Figure 1.
Inhibitor potency and selectivity against caspase family members.
(A) Schematic of divalent tetrapeptide substrate proteolysis to release R110 fluorophore. Removal of both tetrapeptides by caspases is required for signal generation at 535 nm. Concentration-response analysis of compound 3 (B) and VEID-CHO (C) against caspase-6 (green), caspase-3 (black or red) or caspase-7 (blue). The particular divalent R110 peptide substrate used with each enzyme is indicated in the figure key and assay specifics can be found in Experimental Procedures. Potency values for (B–C) can be found in Table S2. Concentration response curves were generated in duplicate and represent 1 of at least 2 experiments with similar results. Each curve is normalized to zero and 100% based on no enzyme or DMSO, respectively. Data represent mean ± standard error of the mean.
Figure 2.
Structure of the N-furoyl-phenylalanine screening hit (2) and the optimized analog 3.
Potency values represent the inhibition of caspase-6 cleavage of (VEID)2R110 substrate.
Figure 3.
Kinetic caspase-6 enzymatic studies with compound 3 show uncompetitive mechanism of inhibition with (VEID)2R110 substrate.
(A) The initial enzyme velocity of caspase-6 was plotted against the indicated concentration of (VEID)2R110 substrate in the presence of 0 nM (DMSO-black), 3 nM (red), 10 nM (orange), 30 nM (green) or 100 nM (blue) compound 3. Double reciprocal plot of this data can be found in Figure S1 and Michaelis-Menten constants can be found in Table S3. (B) Concentration-response analysis of compound 3 when tested in the presence of 0.5 µM (red), 5 µM (black) or 20 µM (blue) (VEID)2R110 substrate. Michaelis-Menten kinetic experiments were performed with single points while concentration-response curves were performed in duplicate. Each data set represents 1 of at least 3 experiments with similar results.
Figure 4.
Compound 3 inhibition of caspase-6 is dependent on the substrate’s amino acid sequence and the P1’ character of the substrate.
(A) Concentration-response analysis of compound 3 against caspase-6 cleavage of divalent R110-containing substrates with VEID (black), DEVD (red), IETD (blue) or WEHD (green) amino acid tetrapeptides. Each assay was performed using substrate concentrations within 3-fold of the Kmapparent. (B) Concentration-response analysis of compound 3 against caspase-6 cleavage of monovalent VEID-based substrates with R110 (black) or AMC (blue) fluorophores conjugated to the C-terminal aspartate residue. (C) The indicated concentration of compound 3 or VEID-CHO was incubated with caspase-6 and GST-Lamin A prior to detection of cleaved Lamin A by western blotting. Only VEID-CHO was capable of inhibiting caspase-6 cleavage of recombinant Lamin A. Concentration response curves were generated in duplicate and represent 1 of at least 3 experiments with similar results. Each curve is normalized to zero and 100% based on no enzyme or DMSO, respectively. Western blot data represents 1 of at least 2 experiments.
Figure 5.
Crystal structure of caspase-6 ternary complex with 3 and covalently bound VEID inhibitor reveals the uncompetitive mechanism of this series of compounds.
(A) Crystal structure of the ternary complex of caspase-6 with zVEID and compound 3 (PDB-ID 4HVA). The caspase-6 dimer is represented as cartoon with the A and B chains colored light blue and grey, respectively, and the L4 loop colored purple. The zVEID inhibitors are represented as sticks and are colored pink. Each inhibitor is covalently bound to the catalytic cysteine (Cys163) in both chain A and B. Two molecules of 3 are shown as ball and stick representation and colored orange. (B) Close up of the active site of chain A colored according to (A) with hydrogen bonds shown as black dashes. (C) Structural comparison of caspase-6 ternary complex with 3 bound (light blue) and caspase-6 binary complex with bound VEID-CHO (wheat) (PDB-ID 3OD5) illustrating the difference in the conformation of the tip of the L4 loop in the two crystal structures (residues 261–271).
Figure 6.
SPR detection of 3 binding to multiple caspase-6 surfaces confirms uncompetitive binding mode.
(A) Catalytically inactive caspase-6 (green), apo-caspase-6 (blue) and caspase-6 saturated with VEID-FMK inhibitor (purple) were captured to chip surfaces and exposed to VEID-AMC, (VEID)2R110 and/or 3 to qualitatively monitor binding. Cooperative binding of 3 and (VEID)2R110 to C163 caspase-6 illustrate formation of the Michaelis-Menten complex. (B) Sensograms representing injections of escalating concentrations of 3 over VEID-FMK inhibitor-blocked caspase-6 surface (black). The inset represents similar injections of 3 over an unblocked apo-caspase-6 surface (blue). (C) Concentration-response analysis of data from (B) when compound 3 was injected over VEID-blocked caspase-6 surface (black) and apo-caspase-6 (blue) surfaces.
Figure 7.
Docking models of caspase-6/VEID-R110/3 ternary complex explains fluorophore-dependent potency of this series of compounds.
(A) Docking model of the Michaelis-Menten complex formed between caspase-6 (light blue), VEID-R110 (green sticks) and 3 (wheat sticks). (B) Docking model of the tetrahedral intermediate between caspase-6, VEID-R110 (green sticks) and 3 (wheat sticks) with substrate covalently bound to Cys163. (C) Depiction of monovalent VEID substrates with R110 or AMC fluorophores.