Substrates and inhibitors for cysteine protease ICH-1

Details Substrates and inhibitors for cysteine protease ICH-1 Substrates and inhibitors for cysteine protease ICH-1

1996-06-18

2001-09-11

Celsa, Bennett (Department: 1627)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S329000, C530S330000, C435S219000

Reexamination Certificate

active

06288037

ABSTRACT:

BACKGROUND OF THE INVENTION
Ich-1 is a human cysteine protease that is a member of a family of cysteine proteases including interleukin-1&bgr; converting enzyme (ICE), a protein essential for the maturational processing of interleukin-1&bgr; (see e.g., Black, R. A., et al. (1988)
J. Biol. Chem.
263:9437-9442; Kostura, M. J., et al. (1989)
Proc. Natl. Acad. Sci. USA
86:5227-5231; Thornberry, N. A., et al. (1992)
Nature
356:768-774; Ceretti, D. P., et al. (1992)
Science
256:97-100) and the
Caenorhabditis elegans
protein Ced-3, a protein required for apoptosis during the development of
C. elegans
(see e.g., Yuan, J., et al. (1993)
Cell
75:641-652). Other members of this family include CPP32/Yama (Fernandes-Alnemri, T., et al. (1994)
J. Biol. Chem.
269:30761-30764; Tewari, M., et al. (1995)
Cell
81:801-809), SCA (Wang, X., et al. (1995)
J. Biol Chem.
270:18044-18050), Ich-2/ICE
rel
II/Tx (Kamens, J., et al. (1995)
J. Biol Chem.
270:15250-15256; Munday, N. A., et al. (1995)
J. Biol. Chem.
270:15870-15876; Faucheu, C., et al. (1995)
EMBO J.
14:1914-1922), ICE
rel
III/Ty (Munday, N. A., et al. (1995)
J. Biol. Chem.
270:15870-15876; Faucheau, C., et al. (1996)
Eur. J. Biochem.
236:297-313), Mch2 (Fernandes-Alnemri, T., et al. (1995)
Cancer Res.
55:2737-2742) and Mch3/ICE-LAP3/CMH-1 (Fernandes-Alnemri, T., et al. (1995)
Cancer Res.
55:6045-6052; Duan, H., et al. (1996)
J. Biol. Chem.
271:1621-1625; Lippke, J. A., et al. (1996)
J. Biol. Chem.
271:1825-1828). The cDNA for Ich-1 has been isolated and the predicted amino acid sequence of Ich-1 displays approximately 30% amino acid identity to ICE (Wang, L. et al. (1994)
Cell
78:739-750). The mouse homolog of Ich-1, termed Nedd2, also has been identified and similarly exhibits approximately 30% amino acid identity to ICE (Kumar, S., et al. (1994)
Genes Dev.
8:1613-1626).
Members of the ICE-related family of proteins are involved in cytokine maturation leading to inflammation and in apoptosis. In particular, Ich-1 has been implicated in apoptosis and neuronal development. For example, Ich-1 (and its murine homolog Nedd2) induces apoptosis when overexpressed in mammalian cells (Kumar (1994) supra; Wang (1994) supra). Additionally, the Nedd2 gene was identified based upon its developmentally down-regulated expression in the adult brain (Kumar, S. et al. (1992)
Biochem. Biophys. Res. Commun.
185:1155-1161). During murine embryonic development, the Nedd2 gene is ]highly expressed in several tissues undergoing a high rate of apoptosis, including the central nervous system (Kumar (1994) supra).
In vitro studies have demonstrated that ICE cleaves prointerleukin-1&bgr; at Asp
116
-Ala
117
to release the fully active 17 kDa form (Black (1988) supra; Kostura (1989) supra). ICE also cleaves prointerleukin-1&bgr; at Asp
27
-Ala
28
to release a 28 kDa form. Cleavage at these sites is dependent upon the presence of aspartic acid in the P1 position (the position immediately amino-terminal to the cleavage site) (Kostura (1989) supra, Howard, A., et al. (1991)
J. Immunol.
147:2964-2969; Griffin, P. R., et al. (1991)
Int. J. Mass. Spectrom. Ion. Phys.
111:131-149). However, an aspartic acid in the P1 position is not sufficient for ICE specificity. For example, several other proteins containing Asp-X bonds, including prointerleukin-1&agr;, are not cleaved by ICE (Howard (1991) supra). An optimal minimal substrate for ICE has been identified as containing the tetrapeptide Tyr-Val-Ala-Asp (SEQ ID NO: 44) (Thornberry (1992) supra).
SUMMARY OF THE INVENTION
This invention provides the amino acid sequence of an optimal minimal substrate for Ich-1, and Ich-1 inhibitors and substrates designed based upon the preferred amino acid sequence specificity of Ich-1. The invention provides compounds of Formula I,
X-Asp-A
3
—A
2
—Z  I
wherein:
X is R
1
—(W)
n
—A
5
, or R
6
, wherein
A
5
is a hydrophobic &agr;-amino acid capable of interacting with the P5 site of Ich-1;
W is any &agr;-amino acid;
n is an integer from 0-15;
R
1
is hydrogen or an N-terminal acyl group selected from the group consisting of amides, carbamates, and ureas;
R
6
is a hydrophobic N-terminal acyl group, selected from the group consisting of amides, carbamates and ureas, capable of interacting with the P5 site of Ich-1;
Asp is an aspartic acid residue;
A
3
is an &agr;-amino acid capable of interacting with the P3 site of Ich-1;
A
2
is an &agr;-amino acid capable of interacting with the P2 site of Ich-1; and
Z is A
1
or Asp-Y, wherein:
A
1
is a residue that imparts Ich-1 inhibitory activity and is an aspartic acid or a glutamic acid residue that is modified at the C-terminus to contain an electrophilic center that is susceptible to nucleophilic attack or nucleophilic displacement by the sulfhydryl group of the active site cysteine residue of Ich-1; and
Asp-Y is a pair of residues that impart Ich-1 substrate activity wherein Asp is an aspartic acid residue and Y is selected from the group consisting of a chromogenic leaving group, a fluorogenic leaving group, —(Q)
m
—[CO
2
H], and —(Q)
m
−
[CONHR
2
], wherein Q is any &agr;-amino acid or a radiolabeled derivative thereof, m is an integer from 1 to 15, and R
2
is selected from the group consisting of H, alkyl, alkenyl, cycloalkyl, (cycloalkyl)alkyl, aryl, arylalkyl, heterocycle, heteroaryl, heterocyclealkyl, heteroarylalkyl, and radiolabeled derivatives thereof.
The compounds of the invention can be incorporated into pharmaceutical compositions. Typically, the composition includes a compound of the invention and a pharmaceutically acceptable carrier.
The inhibitor compounds of the invention can be used, for example, to inhibit the proteolytic activity of Ich-1 by contacting Ich-1 with the inhibitor compound. The substrate compounds of the invention can be used, for example, to detect the presence of Ich-1 in a sample by contacting the sample with the substrate compound and detecting proteolytic cleavage of the substrate as an indicator of the presence of Ich-1 in the sample. In yet other embodiments, an inhibitor compound of the invention can be used to isolate Ich-1. For example, a biotinylated form of the inhibitor compound can be used in affinity purification of Ich-1.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides inhibitor and substrate compounds for Ich-1, and methods of using these compounds, wherein the compounds are designed based upon an optimal minimal amino acid sequence for Ich-1 recognition. The Ich-1 sequence specificity is different from that of ICE, another cysteine protease to which Ich-1 is related. An initial peptide substrate for Ich-1 was identified using a series of peptides corresponding to known or suspected cleavage sites of ICE or ICE-related proteins, as described further in Example 1. This initial peptide was then modified by amino- and/or carboxy-terminal deletions and by amino acid substitutions, to define an optimal minimal substrate recognition site for Ich-1, as described further in Examples 2 and 3. A peptide containing the amino acid sequence Val-Asp-Val-Ala-Asp (VDVAD; SEQ ID NO: 45), corresponding to the P5, P4, P3, P2 and P1 positions, was thus defined as an optimal recognition sequence for Ich-1.
Amino acid substitutions within the optimal VDVAD-containing substrate demonstrated that maintenance of the P4 Asp and the P1 Asp was the most critical for maintenance of efficient cleavage of the substrate by Ich-1, whereas the P5, P3 and P2 positions were more amenable to substitutions (see Example 3). Accordingly, a consensus motif for Ich-1 recognition motif can be defined as A
5
-Asp-A
3
-A
2
-Asp, wherein A
5
, A
3
and A
2
are amino acids that are selected such that the site is recognized by Ich-1. Preferably, A
5
and A
3
are Val, and A
2
is Ala.
Definition of the amino acid sequence of the Ich-1 recognition site allows for the construction of substrates and inhibitors of Ich-1 designed based on this site. For example, peptide aldehyde inhibitors, such as acetyl-VDVAD-[CHO], can be prepared a

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