IL-6 antagonist peptides

Details IL-6 antagonist peptides IL-6 antagonist peptides

2000-11-20

2003-07-29

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S016700, C514S013800, C514S014800, C514S015800, C530S351000, C530S350000, C530S300000, C530S326000, C530S327000, C530S328000, C424S085200

Reexamination Certificate

active

06599875

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to IL-6 antagonist peptides, isolatable from a peptide library through the two-hybrids system by their ability to bind to the intracellular domain of gp130 and containing at least 5 amino acids. In particular, such peptides comprise an amino acid sequence, which is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, as well as salts, functional derivatives, precursors and analogs thereof.
Another object of the present invention is to provide the peptide in substantially purified form, in order to be suitable for use in pharmaceutical compositions, as active ingredient, in pathologies that require IL-6 activity inhibition.
BACKGROUND OF THE INVENTION
The Two Hybrid System (THS) is a method that uses transcriptional activity as a system to detect protein-protein interactions. A gene fusion is constructed to encode the DNA-binding domain of the yeast transcription factor GAL4 as a hybrid with any protein ‘X’(usually a defined mammalian protein being the “bait” binding target). An additional gene fusion construct will encode the transcription activation domain of GAL4 fused to any protein ‘Y’ (usually a library of diverse proteins, the “fish”) (Fields et al., 1994). Whenever an X-Y interaction does occur, it will bring the activation domain close to sites on the DNA recognised by the GAL4 DNA-binding domain, therefore resulting in the expression of a flanking reporter gene regulated by these DNA sites. The reporter genes commonly used include: 1) lacZ, which produces blue colonies on plates or filters containing X-Gal; and 2) His3, a yeast gene involved in histidine biosynthesis, required for growth of host yeast cells.
Recently, Fields and his team have used the THS for screening a library of random peptides, instead of a cDNA library, in order to find peptides capable of binding to the retinoblastoma protein (Rb) (Yang et al., 1995).
The receptor system for Interleukin-6 (IL-6) is composed of two distinct receptor “subunits” designated gp80 and gp130 (see Hirano et al., 1994).
The IL6 type cytokines elicit their signal through receptors that share the gp130 protein. Upon ligand binding gp130 homo- or heterodimerizes with the LIF and OSM receptor, thereby activating associated JAK tyrosine kinases. The JAKs phosphorylate the signal transducer (gp130) and latent transcription factors of the STAT family (Signal Transducer and Activator of Transcription), like STAT1 and STAT3 in the case of IL-6. STAT factors dimerize, translocate to the nucleus and bind to enhancer elements of IL-6 responsive genes (Lüttiken et al., 1993).
Deletion analysis of the intracellular domain of gp130 has defined short stretches of amino acids known as box1 and box2 sufficient to impart both mitogenic activity and binding of JAK proteins (Vanderkuur et al., 1994): these activities were also observed when the binding sites of STATs were deleted. Therefore two functions can be ascribed to JAK kinases: 1) activation of STAT-mediated gene expression; 2) activation of STAT-independent mitogenic activity at least in some hematopoietic cells.
Additional kinases are known to associate with to the intracellular portion of gp130, such as Hck, Fes, Btk and Tec (Matsuda et al., 1995). However these interactions have not been elucidated at the molecular level. Moreover, Tanner et al. have demonstrated that the box1 domain of cytokine receptors is required but not sufficient for interaction with JAK kinases and have suggested that the box1 sequences cooperate with other cytoplasmic domain sequences to effect JAK kinase association (Tanner et al., 1995). Even the molecular counterpart on JAK kinases of box1 and box2 has not been defined.
Synthetic peptides that inhibit IL-6 activity have been described in the International Patent Application WO 97/13781 (YEDA), which relates to peptides derived from the gp80 protein.
DESCRIPTION OF THE INVENTION
As a target in the THS, we have analysed the intracellular portion of the human IL-6 receptor (gp130-ICD). This THS screening should therefore identify candidate peptides which are able to directly interact with gp130-ICD in a phosphorylation-independent manner. Phosphorylation independent-interactions with gp130-ICD are known to occur in the transduction of the signal triggered by IL-6 type cytokines. gp130-ICD interacting counterparts of this type include protein kinases of the JAK family (Darnell et al., 1994).
Therefore, the main object of the present invention is an IL-6 antagonist peptide, isolatable from a peptide library through the two-hybrids system by their ability to bind to the intracellular domain of gp130 and containing at least 5 amino acids. According to a preferred embodiment of the invention, such pepddes contain up to 30 amino acids, more preferably 5-20, most preferably 8-16.
According to the present invention the “bait” (“X”) used in the THS screening is the intracellular domain (ICD) of the gp130 protein. Such domain corresponds to the region from amino acid at position 642 to amino acid at position 918 (Yamasaki K. et al., 1988) of the IL6-R (gp130). The “fish” in the THS screening is a library of random peptides. Such library can be any commercial library or can be produced “in-house” by known methods.
False positives arising from the above screening may be eliminated as described in the literature (Bartel et al., 1993).
According to a further preferred embodiment, such peptides comprise an amino acid sequence, which is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, as well as salts, functional derivatives, precursors and analogs thereof.
When, in SEQ ID NO: 1, Xaa
3
is Gly and Xaa
4
is Leu, the peptide of the invention comprises the amino acid sequence of SEQ ID NO: 2.
“Analogs”, as used in the present application, means those peptides, in which one or more of the amino acids in the above sequences are changed without substantially affecting the IL-6 antagonist activity. In particular, preferred changes for analogs in accordance with the present invention are what are known as “conservative” substitutions. Conservative amino acid substitutions include amino acids replacements with synonymous amino acids within the same group, which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule, Grantham,
Science,
Vol. 185, pp. 862-864 (1974).
The synonymous amino acid groups are those defined in Table I. More preferably, the synonymous amino acid groups are those defined in Table II; and most preferably the synonymous amino acid groups are those defined in Table III.
TABLE I
Preferred Groups of Synonymous Amino Acids
Amino Acid
Synonymous Group
Ser
Ser, Thr, Gly, Asn
Arg
Arg, Gln, Lys, Glu, His
Leu
Ile, Phe, Tyr, Met, Val, Leu
Pro
Gly, Ala, Thr, Pro
Thr
Pro, Ser, Ala, Gly, His, Gln, Thr
Ala
Gly, Thr, Pro, Ala
Val
Met, Tyr, Phe, Ile, Leu, Val
Gly
Ala, Thr, Pro, Ser, Gly
Ile
Met, Tyr, Phe, Val, Leu, Ile
Phe
Trp, Met, Tyr, Ile, Val, Leu, Phe
Tyr
Trp, Met, Phe, Ile, Val, Leu, Tyr
Cys
Ser, Thr, Cys
His
Glu, Lys, Gln, Thr, Arg, His
Gln
Glu, Lys, Asn, His, Thr, Arg, Gln
Asn
Gln, Asp, Ser, Asn
Lys
Glu, Gln, His, Arg, Lys
Asp
Glu, Asn, Asp
Glu
Asp, Lys, Asn, Gln, His, Arg, Glu
Met
Phe, Ile, Val, Leu, Met
Trp
Trp
TABLE II
More Preferred Groups of Synonymous Amino Acids
Amino Acid
Synonymous Group
Ser
Ser
Arg
His, Lys, Arg
Leu
Ile, Phe, Met, Leu
Pro
Ala, Pro
Thr
Thr
Ala
Pro, Ala
Val
Met, Ile, Val
Gly
Gly
Ile
Ile, Met, Phe, Val, Leu
Phe
Met, Tyr, Ile, Leu, Phe
Tyr
Phe, Tyr
Cys
Ser, Cys
His
Arg, Gln, His
Gln
Glu, His, Gln
Asn
Asp, Asn
Lys
Arg, Lys
Asp
Asn, Asp
Glu
Gln, Glu
Met
Phe, Ile, Val, Leu, Met
Trp
Trp
TABLE III
Most Preferred Groups of Synonymous Amino Acids
Amino Acid
Synonymous Group
Ser
Ser
Arg
Arg
Leu
Ile, Met, Leu
Pro
Pro
Thr
Thr
Ala
Ala
Val
Val
Gly
Gly
Ile
Ile, Met, Leu
Phe
Phe
Tyr
Tyr
Cys
Ser, Cys
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp

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