Caspase-5 expression and purification
The p20 subunit (residues 122-311) and p10 subunit (residues 331-418) of wild-type human caspase-5 were separately expressed in E. coli
BL21 (DE3) as inclusion bodies from a pRSET expression vector (Invitrogen, CA). The preparation of inclusion bodies was performed as previously described (16
) with the following modifications. Cells were lysed with a microfluidizer and inclusion-body pellets were collected by centrifuging at 4°C for 30 min. The pellets were washed twice with 50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.25 M guanidine, and 0.5% Triton X-100, followed by two washes using the same buffer without the detergent. Washed pellets were re-suspended in 6 M guanidine–HCl, 20 mM DTT, 0.1 M Tris-HCl, pH 8.0 and frozen at −80 °C. The refolding and purification was carried out using the same procedure as previously described (17
) without using malonate. After purification, the protein fractions were pooled, concentrated, and analyzed by SDS–PAGE.
The screening construct caspase-5 contained five cysteine to alanine mutations denoted C5A (Cys333Ala, Cys370Ala, Cys376Ala, Cys377Ala, Cys378Ala). The mutant was generated by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Stratagene, CA). Two sets of primers were included in a single QuikChange reaction to simultaneously introduce all mutations (extension time of 18 min at 68 °C, 18 cycles). This procedure produced 4 correct clones out of 6 clones sequenced.
Site-directed fragment screening
Disulfide trapping screen was performed following published procedures (10
) with a few modifications. Briefly, purified caspase-5 C5A was freshly diluted to 10 μM in the screening buffer (50 mM Hepes, pH 7.5, 50 mM NaCl, 100 μM β-ME) and was incubated at room temperature for 1 h. with pools of disulfide-containing compounds in 96-well plates. Following the equilibration period, reaction mixtures were analyzed by high-throughput mass spectrometry (LCT Premier, Waters, MA). Hits were identified by comparing the molecular mass of compounds covalently bound to the p10 subunit to the molecular masses of compounds in the pool.
The following two-step procedure was used for parallel re-synthesis of hits. 1) Disulfide dimer formation: in a 4-mL glass vial add EDC (0.11 mmol), the free acid coupling partner (0.10 mmol), a solution of cystamine.2HCl (0.05mmol), HOBt (0.01mmol), triethylamine (0.10 mmol), dH2O (25 μL), and DMF (300 μL). The resulting reaction mixture was stirred overnight. 2) Disulfide exchange: a solution of bis[2-(N,N-dimethylamino)ethyl]disulfide dihydrochloride (0.25 mmol), cysteamine hydrochloride (0.01–0.02 mmol) in water (100 μL) and DMSO (100 μL) was added to the above reaction mixture. Triethylamine (0.7 mmol) is then added and stirred overnight. After reaction, the mixture was diluted with 2:1 DMSO:dH2O to a final volume of 1 mL and injected onto a Waters Xterra 19×50mm Prep MS OBD HPLC column and eluted with a acetonitrile/water (0.05% TFA) gradient (0% to 40% acetonitrile in 8 mins, 40% to 100% in 2 mins, hold at 100% for 2 mins, and decrease to 0% in 1 min).
Measurement of DR50 and β-ME50
To determine the DR50, the testing compound was serially diluted by 2-fold starting at 100 μM before pre-incubated with 2 μM caspase-5 in presence of 100 μM β-ME. For measuring β-ME50, the concentration of the reducing agent was increased by adding freshly prepared β-ME to the reaction mixture containing 2 μM caspase-5 and 50 μM of compound. After 1 h of incubation, the samples were analyzed on LC-MS and the percentage of labeling was calculated based on the ratio of compound-conjugated p10 vs. unconjugated p10. Nonlinear regression was used to calculate DR50 and β-ME5o.
Enzyme kinetics analysis
Caspase-5 or its variants was diluted in assay buffer (50 mM Hepes, pH 7.5, 50 mM KCl, 200 mM NaCl, 100 μM β-ME, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) to 250 nM and incubated with or without compounds at room temperature for 1 h before assaying with fluorescent substrate Ac-WEHD-fmk. The change in relative fluorescence units (RFU) over time was monitored for 10 min using a Spectromax M5 fluorescence plate reader (Molecular Devices, CA) with excitation at 365 nm and emission at 495 nm. Enzyme activity was reported as the rate of change in RFU. All kinetic parameters were determined by fitting with nonlinear regression using the Michaelis-Menten model.