Cell-permeable protein inhibitors of calpain

Details Cell-permeable protein inhibitors of calpain Cell-permeable protein inhibitors of calpain

1999-11-16

2001-09-25

Russel, Jeffrey E. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S013800, C530S324000

Reexamination Certificate

active

06294518

ABSTRACT:

BACKGROUND OF THE INVENTION
Calpains, a group of ubiquitous Ca
2+
-activated cytosolic proteases, are believed to play some role in cytoskeletal remodeling, cellular adhesion, shape change, and motility by cleaving membrane- and actin-associated cytoskeletal proteins (see, e.g., Beckerle et al.,
Cell
51:569-577, 1987; Yao et al.,
Am. J. Physiol.
265(pt. 1):C36-46, 1993; and Shuster et al.,
J. Cell Biol.
128:837-848, 1995). Calpains have also been implicated in the pathophysiology of cerebral and myocardial ischemia, platelet activation, NF-&kgr;B activation, Alzheimer's disease, muscular dystrophy, cataract progression, and rheumatoid arthritis.
Calpastatin is a physiological inhibitor of &mgr;-calpain and m-calpain, which are so named because they require micromolar or millimolar concentrations of Ca
2+
ions, respectively, to achieve half-maximal activity in vitro. Calpastatin has four internally repeated domains, each of which independently binds a calpain molecule in its active, Ca
2+
-bound conformation with high affinity (Mellgren et al.,
The Regulation of Calpains by Interaction with Calpastatins,
and Maki et al.,
Structure
-
Function Relationship of Calpastatins,
both in
Intracellular Calcium
-
Dependent Proteolysis,
Mellgren and Murachi, Eds, CRC Press, Boca Raton, Fla., 1990; and Yang et al.,
J. Biol. Chem.
269:18977-18984, 1994).
There is considerable interest in inhibitors of calpain (Wang et al.,
Trends in Pharm. Sci.
15:412-419, 1994; Mehdi,
Trends in Biochem. Sci.
16:150-153, 1991).
SUMMARY OF THE INVENTION
The invention features methods of inhibiting a calpain (e.g., &mgr;-calpain and/or m-calpain) in a cell (e.g., a eukaryotic cell). The method can be carried out, for example, by contacting the cell with an effective amount of a fusion protein having a first portion and a second portion, the first portion including a signal sequence capable of delivering the fusion protein into the cell and the second portion including a calpastatin peptide or a biologically active variant thereof. A calpastatin peptide (or biologically active variant thereof) will inhibit calpain activity in a standard assay for calpain activity, such as those described herein. Preferably, a calpastatin peptide (or biologically active variant thereof) will inhibit calpain activity by at least 40%, more preferably by at least 60%, and most preferably by at least 80% (e.g., 85%, 90%, 95% or more).
Calpastatin peptides include those described in Table 1. Biologically active variants of these peptides are likely to be those in which conserved amino acid residues (as shown in Table 1; e.g., see the underlined residues in SEQ ID NO:4) are either retained or replaced with an amino acid residue of the same type (i.e., peptides having one or more conservative amino acid substitutions). A conservative amino acid substitution occurs when one amino acid residue is replaced with another that has a similar side chain. Amino acid residues having similar side chains are known in the art and include families with basic side chains (e.g., lysine (Lys/K), arginine (Arg/R), histidine (His/H)), acidic side chains (e.g., aspartic acid (Asp/D), glutamic acid (Glu/E)), uncharged polar side chains (e.g., glycine (Gly/G), asparagine (Asn/N), glutamine (Gln/Q), serine (Ser/S), threonine (Thr/T), tyrosine (Tyr/Y), cysteine (Cys/C)), nonpolar side chains (e.g., alanine (Ala/A), valine (Val/V), leucine (Leu/L), isoleucine (Ile/I), proline (Pro/P), phenylalanine (Phe/F), methionine (Met/M), tryptophan (Trp/W)), beta-branched side chains (e.g., threonine (Thr/T), valine (Val/V), isoleucine (Ile/I)) and aromatic side chains (e.g., tyrosine (Tyr/Y), phenylalanine (Phe/F), tryptophan (Trp/W), histidine (His/H)).
Preferably, the calpastatin peptide includes the sequence Xaa-Xaa-Leu-Gly-Xaa-Xaa-Xaa-Xaa-Thr-Ile-Pro-Pro-Xaa-Tyr-Xaa-Xaa-Leu-Leu-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO:4), wherein
Xaa at position 1 is Glu, Asp, or Lys;
Xaa at position 2 is Lys, Glu, Ala, or Asn;
Xaa at position 5 is Glu, Lys, or Ile;
Xaa at position 6 is Arg, Lys, or Asp;
Xaa at position 7 is Asp, or Glu;
Xaa at position 8 is Asp, Val, Ser, Gly, or Glu;
Xaa at position 13 is Glu, Lys, or Asp;
Xaa at position 15 is Arg, Lys, or Gln;
Xaa at position 16 is Glu, His, Lys, or Leu; and
Xaa at position 19 is Glu, Asp, Asn, Ala, or Val;
Xaa at position 20 is Lys, Asp, Gln, Asn, Thr, or Met;
Xaa at position 21 is Lys, Asp, Glu, Gly, or Asn;
Xaa at position 22 is Thr, Glu, Gly, or Lys;
Xaa at position 23 is Gly, Ala, Glu, Gln, Lys or Asp; and
Xaa at position 24 is Val, Ile, Asp or Gly.
In various preferred embodiments, the amino-terminal end of the second portion is covalently bonded to the carboxy-terminal end of the first portion by a peptide bond; the second portion has the sequence of SEQ ID NO:4; the first portion has the sequence of SEQ ID NO:3; the fusion protein has the sequence of SEQ ID NO:1; the cell is a platelet; the cell is a sickle erythrocyte; the cell is an HIV-infected cell; the cell is an endothelial cell; and the cell is a proliferating cell (e.g., a tumor cell).
In other embodiments, the invention features methods of preventing platelet aggregation. These methods can be carried out, for example, by contacting a plurality of platelets with an effective amount of a fusion protein that includes a first portion and a second portion, the first portion including a signal sequence capable of delivering the fusion protein into the cell and the second portion including a calpastatin peptide or biologically active variant thereof.
In other embodiments, the invention features methods of preventing platelet degranulation. These methods can be carried out, for example, by contacting a plurality of platelets with an effective amount of a fusion protein that includes a first portion and a second portion, the first portion including a signal sequence capable of delivering the fusion protein into the cell and the second portion including a calpastatin peptide or biologically active variant thereof.
In other embodiments, the invention features methods of inhibiting or reversing erythrocyte sickling. These methods can be carried out, for example, by contacting a sickle erythrocyte (i.e., an erythrocyte that displays a sickled morphology or that is susceptible to sickling) with an effective amount of a fusion protein that includes a first portion and a second portion, the first portion including a signal sequence capable of delivering the fusion protein into the cell and the second portion including a calpastatin peptide or biologically active variant thereof.
In other embodiments, the invention features methods of inhibiting a human immunodeficiency (HIV) virus (and thereby treating a patient who has or who is at risk of contracting AIDS). These methods can be carried out, for example, by contacting an HIV-infected cell or a cell that is susceptible to HIV infection with an effective amount of a fusion protein that has a first portion and a second portion, the first portion including a signal sequence capable of delivering the fusion protein into the cell and the second portion including a calpastatin peptide or biologically active variant thereof.
In other embodiments, the invention features methods of treating an inflammatory disorder or an unwanted immune response (e.g., an immune response that culminates in rejection of transplanted tissue). These methods can be carried out, for example, by contacting one or more of a variety of cell types (e.g., B cells, macrophages, dendritic cells or other antigen presenting cells, T cells, endothelial cells, or neutrophils) with an effective amount of a fusion protein that has a first portion and a second portion, the first portion including a signal sequence capable of delivering the fusion protein into the cell and the second portion including a calpastatin peptide or biologically active variant thereof.
In other embodiments, the invention features methods of inhibiting unwanted cellular proliferation or migration (as occurs, for example, in the event of restenosis following balloo

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