Ac-VEID-CHO, Ac-DEVD-CHO, Ac-IETD-CHO, z-VEID-FMK, z-DEVD-FMK, z-VAD-FMK and Q-VD-OPh were obtained from EMD4Biosciences (Gibbstown, NJ). z-VEID-tetrafluorophenoxymethyl (TFPM), z-EID-TFPM and z-ID-TFPM were synthesized by the Discovery Chemistry Department at Genentech, Inc. with procedures described previously ^[30], ^[31]. All inhibitor structures can be found in Figure S1.

SKNAS Cells (ATCC #CRL-2137) were cultured in DMEM medium supplemented with 10% FBS, 2 µM Glutamax I and 1% Pen/Strep under incubator conditions of 37°Celsius, 90% relative humidity with 5% CO₂. Cells were split 1∶5 twice weekly. Caspase-6 KO and wild type mouse fibroblasts were cultured under the same medium and conditions. All tissue culture reagents were purchased from Invitrogen (Carlsbad, CA).

Caspases-3, -6 and -7 were expressed in E. Coli and purified by the Protein Chemistry Department at Genentech, Inc with procedures similar to those previously published ^[32]. Biochemical assays of caspase activity were carried out in 384-well black low-volume assay plates (Perkin Elmer #6008260; Waltham, MA) in 12 µl reaction volumes containing enzyme, fluorogenic substrate and inhibitor. All peptide inhibitors were serially diluted in DMSO prior to cross dilution in assay buffer (50 mM HEPES pH 7.2, 25 mM MgSO₄, 0.5 mM EGTA, 5 mM Glutathione, 0.01% Triton X-100 containing 0.1% Bovine Gamma Globulin (BGG)). Final DMSO concentration in the assay was 1.33%. Rhodamine-labeled bivalent tetrapeptide substrates were obtained from Anaspec (Fremont, CA) and used as follows: 300 pM caspase-3 reacted with 1 µM (Ac-DEVD)₂ -Rh110, 1 nM caspase-6 reacted with 5 µM (Ac-VEID)₂ -Rh110, 5 pM caspase-7 reacted with 1 µM (Ac-DEVD)₂ -Rh110. Enzyme and inhibitor were preincubated for 15 minutes prior to the addition of substrate to initiate the enzymatic reaction. The reaction was permitted to proceed for 40 minutes at room temperature and then read on an Envision (Perkin Elmer) instrument using excitation/emission wavelengths of 485/535 nm. To simplify potency analysis of peptide inhibitors with the capacity to inhibit in a time-dependent manner, the apparent IC₅₀'s determined after combined 15 minutes of preincubation and 40 minute enzyme reaction are reported.

SKNAS cells were seeded in T-25 flasks (Corning; Lowell, MA) at a density of 5x10⁶ cells/flask in growth medium and allowed to adhere overnight. The following day, staurosporine (Enzo; Plymouth Meeting, PA) was delivered directly to medium in the T-25 flask from a 10 mM stock in DMSO resulting in 3 µM final concentration. Medium was aspirated after 6 hours incubation (or indicated time) followed by addition of 250 µl lysis buffer (Cell Signaling; Beverly, MA). Cell lysates were quantified for protein content using a bicinchoninic acid (BCA) protein assay kit (Thermo; Waltham, MA) and 50 µg of cellular lysate was subjected to SDS-PAGE using 4–12% Bis-Tris gel (Invitrogen) for 90 minutes at 125V. Transfer of protein to 0.45 µm nitrocellulose membrane (Invitrogen) was performed using semi-dry transfer apparatus (BioRad; Hercules, CA) for 60 minutes at 15V. Nitrocellulose membranes were blocked with Odyssey blocking buffer (Licor; Lincoln, NE) for 1 hour at room temperature prior to incubation with 1∶1000 dilution of the indicated primary anti-lamin antibody (rabbit polyclonal targeting a neo-epitope on the small subunit of cleaved lamin A/C (Cell Signaling #2035S); rabbit polyclonal targeting a neo-epitope on the large subunit of cleaved lamin A/C (Cell Signaling #2031S); rabbit polyclonal targeting intact lamin A/C (Cell Signaling #2032)) or 1∶5000 dilution of mouse monoclonal anti-β actin antibody (Sigma #A5441) for 60 minutes at room temperature with gentle rocking motion. The membrane was then washed 4 times for 5 minutes with Tris Buffered Saline containing 0.05% Tween-20 (TBST) (Invitrogen) followed by incubation with secondary antibody for 1 hour at room temperature. Dilutions of 1∶2500 (HRP-conjugated donkey anti-rabbit IgG (GE Healthcare #NA934V; Piscataway, NJ)), 1∶10000 (IRDye 800 conjugated affinity purified anti-mouse IgG (Rockland, Gilbertsville, PA)) and 1∶2000 (Alexa Fluor 680 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA)) were used. Finally, the membrane was washed 4 times for 5 minutes in TBST and scanned using a LiCor Odyssey Infrared Scanning instrument (LiCor; Lincoln, NE).

SKNAS or mouse fibroblast (wild type and caspase-6 KO) cells were plated at a density of 20,000 cells per well into poly-D-lysine coated 384-well black clear-bottom cell culture plates (Becton Dickinson #35-4663; Franklin Lakes, NJ) in growth medium and allowed to adhere overnight. The following day, cell culture medium was aspirated and replaced with 20 µl/well basal medium (DMEM with no supplements added). All peptide inhibitors were serially diluted in DMSO prior to cross dilution in basal medium. 5 µl of diluted inhibitor was delivered to each well and incubated for 30 minutes in a cell culture incubator prior to addition of 5 µl/well staurosporine in basal medium. The final DMSO concentration in the assay was 0.1% and the final staurosporine concentration was 3 µM (or indicated concentration). Following 6 hours incubation with staurosporine, 10 µl of 16% paraformaldehyde (Alfa Aesar #43368; Ward Hill, MA) was added directly to each well and incubated at room temperature for 15 minutes before careful aspiration of each well and two 100 µl washes with Phosphate Buffered Saline (PBS) (Invitrogen). Cells were permeabilized by the addition of 50 µl 0.2% Triton X-100 in PBS for 15 minutes at room temperature. Each well was aspirated, washed twice with 100 µl PBS, and nonspecific binding sites blocked with 100 µl blocking agent (Thermo #37535) for 10 minutes at room temperature. The blocking agent was replaced with 20 µl/well rabbit polyclonal primary antibody (Cell Signaling #2035S) delivered at 1∶200 dilution in blocking agent and incubated overnight at 4°Celsius. The following day, primary antibody was aspirated and the wells washed 3 times with 100 µl TBST. Secondary antibody (donkey anti-rabbit IgG, HRP-conjugated (GE Healthcare #NA934V)) was delivered at 1∶2500 dilution in blocking agent (20 µl/well) and incubated 1 hour at room temperature. Following aspiration, the plate was washed 5 times with 100 µl/well TBST, chemiluminescent HRP substrate (Thermo #37074) was added and luminescence measured 5 minutes later using a Perkin Elmer Envision instrument.

Active human recombinant caspase-6 was provided by the Protein Chemistry Department at Genentech, Inc. All other active caspases were purchased from Enzo LifeSciences (Cat # BML-AK010) and used at a final concentration of 5 U/µl (300U total; see manufacturer's guide for description). In each assay, recombinant glutathione S-transferase (GST)-tagged lamin A (17 µg/ml, 170 nM) (Abnova Cat# H00004000-P01; Taipei City, Taiwan) was incubated with active caspase enzyme at 37°C for 2 hours in assay buffer (50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% CHAPS, 10 mM DTT). For caspase-9, the assay buffer was: 50 mM HEPES (pH 7.5), 100 mM NaCl, 0.01% CHAPS, 0.5 M NaCitrate, 1 mM EDTA, 10 mM DTT. Samples were incubated at 100°C. for 5 minutes, resolved by SDS-PAGE (12% Tris-glycine, Invitrogen) and transferred to a nitrocellulose membrane. The blot was probed with horseradish peroxidase (HRP)-conjugated anti-GST antibody (1∶1000) (Sigma-Aldrich; St. Louis, MO). The signal was developed with Chemi-IR™ detection kit using IRDye 800CW rabbit anti-HRP antibody (1∶1000) (Licor) according to manufacturer's recommendation. The membrane was scanned on a LiCor Odyssey Infrared Scanning instrument. Under the same assay conditions, all enzymes were verified in fluorogenic assays to be active in processing their specific tetrapeptide-AMC substrates (data not shown).

The biochemical cleavage assay of lamin A with increasing concentrations of human active caspase-6 (as indicated) was done in 40 mM HEPES (pH 7.2), 100 mM NaCl, 0.1% CHAPS, 80 µM DTT for 2 hours at 37°C. The samples were processed as described above and the signal was quantified using Odyssey software.

The construct for targeting the C57BL/6 caspase-6 locus in ES cells was made using a combination of recombineering ^[33], ^[34] as well as standard molecular cloning techniques.

Briefly, a 6537 bp fragment (assembly NCBI37/mm9, chr3∶129,606,653-129,613,189) from a C57BL/6 mouse BAC (RP23-233B14) was first retrieved into plasmid pBlight-TK ^[34]. Second a loxP-em7-kanamycin-loxP cassette was inserted upstream of exon 2 between position chr3∶129,608,133 and129,608,134. Correctly targeted plasmid was transformed into arabinose-induced SW106 cells (Cre expressing E.coli, ^[35], ^[36]) to remove kanamycin and leave behind a single loxP site. Finally, an frt-PGK-em7-Neo-BGHpA-frt-loxP cassette was inserted downstream of exon 4 between position chr3∶129,609,697 and 129,609,698, resulting in the caspase-6 conditional knock-out (CKO) targeting vector. The final vector was confirmed by DNA sequencing.

The caspase-6 CKO vector was linearized with NotI and C57BL/6 C2 ES cells were targeted using standard methods (G418 positive and gancyclovir negative selection). Positive clones were identified using PCR and taqman analysis, and confirmed by sequencing of the modified locus. Correctly targeted ES cells were transfected with a Flpe plasmid to remove Neo and create the caspase-6 conditional knock-out allele (CKO), or with a Cre plasmid to remove Neo and create the caspase-6 KO allele. Caspase-6 KO or CKO ES cells were then injected into blastocysts using standard techniques, and germline transmission was obtained after crossing resulting chimaeras with C57BL/6N females.

The endpoint absorbance, fluorescent (RFU) or luminescent (RLU) emission in each well was plotted as a function of inhibitor concentration and the 50% inhibition (IC₅₀) values were determined using a nonlinear least squares fit of the data to a four parameter equation using Prism 5.0 software (GraphPad Software, San Diego, CA).

SKNAS cells were plated at a density of 20,000 cells per well into 384-well black clear-bottom cell culture plates in 20 µl/well growth medium and allowed to adhere overnight. The following day, 5 µl of peptide inhibitor diluted in growth medium was delivered to each well 30 minutes prior to addition of 5 µl/well staurosporine in growth medium. The final DMSO concentration in the assay was 0.1% and the final staurosporine concentration was 3 µM. Following 6 hours incubation at 37°C, Caspase-Glo® 6 reagents (Promega; Madison, WI) were prepared according to manufacturer's recommendation, and 30 µl was added directly to each well, followed by 1 hour incubation at 37°C and luminescence recording using a Perkin Elmer Envision.

SKNAS cells were plated at a density of 20,000 cells per well into 384-well black clear-bottom cell culture plates in 20 µl/well growth medium and allowed to adhere overnight. The following day, 5 µl staurosporine diluted in growth medium was delivered to each well and incubated for the indicated time in a cell culture incubator. The final DMSO concentration in the assay was 0.1%. At each timepoint, lactate dehydrogenase (LDH) activity was measured in cell supernatants using a colorimetric LDH activity measurement kit (BioVision; Mountain View, CA) according to manufacturer's recommendation by measuring absorbance at 450 nm using a Spectramax platereader (Molecular Devices; Sunnyvale, CA).

SKNAS cells were seeded in 12-well plates at a concentration of 5×10⁶ cells/mL and allowed to adhere overnight prior to the experiment. Caspase inhibitors (10 µM) in Hank's balance Salt Solution (HBSS; Invitrogen) buffer containing 10 mM HEPES (Invitrogen) and a final DMSO concentration of 0.1% were added in triplicate to the cells. Non specific binding (NSB) samples were prepared by immediately aspirating the dose solution and washing the cells with 1 mL of ice cold HBSS. Cells for the accumulation experiment were incubated for 1 hour at 37°C with inhibitors prior to washing with 1 mL ice cold HBSS. Analytical internal standard (200 nM propranolol (Sigma) in 100% acetonitrile (ACN) with 0.1% formic acid) and blank HBSS buffer were sequentially added to the 12-well plates containing the NSB and accumulation samples. Reference samples for each inhibitor were made by adding internal standard to dose solution and cells incubated for 1 hour at 37°C. The cells from all experiments were scraped from the bottom of the 12 well plates, sonicated for approximately 30 minutes, and centrifuged for 10 minutes at 3000 x g prior to LC/MS/MS analysis.

The LC/MS/MS system consisted of an AB Sciex API4000 mass spectrometer (AB Sciex, Foster City, CA), an Aria LX2 multiplexing LC system (Thermo Fisher Scientific, Waltham, MA) and a CTC Analytics autosampler (Leap Technologies, Carrboro, NC). An ESI Spray source in the positive ion mode and multiple reaction monitoring (MRM) were used to quantitate peak areas. The LC separations were performed on a 50×2.0 mm Gemini C18 analytical column (Phenomenex, Torrance, CA). The mobile phases consisted of water with 0.1% formic acid (mobile phase A) and ACN with 0.1% formic acid (mobile phase B). Mass spectrometer data were processed using Analyst software version 1.4.2.

There are a wide number of commercially available pharmacologic reagents that can be used to inhibit the enzymatic activity of recombinant caspases. Most of these tools are peptide inhibitors based on preferred substrate recognition sequences that possess an electrophilic warhead coupled to the P1 Aspartic acid residue for direct interaction with the enzyme's catalytic cysteine. For example, VEID-aldehyde (Ac-VEID-CHO) is generally inferred as selectively inhibiting caspase-6 while Ac-DEVD-CHO is considered a caspase-3/7 inhibitor. In order to elucidate the ability of these tools to dissect executioner caspase activity in a cellular context, a panel of peptide based caspase inhibitors with divergent amino sequences and warheads was tested for their ability to inhibit the enzymatic activity of caspase-3, -6 and -7 (Table 1). Under the assay conditions tested, aldehyde-containing peptide inhibitors were the most potent against caspase-6 (relative to fluoro-methyl ketone (FMK) derived inhibitors), but the amino acid sequence appears to confer little selectivity. For example, Ac-DEVD-CHO (IC₅₀ = 30.5 nM) and Ac-IETD-CHO (IC₅₀ = 53.2 nM), which are canonically considered selective for caspase-3 and caspase-8, respectively, inhibited caspase-6 with near equal potency as did Ac-VEID-CHO (IC₅₀ = 16.2 nM). This lack of selective inhibition by these tools was seen for each of the executioner caspases tested (Table 1). As an extension to this notion, we monitored the proteolysis of a distinct fluorescent substrate with a VEID recognition motif ((Ac-VEID)2-Rh110) by each of the three executioner caspases. This substrate was readily cleaved under initial rate conditions by caspase-3 and -7 with efficiency comparable to caspase-6 (data not shown) confirming previous findings with similar peptide substrates ^[17], ^[18]. This illustrates the limitation with using any of these reagents as tools to selectivity monitor caspase-6 activity in a cellular context where other executioner caspases are present.

Due to the lack of selectivity of peptide reagents listed above, we sought to identify a full length substrate that might offer a higher degree of selective processing. The nuclear envelope proteins lamin A and lamin C have previously been identified as substrates for caspase-6 and contain the consensus VEID sequence in a helical rod section between N and C terminal globular domains. These two proteins are encoded by a single primary transcript and therefore share the identical first 566 amino acids with the only differences occurring in the C-terminus ^[37]. The efficiency of purified active caspase-6 to process recombinant lamin A was evaluated by monitoring its degradation over a range of enzyme concentrations (Figure 1A). Lamin degradation is evident with as little as 6 nM caspase-6 (30 U).

To assess proteolytic specificity, the capacity of other caspase isoforms to degrade recombinant lamin A was evaluated. Western blot analysis of the reaction products from 300 U active caspases 1–9 reveals that only caspase-6 produced a prominent signal for cleaved lamin (Figure 1B). It was observed that at higher caspase-3 concentrations (>1000 U) slight lamin processing does occur (data not shown), although these enzyme conditions are non-catalytic. Each enzyme was separately evaluated in independent enzymatic experiments to ensure proficient activity and found to possess suitable activity and proper pharmacology (data not shown).

The high degree of selective lamin proteolysis by recombinant caspase-6 prompted us to investigate the use of this substrate as a cellular readout for caspase-6 activity. Staurosporine is a non-selective protein kinase inhibitor that activates caspase-6 ^[38], amongst other caspases, in cells as they progress towards apoptosis. Cell lysates were prepared from SKNAS cells treated with DMSO or 3 µM staurosporine for six hours to activate the caspase machinery. The SKNAS cell line is of human neuroblastoma origin, and was selected for these and following studies due to its robust caspase induction as assessed using Caspase-Glo® 6 assay kit from Promega as well as by observing zymogen conversion to active caspase-6 by western blot detection (data not shown). A panel of three antibodies that bind intact lamin or its proteolytic fragments were monitored for their ability to detect each lamin A/C species via western blot (Figure 2A and 2B). All three antibody preparations detected intact lamin A/C (Lane 5) or its large (Lane 4) or small (Lane 2) proteolytic fragments. As seen in lane 6, the intact lamin A/C antibody also detects the small proteolytic fragment produced upon staurosporine treatment suggesting its binding epitope is on the N-terminus of the protein. On the other hand, the antibodies against the large or small subunit recognize neo-epitopes only exposed subsequent to caspase-6 proteolysis and do not detect intact lamin A/C (Lanes 1, 3). The kinetics of lamin A/C degradation were monitored by inducing an apoptotic state with 3 µM staurosporine for 1–6 hours and identified the abundant appearance of proteolytic fragments after 4–6 hours of staurosporine treatment (Figure 2C). Induction of cellular caspase activity with 6 hours of staurosporine treatment was also observed using the Caspase-Glo® 6 assay kit (data not shown) and provided confidence that this time point was suitable for the remainder of our assay development.

In order to develop an assay more amenable to inhibitor screening, this antibody detection was adapted to a plate-based non-denaturing assay. SKNAS cells cultured in 384-well plates were treated with 3 µM staurosporine for six hours prior to fixation with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100. Binding of the two antibodies that recognize neo-epitopes on the large or small lamin degradation products was detected by horseradish-peroxidase-labeled secondary antibody and subsequent chemiluminescent substrate addition (see methods for details). In this context, the large subunit antibody was unable to produce a signal window (data not shown), while the small subunit antibody generated a robust signal and was utilized for further studies. The concentration dependence of staurosporine required to evoke lamin A/C degradation after a six-hour treatment period indicated that 3 µM staurosporine produced a marked signal:background ratio of 8.8-fold and that higher concentrations could be used to induce a greater response if desired (Figure 3). Under these conditions the assay proved highly robust (Z'≥0.7).

This lamin degradation plate-based assay was used to further investigate the specificity of lamin A/C as a caspase-6 substrate in a cellular environment. Murine embryonic fibroblast cultures were established from tail-tissue-explants derived from caspase-6 knockout and wild-type mice and subjected to increasing concentrations of staurosporine prior to detection of cleaved lamin (Figure 4). The marked response in the wild-type cells compared to the caspase-6 knockout cells supports the biochemical evidence that caspase-6 is the primary caspase responsible for lamin processing, although emergence of lamin degradation products begin to appear slightly at the highest staurosporine concentration tested in the caspase-6 KO fibroblast cells. Under these conditions, the morphological features associated with apoptosis were equally observed in both the wild type and KO cells (data not shown) as would be expected given the ability of staurosporine to function as a universal apoptotic trigger with known capacity to activate the caspase-3 machinery ^[27], ^[39].

The goal of the cell-based assay is to measure inhibition of the intracellular caspase-6 by cell-permeant inhibitors, therefore it is important to establish that cell membrane integrity has not been compromised during caspase activation. To assess the integrity of the SKNAS cell membrane under the staurosporine-induced apoptotic conditions, lactate dehydrogenase (LDH) release was measured upon treatment of cells with a range of staurosporine concentrations and after several incubation time periods. As seen in Figure 5, the concentration dependent release of LDH in these cells is also highly dependent on the incubation time. After 6 hours of treatment, LDH release is detected above background levels with ∼4 µM staurosporine. The same degree of measured LDH release with 30 µM staurosporine only requires 4 hours of treatment. These data also illustrate that under conditions in which maximal lamin cleavage was obtained by wild type but not caspase-6 KO fibroblasts, cytotoxicity as measured by LDH release was minimal. This information can facilitate the selection by the user of incubation time and staurosporine concentration to develop their preferred assay.

The plate-based version of the lamin degradation assay was used to monitor the potency of a panel of peptide inhibitors for their capacity to prevent caspase-6-dependent proteolysis. At inhibitor concentrations up to 100 µM, little or no inhibitory activity was observed with the aldehyde or FMK based tetrapeptide inhibitors possessing a Glu at the second position despite having submicromolar biochemical potencies (Figure 6A and data not shown). The nonselective inhibitor z-VAD-FMK inhibited with 44 µM potency consistent with previously published reports ^[40]. This result might be expected given the highly charged nature of these Glu-containing molecules which prevents their cellular accumulation. Conversely, the pan caspase inhibitor Q-VD-OPh, which possesses a difluorophenoxymethyl warhead, inhibited generation of the small lamin subunit with an IC₅₀ of 940 nM (6-fold lower potency than enzymatic assay) (Figure 6A, Table 2). To this end, three further peptide inhibitors were synthesized each with a similar warhead (tetrafluorophenoxymethyl (TFPM)) with the attempt to facilitate cell activity (Figure S1). These new inhibitors are based off the z-VEID recognition motif and are successively truncated at the N-terminus (z-VEID-TFPM, z-EID-TFPM, z-ID-TFPM). In biochemical caspase-6 assays, each truncation results in a decrease in potency (1.7nM, 8.4nM, 19nM), while the opposite is seen in the cellular assay (Figure 6B, Table 2). In this case, the shortest and least charged inhibitor z-ID-TFPM inhibited lamin degradation most potently with an IC₅₀ = 180nM (∼10-fold less potent than enzymatic inhibition). In order to confirm our hypothesis that lack of cellular activity of these inhibitors is due to their poor membrane permeability, we analyzed the degree of their SKNAS cellular accumulation via mass spectrometry. This analysis revealed that cellular potency is highly correlative with cell penetrance. Q-VD-OPh and z-ID-TFPM were quantified as the two inhibitors most readily able to penetrate into cells and also the two most effective at inhibiting lamin degradation (Table 2). Ac-VEID-CHO is predominantly excluded from accessing the intracellular environment (0.16% cellular accumulation) and lacks any activity in the cellular assay. When the membrane barrier is removed as in the case of the lysate based Caspase-Glo® 6 assay, this peptide inhibitor as well as Ac-DEVD-CHO (also inactive in lamin degradation assay) are both clearly able to inhibit VEIDase activity (Ac-VEID-CHO IC₅₀ = 0.49 µM; Ac-DEVD-CHO IC₅₀ = 1.49 µM) (Figure 7). It is evident from these data that inhibition in the lamin degradation assay is dictated by a compound's absolute binding affinity and its ability to access the intracellular target.