Peptides and peptide analogues designed from binding sites...

Details Peptides and peptide analogues designed from binding sites... Peptides and peptide analogues designed from binding sites...

1997-05-30

2001-07-24

Ulm, John (Department: 1646)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Cyclic peptides

C530S321000, C530S326000

Reexamination Certificate

active

06265535

ABSTRACT:

1. INTRODUCTION
The present invention relates to peptides and peptide analogues designed from a binding loop of a member of the tumor necrosis factor receptor (TNF-R) superfamily, which is involved in binding interactions with its ligand. In particular, it relates to cyclic peptides and peptide analogues designed from three specific binding loops in domains 2 and 3 of TNF-R which inhibit tumor necrosis factor (TNF) binding to its cellular receptors, methods of designing similar peptides and peptide analogues, and methods of using such compounds to inhibit the biological activities of TNF, thereby antagonizing its undesirable clinical effects.
2. BACKGROUND OF THE INVENTION
2.1. Tumor Necrosis Factor and its Pathophysiology
TNF was originally discovered as a molecule which caused hemorrhagic necrosis of mouse tumors (Carswell et al., 1975
, Proc. Natl. Acad. Sci. U.S.A.
72:3666). A second line of investigation of a serum protein known as “cachectin”, thought to be responsible for the condition of cachexia, led to the eventual discovery that cachectin was identical to TNF (Beutler et al., 1989
, Annu. Rev. Immunol.
7:625). TNF has now been established as a broadly active inflammatory mediator involved in diverse clinical conditions.
TNF/cachectin was renamed as TNF-&agr;, and a structurally and functionally related protein previously known as lymphotoxin (LT) was referred as TNF-&bgr; (Vassalli, 1992
, Annu. Rev. Immunol.
10:411). Both molecules are active as homotrimers and mediate similar biological effects by binding to the same two cellular receptors of 55 kD (p55 or complex I) and 75 kD (p75) molecular weight (Smith et al., 1990
, Science
248:1019; Schall et al., 1990
, Cell
61:361). An LT heterotrimer was later discovered, which engaged a third receptor known as TNF-R related protein (rp), but it is not capable of binding to TNF-R p55 and p75 (Browning et al., 1993
, Cell
72:847). This LT has been named as LT-&bgr;, and the LT homotrimer (or TNF-&bgr;) is also referred to as LT-&agr;. Structural comparison of the three TNF-R with several other cell surface receptors has resulted in the classification of these receptors into the TNF-R superfamily (Gruss and Dower, 1995
, Cytokines and Mol. Ther.
1:75).
TNF-&agr; is a 17 kD molecular weight protein produced by several cell types, particularly activated macrophages. Since TNF-R is expressed by numerous cell populations, TNF induces a wide variety of cellular responses, many of which result in deleterious consequences. For example, TNF induces cachexia which is a condition resulting from loss of fat and whole body protein depletion, accompanied by insufficient food intake due to anorexia. Cachexia is commonly seen in cancer patients, and it has also been observed in patients with acquired immunodeficiency syndrome (AIDS).
In addition, injection of high doses of TNF in animals produces most of the symptoms of septic shock. TNF has also been shown to play a role in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, hypersensitivity, immune complex diseases and graft versus host disease as well as transplantation rejection. The involvement of TNF has even been implicated in malaria and lung fibrosis.
2.2. Treatment of TNF-Associated Disorders
Methods for neutralizing the adverse effects of TNF have focused on the use of anti-TNF antibodies and soluble TNF-R. In animal models, treatment of TNF-associated inflammatory disorders with antibodies specific for TNF has shown therapeutic efficacy (Williams et al., 1992
, Proc. Natl. Acad. Sci. U.S.A.
89:9784; Baker et al., 1994
, Eur. J. Immunol.
24:2040; Suitters et al., 1994
, J. Exp. Med.
179:849). Chimeric forms of anti-TNF antibodies have been constructed for use in human clinical trials (Lorenz et al., 1996
, J. Immunol.
156:1646; Walker et al., 1996
, J. Infect. Dis.
174:63; Tak et al., 1996
, Arthritis Rheumat.
39:1077). Additionally, soluble TNF-R fusion proteins have been introduced as TNF-antagonists in human patients (Peppel et al., 1991
, J. Exp. Med.
174:1483; Williams et al., 1995
, Immunol.
84:433; Baumgartner et al., 1996
, Arthritis Rheumat.
39(Suppl.) S74).
While the aforementioned approaches have shown some effectiveness in certain disease conditions, anti-TNF antibodies and soluble TNF-R both suffer from common limitations of macromolecules such as poor bioavailability and stability, induction of immune reactions and ineffective tissue penetration. Thus, there remains a need for improved therapeutic compounds for antagonizing the undesirable effects of TNF.
3. SUMMARY OF THE INVENTION
The present invention relates to peptides and peptide analogues designed from a binding loop of a TNF-R superfamily member. In particular, it relates to peptides and peptide analogues designed from three binding loops of TNF-R. More specifically, the invention relates to peptides and peptide analogues which interfere with the binding interactions between TNF and TNF-R, methods of designing additional peptides and peptide analogues exhibiting inhibitory activities, and methods of using such compounds and pharmaceutical compositions thereof to antagonize the undesirable biological activities of TNF in vivo, as well as methods of using the compounds to detect the presence of TNF in a sample and for inhibiting TNF activities in vitro.
The invention is based, in part, on the Applicants' discovery that peptides designed from three binding loops of TNF-R p55 inhibited TNF-&agr; binding to TNF-R. Such peptides were generated on the basis of the amino acid sequences that form the specific binding loops. Cys residues were added to the peptides to enable their cyclization by formation of disulfide bridges, and hydrophobic aromatic residues were added to their termini to enhance structural stability. Among the peptides that competitively inhibited the binding of TNF-&agr; to its cognate receptor, peptides designed from loop 1 of domain 3 of TNF-R were the most effective.
Generally, a compound of the present invention is a cyclic peptide or peptide analogue which is modified at its termini with hydrophobic moieties. In embodiments wherein the compound is a peptide, the peptide corresponds in primary sequence to a binding loop of a member of the TNF-R superfamily or a portion thereof. In a preferred embodiment, the peptide corresponds in primary sequence to a binding loop of TNF-R p55 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 amino acid 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 has the following formula:
wherein:
AC is a peptide of 3-18 amino acid residues which corresponds in primary sequence to a binding loop of a TNF-R superfamily member, and which may optionally contain one or more conservative amino acid substitutions, or an analogue thereof wherein at least one amide linkage is replaced with a substituted amide or an isostere of amide;
AB
1
is a moiety having a first functional group capable of forming a covalent linkage with one terminus of AC, a second functional group capable of forming a covalent linkage with AB
2
and a third functional group capable of forming a covalent linkage with AA
1
;
AB
2
is a moiety having a first functional group capable of forming a covalent linkage with the second terminus of AC, a second functional group capable of forming a covalent linkage with AB
1
and a third functional group capable of forming a covalent linkage with AA
2
;
AA
1
is a moiety having hydrophobic properties and a functional group capable of forming a covalent linkage with the third functional group of AB
1
;
AA2 is a moiety having hydrophobic properties and a functional

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