Reducing the Peptidyl Features of Caspase-3 Inhibitors:  A Structural Analysis

Abstract

Caspases are cysteine proteases that specifically cleave Asp-Xxx bonds. They are key agents in inflammation and apoptosis and are attractive targets for therapy against inflammation, neurodegeneration, ischemia, and cancer. Many caspase structures are known, but most involve either peptide or protein inhibitors, unattractive candidates for drug development. We present seven crystal structures of inhibited caspase-3 that illustrate several approaches to reducing the peptidyl characteristics of the inhibitors while maintaining their potency and selectivity. The inhibitors reduce the peptidyl nature of inhibitors while preserving binding potency by (1). exploiting a hydrophobic binding site C-terminal to the cleavage site, (2). replacing the negatively charged aspartyl residue at P4 with neutral groups, and (3). using a peptidomimetic 5,6,7-tricyclic system or a pyrazinone at P2-P3. In addition, we have found that two nicotinic acid aldehydes induce a significant conformational change in the S2 and S3 subsites of caspase-3, revealing an unexpected binding mode. These results advance the search for caspase-directed drugs by revealing how unacceptable molecular features can be removed without loss of potency.

Mitochondria as therapeutic targets for cancer chemotherapy

“…Smac/DIABLO), in association with peptidomimetics (for instance, ADD70), able to inhibit specific protein-to-protein interactions. Published crystallographic data allowed the use of the following structures for the construction of this figure: AIF, residues 128-608D540-559 ; Rattus norvegicus HSC70 substratebinding domain, residues 383-540 (Morshauser et al, 1999); caspase-3 heterodimeric catalytic domain with a nicotinic acid aldehyde inhibitor, residues 150-295/320-401 (Becker et al, 2004); two caspase-3 heterodimeric catalytic domains, residues 148-296D (157-160,223,248-253)/310-401 and 148-297D(157-160,223,248-253)/310-401, complexed with XIAP Bir2 domains, residues 127-237 and 135-236D(170-178) (Riedl et al, 2001); Smac/DIABLO monomer, residues from 12 to 184 ; Smac/DIABLO homodimer, residues 1-157, complexed with two XIAP Bir3 domains, residues 256-344 and 256-357 . Owing to the lack of available crystallographic data, ADD70 is represented with the same structure of AIF, but only residues highlighted in orange are actually part of the peptide .…”

New Approaches and Therapeutics Targeting Apoptosis in Disease

“…The most potent tetrapeptide inhibitor identified inhibited caspase-3 in vitro efficiently (IC 50 ϭ 48 nM), whereas it was only weakly effective (IC 50 ϭ 10,000 nM) in cellular assays . Becker et al (2004) therefore reduced the peptide nature of caspase-3 inhibitors by employing a peptidomimetic 5,6,7-tricyclic system or a pyrazinone at P2-P3, by replacing the negatively charged P4 aspartyl with neutral groups and by exploiting a hydrophobic binding site C-terminal to the cleavage site. Merck recently also developed nicotinyl aspartyl ketones.…”

Ursolic Acid, a Natural Nutraceutical Agent, Targets Caspase3 and Alleviates Inflammation‐Associated Downstream Signal Transduction

“…We then analyzed the top scoring poses of UA in all cases, which are displayed as 3D maps ( Figure 3A and B) and 2D maps (Figure 3C and D) indicating the interaction of UA or Q‐VD‐Oph with CASP3. The carboxyl group of UA established hydrogen bonds with the active site amino acids Arg‐207 and Ser‐63, which is the same as Q‐VD‐Oph acting on the S1 subsite 17. Meanwhile, UA may interact with Cys‐163 and His‐121 via a previously proposed Cys‐His catalytic dyad hydrolysis mechanism, wherein the side‐chain of His‐121 approaches Cys‐163 to participate in the catalytic reaction 18.…”