Conformationally restricted calpain inhibitors

Abstract

The cysteine protease calpain-I is linked to several diseases and is therefore a valuable target for inhibition. Selective inhibition of calpain-I has proved difficult as most compounds target the active site and inhibit a broad spectrum of cysteine proteases as well as other calpain isoforms. Selective inhibitors might not only be potential drugs but should act as tools to explore the physiological and pathophysiological roles of calpain-I. α-Mercaptoacrylic acid based calpain inhibitors are potent, cell permeable and selective inhibitors of calpain-I and calpain-II. These inhibitors target the calcium binding domain PEF(S) of calpain-I and -II. Here X-ray diffraction analysis of co-crystals of PEF(S) revealed that the disulfide form of an α-mercaptoacrylic acid bound within a hydrophobic groove that is also targeted by a calpastatin inhibitory region and made a greater number of favourable interactions with the protein than the reduced sulfhydryl form. Measurement of the inhibitory potency of the α-mercaptoacrylic acids and X-ray crystallography revealed that the IC₅₀ values decreased significantly on oxidation as a consequence of the stereo-electronic properties of disulfide bonds that restrict rotation around the S-S bond. Consequently, thioether analogues inhibited calpain-I with potencies similar to those of the free sulfhydryl forms of α-mercaptoacrylic acids.

Fig. 1. Structures of PD150606 (1), PD151746 (2), (Z)-3-(6-bromoindol-2-yl)-2-mercaptoacrylic acid (3) and (2Z,2′Z)-2,2′-disulfanediylbis(3-(6-bromoindol-3-yl)acrylic acid) (4).^,

Fig. 2. (A) Surface representation of chain A of PEF(S) bound to 3 in the hydrophobic pocket that binds Leu 660 (PDB ; 4WQ2). (B) Inhibitory region C of the CAST domain IV bound to PEF(S); Ile 653, Leu 656 and Leu 660 are highlighted (PDB ; 3BOW). (C) 4 bound to chain A of PEF(S); a single binding mode of 4 is represented (PDB ; 4WQ3).

Fig. 3. Comparison of the two conformations 4 (cyan) bound to chain A (yellow) of the PEF(S) homodimer, (A) and (B) respectively, and chain B (green) of the homodimer, (C) and (D) respectively. Residues that form halogen bonds, hydrogen bonds and electrostatic interactions are highlighted and important bonds are indicated by dashed lines, distances are shown in Å (PDB ; 4WQ2 and ; 4WQ3).

Fig. 4. IC₅₀ values (μM) for the α-mercaptoacrylic acid compounds tested in FRET based inhibition assays in the presence (sulfhydryl) and absence (disulfide) of 10 mM DTT.

Fig. 5. Representation of 4 with a dihedral angle of –90.5° bound to chain A of PEF(S).

Fig. 6. (A) Synthetic route to the thioether calpain inhibitors. (i) DABCO, MeOH, 25 °C, 72 h, (ii) NaOH, MeOH, 25 °C, 16 h, (iii) HBr, H₂SO₄, 25 °C, 16 h, (iv) NEt₃, MeCN, 25 °C, 16 h. (B) Symmetric and asymmetric compounds 40–43. (Z)-3-(4-Bromophenyl)-2-((((Z)-2-(4-bromophenyl)-1-carboxyvinyl)thio)methyl)acrylic acid (40), (Z)-3-(3-chlorophenyl)-2-((((Z)-2-(4-chlorophenyl)-1-carboxyvinyl)thio)methyl)acrylic acid (41), (Z)-3-(5-bromoindol-3-yl)-2-(((Z)-2-carboxy-3-(3-chlorophenyl)allyl)thio)acrylic acid (42) and (Z)-3-(6-bromoindol-3-yl)-2-(((Z)-3-(4-bromophenyl)-2-carboxyallyl)thio)acrylic acid (43).