Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Aug 8;56(15):6054-68.
doi: 10.1021/jm4006719. Epub 2013 Jul 22.

Design, synthesis, and optimization of novel epoxide incorporating peptidomimetics as selective calpain inhibitors

Isaac T Schiefer  1 , Subhasish Tapadar, Vladislav Litosh, Marton Siklos, Rob Scism, Gihani T Wijewickrama, Esala P Chandrasena, Vaishali Sinha, Ehsan Tavassoli, Michael Brunsteiner, Mauro Fa', Ottavio Arancio, Pavel Petukhov, Gregory R J Thatcher
Affiliations

Design, synthesis, and optimization of novel epoxide incorporating peptidomimetics as selective calpain inhibitors

Isaac T Schiefer et al. J Med Chem. .

Abstract

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer's disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Overview of nomenclature, mechanism, and design motif of epoxysuccinate peptidomimetic cysteine protease inhibitors
Protease substrates are designated according to their amino acid residues extending from the scissile bond. “Unprimed” and “primed” substrate residues are designated P1, P2, etc. and P1’, P2’, etc., respectively. The design rationale is illustrated using E-64 as a lead.
Figure 2
Figure 2. Reaction between an epoxysuccinate containing peptidomimetic and GSH
HPLC-UV-Vis (λ = 280 nm) chromatogram from incubation of 31 (100 μM) with excess GSH (5 mM) in PBS (50mM, pH 7.4) at 37 °C for 24 hr. Product identity was confirmed using LC-MS/MS and comparison of retention time of authentic sample in the absence of GSH.
Figure 3
Figure 3. Examination of modified Cal1cat active site using LC-MS/MS
Confirmation of covalent modification of Cal1 active site Cys by epoxysuccinate inhibitors. (A) After in gel digest and capping of free cysteines with iodoacetamide (IAA), peptide fragments were analyzed by LC-MS/MS. (B) Total ion chromatograms (TIC) for the incubation of recombinant Cal1cat (5 μM) in the absence or presence of inhibitors (10 μM). A non-active site and IAA-capped peptide fragment (Rt = 41.3 min) was used as an internal standard for EIC quantitation. (C) Active site Cys modification of Cal1cat was concentration dependent. (D) Co-incubation of Cal1cat (1 μM) with an inhibitor mixture (5 μM) showed competitive modification of the active site by 22a and E-64.
Figure 4
Figure 4. Kinetic analysis of papain and Cal1 enzyme inhibition by selected inhibitors
Plots of secondary and primary kinetic data for inhibition of papain (A) and Cal1 (B) used to derive detailed kinetic parameters reported in Table 2: E-64 (diamonds – solid green); 22a (closed circles – dashed red); 31 (squares – solid orange); 36 (open circles – dashed blue). The observed rate constants (kobs) shown in the double reciprocal plots were measured from non-linear fitting of “progress curves” for product formation from substrate. Exemplar progress curves are shown for Cal1 inhibition by E-64.
Figure 5
Figure 5. Representative proposed binding modes of selected inhibitors
Selected inhibitors (compounds 22a, 28a, and 31) were docked within the WR-18 x-ray structure of Cal1cat [PDB: 2NQG]. (A) Structural overlay of putative docking poses; (B) Magnified S2 pocket illustrating proposed H-bond formed between the conserved H2O molecule and the P2 moiety. Docking was carried out using GOLD docking platform. Docking poses were rendered using UCSF Chimera molecular modeling software.
Figure 6
Figure 6. Model of active site describing the bond distances and angles associated with active site modification by epoxysuccinate containing peptidomimetics
Crystal structures of epoxide inhibitors bound at the active site of Cal1 show a relaxed, ring-opened structure, whereas a transition state for epoxide ring-opening requires the Cys nucleophile and His general acid to be aligned for SN2 substitution. Triazole-based inhibitors were designed based upon docking scores biased towards d2 = ~2Å. For comparison, in the crystal structures: 2NQG d1= 4.606 and d2= 1.810; and 1TLO (shown with E-64, His, and Cys) d1= 4.775, d2= 1.833.
Scheme 1<sup>a</sup>
Scheme 1a
Scheme 2
Scheme 2
Scheme 3<sup>a</sup>
Scheme 3a
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Goll DE, Thompson VF, Li H, Wei WEI, Cong J. The Calpain System. Phys. Rev. 2003;83:731–801. - PubMed
    1. Perlmutter LS, Siman R, Gall C, Seubert P, Baudry M, Lynch G. The ultrastructural localization of calcium-activated protease "calpain" in rat brain. Synapse. 1988;2:79–88. - PubMed
    1. Veeranna, Kaji T, Boland B, Odrljin T, Mohan P, Basavarajappa BS, Peterhoff C, Cataldo A, Rudnicki A, Amin N, Li BS, Pant HC, Hungund BL, Arancio O, Nixon RA. Calpain mediates calcium-induced activation of the erk1,2 MAPK pathway and cytoskeletal phosphorylation in neurons: relevance to Alzheimer's disease. Am. J. Pathol. 2004;165:795–805. - PMC - PubMed
    1. Shea TB. Restriction of microM-calcium-requiring calpain activation to the plasma membrane in human neuroblastoma cells: evidence for regionalized influence of a calpain activator protein. J. Neurosci. Res. 1997;48:543–550. - PubMed
    1. Di Rosa G, Odrijin T, Nixon RA, Arancio O. Calpain inhibitors: a treatment for Alzheimer's disease. J. Mol. Neurosci. 2002;19:135–141. - PubMed

Publication types