Inhibition of human μ-calpain by conformationally constrained calpastatin peptides

Abstract

The 27-mer peptide CP1B-w1-27x derived from exon 1B of calpastatin stands out among the known inhibitors for m-and m-calpain due to its high potency and selectivity.By systematical truncation, a 20-mer peptide, CP1B-w4-23x, was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-w1-27x. Starting with this peptide, the turn-like region Glu 10 (i)-Leu 11 (iq1)-Gly 12 (iq2)-Lys 13 (iq3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position iq1 and iq2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and iq3, a series of conformationally constrained variants of CP1B-w4-23x were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(iq3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu 10 -Lys 13 upon binding to m-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site.

Calpains and neuronal damage in the ischemic brain: The swiss knife in synaptic injury

“…Compound Name* Reference calpastatin and it synthetic derivatives Calpastatin, CP1B, PCP1B, 7-mer-PCP1B, 11R-CS, CP1B-[4-23] (Betts et al, 2003;Fiorino et al, 2007;Gil-Parrado et al, 2003;Hanna et al, 2008;Pfizer et al, 2008;Wu et al, 2003) Peptidomimetic calpain inhibitors Inhibitors with modification at the P2 position Compounds 2 to 7 (Donkor and Korukonda, 2008;Donkor et al, 2000;Sanders and Donkor, 2006) Inhibitors with modification at the P3 position Compounds 8 to 10 (Jones et al, 2008) Inhibitors with modification in the P1 -P3 region…”

Calpains: Attractive Targets for the Development of Synthetic Inhibitors

“…The potency and selectivity of these peptides is unaffected by such conjugation or extension of CP1B ( Table 5). Systematic truncation of CP1B (= CP1B- ) led to the identification of a 20-mer peptide, CP1B- [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23], which is the core sequence required to maintain the affinity and selectivity profile of CP1B- [150]. Derivatives (3)-(5) ( Table 5), in which residues 7 (Glu) and 10 (Lys, Orn or Dab) of CP1B- [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] are conformationally constrained by macrolactamization, are equipotent and as selective as CP1B- [4][5][6][7]…”

“…In addition, an 18-mer peptide analogue of CP1B, which contains residues 6-23 (CP1B- [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]), has been shown to be the minimum sequence for calpain inhibition [151]. This compound displays a K i value of 93 nM for the inhibition of calpain 1 ( Table 5), with a decrease in selectivity by one or two orders of magnitude compared to CP1B- [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] and CP1B- , respectively [150]. Further truncation of CP1B results in much reduced selectivity and poor inhibition with micromolar affinity [150].…”

“…This compound displays a K i value of 93 nM for the inhibition of calpain 1 ( Table 5), with a decrease in selectivity by one or two orders of magnitude compared to CP1B- [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] and CP1B- , respectively [150]. Further truncation of CP1B results in much reduced selectivity and poor inhibition with micromolar affinity [150].…”

“…e Taken from ref. [150]. constrained macrocyclic inhibitors as discussed in the next section [172,[177][178][179].…”