Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-12-18
2001-09-04
Low, Christopher S. F. (Department: 1654)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S326000
Reexamination Certificate
active
06284732
ABSTRACT:
1. INTRODUCTION
The present invention relates to peptides and peptide analogues designed from HFE protein. In particular, it relates to peptides and peptide analogues designed from an alpha-1 region of HFE protein which lowers the binding affinity of transferrin receptor for transferrin. Such compounds mimic HFE protein function, and reduce iron uptake and/or accumulation by a cell.
2. BACKGROUND OF THE INVENTION
Hereditary hemochromatosis (HH) is a common genetic disorder characterized by excess iron deposition in the major organs of the body (Dadone et al., 1982
, AM. J. Clin. Pathol
. 78:196-207; Edwards et al., 1988
, N. Engl. J. Med
. 18:1355-1362; McLaren, et al., 1995
, Blood
86:2021-2027; Bothwell et al., 1995
, The metabolic and molecular basis of inherited disease
(ed. C. R. Scriver, E. A.) 2237-2269, McGraw-Hill, New York; Bacon et al., 1996
, Hepatology, A textbook of liver disease
(eds. Zakim, D. & Boyer, T. D.) 1439-1472, W. B. Saunders, Philadelphia). A candidate gene linked to this disease, HFE, was identified by positional cloning (Feder et al., 1996
, Nature Genetics
13:399-408). The gene, a novel member of the major histocompatibility complex (MHC) class I family, was found to have a mutation, cysteine 282→tyrosine (C282Y), in 83% of patient chromosomes (Feder et al., 1996
, Nature Genetics
13:399-408). This mutation eliminates the ability of HFE to associate with &bgr;
2
-microglobulin (&bgr;
2
m) and prevents its expression on the cell surface (Feder, et al., 1997
, J. Biol. Chem
. 272:14025-14028).
Recently, the HFE protein was found to bind the transferrin receptor (TfR) at high affinity, and such binding, in turn, lowers TfR affinity for transferrin, the major iron-binding protein found in the serum (Feder et al., 1998
, Proc. Natl. Acad. Sci. U.S.A
. 95:1472-1477; Gross et al., 1998
, J. Biol. Chem
. 273:22068-22074). It has been shown that by lowering the TfR affinity for transferrin, the actual amount of iron taken up by the cell is decreased, as reflected by the disappearance of the cellular iron storage protein, ferritin. These observations suggest that HFE may be important in controlling iron homeostasis by interacting with the TfR.
A murine MHC class I molecule has been shown to interact with the insulin receptor on the cell surface. In addition, a 25 amino acid peptide derived from the alpha-1 domain (residues 61-85) of the murine MHC class I molecule H-2D
k
altered the function of the insulin receptor (Stagsted et al., 1990
, Cell
62:297-307) by increasing rat adipocyte glucose uptake. This peptide was believed to act as a competitive inhibitor of intact MHC molecules on the cell surface. Additional studies have shown that by inhibiting insulin receptor internalization, the peptide activated a signal-transducing protein on the cell surface which led to increased glucose metabolism. A shortened version of the peptide containing 17 amino acids (residues 69-85) also exhibited similar activities (Stagsted et al., 1993
, Proc. Natl. Acad. Sci. U.S.A
. 90: 7686-7690). However, prior to the present invention, it was not known if a peptide derived from the HFE protein would mediate a biologic effect on the TfR.
3. SUMMARY OF THE INVENTION
The present invention relates to peptides and peptide analogues designed from HFE protein. In particular, it relates to peptides and peptide analogues designed from an alpha-1 region of HFE protein. More specifically, the invention relates to peptides and peptide analogues which lower the binding affinity of TfR for transferrin, methods of designing additional peptides and peptide analogues exhibiting similar activities, methods of using such compounds and pharmaceutical compositions thereof to inhibit iron uptake and/or accumulation by cells, as well as methods of using the compounds to treat iron overload diseases.
The invention is based, in part, on the Applicants' discovery that a peptide designed from an alpha-1 region of HFE protein inhibited cell-associated binding of transferrin. This peptide was generated on the basis of an amino acid sequence of HFE protein that made contact with TfR. The peptide lowers TfR binding affinity for transferrin, and enhances HFE inhibitory activities. The crystal structure of HFE protein shows that a tryptophan at position 81 (W81) of HFE could be at the TfR binding site (Lebron et al., 1998
, Cell
93:111-123). This suggestion was confirmed by subsequent site directed mutagenesis, such that mutation of this residue to an alanine reduced the ability of HFE to bind TfR. Similarly, mutation of a peptide from Trp to Ala at residue 81 also reduced the ability of the peptide to inhibit cell-associated binding of transferrin. It is noted that the numbering of amino acid positions disclosed herein is based on the mature HFE protein. In contrast, the position of an amino acid mutation at residue 282 responsible for HH in the majority of patients is based on the precursor protein, which includes the leader peptide.
Generally, a compound of the present invention is a peptide or peptide analogue which can be 17 amino acids in length. In embodiments wherein the compound is a peptide, the peptide comprises an amino acid sequence that corresponds in primary sequence to an alpha-1 domain of HFE or a portion thereof. In certain embodiments, one or more amino acid residues within the peptide are substituted with other amino acid residues. Typically, such substitutions are conservative, i.e., the amino acid residues are replaced with other amino acid residues having similar physical and/or chemical properties. In embodiments wherein the compound is a peptide analogue, the analogue is obtained by replacing at least one amide linkage in the peptide with a substituted amide or an isostere of amide.
In an illustrative embodiment, a compound of the invention comprises the following formula:
Z
1
-X
1
-X
2
-X
3
-X
4
-X
5
-X
6
-X
7
-X
8
-X
9
-X
10
-X
11
-X
12
-X
13
-X
14
-X
15
-X
16
-X
17
-Z
2
(I)
wherein:
X
1
is an apolar residue;
X
2
is a hydrophobic residue;
X
3
is an acidic or an aliphatic residue;
X
4
is a basic residue;
X
5
is an apolar residue;
X
6
is an aromatic residue;
X
7
is a polar residue;
X
8
is an aliphatic residue;
X
9
is an acidic or an aliphatic residue;
X
10
is an aromatic residue;
X
11
is an aromatic residue;
X
12
is a polar residue;
X
13
is Ile;
X
14
is an apolar residue;
X
15
is an acidic residue;
X
16
is a polar residue;
X
17
is a basic or an aliphatic residue;
Z
1
is H
2
N—, RHN— or, RRN—;
Z
2
is —C(O)R, —C(O)OR, —C(O)NHR, —C(O)NRR where each R is independently (C
1
-C
6
) alkyl, (C
1
-C
6
) alkenyl, (C
1
-C
6
) alkynyl, substituted (C
1
-C
6
) alkyl, substituted (C
1
-C
6
) alkenyl or substituted (C
1
-C
6
) alkynyl; and
“-” is a covalent linkage.
In a preferred embodiment of the compounds of formula (I), X
1
-X
17
is a peptide which corresponds in primary amino acid sequence to an alpha-1 domain of HFE protein, and which optionally may contain one or more conservative amino acid substitutions, or an analogue thereof. In a particularly preferred embodiment, the peptides and peptide analogues lower the binding affinity of TfR for transferrin. Therefore, such compounds are useful in inhibiting iron uptake and/or accumulation by cells. In yet another embodiment, the peptides and peptide analogues bind TfR and inhibit HFE binding to TfR, thereby increasing iron uptake by cells.
REFERENCES:
patent: 5712098 (1998-01-01), Tsuchihashi et al.
patent: WO 97/38137 (1997-10-01), None
patent: WO 98/56814 (1998-12-01), None
Ngo et al., ‘Computational Complexity, Potein Structure Prediction, and the Levinthal Paradox,’ The Protein Folding Problem and Tertiary Structuer Prediction. Ed. K. Merz and L. Le Grand. BirkHauser, Boston MA. pp. 491-495, 1994.*
Rudinger, J. (1976). Peptide Hormones (ed. J.A. Parsons). University Park Press. Baltimore. pp. 1-7, 1976.
Bennett Melanie
Bjorkman Pamela J.
Feder John N.
Lebron Jose
Schatzman Randall C.
Bio-Rad Laboratories, Inc.
Gupta Anish
Low Christopher S. F.
Pennie & Edmonds LLP
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