Peptide ligands for the erythropoietin receptor

Details Peptide ligands for the erythropoietin receptor Peptide ligands for the erythropoietin receptor

2000-09-20

2003-11-04

Celsa, Bennett (Department: 1639)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S326000, C435S375000, C930S090000

Reexamination Certificate

active

06642353

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the fields of pharmacology and drug discovery. More particularly, the invention relates to novel peptide compositions that can bind and activate the human erythropoietin receptor, and to methods of making small molecule agonists of the erythropoietin receptor using such peptide compositions as design templates.
BACKGROUND OF THE INVENTION
Drug discovery traditionally has relied upon high-throughput screening of large numbers of chemical compounds to identify novel drug leads. More recently, combinatorial libraries constructed by chemical or biological means have greatly expanded the number of compounds available for screening. Biological libraries, such as phage displayed peptide libraries, of random directed semi-random sequences represent particularly rich sources of molecular diversity and advantageously possess the ability to self-replicate. With a self-replicating system, the search for high affinity leads is not limited to members that happen to be present in the initial library. As discussed more fully below, desired characteristics of initial sequences can be greatly improved by employing successive rounds of mutagenesis, affinity selection, and amplification. These approaches recently have been used to discover small peptides capable of binding several cytokine receptors.
Erythropoietin (EPO) is a cytokine that stimulates the formation of red blood cells by inducing the growth and differentiation of progenitor cells. The recombinant version of human EPO is a valuable therapeutic agent useful for treating anemia that is associated with several pathological conditions, including chronic renal failure, malignancy or the effects of chemotherapy, HIV and rheumatoid arthritis. When used therapeutically, EPO must be administered either by intravenous or subcutaneous injection. The fact that EPO is a relatively large glycoprotein adversely impacts the cost of manufacture, the pharmacological properties of molecule, and the mode of delivery of this therapeutic agent.
The erythropoietin receptor (EPOR) belongs to the cytokine receptor superfamily whose members share common structural features including an extracellular ligand binding domain, a single transmembrane-spanning region, and. an intracellular cytoplasmic domain. The
e
xtra
c
ellular
d
omain (ECD) is sufficient to mediate receptor-ligand binding. It is therefore possible through recombinant DNA techniques to synthesize DNA encoding the ECD as a fusion with secreted proteins to produce reagents useful for identifying receptor binding molecules, for example by screening a phage display library.
Phage display libraries expressing fusions of random or semi-random peptides and bacteriophage coat proteins represent convenient versions of combinatorial libraries that can be screened to identify receptor ligands. Upon infection and assembly of phage particles, the random polypeptides are outwardly disposed for interaction with antibodies or other receptor probes. Since the phage particles contain the nucleic acid that encodes the fusion protein, the genetic information which identifies the sequence of the fusion protein is physically linked to the fusion protein. Construction and screening of such phage expression libraries are well known and have been described previously, such as, for example, in Sawyer et al.,
Protein Engineering
4:947-953 (1991); Akamatsu et al.,
J. Immunol
. 151:4651-59 (1993), Smith et al.,
Methods in Enzymol
. 217:228-257 (1993); Clackson et al.,
Trends Biotechnol
. 12:173-184 (1994), and U.S. Pat. No. 5,427,908 to Dower et al.
In one example of a screening procedure, a soluble form of the EPOR was used to probe a phage display library to identify candidate peptides having EPO-like properties. Wrighton et al., in
Science
273:458 (1996) described the use of a fusion protein comprising the EPOR extracellular domain and human placental alkaline phosphatase in a library screening protocol. The library used for this purpose displayed cyclic 8-residue peptides as fusions with the pVIII coat protein of a filamentous phage. Peptides having higher affinity for the EPOR were subsequently isolated from mutagenesis libraries that displayed pIII protein fusions. This approach led to the identification of several peptides that stimulated erythropoiesis in mice. These agonists were disulfide-bonded cyclic peptides having the minimum consensus sequence YXCXXGPXTWXCXP (SEQ ID NO:1), where X is a position that can be occupied by any of several amino acids.
Despite this progress toward identifying EPOR ligands, there remains a need to identify ligands exhibiting superior receptor-binding properties as well as ligands that bind to the receptor using previously unknown contact sites.
SUMMARY OF THE INVENTION
According to one aspect of the invention, there is disclosed an isolated polypeptide capable of binding to a human erythropoietin receptor, having the formula:
X
n
-C-X
1
-X
2
-G-W-V-G-X
3
-C-X
4
-X
5
-W-X
C
wherein X
n
is an amino-terminal peptide of from 2 to 4 natural &agr;-amino acids in length; X
C
is a carboxy-terminal dipeptide; and X
1
, X
2
, X
3
, X
4
and X
5
are independently selected from the group consisting of natural a-amino acids.
In preferred embodiments of the isolated polypeptide, the amino-terminal X
n
is X
n1
-X
n2
-X
n3
-X
n4
, wherein X
n1
is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural a-amino acids, or optionally X
n1
is absent; X
n2
is selected from the group consisting of neutral and polar, neutral and hydrophobic, and basic natural a-amino acids, or optionally X
n2
is absent if X
n1
is absent; X
n3
is selected from the group consisting of natural &agr;-amino acids; and X
n4
is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural &agr;-amino acids. More preferably, X
n1
is E, G, N, S, D, Q, L, Y or A; X
n2
is F, V, A, K, R, G, S, I or L; X
n3
is H, Q, E, G, D, A, or V; and X
n4
is E, G, V, A, P, D, T or M. In other preferred embodiments, X
n1
is absent; X
n2
is F, V, A, K, R, G, S, I or L; X
n3
is H, Q, E, G, D, A, or V; and X
n4
is E, G, V, A, P, D, T or M. In one embodiment X
n1
is an acidic amino acid. In another embodiment, X
n2
is a branched-chain amino acid or K. In yet another embodiment, X
n3
is an acidic amino acid or V. In one embodiment, X
n4
is V or G. In preferred embodiments, X
1
is a neutral and hydrophobic, neutral and polar, or basic amino acid; more preferably X
1
is R, I, G, Q, L, T or S; and most preferably, is R or I. In preferred embodiments, X
2
is a neutral and hydrophobic, neutral and polar, or basic amino acid; more preferably, X
2
is R, P, W, G, L or N; and most preferably is R. In preferred embodiments, X
3
is a neutral and polar, or basic amino acid; and more preferably X
3
is H, Q or N. In preferred embodiments, X
4
is a neutral and polar, basic, or acidic amino acid; more preferably X
4
is Q, N, S, K or E; and most preferably is K or N. In preferred embodiments, X
5
is a neutral and polar, neutral and hydrophobic, or acidic amino acid; more preferably is V, A, Y, D or E; and most preferably is D or E. In preferred embodiments, the carboxy-terminal X
c
is X
c1
-X
c2
and wherein X
c1
is a neutral and polar, or neutral and hydrophobic amino acid, and X
c2
is a neutral and polar, neutral and hydrophobic, or basic amino acid. In preferred embodiments, X
c1
is L, I, P, F, M, Q or G; and more preferably is L or Q. In preferred embodiments, X
c2
is M, W, T, S, G, N or R; and, more preferably, X
c2
is G or R.
Other aspects of the invention are isolated polypeptides having the amino acid sequences of SEQ ID NO:81 and SEQ ID NO:82.
According to another aspect of the invention, there is disclosed a method of activating a human erythropoietin receptor, comprising the steps of contacting a cell having an erythropoietin receptor on it surface with a peptide mimetic of erythropoietin, wherein the peptide mimetic is a compound having the general formula
X
n
-C-X
1
-X
2
-G-W

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