Engaging a Non-catalytic Cysteine Residue Drives Potent and Selective Inhibition of Caspase-6

Abstract

Caspases are a family of cysteine-dependent proteases with important cellular functions in inflammation and apoptosis, while also implicated in human diseases. Classical chemical tools to study caspase functions lack selectivity for specific caspase family members due to highly conserved active sites and catalytic machinery. To overcome this limitation, we targeted a non-catalytic cysteine residue (C264) unique to caspase-6 (C6), an enigmatic and understudied caspase isoform. Starting from disulfide ligands identified in a cysteine trapping screen, we used a structure-informed covalent ligand design to produce potent, irreversible inhibitors (3a) and chemoproteomic probes (13-t) of C6 that exhibit unprecedented selectivity over other caspase family members and high proteome selectivity. This approach and the new tools described will enable rigorous interrogation of the role of caspase-6 in developmental biology and in inflammatory and neurodegenerative diseases.

Figure 1

Examples of substrate-like tool compounds (left) and drug candidates (right) that engage the catalytic cysteine of caspases.

Figure 2

Summary of a targeted approach to the discovery of potent and selective caspase-6 inhibitors and chemical probes.

Scheme 1. Synthesis of Enantiomers 2a and 2b

Figure 3

Structures of 2a, 3a, and Ac-VEID-CHO bound to caspase-6. (A) Superposition of crystal structures of 2a (cyan) and 3a (green) bound in the active site of caspase-6. The catalytic cysteine C163 is on the large subunit (at right), while inhibitor-modified C264 is on the small subunit (at left). (B) Superposition of 3ou for your a (green, light green protein) and substrate analogue Ac-VEID-CHO (violet, light violet protein). The side chain of R220 moves 4.2 Å to accommodate the trifluoromethoxyphenyl substituent on 3a. PDBIDs: 8EG6 (2a/C6); 8EG5 (3a/C6); 3OD5 (VEID/C6).

Scheme 2. Synthesis of (±)-3-t and (±)-3-c, and the Corresponding Non-Racemic Stereoisomers 3a–d

Compounds 3a–d were prepared from the individual stereoisomers 9a–d, which were separated by silica gel chromatography, followed by chiral-SFC. The acrylamide analogue (±)-10-t was synthesized from (±)-8-t, as shown at lower right

Figure 4

Biochemical activities of 3a. (A) 3a inhibits wild-type (WT) caspase-6 (C6) but not C264A C6, caspase-2 (C2), or caspase-3 (C3). Error bars represent the standard deviation of thre replicate measurements. (B) Inhibition at 10 μM 3a of ten human caspases (C1–C10). Activity measured by the cleavage of aminofluorocoumarin-labeled peptides (see the Supporting Information for sequences). C = caspase.

Figure 5

Cell-based activity of 2a and 3a. Inhibition of lamin A cleavage in SK-N-AS cells after staurosporine induction of caspases. Cells were treated with the indicated compound for 1 h before the addition of 10 μM staurosporine for 4 h. Error bars indicate the standard deviation from n = 4 replicate samples.

Figure 6

Targets of (±)-13-t (1 μM) in HEK293 cells transfected with caspase-6. (A) In gel fluorescence showing that a protein the size of the caspase-6 small subunit is a primary target of 13-t, and this target is competed off by the presence of 3a. (B) Volcano plot showing 336 total proteins identified, 141 of which were statistically significant (p < 0.05) targets of 13-t that were competed for binding with 3a. Red circles, competition > log 2 and p value <0.05; blue diamond, a small subunit of caspase-6. Keratin proteins are not highlighted because these proteins are likely present due to contamination. Data shown are representative of two independent experiments.

Scheme 3. Synthesis of (±)-13-t, a Prototype Activity-Based Probe of Caspase-6, for Cellular and Chemoproteomic Studies