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. Author manuscript; available in PMC: 2011 Nov 24.
Published in final edited form as: Chem Biol. 2010 Nov 24;17(11):1189–1200. doi: 10.1016/j.chembiol.2010.08.014

Identification and Evaluation of Novel Small Molecule Pan-Caspase Inhibitors in Huntington’s Disease Models

Melissa J Leyva 1, Francesco DeGiacomo 2, Linda S Kaltenbach 3, Jennifer Holcomb 2, Ningzhe Zhang 2, Juliette Gafni 2, Hyunsun Park 4, Donald C Lo 3, Guy S Salvesen 5, Lisa M Ellerby 2, Jonathan A Ellman 6
PMCID: PMC3035168  NIHMSID: NIHMS249230  PMID: 21095569

SUMMARY

Huntington’s Disease (HD) is characterized by a mutation in the huntingtin gene encoding an expansion of glutamine repeats on the N-terminus of the huntingtin (Htt) protein. Numerous studies have identified Htt proteolysis as a critical pathological event in post mortem human tissue and mouse HD models, and proteases known as caspases have emerged as attractive HD targets. We report the use of the substrate activity screening method against caspases-3 and -6 to identify three novel, pan-caspase inhibitors that block proteolysis of Htt at caspase-3 and -6 cleavage sites. In HD models, these irreversible inhibitors suppressed Hdh111Q/111Q-mediated toxicity and rescued rat striatal and cortical neurons from cell death. In this study the identified nonpeptidic caspase inhibitors were used to confirm the role of caspase-mediated Htt proteolysis in HD. These results further implicate caspases as promising targets for HD therapeutic development.

INTRODUCTION

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder characterized by progressive deterioration of neurons in the striatum and cortex. Symptoms can occur at any age but usually occur with an adult-onset and patients exhibit progressive loss of cognitive and motor function. HD is caused by a mutation in the Htt gene with a trinucleotide (CAG) expansion encoding glutamine repeats in the N-terminus of huntingtin (Htt), a scaffold protein with many interacting proteins which has been shown to be involved in vesicular trafficking (HDCRG, 1993). A neuropathological hallmark of HD in human and mouse models is the accumulation of N-terminal Htt fragments leading to cytotoxicity, suggesting that Htt proteolysis is a critical event in pathogenesis (Davies et al., 1997; DiFiglia et al., 1997; Gafni and Ellerby, 2002; Gafni et al., 2004; Goldberg et al., 1996; Gutekunst et al., 1999; Martindale et al., 1998).

Numerous studies have demonstrated the cleavage of Htt by multiple proteases, including aspartyl proteases, calpains, and caspases (Gafni and Ellerby, 2002; Gafni et al., 2004; Goldberg et al., 1996; Lunkes et al., 2002; Wellington et al., 1998). Caspases are cysteine proteases characterized by their high specificity for substrates with an aspartic acid at the site of cleavage in the P1 position and play a prominent role in apoptosis (Pop and Salvesen, 2009). Dysregulation of apoptosis has been implicated in stroke, neuronal degeneration, liver disease, cancer, and autoimmune disorders (Reed John, 2002). Due to the dramatic neuronal cell death in HD, it is not surprising that Htt was the first neuronal protein identified as a caspase substrate (Goldberg et al., 1996). A number of studies have defined the cleavage sites of Htt for caspase-3 at amino acids 513 and 552, for caspase-2 at amino acid 552, and for caspase-6 at amino acid 586 (Wellington et al., 1998; Wellington et al., 2000). Recently, caspase-6 cleavage of mutant Htt and activation of caspase-6 has been shown to play a significant role in HD pathogenesis in HD mouse models and is also activated in HD postmortem tissue (Graham et al., 2006; Hermel et al., 2004).

Although the aforementioned studies clearly implicate caspase-mediated cleavage of Htt in HD, few studies have evaluated the effects of caspase inhibition on cell death. The non-specific irreversible, peptidic fluoromethyl ketone inhibitor, zVAD-fmk, protected striatal neurons against malonate-induced excitoxicity (Schulz et al., 1998), and the reversible caspase-3 specific inhibitor, M826, significantly displayed neuroprotection against malonate-induced striatal injury in a rat model of HD (Toulmond et al., 2004). Additionally, through indirect means minocycline, a tetracycline derivative with the ability to cross the blood-brain barrier, inhibits caspase-1 and caspase-3 transcriptional upregulation and delays cell death in HD transgenic mouse models (Chen et al., 2000). The development of small molecule, nonpeptidic inhibitors of both caspase-3 and -6 and their evaluation in HD biology would provide useful tool compounds to the field. Unfortunately, while numerous caspase inhibitors have been developed, (Cornelis et al., 2007; O’Brien and Lee, 2004) most are peptidic in nature and efficacy in cells and animals is compromised due to poor cell penetration and ADME (Absorption, Distribution, Metabolism, and Excretion) properties, respectively (Talanian et al., 1997). We therefore applied a substrate-based fragment approach called substrate activity screening (SAS) to the development of nonpeptidic inhibitors of caspases-3 and -6. In the SAS method, which has previously been applied to proteases of the papain family, (Brak et al., 2008; Inagaki et al., 2007; Patterson et al., 2006; Wood et al., 2005) weak binding nonpeptidic substrate fragments are identified, optimized, and then converted to potent inhibitors. Herein, we report the identification of three novel, nonpeptidic pan-caspase irreversible inhibitors that blocked proteolysis of Htt at caspase-3 and caspase-6 sites, suppressed Hdh111Q/111Q-mediated toxicity, and rescued HttN90Q73-induced degeneration of rat striatal and cortical neurons.

RESULTS AND DISCUSSION

Substrate Library Synthesis and Screening

To target caspase-3, we initially set out to create a library that incorporated the P1 aspartic acid required for caspase recognition and amide bond hydrolysis. To satisfy the goal of identifying nonpeptidic fragments, we synthesized an initial library of 1,4-disubstituted-1,2,3-triazole substrates 1 (Figure 1) because triazoles have been demonstrated as efficient amide bond replacements in the development of protease inhibitors (Brik et al., 2005; Wood et al., 2005), and diverse functionality could readily be introduced into the substrates using solid-phase methods (see Supplemental Data). Substrates were screened in a high-throughput fluorometric assay to detect caspase-3-catalyzed amide bond proteolysis and liberation of the fluorophore, 7-amino-4-methylcoumarin-3-acetic acid (AMCA) (Figure 1).

Figure 1.

Figure 1

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Screening of 7-Amino-4-methylcoumarin-3-acetic acid (AMCA) Substrates 1 First step in SAS method, screening diverse AMCA substrates against caspase to yield a fluorescent AMCA reporter group.

Structure-Activity Relationship (SAR) of AMCA Substrates Against Caspase-3

Figure 2A exhibits the relative cleavage efficiencies of a subset of initial substrates that were evaluated. Substrate 2 with a phenylethyl group at the R position had the lowest cleavage efficiency of all of the substrates for which turnover was detected and was assigned a relative cleavage efficiency value of 1. Substrates for which no turnover was detected were given a relative cleavage efficiency value of 0. Substrates 8 and 9 with α-branching at the R position as provided by the cyclohexyl moiety along with hydroxyl and amide groups, respectively, that are both capable of H-bonding, showed the highest activity. Substrate 9 was particularly appealing because the amine functionality provides the opportunity to introduce a variety of additional functionality through amine acylation and reductive amination chemistry.

Figure 2.

Figure 2

Figure 2

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Caspase-3 Cleavage Efficiencies of AMCA Substrates

(A) Relative kcat/Km of initial substrate library incorporating alkyl and polar R groups. The relative kcat/Km of the least efficiently cleaved substrate for which turnover could be detected was assigned a value of 1.0. For caspase-3, substrate 2 was assigned a value of 1.0.

(B) Relative kcat/Km of substrate set incorporating α-substituted amide R structures.

(C) Relative kcat/Km of N-substituted amide substrates with (S)-configured methyl and cyclohexyl substituents.

See also Figure S1.

To further explore the effect of α-branched substituents within the amide framework present in substrate 9, an additional collection of substrates was prepared and evaluated (Figure 2B). When the cyclohexyl group, that introduces α,α-dibranching in substrate 9 was replaced by the α-monobranched structures in substrates 10-14, no substrate turnover was observed, which suggests the importance of maintaining dibranched functionality at this position. Similarly, dibranched substituents with the smaller methyl, ethyl, and isopropyl groups all displayed reduced cleavage efficiency (substrates 15-17). In contrast, substrate 18, which incorporated methyl and cyclohexyl substituents with an (S)-configuration, showed a greater than five-fold increase in cleavage efficiency relative to the initial cyclohexyl amide substrate 9. It is noteworthy that caspase-3 showed very strong chiral recognition with no cleavage observed for substrate 19, which is the (R)-epimer of substrate 18. The strong preference for the (S)-epimer suggests the importance of this configuration for proper orientation and enhanced binding in the S2 pocket of caspase-3.

With substrate 18 incorporating cyclohexyl and methyl substitituents with (S)-configuration providing the highest cleavage efficiency, we next chose to evaluate the replacement of the acetamide group with a variety of different amides and amines to identify key binding interactions for this region of the substrate (representative structures shown in Figure 2C). Replacing the acetamide in substrate 18 with the N,N-diethyl moiety in substrate 20 almost completely eliminated substrate turnover suggesting the importance of the H-bond donor or accepting capability of the amide functionality. In contrast, replacing the acetamide in substrate 18 with the benzamide in substrate 21 resulted in only a modest reduction in cleavage efficiency demonstrating that groups considerably larger than acetamide can be accommodated in the enzyme active site. The isosteric secondary amine in substrate 22 resulted in a >10-fold reduction in cleavage efficiency of the substrate again confirming the importance of the amide functionality either for orientation or for hydrogen bonding interactions. The phenyl acetamide present in substrate 23 resulted in a modest increase in cleavage efficiency while the isosteric phenylmethylsulfonamide present in 24, which maintains hydrogen-bond donor and accepting capability, provided a considerable boost in cleavage efficiency. Finally, introduction of a second carboxylic acid in substrates 25-26 resulted in a considerable increase in cleavage efficiency with substrate 25 providing the highest cleavage efficiency out of all the substrates tested. This result is consistent with the known peptide substrate specificities for many of the caspases, which prefer a second aspartic acid residue at the P4 position, i.e., four amino acids away from the cleavage site (Talanian et al., 1997).

Substrate Screening and Optimization against Caspase-6

For peptide substrates, caspases-3 and -6 both require an aspartic acid residue at the site of cleavage but differ in preferred amino acid side chains two (P2) and four (P4) amino acids from the cleavage site (Thornberry et al., 1997). In P2, caspase-3 shows a strong preference for small hydrophobic residues, while caspase-6 tolerates larger residues. In P4, caspase-3 demonstrates an almost absolute requirement for an aspartic acid residue, while larger aliphatic residues are tolerated by caspase-6. Therefore, all of the previously synthesized caspase-3 substrates were assayed against caspase-6 with key substrate activity relationships represented by the substrates shown in Figure 3A. No substrate turnover was observed for α-monobranched substrates as exemplified by substrates 10 and 13 therefore they were given the relative cleavage efficiencies of 0. Out of all the α,α -dibranched substrates, only substrates 18 and 19 with methyl and cyclohexyl substituents resulted in cleavage. Substrate 19 gave the lowest cleavage efficiency out of all the substrates for which turnover was detected for caspase-6 and was assigned a relative cleavage efficiency of 1. The (S)-epimer of substrate 18 provided a five-fold increase in cleavage efficiency relative to substrate 19 consistent with the stereochemical preference observed for caspase-3. For substrate 25 incorporating the carboxylic acid functionality resulted in a greater than two-fold increase in cleavage efficiency, while no substrate turnover was detected for substrate 26, which also contains the carboxylic functionality but with a shorter chain length. The importance of the H-bond accepting capability of the amide carbonyl is apparent by comparing the cleavage rate of amide substrate 21 and amine substrate 22. The phenylacetamide substrate 23 provided a considerable increase in cleavage efficiency with the corresponding isosteric phenylmethylsulfonamide substrate 24 also serving as a good substrate but with ~3-fold lower efficiency than 23.

Figure 3.

Figure 3

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Caspase-3 and -6 Cleavage Efficiencies of AMCA Substrates

(A) Relative kcat/Km of a select number of previously synthesized AMCA substrates against caspase-3 and -6. The relative kcat/Km of the least efficiently cleaved substrate for which turnover could be detected was assigned a value of 1.0. For caspase-3, substrate 2 was assigned a value of 1.0 (Figure 2A). For caspase-6, substrate 19 was assigned a value of 1.0.

(B) Relative kcat/Km of newly synthesized substituted phenylamide substrates against caspase-6.

Figure 3A displays notable differences in structure-activity relationships between caspase-3 and caspase-6 substrates. The chiral preference for (S)-epimer 18 was stronger for caspase-3 over caspase-6. This can be explained by the difference in size of the S2 pocket between caspase-3 and caspase-6. Various substitutions are tolerated in P2 for both caspases but caspase-6 is able to accommodate larger aliphatic and aromatic residues in contrast to caspase-3. A difference in relative cleavage efficiencies between benzamide 21 and phenylacetamide 23 was observed for caspase-3 and caspase-6. Substrate 23 showed only a modest increase in cleavage efficiency compared to substrate 21 for caspase-3, while 23 displayed a >18-fold increase compared to 21 for caspase-6. Consistent with the known peptidic substrate specificity data for caspases-3 and -6 (vide infra), substrate 25 with an acidic side chain capable of binding in the S4 pocket was the most efficiently cleaved by caspase-3; while substrate 23 displaying hydrophobic functionality was the most efficiently cleaved by caspase-6.

Because substrate 23 incorporating the phenylacetamide showed by far the greatest cleavage efficiency by caspase-6, analogues with substitution on the phenyl ring were prepared and evaluated (Figure 3B). Methyl and phenyl electron donating substituents at the meta position in substrates 27 and 28 decreased cleavage efficiency. The considerably lower cleavage efficiency of substrate 28 can be attributed to unfavorable steric interactions with the large phenyl substituent. Modest reductions in cleavage efficiency were also observed for substrates with meta-substitution with the more strongly electron donating phenoxy and methoxy substituents, 29 and 30, respectively, as well as with the electron-withdrawing trifluoromethyl substituent (substrate 31). The electron-donating methoxy substituent at the para position provided an ~eight-fold drop in cleavage efficiency (substrate 32). A slight decrease in cleavage efficiency was also observed for the electron-withdrawing chloro substituent at both the para and ortho positions, present in substrates 33 and 34, respectively.

Inhibitor Synthesis and Screening

The final step in the SAS method is conversion of the most active substrates to inhibitors. Substrate cleavage is only observed when the amide carbonyl of the N-acyl aminocoumarin is properly positioned in the enzyme active site, thus allowing for the aminocoumarin group to be replaced by a mechanism-based pharmacophore to produce reversible or irreversible inhibitors. Pharmacophore selection was based upon a class of peptidic pan-caspase inhibitors containing the 2,3,5,6-tetrafluorophenoxy ketone irreversible inhibitor pharmacophore developed by Idun Pharmaceuticals because members of this inhibitor series have advanced the farthest in clinical development.(Linton et al., 2004; Linton et al., 2005). Indeed, inhibitor IDN-6556 (Figure 4A) was determined to be nontoxic in mouse models and has entered Phase II clinical trials for the treatment of human liver preservation injury (Baskin-Bey et al., 2007). The 2,3,5,6-tetrafluorophenoxyketone pharmacophore was incorporated into three distinct substrates, 23-25, that showed good cleavage efficiency by both caspase-3 and -6 to provide inhibitors 35-37 (see Supplemental Data for inhibitor synthesis and characterization). The inhibitory activity of these compounds was then characterized against a full panel of caspases-1, -2, -3, -6, -7, -8, and -9 (Figure 4B). All three inhibitors provided potent inhibition of caspases-3 and -6 with kinact/Ki values of >20,000 M−1s−1 for each enzyme. Each of the inhibitors also provided potent inhibition of caspases-1, -7, -8 and -9 as would be expected by the similar substrate specificities across these enzymes. In contrast to the other caspases, caspase-2 has an extended binding pocket, and a P5 residue has shown to be critical for increased cleavage efficiency (Schweizer et al., 2003; Talanian et al., 1997). Due to this extended binding requirement, it is not surprising that for inhibitors 35-37 poor inhibition of caspase-2 was observed with kinact/Ki values of <33 M−1s−1.

Figure 4.

Figure 4

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Irreversible 2,4,5,6-Tetrafluorophenoxymethyl Inhibitors

(A) Pan caspase inhibitor incorporating a 2,4,5,6-tetrafluorophenoxymethyl ketone pharmacophore developed by Idun Pharmaceuticals.

(B) Inactivation rates of irreversible 2,4,5,6- tetrafluorophenoxymethyl inhibitors against caspases -1, -2, -3, -5, -6, -7, -8, and -9, cathepsins B, S, and V, and legumain. aAssays determining kinact/Ki (M−1s−1) were performed in duplicate with SD values included. bCaspase-2 assays determining kass (M−1s−1) were performed in duplicate with SD values included. c No inhibition after 15 min of preincubation with various concentrations of inhibitor of up to 400 μM. d Very weak inhibitory activity prevented determination of kinact/Ki values, but IC50 values of 200-350 mM were obtained after 15 min of preincubation. See also Figures S2, S3 and S4.

Recent studies have demonstrated that incorporation of P1 aspartyl sidechain functionality in caspase inhibitors does not necessarily afford selective caspase inhibition. Aspartyl peptidyl flouromethyl and choloromethyl ketones have been reported to efficiently inhibit other cysteine proteases such as cathepsin B, S and V as well as legumain (Rozman-Pungercar et al., 2003). More recently, aspartyl peptidic acyloxymethyl ketones have been utilized as efficient cell-permeable activity-based probes for legumain (Sexton et al., 2007). Due to the aforementioned studies, all three inhibitors were screened against multiple cathepsins and legumain to evaluate cross-reactivity. Inhibitors 35, 36, and 37 displayed no inhibition of cathepsins B and V. Weak inhibition was observed for cathepsin S with kinact/Ki values of < 1,138 M−1s−1 Inhibition of legumain by 35 and 36 was so poor (IC50 = 200-350 μM at 15 min preincubation) that kinact/Ki values could not be calculated, and for inhibitor 37 absolutely no inhibition of this enzyme could be detected. Overall, the caspase-designed aryloxymethyl ketone irreversible inhibitors showed little to no inhibition against legumain, cathepsin B, S, and V.

Evaluating Inhibitors in Huntington’s Disease Models

In vitro, huntingtin is preferentially cleaved at amino acid 552 by caspase-2, 513 by caspase-3, and 586 by caspase-6 (Hermel et al., 2004; Wellington et al., 2000). Inhibitors 35, 36 and 37 were each evaluated for Htt proteolysis by caspases-2, -3 and -6 with neoepitope antibodies that recognize the specific Htt cleavage products using an in vitro assay. Figure 5 shows western blot analysis of cellular lysates expressing full-length Htt23Q and Htt148Q treated with caspase-2, -3, and -6 and the three inhibitors at various concentrations using the neo-specific Htt antibodies. While minimal protection against cleavage of Htt by caspase-2 was observed as expected (Figure 5A), inhibitors 35, 36, and 37 each effectively blocked proteolysis by caspase-3 at amino acid 513 at all of the tested concentrations (0.1, 1.0 and 10 μM, Figure 5B). For full-length Htt treated with caspase-6, all three inhibitors, 35, 36, and 37 also showed complete protection from cleavage at amino acid 586 at concentrations of 1.0 and 10 μM (Figure 5C). To gain insight into the efficiency of the inhibitors for blocking caspase cleavage of Htt, we determined the kcat/Km of full-length Htt with caspase-3 and caspase-6 (Supplemental Table S1), and it appears that Htt in lysates is less efficiently cleaved by caspase-6 than caspase-3. This may correlate with the higher dose required to inhibit caspase-6 cleavage of Htt; however without determining Km of Htt we cannot compare the efficiency of the inhibitors for the caspases.

Figure 5.

Figure 5

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Inhibitors 35, 36 and 37 Block Caspase-6 or Caspase-3 Mediated Cleavage of Htt

Full-length c-myc-Htt23Q and c-myc-Htt148Q were transiently transfected in HEK 293T cells for 72 h. Western blots of cellular lysates treated with recombinant caspase-2, -3, or -6 and inhibitors were probed with the indicated neo-specific antibody recognizing the Htt cleavage fragment. * indicate non-specific band. The lysates contain endogenous wild-type Htt so this is also detected in right panel A and B. For Panel B it should be noted that the inhibitors are less effective on caspase-2 cleavage of Htt. Because these are cellular lysates it is possible that addition of caspase-2 activates some of the other caspase members yielding a slight effect on full length Htt. See also Table S1.

Next, each inhibitor was evaluated in a HD cellular toxicity model by measuring caspase activity and Htt proteolysis. Since HD primarily affects striatal and cortical neurons, we used immortalized mouse striatal cell Hdh7Q/7Q and Hdh111Q/111Q lines to measure cellular toxicity and Htt proteolysis (Trettel et al., 2000). We cultured Hdh7Q/7Q and Hdh111Q/111Q cells and evaluated Htt proteolysis 24 h after serum withdrawal. We found neoHtt513 antibody detected proteolysis of Htt in cells and lysates (Figure 6A,B). Qualitatively, the levels of Htt proteolysis are higher in the Hdh111Q/111Q when compared to Hdh7Q/7Q lines (Figure 6B). Caspases are known to cleave the huntingtin protein containing the polyglutamine expansion and evidence suggests that these N-terminal cleavage products lead to cellular toxicity and apoptosis (Wellington et al., 1998). During serum withdrawal, we found that Hdh111Q/111Q cells have a 7-fold increase in caspase activation compared to control Hdh7Q/7Q. Cell death occurred at 48 hours after serum withdrawal (Supplemental Figure S5). All three irreversible inhibitors, 35, 36, and 37 at concentrations of 1-100 μM were evaluated in Hdh7Q/7Q and Hdh111Q/111Q cells and were found to significantly block caspase activity in both the normal and expanded huntingtin cell model 24 h after serum deprivation (Figure 6C). Further, the irreversible inhibitors 35 and 37 at concentrations of 10-100 μM blocked the proteolysis of Htt as detected by neoHtt513 (Figure 6D). Interestingly, inhibitor 36, which incorporates additional acidic carboxylic acid functionality and thus has distinct physicochemical properties relative to inhibitors 35 and 37, showed minimal effect. To determine the IC50 values for inhibitors 35-37, DEVDase activity was measured as a function of concentration for each inhibitor (Figure 6E). Of note, the concentration needed to block caspase activity was lower in the HD cells compared to WT. Perhaps doses of caspase inhibitors can be utilized in therapeutics that do not impact normal cellular function. We do not know the exact mechanism for the lower dose required to inhibit the caspases in the Hdh111Q/111Q cells but two possible explanations are: (1) the Hdh7Q/7Q caspase-3 activity is from the proform of the enzyme which has a distinct kcat and Km when compared to active caspase-3 (Hdh111Q/111Q could have more), or (2) the endogenous inhibitors of caspases are lower in the disease HD cells due to activation of degradative pathways such as proteasome or autophagy.

Figure 6.

Figure 6

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Inhibitors 35, 36 and 37 Block Caspase Activation in Mouse Striatal Hdh7Q/7Q and Hdh111Q/111Q Cells

(A) Striatal Hdh7Q/7Q and Hdh111Q/111Q cells were cultured for 48 h and then subjected to analysis 24 h after serum withdrawal. Immunochemistry of Hdh7Q/7Q and Hdh111Q/111Q cells with neoHtt513 demonstrates cleavage of Htt. Qualitatively, more Htt cleavage is found in the Hdh111Q/111Q cells.

(B) Western blot analysis using neoHtt513 antibody demonstrates proteolysis of Htt at the caspase-3 cleavage site located at amino acid 513 after serum withdrawal.

(C) Striatal Hdh7Q/7Q and Hdh111Q/111Q cells where cultured for 48 h and then caspase activity was measured 24 h after serum withdrawal. Hdh111Q/111Q cells have a 7-fold increase in caspase activation compared to control Hdh7Q/7Q. Inhibitors 35, 36, 37 blocked caspase activity at the doses indicated. The experiments were repeated three times, each time with an n= 3. ANOVA analysis indicates the statistical significance *p < .05, ** p < .01, *** p < .005.

(D) Striatal Hdh7Q/7Q and Hdh111Q/111Q cells were cultured for 48 h and 24 h after serum withdrawal cells lysates were prepared. Inhibitors 35, 36, 37 blocked the proteolysis of Htt at the caspase-3 cleavage site located at amino acid 513 at the indicated doses.

(E) Dose response curve of relative caspase activity (Y-axis) in striatal Hdh7Q/7Q and Hdh111Q/111Q cells with the indicated inhibitor provided IC50 values. Analysis was carried out using Prism 4 Graph Software with a sigmoidal dose response curve.

See also Figures S5 and S6.

Next, we evaluated these inhibitors ex vivo, using primary co-cultures of striatal and cortical neurons expressing normal and expanded Htt N-terminal fragments-Htt N90Q73, which induces degeneration of rat striatal and cortical neurons in primary co-cultures. We use co-cultures because in HD it is known that non-cell autonomous interactions and deficits in cortico-striatal circuitry occur. We use a fragment HD model because we have found these toxic Htt fragments cause the activation of the same proteases involved in cleaving full-length Htt and producing neurotoxicity. The percentage of healthy striatal and cortical neurons after treatment with varied concentrations of the irreversible inhibitors is shown in Figure 7 (see Supplemental Figure S6 for relative reversibility of neuronal effects upon removing irreversible inhibitors). All three inhibitors rescued striatal neurons from cell death, and treatment with inhibitor 35 resulted in the most significant increase of healthy striatal neurons at 0.4, 1.2, and 3.7 nM. The same trend is observed for the treatment of cortical neurons, with 35 resulting in a substantial increase (25-175%) in healthy neurons. The control is transfection of the fluorescent protein-encoding plasmid and empty plasmid in place of Htt plasmid. For the striatal, the control is 237% +/− 25. For the cortical, control is 311% +/−60. This suggests that some doses of the inhibitors completely rescued the cell death mediated by mutant Htt. The efficacy of the inhibitors at even single digit nanomolar concentrations is likely due to the greater sensitivity of primary cultures to cell death when compared to the immortalized cells. Primary cultures are terminally differentiated and the culture conditions are distinct when compared to the immortalized mouse striatal Hdh7Q/7Q and Hdh111Q/111Q cell system.

Figure 7.

Figure 7

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Protective Effects of Caspase Inhibitors in HttN90Q73-Induced Degeneration of Rat Striatal and Cortical Neurons in Primary Co-Culture

Rat striatal and cortical neurons expressing HttN90Q73 together with YFP or mCherry respectively, combined in primary co-culture and treated with DMSO or caspase inhibitors at the indicated doses. YFP positive striatal neurons and mCherry positive cortical neurons were quantified at six days post transfection by automated fluorescent object detection on the Cellomics ArrayScan VTI. All conditions were normalized against the DMSO condition set to 100%. N=6 for each condition; error bars are standard deviation. Asterisks indicate significant difference from the DMSO condition using one-way ANOVA followed by Dunnett post hoc comparison. Analysis indicates the statistical significance as: *p < .05, ** p < .01, *** p < .005.

SIGNIFICANCE

HD is an incurable disease caused by a mutation encoding a polyglutamine expansion in the N-terminus of the huntingtin protein. Major research efforts have focused on the relationship of polyglutamine repeats and HD pathogensis, but the molecular mechanisms leading to specific neuronal death are not fully understood. Proposed mechanisms leading to neuronal dysfunction and death include formation of polyglutamine aggregates and inclusions (Arrasate et al., 2004; Sawada et al., 2007), altered confirmation of mutant huntingtin leading to transcriptional dysregulation (Dunah et al., 2002; Li et al., 2002; Zhai et al., 2005), excitotoxic neuron damage by excessive stimulation of glutamate receptors (Charvin et al., 2008; Fan et al., 2007; Stack et al., 2007), and induction of apoptosis and proteolysis. A great amount of work has focused on the causative polyglutamine expanded Htt protein and its proteolysis. Evidence has emerged that suggest Htt fragments lead to neuronal toxicity but the exact mechanism remains unknown. The proteolytic cleavage of Htt, and the enzymes involved in this process have emerged as potential therapeutic targets for treating HD.

Caspases are proteases involved in programmed cell-death and have been found to cleave Htt. Specifically, accumulation of caspase-cleaved Htt fragments represents a neuropathological hallmark of HD. Furthermore, Graham et al. (2006) showed that mice expressing mutant Htt resistant to cleavage by caspase-6 and not caspase-3, exhibit normal neuronal function and do not develop neurodegeneration. Therefore, inhibitors that block caspase cleavage provide a promising starting point for the study of Htt proteolysis.

A substrate-based fragment approach was used to develop three nonpeptidic irreversible inhibitors displaying pan-caspase activity. In vitro, these inhibitors blocked Htt cleavage by caspase-3 and -6. Results from a HD cellular toxicity model showed that all three inhibitors dramatically blocked caspase activation in disease model cells. Ex vivo studies using cultures of striatal and cortical neurons expressed mutant Htt, demonstrated the rescue of neuronal death after treatment with inhibitors. Overall, the identified inhibitors have validated the correlation between blocking caspase Htt cleavage and rescue of HD-mediated neurodegeneration.

EXPERIMENTAL PROCEDURES

Fluorogenic Substrate Screen Against Caspase-3 and -6

Caspases-3, -6, -7, -8, and -9 were obtained by expression in E. coli as previously described (Stennicke and Salvesen, 1999). Caspase-1, cathepsin B, L, and V were purchased from EMD Biosciences (San Diego, CA), caspase-2 was purchased from Biomol International (Plymouth Meeting, PA) and Legumain was purchased from R&D Systems (Minneapolis, MN). Assays were conducted at 37 °C in duplicate with and without the enzyme in Dynatech Microfluor fluorescence 96-well microtiter plates, and readings were taken on a Molecular Devices Spectra Max Gemini XS instrument according to previously reported protocols [see Supplemental Data for full assay details for each enzyme]. Caspases-3 and -6 were used at concentrations 2.7 μM and 3.0 μM, respectively. Relative fluorescent units (RFU) were measured at regular intervals over a period of time (maximum 15 min). A plot of RFU versus time was made for each substrate with and without enzyme. The slope of the plotted line gave relative kcat/Km of each substrate.

Inhibitor Kinetic Assays

Caspase substrates Ac-DEVD-AMC (caspases-3, -6, -7, -8), Ac-VDVAD-AFC (caspase-2), and Ac-LEHD-AFC (caspase-9) were purchased from EMD Biosciences (San Diego, Ca) and Ac-VDVAD-AMC (caspase-2) was purchased from Biomol International (Plymouth Meeting, PA). Cathepsins B and V substrate Cbz-FR-AMC was purchased from EMD Biosciences (San Diego, CA). Cathepsin S substrate Cbz-LR-AMC and legumain substrate Cbz-AAN-AMC were purchased from Bachem (Torrance, CA). Assay wells contained a mixture of inhibitor and substrate in buffer [see Supplemental Data for full assay details for each enzyme]. Aliquots of enzyme were added to each well to initiate the assay. Hydrolysis of the fluorescent substrate was monitored fluorometrically for 45 min. The kinact/Ki for inhibitors was determined under pseudo-first order conditions using the progress curve method (Bieth, 1995; Ekici Ozlem et al., 2006). Inhibition was measured in triplicate and the average and standard deviation is reported. Detailed procedures for each enzyme, including buffer conditions and enzyme, inhibitor and substrate concentrations are provided in the Supporting Information.

Huntingtin Neoepitope Antibody Production

Antibodies specific for the C-terminal ends of Htt caspase cleavage products ending at amino acid 513, 552 and 586 were prepared using the immunizing peptides KLH-DHTLQADSVD, KLH-DSDPAMDLND and KLH-COSDSSEIVLD. Peptide sequences were injected into rabbits, antibody was purified to the injected peptide and a bridging peptide was used to remove antibodies reacting to full-length Htt (Open Biosystems). Antibodies were affinity purified as previously described with minor modifications (Wellington and Ellerby et al, 2002, Gervais et al., 1999).

Htt Constructs

Htt expression constructs used in these studies included a normal (23Q) and expanded (148Q) full-length Htt construct.

Cell Culture

Superfect reagent (Qiagen) was used for transient transfections of human embryonic kidney (HEK) 293T cells with Htt constructs. Cells were collected 60 h post-transfection for analysis. 293T cells or striatal Hdh7Q/7Q and Hdh111Q/111Q cells were cultured in Dulbecco’s modified eagle medium (DMEM; Cellgro) with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 ug/mL streptomycin.

Western Analysis

293T cell pellets were lysed in M-PER (Mammalian Protein Extraction Reagent, Pierce) with protease inhibitors (Mini Complete, Roche), lysates were sonicated and then spun to remove debris (16,000Xg, 20 min). Protein concentration was determined by BCA Protein Assay kit (Pierce). Htt-expressing lysates were in some cases treated exogenously with caspases-2, -3 or -6 (ENZO, 100 U, 2 h, 37 °C) with and without inhibitors 35, 36 and 37. Samples were resolved by SDS-PAGE on NuPAGE 4-12% BisTris gel (Invitrogen) in MES running buffer (Invitrogen) for 55 min at 200V. Proteins were transferred to Optitran BA-S nitrocellulose (Schleicher & Schuell) for 14 h at 20 V at 4 °C. Membranes were blocked in 5% milk in Tris-buffered saline Tween-20 and probed overnight with polyclonal neoHtt513, neoHtt552 and neoHtt586 (Open Biosystems; 1:1000 or 1:100 respectively, produced in collaboration with CHDI/Open Biosystems) or monoclonal α-Huntingtin 2166 (Chemicon, 1:1000). Secondary anti-rabbit or anti-mouse antibodies (1:3000, Amersham Biosciences/GE Healthcare) and enhanced chemiluminensce (ECL) reagent (Pierce) were used for detection. We utilized anti-GAPDH as the loading control (Fitzgerald #10R-G109a, 1:1000).

kcat/Km of Htt with Caspases

293T cells over-expressing myc-tagged 23Q human full-length Htt were lysed in M-PER (Mammalian Protein Extraction Reagent, Pierce), sonicated and spun to remove debris (16,000Xg, 20 min). Protein was determined using a BCA assay kit (Pierce). Purified human poly-(ADP-ribose) polymerase (PARP) was obtained from Trevigen (Gaithersburg, MD). Htt lysate (15 ug) was incubated with caspase-3 (4 nM; ENZO) or caspase-6 (48 nM, ENZO) for 60 min at 37°C in caspase assay buffer (20 mM PIPES, pH 7.2, 100 mM NaCl, 1% CHAPS, 10% sucrose). Purified PARP (35 ng) was incubated with caspase-3 (7.2-144 nM) under identical conditions. NuPAGE LDS sample buffer (Invitrogen) and 50 mM DTT were added and samples were boiled and separated using SDS-PAGE. Following transfer to nitrocellulose membranes, Htt protein blots were incubated in monoclonal Htt 2166 (1:500; Millipore), PARP protein blots were incubated in polyclonal PARP 253 (1:300; ENZO) and densitometry of bands was performed. All reactions were carried out using subsaturating levels of substrate, where the cleavage is assumed to be a first order process. Values of kcat/Km were calculated from the relationship St/So = e −kobs*t where St=concentration of substrate remaining at time t, So=initial substrate concentration and kobs = kcat*[enzyme]/Km (Gervais et al., 1998).

Caspase Activity in Striatal Hdh7Q/7Q and Hdh111Q/111Q cells

Striatal Hdh7Q/7Q and Hdh111Q/111Q cells were maintained at 33 °C in a humidified atmosphere of 95% air and 5% CO2, in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were fed with fresh medium every 2–3 d. Striatal cells were plated at 5000 cells/well in collagen coated 96-well plates and were grown for 48 h. Cells were maintained in serum DMEM for 24 h with or without caspase inhibitors and an additional 24 h without serum with same inhibitor treatment. Caspase 3/7 activity was measured using kit as per manufacturer’s direction (Mountain View, CA) and normalized by protein concentration. Briefly, cells were lysed in working cell lysis buffer (50 μL, 1:1 lysis buffer: serum free DMEM) by shaking at 700 rpm for 5 min and a 20 μl aliquot was measured for protein concentration. Afterwards 70 uL of working lysis buffer with DTT (15 mM), and caspase substrate (1x) was added to remaining lysed cells, shaken briefly and then read continuously at 37 °C for 90 minutes with excitation at 488nm and emission at 530nm.

Immunocytochemistry

Striatal Hdh7Q/7Q and Hdh111Q/111Q cells cultured on cover slips with or without serum withdrawal were fixed with 4% paraformaldehyde for 20 min at room temperature. The fixed cells were then permeabilized with 0.1% TritonX-100 for 10 min and blocked with 5% donkey serum for 45 min. The incubation with the primary antibody (rabbit polyclonal neoHtt513, 1:100) at 4°C overnight was followed by secondary antibody (donkey anti rabbit Alexa546, 1:1000, Invitrogen) incubation at room temperature for 1.5 h. Finally cells were mounted to a slide with droplets of Prolong Gold with DAPI (Invitrogen) on it before microscopy.

Primary cortico-striatal co-culture

Striatum and cortex from E18 rat embryos were micro-dissected, enzymatically treated with papain and dissociated. Five million cells of each type were electroporated separately (Amaxa Biosystems) with DNA constructs expressing either YFP in striatal neurons or mCherry in cortical neurons together with a plasmid expressing mutant polyQ expanded Htt exon1 (HttN90Q73), then plated together on a layer of astrocytes which was prepared three days prior. Our method for assessing culture health in experiments is by determining the numbers and morphology of GFP expressing cells. In developing the assay we found with vital stains that about 50% of cells die immediately after the electroporation. The neurons that survive the electroporation appear to be healthy, extending neurites and forming functional synapses as assessed by synaptic marker staining and electrophysiology. Neurons and astrocytes were grown in Neurobasal media (Invitrogen) supplemented with 5% fetal calf serum (Sigma), 2 mM glutamine (glutamax, Invitrogen), 10 mM potassium chloride and 5 ug/ml gentamycin. Astrocytes were isolated from E18 embryos and expanded for three passages before plating into 96-well plates. Co-cultures were incubated in 95% O2/5% CO2 at 37°C for six days before analysis. Compounds were dissolved in DMSO and dosed as a single dose shortly after neuron plating. The control condition was HttN90Q73 with DMSO alone. For quantification of neurons using the Cellomics ArrayScan VTI, fluorescent images were acquired at 10x magnification from 9 fields per well using YFP and dsRED filter sets and analyzed using the Target Activation algorithm. The algorithm was optimized for object size, object shape and fluorescence intensity to identify specific neuron cell bodies. Healthy neurons are easily identified by the size of the cell body and neuritic morphology. Graphical analysis and statistical computations were performed with GraphPad Prism.

CONCLUSION

The substrate activity screening method was applied to caspases-3 and -6 leading to the identification of 1,2,3-triazole-based nonpeptidic substrates with high cleavage efficiencies. Subsequent replacement of the aminocoumarin reporter group with the 2,3,5,6-tetrafluorophenoxymethyl ketone pharmacophore in the most efficient substrates led to three novel nonpeptidic irreversible inhibitors showing strong potency against caspases and weak to no inhibition of other cysteine proteases previously reported to have cross reactivity with published caspase inhibitors. All three inhibitors, 35, 36 and 37, blocked proteolysis of Htt at the caspase-3 and -6 cleavage sites, amino acids 513 and 586, respectively. Moreover, in a Huntington’s disease model, all three inhibitors rescued cell death in striatal and cortical neurons at nanomolar concentrations. These pan-caspase inhibitors continue to be applied as tool compounds in studies of Htt proteolysis and toxicity. Future work will be directed at evaluating these compounds in mouse models of HD to determine their effect on the pathogenesis of Huntington’s disease.

Supplementary Material

01

ACKNOWLEDGEMENTS

This research was supported by grants from NIH: GM54051 (JAE), NS40251 (LME), CHDI (LME, DCL), and RGP0024/2006 from the Human Frontier Science Program (GSS). Neo Htt epitope were produced in collaboration with Jacqueline Duke and Matt Baker at OpenBiosystems. We thank both of them for their expert advice and help.

Footnotes

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SUPPLEMENTAL DATA AND FIGURES

Supplemental Data include the synthetic procedures and characterization data for all AMCA substrates and inhibitors described in this report as well as the full assay protocols. Two figures are included that show (1) the removal of the inhibitors 22 hours prior to assaying for caspase-3 activity from striatal Hdh111Q/111Q still results in reduction in activity; (2) cell death of striatal cells 48 hours after serum withdrawal.

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