Growth factor analogs

A peptide having growth factor type activity (mitogenic and agonist/antagonist for the EGF receptor) which is a TGF-.alpha. analog but contains a bicyclic Pro analog (for instance D-2-azabicyclo[2,2,1]heptane-3-carboxylic acid), an N-terminal Phe or Trp residue, and a disulfide bridge.

The present invention relates to peptides having growth factor receptor 
activity, and in particular to peptides which are analogs of Transforming 
Growth Factor-Alpha (TGFA). The invention further relates to uses of such 
peptides. 
Growth factors represent a group of polypeptides which induce inter alia 
cell division. TGFA and the closely related Epidermal Growth Factor (EGF) 
have a role in a number of normal physiological processes such as wound 
healing. 
The sequence and three dimensional structure of both EGF and TGFA have been 
determined (Campbell et al, Prog. Growth Factor Res. 1989, 1, 13-22). EGF 
is a 53 amino acid polypeptide (Sequence I.D. No. 1). TGFA is a 50 amino 
acid polypeptide (Sequence I.D. No. 2) having about 40% homology of 
residues with EGF. Both peptides are characterised by three well defined 
loops (denoted A, B and C) and have three intramolecular disulphide bonds. 
Several growth factors, including TGFA and EGF, are believed to exert their 
biological effects via interaction with the Epidermal Growth Factor 
Receptor (EGF Receptor). The EGF Receptor is a Type 1 receptor tyrosine 
kinase. The EGF Receptor and its ligands are of interest for their roles 
in normal physiological processes as well as in hyperproliferative and 
neoplastic diseases. 
A number of studies have been made to establish structure-function 
relationships within EGF and TGFA so as to be able to design suitable 
investigative or therapeutic agents (such as EGF Receptor agonists and 
antagonists). These studies have been made on the well established basis 
that the sequence of amino acids from which a polypeptide or protein is 
composed (or primary structure) has a profound effect on its three 
dimensional (secondary, tertiary and quarternary) structure. The three 
dimensional structure will in turn affect the function and activity of the 
polypeptide or protein. 
Thus by altering (either randomly or rationally) the number or sequence of 
amino acids in a peptide, properties such as its kinetics, stability, 
protease or thermal resistance, specificity and ligand-receptor 
interactions may be controlled. 
Thus in EGF it has been reported that synthetic fragments corresponding to 
the B-loop bind to the EGF Receptor and have weak mitogenic activity 
(Komoriyam et al, Proc. Natl. Acad. Sci. USA 1984, 81, 1351). Analogs of 
the C-loop also have biological activity (Bailie, J. R. et al, Int J, 
Peptide Protein Res. 1994, 43, 225). Residue 47 (leucine) near the 
C-terminus of the peptide has also been shown to be important in binding 
and mitogenesis (Ray, P. et al, Biochemistry 1988, 27, 7289). 
In TGFA it has been reported that synthetic peptides corresponding to the 
B-loop bind only weakly to the EGF Receptor and do not invoke a mitogenic 
response (see, eg Defeo-Jones, D. et al Mol. Cell. Biol. 1988, 8, 
2999-3007). It has also been found that residue 15 (phenylalanine) is an 
important receptor contact; Mol. Cell. Biol. 1988, 8, 2999-3007. 
Unfortunately none of the synthetic peptides reported above have been shown 
to possess significant potential as EGF Receptor agents. Thus there 
currently exists a need for novel growth factor analogs (or other growth 
factor receptor ligands) for use in the investigation or manipulation of 
growth factor mediated processes and diseases. 
The present invention has now provided novel growth factor analogs and 
methods of preparation of such analogs that address shortcomings of the 
prior art. 
According to a first aspect of the present invention there is provided a 
peptide comprising an amino acid sequence corresponding to Sequence I.D. 
No 3 or a sequence wherein the amino acids of Sequence I.D. No 3 are 
replaced by conservative substitutions or a sequence having 90% homology 
to either of these peptide sequences characterised in that the amino acid 
at position 10 of Sequence I.D. No 3 or the sequences conservatively 
substituted or homologous thereto is a bicyclic proline analog and in that 
a disulphide bridge is provided between the cysteine residues at positions 
1 and 12. 
By bicyclic proline analog is meant any azabicycloalkane carboxylic acid or 
azabicycloalkene (monounsaturated) carboxylic acid or derivatives thereof. 
A comparison of Sequence I.D. No 3 and Sequence I.D. No 2 shows that the 
peptides of the current invention broadly correspond to the central 
portion of authentic TGFA (which forms a part of the B-loop). However they 
differ in a number of important respects. It is these important 
differences which confer unexpected and industrially applicable properties 
on the peptides including, inter alia, growth factor receptor activity. 
By `growth factor receptor activity` is meant either the ability to induce 
mitogenic activity, or to interact with the EGF Receptor (or both). 
By `mitogenic activity` is meant the ability to induce increased DNA 
synthesis in cell lines, and in particular in fibroblasts for example 
human fibroblasts. Methods for assaying mitogenic activity are well known 
to those skilled in the art (see, for example, Davies, D. E. et al, Br. J. 
Cancer 1994, 70, 263-269). 
By `interact with the EGF Receptor` is meant the ability to act as either 
an agonist or antagonist (with respect to authentic TGFA) therefor. 
Agonist activity may be deduced inter alia from studying the mitogenic 
response of a cell line responsive to EGF or TGFA to the novel peptides, 
or assaying second messengers associated with this response. It may also 
include tyrosine phosphorylation, for example EGF-R tyrosine 
phosphorylation, but it is not necessary for it to do so. 
Antagonist activity may be deduced inter alia by binding studies with 
labelled TGFA, or by observations of the TGFA-induced response in presence 
of the peptides. 
Significantly--it has not been possible to demonstrate growth factor 
receptor activity with the `authentic` B-loop TGFA fragments of the prior 
art. 
One way in which the peptides made available by the present invention 
differ from those in the prior art (both EGF and TGFA fragments) is that 
they contain a synthetic (ie non-naturally occurring) amino acid. 
It is well known that there are twenty different types of naturally 
occurring amino acid commonly found in proteins and polypeptides. Each 
(apart from glycine) has an assymetric carbon atom bonded to a hydrogen 
atom, an amino group or imino group, a carboxyl group, and a distinctive 
side-chain, and each occurs naturally in the L-form. The peptide bond 
shows a high degree of resonance stabilisation such that they have a 
degree of double bond character and cannot rotate freely. These peptide 
bonds generally exist in the trans- form with respect to the assymetric 
carbons. However, proline is an exception to this wherein the pyrollidine 
structure and the N-alkylated amide bond lead to a smaller rotational 
barrier giving rise to cis- and trans- peptide bonds of comparable energy. 
The bicyclic proline analogs incorporated into the peptides of the current 
invention differ from proline proper in that they have a bicyclic 
side-chain. Examples of possible analogs include 
2-azabicyclo[2,2,1]heptane-3-carboxylic acid and 
2-azabicyclo[2,2,2]octane-3-carboxylic acid, and substituted derivatives 
thereof. 
Bicyclic and monocyclic proline analogs having unnatural ring sizes or 
substitutions at the 3,4 and 5 position have been prepared but few have 
been incorporated into peptides and none into EGF or TGFA analogs. 
Preferably the bicyclic proline analog is 
2-azabicyclo[2,2,1]heptane-3-carboxylic acid and more preferably it is in 
the D-form (1R, 3R, 4S form) (hereinafter referred to as `DABHC`). 
However, any bicyclic proline analog wherein the rigidity of the side chain 
is enhanced with respect to proline proper, thereby limiting rotation 
about one or both of the peptide bonds which form adjacent to the analog 
may be suitable. 
Analogs wherein the interactions between the side-chain and the peptide 
bond formed by the imino group are such as to lower the energy associated 
with the cis form of the peptide bond formed by the imino group are 
particularly suitable. By stabilising the cis form of the peptide bond, 
the peptide may be constrained into (or favour) a conformation which would 
not ordinarily be favoured by peptides containing proline proper or other 
amino acids. 
These peptides further differ from the prior art peptides in that they are 
cyclised by means of a disulphide bridge between residues 1 and 12 of 
Sequence I.D. No 3. This bridge is not present in authentic TGFA, but has 
been included in these peptides for the purpose of enhancing their growth 
factor receptor activity. 
The invention embraces peptides comprising an amino acid sequence 
corresponding to sequences wherein the amino acids of Sequence I.D. No 3 
are replaced by conservative substitutions. 
By `conservative substitution` is meant the substitution of an amino acid 
by another one of the same class; the classes being as follows: 
______________________________________ 
Class Examples of Amino Acid 
______________________________________ 
Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp 
Uncharged polar Gly, Ser, Thr, Cys, Tyr, Asn, Gln 
Acidic Asp, Glu 
Basic Lys, Arg, His 
______________________________________ 
The above table includes D and L amino acids. 
Other substitutions may also be made according to the following table: 
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Class Examples of Amino Acid 
______________________________________ 
Aromatic Phe, Tyr, His 
Proton Donor Asn, Gln, Lys, Arg, His, Trp 
Proton Acceptor Glu, Asp, Thr, Ser, Tyr, Asn, Gln 
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As is well known to those skilled in the art, altering the primary 
structure of a peptide by a conservative substitution may not 
significantly alter the activity of that peptide because the side-chain of 
the amino acid which is inserted into the sequence may be able to form 
similar bonds and contacts as the side chain of the amino acid which has 
been substituted out. This is so even when the substitution is in a region 
which is critical in determining the peptides conformation. Indeed, such 
changes may confer slightly advantageous properties on the peptide. 
Suitable conservative substitutions within Sequence I.D. No 3 would 
include the substitution of arginine 2 for lysine, glutamine 6 for 
asparagine or alanine 11 for valine. 
The invention embraces peptides comprising an amino acid sequence having 
90% homology to Sequence I.D. No 3 (or a sequence wherein the amino acids 
of Sequence I.D. No 3 are replaced by conservative substitutions as 
discussed hereinbefore). As is well known to those skilled in the art, 
substitutions to regions of a peptide which are not critical in 
determining its conformation may not greatly affect its activity because 
they do not greatly alter the peptide's three dimensional structure. 
Hence, such peptides (provided that they still contain the bicyclic 
proline analog and cysteine bridge) are encompassed by the present 
invention. 
The invention also comprises a peptide comprising any of the amino acid 
sequences described above ie wherein the N- or C-terminus has been 
extended. Extension of the peptides above may confer additional desirable 
properties on them, for instance stronger binding affinity for the EGF 
Receptor. Suitable extensions include sequences corresponding to (or 
mimicing) other structural features or motifs of EGF or TGFA proper, for 
instance the A or C loops, or N- or C-termini. 
The invention further makes available a peptide comprising an amino acid 
sequence corresponding to Sequence I.D. No 4 or a sequence wherein the 
amino acids of Sequence I.D. No 4 are replaced by conservative 
substitutions or a sequence having 90% homology to either of these peptide 
sequences characterised in that the amino acid at position 1 of Sequence 
I.D. No 4 or the sequences conservatively substituted or homologous 
thereto is phenylalanine or tryptophan and at position 11 is a bicyclic 
proline analog and in that a disulphide bridge is provided between the 
cysteine residues at positions 2 and 13. 
Thus these peptides further differ from those in the prior art in that they 
contain phenylalanine or tryptophan at position 1 (which corresponds to 
threonine 20 in authentic TGFA). Preferably the residue at position 1 is 
phenylalanine. The aromatic residue is incorporated into these peptides 
for the purpose of enhancing their growth factor receptor activity. 
Alternatively, instead of phenylalanine or tryptophan then tyrosine may be 
substituted at position 1. 
The invention further encompasses use of any of the above peptides wherein 
the growth factor receptor activity comprises mitogenic activity in 
promoting mitogenesis, and in particular in promoting cell replication. 
Such use may have application in research or in accelerated wound healing. 
Similarly, the invention further encompasses use of any of the above 
peptides which have EGF Receptor-agonist activity as agonists for the EGF 
Receptor. 
The invention further encompasses use of any of the above peptides which 
have EGF Receptor-antagonist activity as antagonists for the EGF Receptor. 
Such use may have application in research or in the treatment of EGF 
Receptor related disorders, or neoplastic or hyperproliferative diseases. 
Also embraced by the current invention are therapeutic compositions for 
these purposes which comprise the peptides above. 
Thus the invention makes available peptides which have a wide application 
in the research or manipulation of growth factor mediated processes. Such 
peptides can be readily manufactured using processes based on those 
already used by those skilled in the art. 
The peptides and processes of the present invention will now be described, 
by way of illustration only, through reference to the following examples. 
Other embodiments falling within the scope of the invention will occur to 
those skilled in the art in the light of these.

SEQUENCE LISTINGS 
Sequence I.D. No 1 corresponds to human EGF. 
Sequence I.D. No 2 corresponds to human TGFA. 
Sequence I.D. No 3 corresponds to a first novel peptide of the present 
invention wherein the amino acid at position 10 is a bicyclic proline 
analog, and a disulphide bridge is provided between the cysteine residues 
at positions 1 and 12. 
Sequence I.D. No 4 corresponds to a second novel peptide of the present 
invention wherein the amino acid at position 1 is phenylalanine or 
tryptophan and at position 11 is a bicyclic proline analog, and a 
disulphide bridge is provided between the cysteine residues at positions 2 
and 13. 
EXAMPLES 
Example 1 
Synthesis of Peptide in Accordance with Sequence I.D. No 4 
A peptide according to Sequence I.D. No 4 wherein X1 was phenylalanine and 
X11 was DABHC was manufactured as follows: 
DABHC SYNTHESIS 
DABHC was synthesised in accordance with methods known in the art (see, for 
example, Gaitanopoulos, D. E. et al, J. Heterocycl. Chem. 1985, 22, 4, 
957-959; patent DE 3246757 (Henning R., et al); patent FR 2525604 (Vincent 
M., et al); patent JP 78143278 (Sanraku-Ocean Co., Ltd). 
PEPTIDE SYNTHESIS 
The peptide was synthesised by linear assembly via solid phase methods 
using 9-fluorenylmethoxycarbonyl chemistry followed by construction of the 
disulphide bond linking the Cys residues. Assembly followed standard 
procedures using the following protecting groups, Cys (trityl), Arg 
(pentamethylchroman sulphonyl), Asp and Glu (t-butoxy) and Lys 
(t-butoxylcarbonyl). The peptide was cleaved from the 
4-hydroxymethylphenoxymethyl resin with 95% triflluoroacetic acid (TFA). 
The linear peptide was converted to a cyclic peptide by air oxidation in 
ammonium bicarbonate solution at 38.degree. C. 
PEPTIDE PURITY 
Final purification by HPLC was achieved by gradient elution on 
Reverse-Phase HPLC using TFA/H.sub.2 O TFA/MeCN 5% to 95%. The peptide was 
homogenous by HPLC. Mass spectroscopy established the required molecular 
weight and amino acid analysis showed the presence of all residues apart 
from DABHC and Cys. The sequence was confirmed by 2-D NMR. 
Example 2 
Growth Factor Receptor Activity of Example 1 Peptide 
The mitogenic and EGF Receptor activity of the Example 1 peptide was 
studied as follows: 
MATERIALS 
Materials came from the following sources: Recombinant hEGF (Life 
Technologies, UK); recombinant hTGFA (British Biotechnology); NR6/HER 
cells [NR6 fibroplasts transfected with hEGF Receptor] (Ludwig Institute 
for Cancer Research, UK). 
CELL CULTURE 
NR6 and NR6/HER cells (NR6 mouse fibroblasts transfected with human EGF-R 
expression plasmid) and HN5 squamous carcinoma cells (see Cowley, G. P. et 
al, Br. J. Cancer 1986, 53, 2230229) were routinely cultured in Dulbecco's 
Modified Eagles Medium (DMEM) containing 10% foetal bovine serum (FBS), 2 
mM glutamine, non-essential amino acids, 10 U/ml penicillin and 10 
.mu.g/ml streptomycin at 37.degree. C. in a humidified atmosphere of air 
containing 5% CO.sub.2. G418 (100 .mu.g/ml) was also included in the 
NR6/HER growth medium as selection for EGF Receptor encoding 
transformants. 
MITOGENIC ACTIVITY 
The ability of the Example 1 peptide to induce DNA synthesis in confluent 
and quiescent cells was assayed as described in Davies, D. E. et al, Br. 
J. Cancer 1994, 70, 263-269 but using NR6/HER or NR6 cells in place of 
human foreskin fibroplasts. The results are shown in FIGS. 1 and 2. 
The Example 1 peptide (incorporating DABHC) stimulated DNA synthesis with 
an EC.sub.50 value of 130 .mu.M (FIG. 1(a)). Corresponding synthesis and 
testing of the equivalent peptide incorporating LABHC yielded a value of 
330 .mu.M (FIG. 1(b)). Corresponding peptides using D- or L-proline proper 
were inactive (FIGS. 1(c) and (d) respectively). All four peptides were 
inactive in linear, free sulphydryl form. These results clearly 
demonstrate the usefulness of the peptides provided by the present 
invention, as mitogenic agents and the importance of the bicyclic proline 
analog and the disulphide bridge. 
The Example 1 peptide induced a similar maximal mitogenic response to a 
maximal dose of authentic TGFA. At suboptimal doses of TGFA, the Example 1 
peptide produced an additive response up to the plateau level of 
stimulation by TGFA (FIG. 2(a)). However, at maximal doses of TGFA, the 
Example 1 peptide reduced the mitogenic response (FIG. 2(b)). Similar, but 
less pronounced, results were found for the corresponding peptide 
incorporating LABHC. Corresponding peptides using D- or L-proline proper 
did not yield the same effect. These results clearly indicate the 
usefulness of the peptides of the current invention as EGF Receptor 
agonist and antagonists. 
In order to establish that the DABHC peptide interacted with EGF-R its 
activity was assessed on the parental NR6 cell line which lacks EGF-R 
(FIG. 3). 
The NR6 cells showed a mitogenic response to doses of DABHC or TGF.alpha. 
which were maximally stimulating for NR6/HER cells. The responsiveness of 
the cells to a mitogenic stimulus was confirmed in the presence of PDGF. 
EGF Receptor Tyr PHOSPHORYLATION 
EGF Receptor Tyr phosphorylation assays were performed using HN5 cells at 
approximately 50% confluence in 96-well plates. Cells were plated in 10% 
FBS/DMEM and allowed to adhere overnight. Medium was removed and wells 
washed twice with DMEM. The peptide was diluted in DMEM and added in 50 
.mu.l volume and cells incubated for 10 minutes at room temperature. At 
the end of incubation, reagents were removed and the cells washed once 
with 50 .mu.l/well buffered saline and then rapidly solubilised with 50 
.mu.l/well.times.1 sample buffer containing phosphatase and protease 
inhibitors (1 mM NaF, 1 mM Na.sub.3 VO.sub.4, 100 .mu.M PMSF). Enhanced 
phosphorylation of the 170 kDa EGF-R was determined by Western Blotting 
after SDS polyacrylamide gel electrophoresis as in Holt, S. J. et al, 
Biochem. Pharmacol. 1994, 47, 117-126. 
Neither the Example 1 peptide nor the corresponding peptide incorporating 
LABHC appeared to lead to increased EGF Receptor phosphorylation. This 
demonstrates the possible basis for their antagonist activity. EGF-R 
tyrosine phosphorylation is not a pre-requisite for induction of DNA 
synthesis (Decker, 1993, J. Biol. Chem. 268, 9176-79). 
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# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 4 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 53 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens 
- - (ix) FEATURE: 
(A) NAME/KEY: Disulfide-bo - #nd 
(B) LOCATION: 6..20 
- - (ix) FEATURE: 
(A) NAME/KEY: Disulfide-bo - #nd 
(B) LOCATION: 14..31 
- - (ix) FEATURE: 
(A) NAME/KEY: Disulfide-bo - #nd 
(B) LOCATION: 33..42 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - Asn Ser Asp Ser Glu Cys Pro Leu Ser His As - #p Gly Tyr Cys Leu 
His 
1 5 - # 10 - # 15 
- - Asp Gly Val Cys Met Tyr Ile Glu Ala Leu As - #p Lys Tyr Ala Cys Asn 
20 - # 25 - # 30 
- - Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gl - #n Tyr Arg Asn Leu Lys 
35 - # 40 - # 45 
- - Trp Trp Glu Leu Arg 
50 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 50 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens 
- - (ix) FEATURE: 
(A) NAME/KEY: Disulfide-bo - #nd 
(B) LOCATION: 8..21 
- - (ix) FEATURE: 
(A) NAME/KEY: Disulfide-bo - #nd 
(B) LOCATION: 16..32 
- - (ix) FEATURE: 
(A) NAME/KEY: Disulfide-bo - #nd 
(B) LOCATION: 34..43 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Val Val Ser His Phe Asn Asp Cys Pro Asp Se - #r His Thr Gln Phe Cys 
1 5 - # 10 - # 15 
- - Phe His Gly Thr Cys Arg Phe Leu Val Gln Gl - #u Asp Lys Pro Ala Cys 
20 - # 25 - # 30 
- - Val Cys His Ser Gly Tyr Val Gly Ala Arg Cy - #s Glu His Ala Asp Leu 
35 - # 40 - # 45 
- - Leu Ala 
50 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (ix) FEATURE: 
(A) NAME/KEY: Disulfide-bo - #nd 
(B) LOCATION: 1..12 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - Cys Arg Phe Leu Val Gln Glu Asp Lys Xaa Al - #a Cys 
1 5 - # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (ix) FEATURE: 
(A) NAME/KEY: Disulfide-bo - #nd 
(B) LOCATION: 2..13 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - Xaa Cys Arg Phe Leu Val Gln Glu Asp Lys Xa - #a Ala Cys 
1 5 - # 10 
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