(Received for publication, May 4, 1995; and in revised form, June 22, 1995)
From the
Two proline mimetics, the enantiomers of
2-azabicyclo[2,2,1]heptane-3-carboxylic acid, have been
incorporated in place of Pro into synthetic peptides based
on the B-loop
-sheet sequence of human transforming growth
factor-
(TGF-
) (residues Cys
-Cys
).
The peptides were further modified by inclusion of an N-terminal
phenylalanine and constrained by formation of an intramolecular
disulfide bond. While no mitogenic response was observed in the
parental NR6 cell line, the peptides stimulated DNA synthesis in
NR6/HER cells (NR6 fibroblasts transfected with the human epidermal
growth factor receptor). Induction of DNA synthesis was dose dependent,
with EC
values in the range 130-330 µM;
in the presence of low doses of TGF-
, the mitogenic effect of the
peptides was additive, up to the plateau response achieved by maximal
doses of TGF-
alone. These effects are consistent with the
peptides acting via the same mechanism as TGF-
. Analysis of the
structure of the peptides by NMR indicated that the presence of the
mimetics significantly increased the propensity of the peptidyl-proline
bond to adopt the cis conformation. These data confirm the
role of the
-sheet in receptor activation, and emphasize the
importance of presentation of peptides in an appropriate conformation
for recognition.
Epidermal growth factor (EGF) ()and transforming
growth factor-
(TGF-
) belong to a family of EGF-like growth
factors (1) that stimulate a variety of epithelial and
mesenchymal cells. The biological effects of these growth factors are
mediated via the transmembrane epidermal growth factor receptor (EGF-R) (2) , a type 1 receptor tyrosine kinase(3) . The EGF-R
and its ligands are of considerable interest not only in normal
physiological processes such as wound healing (4) and
embryogenesis (5) but also through their involvement in the
pathology of hyperproliferative (6) and neoplastic (7) diseases. Thus, the identification of small bioactive
molecules may have several clinical applications.
NMR studies have
identified the major structural element in EGF and TGF- as a
double stranded anti-parallel
-sheet (8) which includes
the 10 residues between the third and fourth cysteines of the growth
factors. Mutational analysis of Ile
(9) and
Leu
(10) in EGF has implicated the
-sheet in
the ligand:receptor interaction. In TGF-
, we have recently
identified this same region as one which interacts with the chicken
EGF-R(11) . These observations confirm our previous studies
using an epitope mapping technique which identified a putative receptor
binding cavity in TGF-
(12) . The cavity was formed from
the B-loop
-sheet, as well as the C-loop and flexible C-terminal
tail of TGF-
. The side chain of Phe
, a known receptor
contact residue, was not identified by the mapping technique, but was
found to occupy a central position within this cavity.
Previous
studies have shown that synthetic peptide fragments of EGF (13, 14, 15, 16) and TGF- (17, 18, 19) bind only weakly to EGF-R,
nevertheless, only peptides corresponding to the B-loop segment of EGF
have been consistently shown to bind to the EGF-R and invoke a weak
mitogenic response. While low activity of synthetic peptide fragments
may reflect the complex interaction of the ligand with EGF-R, it may
also be due to failure of these peptides to adopt a conformation
similar to that of the native ligand. Hence, some workers have used
peptidomimetics (20) or cyclization (21) to apply
additional conformational constraints.
In the present study, we have
generated constrained peptides based on the -sheet segment of
TGF-
and have exploited the presence of proline in this peptide.
This amino acid has a pyrrolidine structure and an N-alkylated
amide bond which gives rise to cis and trans rotamers
of comparable energy(22) . In view of these structural
implications, it represented an attractive site for modification and
development of TGF-
analogue peptides.
Initial studies focused on TGF- residues
Cys
-Cys
which were constrained by
introduction of an intramolecular disulfide bond. Although these two
cysteines are not disulfide-bonded to each other in the intact growth
factor (Fig. 1a), molecular modeling using NMR-derived
conformations of TGF-
revealed that they were sufficiently close
(5 Å) to form an undistorted disulfide bond without significant
changes in the conformation of the
-sheet. The peptide was further
modified by introduction of an N-terminal phenylalanine to mimic the
presence of Phe
, a known receptor contact residue whose
side chain lies close to Cys
and Cys
(Fig. 1, b and c). This cyclic peptide
was designated N-pheTGF-
[21-32].
Figure 1:
Structure
of TGF- and derivation of TGF-
analogue peptides. a,
the primary structure of TGF-
showing the three-looped motif with
the residues incorporated into N-pheTGF-
[21-32]
highlighted; b, the primary structure of
N-pheTGF-
[21-32] highlighting Pro
; c, the three-dimensional structure of TGF-
visualized
using RasMol v2.4 and NMR coordinates obtained from Dr. T. P. Kline (39) ; the TGF-
structure is displayed as a ribbon with
the region between residues 21 and 32 in boldface; the
locations of the side chains of the six cysteines and Phe
are identified in stick representation, and the spheres identify the sulfurs of Cys
and
Cys
.
N-pheTGF-[21-32] and three analogue peptides in
which L-proline was replaced by D-proline or by the
two enantiomers of 2-azabicyclo[2,2,1]heptane-3-carboxylic
acid (N-pheTGF-
[21-32]
,
N-pheTGF-
[21-32]
, and
N-pheTGF-
[21-32]
,
respectively) were synthesized as described elsewhere (25) and
were purified by HPLC, both before and after cyclization. The
authenticity of the peptides was established by a variety of criteria:
all were homogeneous by HPLC (Fig. 2), mass spectroscopic
results established the required molecular masses, amino acid analysis
demonstrated the presence of all residues other than the unnatural
amino acids and cysteine, and the amino acid sequence was confirmed by
two-dimensional NMR.
Figure 2:
Analytical HPLC profiles of cyclic
N-pheTGF-[21-32] peptides. Each peptide was
analyzed on a Dynamax C
analytical column using a gradient
of acetonitrile, 0.1% trifluoracetic acid in 0.1% trifluoroacetic acid
in H
O as shown. The profiles show:
N-pheTGF-
[21-32] (a),
N-pheTGF-
[21-32]
(b),
N-pheTGF-
[21-32]
(c), and
N-pheTGF-
[21-32]
(d).
NMR spectra of the free SH and ACM-blocked
linear peptides were virtually identical, and their sequential
assignment was straightforward using the TOCSY and ROESY data acquired.
Resonance heterogeneity was apparent for several protons, being
greatest for those closest to the prolyl amide bond (Fig. 3a). This was consistent with cis-trans isomerization, which was estimated for each compound and shown to
be greatest for linear
N-pheTGF-[21-32]
(Table 1).
Figure 3:
TOCSY spectra showing the NH region of (a) linear ACM-blocked and (b) cyclic
N-pheTGF-[21-32]
recorded in 90% H
O/D
O. In (a),
spin systems have been identified according to residue using the single-letter code; prime (`) characters denote a
secondary conformation arising from the presence of proline cis and trans isomers, while additional peaks arising from
racemization of Cys
are identified as X. The
partial assignment in (b) highlights similarities with (a).
In contrast to the simple appearance of the linear peptides, the spectra of the cyclic peptides were substantially more complex (Fig. 3b). For each peptide, up to four resonances per proton were detectable. From these resonances, spin systems similar to the major (trans-prolyl) conformation in the spectra of the linear peptides could be conveniently located with little change in their relative proton chemical shifts. Similarly, resonances corresponding to the cis-prolyl conformation were also evident but the disulfide constraint induced greater resonance heterogeneity and discrete resonances were apparent for most backbone protons. The remaining peaks were suggestive of a separate family of conformations, with cis- and trans-pro subfamilies. Assignment of spin-systems for each of the conformations was hampered by the degree of degeneracy between chemical shifts, making unambiguous assignment of particular rOes difficult. Furthermore, the rOe intensity was poor due to the large number of different chemical shifts displayed by each proton, consequently very few long range (nonsequential) rOes were observed. Although a tentative sequential assignment of the major conformation of each peptide was possible, the number of rOe-derived constraints available was insufficient to restrict conformational space and so produce a reliable picture of the peptide conformation.
The
extent of the resonance heterogeneity in spectra of the cyclic
compounds was unexpected. We considered two possible explanations for
this: slow conformational exchange, perhaps as a result of disulfide
bond isomerization, or the presence of an impurity arising, for
example, from racemization of a chiral center. Although our careful
analytical procedures had indicated that the peptides were homogeneous,
disulfide bond isomerization is unlikely to lead to separate resonances
in view of previous reports which indicate that this is rapid in all
but the most severely constrained peptides(30, 31) .
This led us to consider the possibility of racemization. During Fmoc
peptide synthesis, racemization of the C-terminal residue during
coupling to certain resins is a minor problem(32) . However, it
has also been observed that when this residue is cysteine, further
racemization can occur during chain elongation(33) . Further
inspection of the NMR spectra of the linear compounds revealed two or
three weak cross-peaks (identified as X in Fig. 3a) which could not be readily explained by cis-trans isomerization at the prolyl residue. Comparison of
their chemical shifts and rOes (where visible) identified them with the
two C-terminal residues. For the cyclic compounds, we conclude that the
second family of resonances apparent in the NMR spectra represent trans- and cis-conformers of the TGF- peptide
containing a C-terminal D-cysteine residue. These are more
noticeable in the spectra of the cyclic compounds because the presence
of a disulfide constraint leads to a greater perturbation in the local
environment. Although separation of the D-cysteine containing
peptides was not possible, we estimated that our syntheses all
contained about 20% D-cysteine. Therefore, different ratios of D- and L-cysteine could not be the sole explanation
for any differences in biological activity between compounds.
The
peptides were tested for their ability to induce DNA synthesis in
murine fibroblasts stably transfected with the human EGF-R (NR6/HER
cells). None of the linear precursor peptides exhibited any biological
activity. In contrast, when constrained by introduction of an
intramolecular disulfide bond, the two peptides containing the proline
analogues, N-pheTGF-[21-32]
and N-pheTGF-
[21-32]
stimulated DNA synthesis with EC
values of 130 and
330 µM, respectively (Fig. 4a); the
equivalent L-Pro- or D-Pro-containing cyclic peptides
were inactive. When the N-pheTGF-
[21-
32]
and
N-pheTGF-
[21-32]
peptides were assayed alone, incorporation of label into DNA
approached that obtained with a maximal dose of TGF-
. In presence
of TGF-
, N-pheTGF-
[21-32]
produced an additive response (data not shown), but this did not
exceed the maximal level of stimulation achieved by TGF-
alone (Fig. 4b, left side).
Figure 4:
Mitogenic activity of cyclic
N-pheTGF-[21-32] peptides. a, the
activity of N-pheTGF-
[21-32] (
),
N-pheTGF-
[21- 32]
(
),
N-pheTGF-
[21-32]
(
),
and N-pheTGF-
[21- 32]
(
), was measured by incorporation of the thymidine
analogue,
I-UdR, into DNA in NR6/HER cells during S-phase
as described under ``Experimental Procedures.'' In this
experiment, maximal incorporation of label in the presence of TGF-
was 7.5
10
cpm. Similar results were obtained in
three separate assays. b, comparison of the mitogenic activity
of N-pheTGF-
[21-32]
(0.5
mg/ml), TGF-
(6 ng/ml, a maximally stimulating dose) and PDGF (2.5
IU/ml) on NR6/HER cells (hatched bars) or NR6 cells (cross-hatched bars). Basal incorporation of label was 600 and
2040 cpm in the NR6/HER and NR6 cells, respectively. Results are mean
of triplicate determinations.
Even though
N-pheTGF-[21-32]
elicited a mitogenic response, no detectable increase in EGF-R
phosphorylation was observed when the peptide was tested on the EGF-R
overexpressing squamous carcinoma cell line, HN5 (data not shown).
Therefore, to confirm that the peptide was acting via the EGF-R it was
tested on the parental NR6 cell line which lacks EGF-R. Fig. 4b shows that whereas both
N-pheTGF-
[21-32]
and
TGF-
were mitogenic for NR6/HER cells, neither stimulated DNA
synthesis in NR6 cells, even though these cells were responsive to
PDGF.
In recent years, generation of synthetic peptides with
improved properties for probing ligand:receptor interactions has been
greatly facilitated by use of modifications such as introduction of
unnatural amino acids and other conformational constraints to mimic
receptor bound ``bioactive'' conformations. By exploiting our
knowledge of the three-dimensional structure of TGF- and by use of
unnatural amino acids, we report for the first time the ability of an
analogue peptide based on the B-loop
-sheet segment of TGF-
to exhibit EGF-R activity. In relative terms, the peptide still
exhibits low activity by comparison with TGF-
(
10
times less active). However, given that residues from the
C-domain (Arg
and Leu
in TGF-
)
contribute about half of the binding free energy of the growth factor
with EGF-R(34, 35, 36, 37) , we
might reasonably expect that a B-loop peptide would have an affinity
10
times less than TGF-
(10
M
) and a correspondingly low
mitogenic activity.
The specificity of the peptides for the EGF-R
was evident from their ability to stimulate NR6/HER but not NR6 cells.
Furthermore, in the presence of varying doses of TGF-, induction
of mitogenesis by the peptides never exceeded the plateau level of
stimulation caused by maximal doses of TGF-
alone. Failure to
observe EGF-R autophosphorylation in response to the peptides may
reflect the fact that there is not an absolute requirement for receptor
phosphorylation to initiate mitogenic signaling(38) . Indeed,
EGF mutants with modified C-terminal tails also have impaired ability
to activate EGF-R autophosphorylation, even though they are still
mitogenically active (35) . For the TGF-
B-loop analogue
peptides, which completely lack this region of the growth factor, it is
perhaps not surprising that their ability to activate the kinase is
low.
We believe previous studies failed to generate bioactive
peptides because they used the entire TGF- B-loop sequence
constrained only by a disulfide bond in the native pairing (i.e. between cysteines two and four). To yield mitogenic compounds, we
found it necessary both to reduce the size of the peptide and to
introduce some additional conformational constraints. Our choice of
sequence, with a non-native disulfide bond between cysteines three and
four, was based on the observation that the N-domain is more properly
described as being formed by three segments (A, J (joining),
and B) rather than by the conventional two loop designation (i.e. A-loop and B-loop, see Fig. 1). The A-segment is comprised
of the part of the A-loop between the first and second cysteines, the
J-segment is common to the A- and B-loops and lies between the second
and third cysteines, while the B-segment forms the
-sheet in the
B-loop between the third and fourth cysteines (Fig. 5a). This definition emphasizes the importance of
the disulfide bonds which act as nodes from which the three segments
are presented (Fig. 5b). These nodes lie sufficiently
close together so that any one of the three segments can be
independently modelled by linking its ends to create a cycle. In the
case of the TGF-
peptides, we studied the B-segment which was
closed by a non-native disulfide bond.
Figure 5:
Diagrammatic representations of the
N-terminal domain of TGF-. In a, the chain path and the
residue numbers of the cysteines in the disulfide cross-links are
identified; b shows the topological equivalent of a,
illustrating the arrangement of the A-, J- and B-segments and the
location of the nodes.
Our data indicate that both
cyclization and substitution with prolyl mimetics were important for
activity. It seems likely that the beneficial effect of the cyclic
constraint arises from stabilization of peptide conformations more
suitable for binding to the EGF-R. In native TGF-, residues
21-32 form a
-sheet (8, 39) and this has
been implicated in receptor binding(11, 12) . However,
in our TGF-
peptides, the disulfide constraint appeared unable to
maintain an anti-parallel alignment of the peptide main chain as no
nonsequential rOes characteristic of this structural element were
evident in their NMR spectra. Nevertheless, a restrictive effect of the
disulfide was apparent, where it induced additional resonance
heterogeneity in the cyclic compared to the linear compounds.
The contribution of the bicyclic proline analogues to peptide activity may also be related to their effect on peptide conformation, mostly likely prolyl amide cis-trans isomerization. Differences in the degree of isomerization were evident in the linear compounds (Table 1) and the highest proportion of the cis isomer was observed in the precursor of the most active cyclic compound. Although NMR spectra of the cyclic compounds showed clear evidence for the cis prolyl isomer, quantitation of the ratio of trans:cis was precluded by the complexity of these spectra.
One problem which became evident from the NMR studies was the apparent racemization of the C-terminal cysteine. Although not frequently reported, this problem may be quite common in peptides with C-terminal cysteine, being particularly difficult to detect using conventional analytical quality control; mass spectroscopy cannot identify this impurity, and it represents the most stringent test for HPLC since it leads to inversion of the hydrogen and the carboxyl group at the C terminus where it has minimal effects on conformation. For our peptides, the degree of racemization was similar, as would be expected since they were synthesized under identical conditions and differed only in the presence of the individual proline analogues. Thus, it is unlikely that racemization per se could account for the differing activities of the peptides. It remains to be determined whether biological activity resides with one or both isoforms.
In
conclusion, we have shown that appropriately constrained B-segment
fragments of TGF- are mitogenically active on cells expressing
EGF-R. The relative activity of the peptides correlated with the
proportion of the cis-proline isomer in their linear
precursors and this may have contributed to their biological potency
after cyclization. Our observation that the ABHC proline mimetics can
be readily incorporated into peptides and used to enhance the
population of cis-propeptidyl bonds may have wider synthetic
applications in the development of other bioactive peptides.