating and shipping, quality control (QC) testing at BioFocus DPI confirmed compound solubility in DMSO, the presence of the expected molecular ion in liquid chromatography-mass spectrometry (LC-MS), and >90% purity by evaporative light scattering or ultraviolet at 214 nm. On arrival, plates were stored in a desiccator (<20% relative humidity) at −25°C. After storage for approximately 3 months, plates were allowed to warm to room temperature in a desiccator. The thawed plates were used to prepare dilution plates for single-compound and mixture HTS as described below and then refrozen.
Preparation of plates for single-compound and mixture HTS
For single-compound HTS, a set of 0.5 mM dilution plates were prepared on a Perkin Elmer (Boston, MA) EP3 workstation using a 384-tip pipetting head. Two microliters from each BioFocus DPI plate was pipetted into a polypropylene V-bottom plate containing 18 μl per well of DMSO and mixed by repeated aspiration and dispense (15 μl). Plates were heat-sealed and stored at room temperature for 1–2 weeks in a desiccator prior to use. Plates for mixture HTS were prepared as shown in . Two hundred plates containing the 64,000 compounds from BioFocus DPI were arranged in two 10 × 10 grids of 100 plates each. Sets of 10 plates were pooled to give a single mixture plate as shown, resulting in 20 mixture plates per 100 single-compound plates. An automated plate pooling protocol was set up on a Thermo-CRS (Burlington, ON, Canada) screening deck, controlled by Polara operating software. The heat seals were removed by hand from each set of 10 single-compound plates and replaced with loose-fitting polystyrene plate lids. Each plate in turn was delidded and placed on a Perkin Elmer EP3 workstation. Two microliters of compound in DMSO from each plate was transferred by pipetting head (384 disposable tips) into a single mixture plate, giving a final mixture volume of 20 μl. The concentration of each mixture was 10 mM in DMSO (1 mM per compound). After pipetting was complete each plate was immediately heat-sealed to minimize exposure to atmospheric moisture. In a separate protocol the mixture plates were then diluted fourfold by transfer of 5 μl of each mixture into 15 μl of DMSO to give mixtures 2.5 mM in DMSO (250 μM per compound). Plates were heat-sealed and stored at room temperature for 2–3 days in a desiccator prior to use.
FIG. 1. Orthogonal pooling of 100 compound plates to give 20 mixture plates. Each compound is in two locations in the mixture plates, mixed with a different set of nine other compounds in each location. For example, the single compound shown in plate C7 (, (more ...)
Preparation of plates for 50% inhibitory concentration (IC50) testing
Hits selected from single-compound and mixture HTS were cherry-picked from the set of 10 mM compound plates supplied by BioFocus DPI, and each compound was transferred to a single well in row A of a 384-well V-bottom polypropylene plate. Frozen compound plates were allowed to warm to room temperature in a desiccator. Compound pipetting was carried out using disposable liquid level sensing tips on a Perkin Elmer Janus four-tip workstation. Ten microliters of DMSO was added to row A of 384-well V-bottom polypropylene plates, and 10 μl of each selected compound was transferred to one well out of A3 to A22. An additional 10 μl of DMSO was added to wells A1, A2, A23, and A24, followed by 20 μl per well of DMSO added to the entire plate using a Thermo Fisher Scientific (Waltham, MA) Multidrop. The compounds were twofold serially diluted by transfer of 20 μl row by row from row A to row P using a single row of disposable tips on a Perkin Elmer EP3 384-tip pipetting head, after which 20 μl was discarded from row P. The resulting dose–response plates contained 16 twofold dilutions (2.5 mM–50 nM) of each compound, arranged one compound per column in columns 3–22. Plates were heat-sealed and stored at room temperature for 1–2 days in a desiccator prior to use.
LC-MS analysis of hits
Purity and integrity of compounds identified as hits in the mixture and single-compound HTS were analyzed by LC-MS on a Waters (Milford, MA) ZQ system. Forty microliters of a 100 μM solution of each compound in DMSO was eluted from a SunFire™ (Waters) C18 column (4.6 × 50 mm) with a 5-min gradient of 90:10 to 10:90 water (0.05% formic acid):acetonitrile (0.05% formic acid). Compound integrity and purity were determined by identification of the expected molecular ion and peak integration by evaporative light scattering and ultraviolet absorbance at 214 nm.
Determination of substrate Km
The assay buffer consisted of 100 mM sodium-potassium phosphate (pH 6.8) (86 mM potassium phosphate, monobasic; 7 mM sodium phosphate, monobasic; and 7 mM sodium phosphate, tribasic), 1 mM EDTA, and 2 mM dithiothreitol (DTT). Z-Arg-Arg-AMC substrate was serially diluted from 400 to 6 μM in assay buffer containing 4% DMSO, and 5 μl per well of each dilution was transferred by multichannel pipette into four columns of a low-volume 384-well assay plate. Cathepsin B was activated prior to addition to the assay plate by incubating in assay buffer for 15 min. The assay was started by addition of 5 μl of activated enzyme to two columns to give final concentrations of enzyme, substrate, and DMSO of 2.36 nM, 3–200 μM, and 2%, respectively. The remaining two columns consisted of control wells at each substrate concentration containing 5 μl of assay buffer without enzyme. The mixture was allowed to incubate at room temperature, and fluorescence was read in a Perkin Elmer EnVision™ microplate fluorimeter (excitation 355 nm, emission 460 nm) at 1-min intervals for 1 h. Fluorescence values were corrected for background readings from the control wells containing substrate in buffer but no enzyme. Rate of change of fluorescence was calculated from the initial linear timecourse over the first 15 min and converted to rate of AMC release in μM s−1 using a standard curve of AMC fluorescence. Rate of AMC release versus substrate concentration was plotted and fit in XLfit® (IDBS, Guildford, UK) using fit model 350 (Michaelis-Menten equation).
Compound mixtures were added by pintool transfer on a Perkin Elmer EP3 workstation. A 384-pin pintool (V&P Scientific, San Diego, CA) was used to transfer 200 nl of a 2.5 mM solution in DMSO into assay plates containing 4 μl of water per well, giving a final concentration of each compound in the assay of 5 μM in 2% DMSO. Single compounds were added likewise from a 0.5 mM solution in DMSO giving a final concentration in the assay of 10 μM in 2% DMSO. QC plates contained the cysteine protease inhibitor E-64 at a concentration of 50 nM, obtained by addition of 200 nl of a 2.5 μM solution in DMSO. The assay buffer consisted of 100 mM sodium-potassium phosphate (pH 6.8) (86 mM potassium phosphate, monobasic; 7 mM sodium phosphate, monobasic; and 7 mM sodium phosphate, tribasic), 1 mM EDTA, and 2 mM DTT. Cathepsin B was activated prior to addition to the assay plate by incubating in assay buffer for 15 min. The assay was started by addition of substrate (1 μl) and activated enzyme (5 μl) using Multidrop reagent dispensers to give final concentrations of 2.36 nM cathepsin B, 15 μM Z-Arg-Arg-AMC substrate, and 2% DMSO in 10 μl of assay buffer. After 1 h at room temperature fluorescence was read in an EnVision microplate fluorimeter (excitation 355 nm, emission 460 nm).
This assay was identical to the HTS assay, except that 16 twofold dilutions of each compound were tested. Compounds were twofold serially diluted in DMSO from 2.5 mM to 50 nM and transferred by pintool into assay plates to give a final concentration range of 50 μM–1.5 nM in 2% DMSO. For the IC50 assay in the presence of cysteine, DTT in the cathepsin B assay buffer was replaced with 2 mM cysteine. Enzyme was activated in the cysteine-containing buffer for 1 h and then assayed as described above.
HTS data analysis
Data were analyzed using ActivityBase (IDBS). Each HTS plate contained individual compounds (10 μM
) or mixtures (10 compounds at 5 μM
each) in columns 3–22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. Percentage inhibition was calculated for each compound from the signal in fluorescence units and the means of the plate controls and plate blanks using the following equation:
Compounds from the single-compound HTS that gave >33% inhibition were selected as hits and retested in dose–response. Percentage inhibition results from the mixture HTS were retrieved in SARgen (IDBS) together with the identity of the individual compounds within each mixture. As each compound was present in two mixtures the data were rearranged using a custom Excel (Microsoft, Redmond, WA) macro (developed and generously contributed by Dr. Mandar Ghatnekar and Rajaram Gurumurthi, Infosys Technologies Ltd., Bangalore, India) to align both percentage inhibition values associated with each compound. Compounds that gave >20% inhibition in both mixture locations were selected and retested individually in dose–response.
IC50 data analysis
plates contained one compound per column in columns 3–22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. Each column from 3 to 22 contained 16 twofold dilutions of a single compound, ranging in concentration from 50 μM
to 1.5 nM
. Percentage activity was calculated for each dilution of each compound from the signal in fluorescence units and the means of the plate controls and plate blanks using the following equation:
Dose–response curves of percentage activity were fit in XLfit, using a four-parameter logistic fit (equation 205 with maximum percentage activity and minimum percentage activity fixed at 100% and 0%, respectively).