(Received for publication, August 15, 1994; and in revised form, January 6, 1995)
From the
Small monomeric cyclic analogs that mimic the -turn regions
of nerve growth factor (NGF) were designed and synthesized. Potent
competitive antagonists were derived from the NGF
-turn C-D, which
inhibited [
I] NGF binding to TrkA receptors and
specifically inhibited optimal NGF-mediated neurite outgrowth in PC12
cells. The cyclic
-turn A`-A" analog also inhibited NGF binding to
TrkA receptors but with lower potency. These data indicate that
-turns C-D and A`-A" are critical for TrkA binding and may confer
neurotrophin receptor specificity. Furthermore, structural requirements
for binding are absolute, because unconstrained analogs derived from
the same regions had no effect. Compounds that mimic NGF will be useful
in deciphering the interactions of NGF and its receptors and in
rational drug design.
Nerve growth factor (NGF) ()is a polypeptide growth
factor member of the neurotrophin family, which includes brain-derived
neurotrophic factor, neurotrophin 3, and neurotrophin 4/5. Two cell
surface NGF receptors have been characterized on the basis of binding
affinity and signal transduction properties, namely the p75 low
affinity NGF receptor and p140 TrkA.
The p75 receptor (K = 10
M) (Radeke et al., 1987; Johnson et
al., 1986) is a 75-kDa glycoprotein member of the tumor necrosis
factor receptor/Fas/CD40 family of receptors (Itoh et al.,
1991). p75 contains no intrinsic catalytic activity but can associate
with the Erk family of soluble kinases (Volonté et al., 1993) and plays a role in protection from neuronal
apoptotic death (Rabizadeh et al., 1993). p75 is also the low
affinity receptor for brain-derived neurotrophic factor and
neurotrophin 3 (Rodriguez-Tebar et al., 1990, 1992), but these
latter growth factors each have distinct Trk receptors.
The p140
TrkA receptor is a 140-kDa glycoprotein encoded by the trk proto-oncogene (Kaplan et al., 1991; Klein et
al., 1991). Scatchard analysis of cells expressing only TrkA
receptors showed either a curvilinear plot with K values of
10
and 10
M (Jing et al., 1992) or a single K
of
10
M (Mahadeo et al., 1994). The TrkA receptor has intrinsic
tyrosine kinase activity and is capable of evoking cellular
neurotrophic responses in vitro in the absence of p75 low
affinity NGF receptor (Hempstead et al., 1991; Loeb and
Greene, 1993).
Co-expression of both p75 and p140 affords a K of
10
M (Hempstead et al., 1989; Bothwell, 1991; Jing et
al., 1992; Mahadeo et al., 1994). Hence, p75 can
associate with different Trk receptors to form high affinity binding
sites, but neurotrophin binding specificity is mediated by distinct Trk
receptors (Ip et al., 1993).
The structure of mouse NGF has
been resolved from crystallographic data at 2.3-Å resolution
(McDonald et al., 1991). In the crystals, the NGF molecule is
a tightly associated dimer made up of parallel protomers of 118 amino
acids. Each protomer has seven -strands forming three antiparallel
pairs. The
-strands are linked by four exposed regions: three
-turns (termed A`-A", A‴-B, and C-D) and one series of
three contiguous reverse turns (termed B-C).
The -turn and
reverse turn regions have been noted for their hydrophilic nature
(Meier et al., 1986; Ebendal et al., 1989), and,
unlike the mostly conserved buried residues of the
-strands, these
regions have little conservation between different neurotrophins
(Hallbook et al., 1991). The variability and hydrophilicity of
these turn regions have prompted the hypothesis that they may be
involved in determining neurotrophin receptor specificity, because
several dimeric molecules use
-turns as critical binding
surface(s). Similarly, antibodies and other members of the
immunoglobulin gene superfamily (Chothia and Lesk, 1987; De la Paz et al., 1986) and other globular proteins (Sibanda and
Thorton, 1985; Sibanda et al., 1989) use
-turns to
interact with complementary sequences with high affinity and
specificity.
Experimental evidence using mutagenesis and chimeric
molecules has sustained the hypothesis concerning -turns of NGF
(Ibáñez et al.,
1991, 1992, 1993). However, attempts to create analogs of the
-turns of NGF that mimic its activity have been less successful
(Longo et al., 1990; Murphy et al., 1993). Linear
peptides derived from NGF showed limited biological antagonistic
activity against suboptimal concentrations of NGF and did not affect
the binding of radiolabeled NGF. It is likely that the linear peptides
do not adopt the native
-turn structure from which they were
derived and that the peptides were sequence analogs rather than
structural analogs of NGF.
Several studies have contributed to defining important regions of the NGF molecule. However, the exact combination of amino acids of NGF and the structure(s) that participate in binding to p75 or TrkA receptors and cause biological effects remains to be determined. We have approached this question by producing structural NGF analogs after designing structural constraints to maintain the desired conformation (Saragovi et al., 1991, 1992; Saragovi and Greene, 1992; Chen et al., 1992).
Small
(average molecular weight 1,500) structural analogs of
-turns
or reverse turns of NGF were designed, synthesized, and constrained by
cyclization, whereas linear and randomized analogs served as controls.
Conformationally constrained analogs had significant NGF antagonistic
activity in biological and binding assays, and the analogs were used to
map receptor-ligand interactions. These data further support the
hypothesis that
-turns are critical in NGF binding to its
receptors and show that a large macromolecule such as NGF can be
reduced to small functional units if the structure of the native
molecule is retained.
Cyclic analogs C(92-96) and C(92-97), which were derived
from -turn C-D, and C(43-48) and C(44-48), which were
derived from
-turn A‴-B of NGF, demonstrated antagonistic
activity and significantly inhibited NGF-mediated PC12 cell neurite
projections (Fig. 1, C and E) but did not
affect bFGF-mediated responses (Fig. 1, D and F). In contrast, other cyclic analogs (e.g.
-turn A`-A" analog C(30-35); Fig. 1, G and H) and the linear or randomized analogs (data not
shown) did not affect either NGF- or bFGF-mediated neurite outgrowth
(for a summary see Table 2).
Figure 1:
Antagonistic activity of NGF analogs.
The effect of the analogs on NGF-dependent neurite outgrowth was
determined in PC12 cells. Cells were incubated with 50 ng/ml NGF (A, C, E, and G), with no growth
factor (A, inset), or with 50 ng/ml bFGF (B, D, F, and H). The indicated analogs were
then added to a concentration of 10 µM.
Representative results for three analogs are shown. For a summary and
complete list of the results from these biological tests see Table 2.
Controls ruled out toxicity and
demonstrated the specificity of the analogs as antagonists of NGF.
First, no cell death was observed after culture with the compounds
(exceptions that did cause necrosis were C(29-35D30A) from
-turn A`-A" and C(61-66) from
-turn B-C; they were not
used further). Second, when the analogs were removed PC12 cells
responded normally to NGF. Third and most importantly, the NGF analogs
did not affect PC12 cell neurite outgrowth in response to bFGF,
suggesting that the analogs were specific for NGF receptors.
Two of the inhibitory cyclic analogs, C(32-35) and C(44-48), caused enlargement of the cell size and clumping (Table 2). However, non-inhibitory analogs L(42-49) and R(59-67) caused a similar effect, suggesting that this was unrelated to activity. We continue to investigate the significance of this observation.
Analogs from the C-D
-turn region were effective in inhibiting
[
I]NGF binding to E25, R7, and PC12 cells.
Inhibition of binding was roughly comparable with that obtained with
0.1 µM unlabeled NGF (Table 3). These analogs were
more efficient at inhibiting NGF binding to E25 cells than to R7 cells.
Furthermore, although analogs C(92-96) and C(92-97)
inhibited NGF binding to E25 cells to similar degrees (71.3 and 64.9%,
respectively), C(92-96) was more effective than C(92-97) in
blocking NGF binding to R7 cells (51.1 versus 24.3%,
respectively).
Analogs C(30-35) and C(32-35) that were
derived from the -turn A`-A" region inhibited NGF binding to E25
cells (albeit less efficiently than the C-D region analogs) but did not
affect NGF binding to R7 cells. This suggests that the A`-A" region
binds to TrkA receptors that are not in association with p75 or that
p75 association to TrkA changes the conformation of TrkA such that the
binding site for the analog is not available or stable.
None of the
other cyclic, linear, or randomized analogs had significant effects in
[I]NGF binding (e.g. C(43-48) or
L(91-99); Table 3). Note that two biologically active
cyclic analogs (A‴-B region analogs C(43-48) and
C(44-48), Table 2) did not inhibit
[
I]NGF binding in these assays; they may
function through a different mechanism.
Dose-response studies using increasing amounts of analogs
(0.4-200 µM) or unlabeled NGF (4 nM to 2
µM) were performed in the presence of a constant 200
pM concentration of [I]NGF (Fig. 2A). Averages of similar experiments showed for
analog C(92-96) an IC
of 23.5 ± 16 µM compared with that of unlabeled NGF (IC
= 2.65
± 0.35 nM).
Figure 2:
Competitive inhibition of
[I]NGF binding to TrkA by designed NGF analogs.
Saturation binding assays were performed with TrkA receptor-expressing
E25 cells as described under ``Materials and Methods.'' A, increasing concentrations of inhibitors were tested for
their ability to inhibit a constant amount of
[
I]NGF (200 pM). B, a
constant amount of inhibitors (
45 µM C(92-96)
analog or a 2000-fold excess of unlabeled NGF) was added to increasing
concentrations of
[
I]NGF.
Saturation analyses using increasing
concentrations of [I] NGF (0-1.13
nM) versus a constant concentration of analog
C(92-96) (45 µM) or a 2,000-fold excess of unlabeled
NGF were performed (Fig. 2B). NGF receptor saturation
by [
I]NGF was displaced by both the analog and
the unlabeled NGF by reducing receptor availability rather than
receptor affinity, suggesting that the inhibition is of a competitive
nature.
Structural requirements for the binding of NGF to its defined
cell surface receptors were studied. To investigate the role of
-turn regions of NGF, we used the approach of designing and
synthesizing small cyclic structural analogs of these regions to be
used as probes. We hypothesized that cyclic analogs that conserve and
closely mimic the three-dimensional structure of the
-turn regions
might bind to NGF receptors. In contrast, their linear counterparts
would seldom adopt the appropriate configuration required to fit the
ligand docking site.
The data presented are the first direct
demonstration of the involvement of a defined -turn region of a
polypeptide ligand in binding to a defined neurotrophin receptor.
Overall, these data provide support for the notion that
-turn
regions are crucial for certain ligand-receptor interactions, and this
concept may now be applied to other members of the neurotrophin family.
Cyclic analogs C(92-96) and C(92-97) derived from the
C-D -turn region of NGF were potent antagonists for TrkA
receptors. Saturation binding and classical binding displacement
experiments indicated that the antagonism was of a competitive nature.
We have estimated that the C(92-96) analog has an affinity of
10
M for TrkA receptors. C-D region
analogs C(92-96) and C(92-97) differ by one Ala. This
difference alters the loop size, the type of
-turn mimic, and
possibly the orientation of amino acid side chains, changes likely to
confer the increased activity against TrkA seen for C(92-96)
compared with (C92-97).
Analogs derived from the -turn
A`-A" region also inhibited NGF binding to TrkA-expressing E25 cells,
albeit with lower potency than
-turn analogs derived from the C-D
region. We have not yet measured the IC
of A`-A" region
analogs, but we expect them to be of lower affinity. A`-A" region
analogs did not affect NGF binding to cells that express both p75 and
TrkA at all, suggesting that they only bind TrkA receptors that are not
in association with p75.
Lack of inhibition by A`-A" region analogs on cells expressing both p75 and TrkA can be the result of higher receptor affinity for the ligand. Additionally, a receptor conformational change or masking of the docking site can occur upon heterodimerization of p75 and TrkA (Mahadeo et al., 1994; Verdi et al., 1994). Theoretical models of functional NGF receptors (Bothwell, 1991; Chao, 1992; Klein et al., 1991; Jing et al., 1992) are consistent with the possibility that the Trk-docking site of the analogs may be masked upon association of p75 and TrkA, but concomitant p75 binding by the analogs could not be formally ruled out.
Analogs C(43-48) and C(44-48)
derived from NGF -turn A‴-B were effective in inhibiting
biological assays in PC12 cells without being effective at all in
binding assays in E25 cells. The biological effects of C(43-48)
must be mediated by the NGF receptor, because bFGF was not affected.
Perhaps this analog can prevent TrkA receptor dimerization, TrkA
receptor internalization, or NGF stability without actually affecting
NGF binding. Another explanation is that this inconsistency reflects
differences between rat and human TrkA receptors, which are expressed
in neuronal or fibroblastoid cell lines, respectively. Inconsistencies
between biological and binding responses in transfected fibroblasts versus PC12 cells have been reported (Ip et al.,
1993). The mechanism of inhibition by NGF
-turn A‴-B
analogs will be resolved by further studies.
Taken together, these
data demonstrate that cyclic sequence analogs of NGF -turns C-D
and A`-A" likely mimic the native architecture and are therefore able
to bind to TrkA receptors. Secondary structure requirements for
antagonistic activity proved to be absolute because linear and random
compounds with primary sequences from all
-turns had no effect at
the concentrations tested. Thus, the conformation of the analog must
retain some if not many of the features found in the original ligand.
This experience is emphasized by previous reports of low affinity or
inactive linear analogs of NGF (Longo et al., 1990; Murphy et al., 1993).
Analogs derived from -turn C-D
inhibited neurite outgrowth induced by NGF in PC12 cells and inhibited
NGF binding to several receptor-expressing cells. Because all
neurotrophins (except NGF) have an extra amino acid in
-turn C-D,
binding specificity of NGF for TrkA may be partly explained
(Ibáñez et al.,
1993). This region is likely to confer added specificity to NGF in
binding to TrkA and perhaps to other neurotrophins in binding to their
specific high affinity receptors.
It is unlikely that the mechanism of biological antagonism was to hinder NGF but not bFGF signal transduction, because transduction is Ras-dependent for both ligands (Kremer et al., 1991). Therefore, the analogs mediate their action by directly binding to the extracellular domain of NGF receptors, and in this regard they are different than K252 molecules, which mediate their action by inhibition of the kinase activity of TrkA (Berg et al., 1992).
NGF analogs did not behave as agonists
of PC12 cells. For agonistic activity the ligand must either engage
more than one site on a given receptor or possess the ability to induce
receptor dimerization (Bernd and Greene, 1984). Because the analogs are
structurally equivalent to only one -turn region of NGF, they
would not induce receptor dimerization and would be expected to behave
as antagonists in biological assays. Preliminary studies testing
combined analogs from NGF
-turns A`-A" and C-D showed additive
(but not synergistic) effects in NGF binding assays (data not shown).
We expect that appropriate coupling of these analogs as homodimers or
heterodimers will likely reveal synergy or agonistic function.
Previous studies have implicated the variable domains in the loop regions we have studied as mediators of binding and biological activity (Ibáñez et al., 1993). In addition, the amino terminus of NGF comprising amino acids 1-9 was also implicated in binding to TrkA receptors (Kahle et al., 1992; Nanduri et al., 1994; Ibáñez et al., 1993). However, the amino termini are highly susceptible to proteolytic cleavage (Server and Shooter, 1977), and this region was not resolved crystallographically (McDonald et al., 1991).
The study of the mechanism of binding by the analogs to cells that express only TrkA versus cells that express both p75 and TrkA will provide more information concerning receptor-ligand interactions. Furthermore, by creating homodimeric and heterodimeric forms of the analogs described herein, we will attempt to generate agonistic ligands that permit receptor dimerization.