©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Small Peptide Mimics of Nerve Growth Factor Bind TrkA Receptors and Affect Biological Responses (*)

(Received for publication, August 15, 1994; and in revised form, January 6, 1995)

Lynne LeSauteur (§) Ling Wei Bernard F. Gibbs (1) H. Uri Saragovi (¶)

From the Department of Pharmacology and Therapeutics and McGill Cancer Centre, McGill University, Montreal, Quebec H3G 1Y6 and the Biotechnology Research Institute, Montreal, Quebec H4P 2R2, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Small monomeric cyclic analogs that mimic the beta-turn regions of nerve growth factor (NGF) were designed and synthesized. Potent competitive antagonists were derived from the NGF beta-turn C-D, which inhibited [I] NGF binding to TrkA receptors and specifically inhibited optimal NGF-mediated neurite outgrowth in PC12 cells. The cyclic beta-turn A`-A" analog also inhibited NGF binding to TrkA receptors but with lower potency. These data indicate that beta-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.


INTRODUCTION

Nerve growth factor (NGF) (^1)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 = 10M) (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 10M (Jing et al., 1992) or a single K of 10M (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 10M (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 beta-strands forming three antiparallel pairs. The beta-strands are linked by four exposed regions: three beta-turns (termed A`-A", A‴-B, and C-D) and one series of three contiguous reverse turns (termed B-C).

The beta-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 beta-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 beta-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 beta-turns to interact with complementary sequences with high affinity and specificity.

Experimental evidence using mutagenesis and chimeric molecules has sustained the hypothesis concerning beta-turns of NGF (Ibáñez et al., 1991, 1992, 1993). However, attempts to create analogs of the beta-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 beta-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 beta-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 beta-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.


MATERIALS AND METHODS

Peptide Synthesis and Characterization

Mouse NGF analogs were synthesized using standard Fmoc synthetic procedures (Fields and Noble, 1990). To synthesize the large number of analogs required, the multi-pin cleavable peptide synthesis system (Chiron Mimotopes, Australia) was used. This method allows simultaneous synthesis of 96 peptides on diketopiperazine linker-derivatized polyethylene pins. Cleavage was performed under mild base conditions using 0.15 M ammonium bicarbonate, pH 8.4. All peptides were synthesized and tested either with the amino termini acetylated or with the Fmoc group intact and with the diketopiperazine linker present at the carboxyl terminus. For cyclization, NH(3)-X-Cys and Cys-X-COO were added to the termini of the indicated peptides, where X represented Gly when Fmoc amino termini were present and represented Tyr or His when they were absent (see Table 1). Following cleavage the peptides were cyclized by oxidation of the Cys residues. Peptides were lyophilized, resuspended in water, and purified in a Waters high pressure liquid chromatograph (0.1% trifluoroacetic acid and 10-40% acetonitrile gradient for 40 min) using a Vydac 218TP C18 (1 times 25 cm) column. All peptides were characterized by amino acid analysis (Beckman model 6300 analyzer) and by mass spectrometry (SCIEX API III).



Cell Cultures

PC12 pheochromocytoma cells (Greene and Tischler, 1976) were grown in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum, 10% horse serum, and antibiotics (Life Technologies, Inc.). PC12 cells used for experiments were grown on collagen-coated dishes. The NIH-3T3 fibroblast transfectants R7 (p75 and p140 trkA double transfectant) and E25 (p140 trkA transfectant) (Jing et al., 1992) were grown in RPMI 1640 medium supplemented with 5% fetal bovine serum, antibiotics, and the appropriate drug selection.

Neurite Outgrowth Assay

Analogs were tested for their ability to affect NGF-induced neurite outgrowth in PC12 cells. PC12 cells were incubated in complete media containing optimal concentrations of NGF (50 ng/ml; approximately 2.0 nM) or basic fibroblast growth factor (bFGF) (50 ng/ml; Prince Labs, Toronto, Canada). NGF analogs or control analogs were then added to a final concentration of 10 µM, cells were incubated, and neurite outgrowth was measured every 24 h for 3 days. All analogs were tested in at least three separate assays from three different syntheses. Samples were coded, and analyses were performed blind.

NGF Binding Studies

Analogs were tested for their ability to affect binding of [I]NGF (73.1 µCi/µg; DuPont NEN). 0.4-1 times 10^6 cells were incubated in binding buffer (Hanks' balanced salt solution, 1% bovine serum albumin, 0.05% sodium azide, pH 7.4) at 4 °C with the indicated concentration of cyclic NGF analogs, control analogs, a titration of unlabeled NGF, or nothing. After 15 min, [I]NGF was added to a final concentration of 0.4-2 nM (40,000 cpm), and the mixtures were further incubated for 40 min at 4 °C. Cells were then washed in binding buffer and pelleted, and cell-associated [I]NGF was determined. Background binding was measured by the addition of 2000-fold excess unlabeled NGF, which resulted in less than 10% of maximum binding, or by measuring binding to NGF receptor negative NIH-3T3 cells (5%) (data not shown). Assays were performed >4 times, each from at least three different syntheses (inhibitory analogs), or 1-3 times, each from two different syntheses (non-inhibitory analogs). Receptor saturation experiments were similarly performed with increasing concentrations of [I]NGF ligand and a constant dose of inhibitor.


RESULTS

Design and Synthesis of NGF Analogs

Four regions of the NGF primary sequence were defined that differ significantly from the primary sequence of other neurotrophins, corresponding to amino acid numbers 28-36 (beta-turn A`-A"), 42-49 (beta-turn A‴-B), 59-67 (turn B-C), and 91-99 (beta-turn C-D) of mouse NGF (Table 1). Cyclic peptides were synthesized, and for each a linear (unconstrained sequence) control and a scrambled (randomized sequence) control were also made.

Inhibition of NGF Function in Biological Assays

Rat PC12 pheochromocytoma cells in culture can differentiate and produce neurites in response to NGF or bFGF. None of the analogs were able to induce neurite projections when added to PC12 cell cultures, suggesting that by themselves they lacked NGF agonistic activity at the concentrations used in these assays (data not shown).

Cyclic analogs C(92-96) and C(92-97), which were derived from beta-turn C-D, and C(43-48) and C(44-48), which were derived from beta-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. beta-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(29-35DeltaD30A) from beta-turn A`-A" and C(61-66) from beta-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.

Direct Inhibition and Mapping of NGF Binding Sites

To determine the mechanism of biological antagonistic activity by the cyclic analogs, [I]NGF binding measurements were performed with cell lines expressing either both p75 and TrkA receptors (PC12 cells and R7 transfectants) or only TrkA receptors (E25 transfectants) (Table 3). Analogs were used at a concentration of 40 µM.



Analogs from the C-D beta-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 beta-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.

C-D Region Analogs Are Competitive Inhibitors of NGF by Binding TrkA Receptors

In order to determine the nature of the inhibition of [I]NGF binding to TrkA receptors by the C-D region analogs, we performed dose-response and saturation studies.

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.


DISCUSSION

Structural requirements for the binding of NGF to its defined cell surface receptors were studied. To investigate the role of beta-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 beta-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 beta-turn region of a polypeptide ligand in binding to a defined neurotrophin receptor. Overall, these data provide support for the notion that beta-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 beta-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 10M 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 beta-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 beta-turn A`-A" region also inhibited NGF binding to TrkA-expressing E25 cells, albeit with lower potency than beta-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 beta-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 beta-turn A‴-B analogs will be resolved by further studies.

Taken together, these data demonstrate that cyclic sequence analogs of NGF beta-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 beta-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 beta-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 beta-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 beta-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 beta-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.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council of Canada, the National Cancer Institute of Canada, and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (Québec) (to H. U. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Medical Research Council of Canada.

To whom correspondence should be addressed: 3655 Drummond St. #1314, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-3628; Fax: 514-398-6690; uri{at}pharma1.pharma.mcgill.ca.

(^1)
The abbreviations used are: NGF, nerve growth factor; Fmoc, N-(9-fluorenyl)methoxycarbonyl; bFGF, basic fibroblast growth factor.


ACKNOWLEDGEMENTS

We are grateful to Dr. M. Barbacid (Brystol-Myers-Squibb) for the gift of E25 and R7 cell lines, to Drs. M. Szyf and P. B. S. Clarke (McGill) for reviewing this manuscript, and to N. Lavine for technical assistance.


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