|Home | About | Journals | Submit | Contact Us | Français|
Neurotrophins are a family of growth factors that regulate the peripheral and central nervous system. We designed and tested a mini-library of small molecules peptidomimetics based on β-turns of the neurotrophin growth factor polypeptides NT-3, which is the natural ligand for TrkC receptors. Biological studies identified a peptidomimetic 2Cl that exhibited selective antagonism of TrkC. 2Cl reduces TrkC-activation and signaling promoted by NT-3, and selectively blocks ligand-dependent cell survival. 2Cl also blocks ligand-independent TrkC activation and signals that take place when the receptor is over-expressed. This work adds to our understanding of how the neurotrophins function through Trk receptors, and demonstrates that peptidomimetics can be designed to selectively disturb neurotrophin-receptor interactions, and receptor activation.
Neurotrophins are dimeric polypeptide growth factors that regulate the peripheral and central nervous systems and other tissues. Neurotrophins (Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), and Neurotrophin-3 (NT-3)), as well as their cell surface receptors (p75, TrkA, TrkB, and TrkC) are validated targets for therapeutics in a variety of pathologies ranging from cancer to neurodegeneration [19, 20].
All the neurotrophins bind the p75 receptor , a member of the tumor necrosis factor (TNF) receptor superfamily. The p75 receptor has multiple functions depending on the cells in which it is expressed, whether a ligand engages it, and many other factors [3, 4]. This makes its function difficult to predict and control pharmacologically.
On the other hand, many neurotrophic activities arise from ligand binding preferentially and with selectivity to the Trk family of receptors. For example, NGF docks with TrkA, BDNF binds preferentially to TrkB [1, 21], whereas NT-3 interacts preferentially with TrkC but can also bind to TrkA . Trk receptors are typical receptor tyrosine kinases (RTKs), with an ectodomain, a single transmembrane region, and an intracellular tyrosine kinase catalytic domain. The extracellular domain of Trk binds the ligand, leading to activation of the tyrosine kinase, phosphorylation (pTyr) of the Trk intracellular domain, and signal transduction cascades involving associated partners such as MAPK and Akt .
However, over-expression of Trk receptors can also cause activation of signals in a ligand-independent manner . This has been reported for Trks and for many other RTKs, and over-expression is thus pro-oncogenic [19, 20]. Thus it would be important to develop Trk antagonists as tools to better understand how these receptors function.
Previously we used protein mimicry of neurotrophin β-turns to develop β-turn cyclic peptides and organic peptidomimetics of neurotrophins [2, 11, 12, 14, 15]. To date, we have reported on selective agonists for TrkA  or TrkC . Here, we sought to develop antagonists by mimicry of NT-3. We designed a library of peptidomimetics with an organic backbone, and a pharmacophore based on β-turn NT-3 structure. The aim was to target the extracellular domain of the TrkC and TrkA receptors, because NT-3 can bind to both TrkC and TrkA .
The resulting new agents were tested in biological screens which lead to the identification of a small molecule NT-3 peptidomimetic 2Cl that is a selective antagonist of TrkC receptors, but which does not affect TrkA receptors. This antagonist blocks ligand-dependent as well as ligand-independent TrkC activity. This reagent may help us understand how the neurotrophins function via Trk receptors.
For a view of the peptidomimetic structures see Figure 1. The pharmacophores are described in Table 1. The syntheses protocols of the compounds are published [8, 9, 24], and are described below in sections 1.1.1, 1.1.2, and 1.1.3; and schematically in scheme 1 and scheme 2.
Polystyrene resin functionalized with a 4-hydroxybenzyl linker (800 mg, 1.30 mmol/g, 1.04 mmol), template 1 (500 mg, 1.1 mmol), diisopropylcarbodiimide (172 mg, 1.1 mmol), HOBt (148 mg, 1.1 mmol), and N,N-diisopropylethylamine (348 µL, 2.0 mmol) in 8 mL of 4:1 dichloromethane/N,N-dimethylformamide were gently stirred at 25 °C for 12 h. The resin was filtered, washed, and treated with 10 equiv of SnCl2•2H2O in DMF for 1 day. After washing and drying, the loading of the resin was determined to be 0.78 mmol/g by UV analysis (405 nm, ε = 37000) of the trityl content of a known amount of resin in 10% trifluoroacetic acid in dichloroethane. Subsequent couplings of amino acids were performed following the general procedure given for peptidomimetics below.
Rink resin (1.4 g, 1.0 mmol/g) was swelled in CH2Cl2 (10 mL/g) in a fritted syringe for 30 min. The FMOC group on the resin was removed by treating the resin with 20% piperidine in DMF (2×5 mL, 10 min and then 15 min). The resin was washed, after which templates 4 (3 equiv), HBTU (3 equiv), HOBt (3 equiv), and DIEA (5 equiv) in DMF (2.0 mL) were added. After 1 h of gentle shaking, a ninhydrin test on a small sample of beads gave a negative result. The reaction mixture was drained, and the resin was subjected to the above washing cycle. The loading of the resin was determined to be 0.46 mmol/g by UV analysis.
The resin containing template 1 was treated with FMOC-Lys-(Boc)-OH (4 equiv), PyBroP (4.8 equiv), and 2,6-lutidine (15 equiv) in CH2Cl2 (1.5 mL) for 24 h. After washing and Fmoc deprotection, the resin was then treated with FMOC-Gly-OH (3 equiv), PyBOP (3 equiv), HOBt (3 equiv), and DIEA (5 equiv) in DMF (3 mL) for 5 h. The washing cycle and Fmoc deprotection were repeated, and 2-fluoro-5-nitrobenzoic acid moiety was introduced by treating the resin with 2-fluoro-5-nitrobenzoic acid (3 equiv) PyBOP (3 equiv), HOBt (3 equiv), and 2,6-lutidine (15 equiv) in CH2Cl2/DMF (1:1, 3 mL) for 5 h. The side chain protecting group (Trt) of the template was removed by treatment with 1% TFA and 5% HSiiPr3 in CH2Cl2 (7×2 min, or until color disappeared). After the resin was washed, the macrocyclization step was carried out by treating the supported peptide with K2CO3 (10 equiv) in DMF at 25 °C. After gentle shaking for 2 d, the peptide-resin was washed then dried in vacuo for 4 h. The peptide was cleaved from the resin by treatment with a mixture of 90% TFA, 5% HSiiPr3, and 5% H2O for 3 h. The cleavage solution was separated from the resin by filtration. After most of the cleavage cocktail (about 90%) was evaporated in vacuo, the crude peptide was triturated using anhydrous ethyl ether, dissolved in H2O, and then lyophilized to give the crude product. Preparative HPLC (Beckman System, 10–80% MeCN in H2O + 0.1% TFA in 40 min) was carried out to provide a yellowish powder of 2Ag (5 mg, 43%) obtained as an ammonium salt. In general, all the products were in their protonated forms.
NIH-3T3 cells are mouse fibroblasts that do not express any neurotrophin receptors. Parental NIH-3T3 cells were stably transfected with the indicated receptors, using plasmids also encoding for neomycin resistance. After long-term neomycin drug selection and sub-cloning, the cells were characterized to express the appropriate functional cell surface receptor. Stable clones of NIH-TrkC express ~100,000 TrkC receptors/cell, NIH-TrkA express ~200,000 TrkA receptors/cell, and NIH-IGF-1R express ~100,000 IGF-1R receptors/cell. These cells, and their functional responses to the appropriate growth factor have been reported .
Cell survival was measured quantitatively by the MTT assay and optical density (OD) readings, as previously described [14, 15], in 96-well plates. Cells in SFM die by apoptosis, but can be rescued if supplemented with the appropriate growth factor if they express functional receptors. Wells were not supplemented (negative control), or were supplemented with suboptimal or optimal concentrations of the indicated growth factor. NIH-TrkA cells respond to NGF, NIH-IGF-1R cells respond to IGF-1, and NIH-TrkC cells respond to NT-3. Test peptidomimetics or vehicle control were added to the two culture conditions above, namely, in SFM or SFM supplemented with growth factor. Cellular controls are provided by lack of effect on NIH-TrkA or NIH-IGF-1R with effect on NIH-TrkC, as evidence of selectivity. Lack of general toxicity was assessed by testing compounds on all cells growing in normal serum conditions (data not shown). All assays were repeated independently at least three times (n = 4 for each assay). MTT data are standardized to optimal dose of neurotrophin = 100% survival, and SFM = 0% survival, using the formula [(ODtest − ODSFM) × 100/(ODoptimal NTF − ODSFM)].
Cells were stimulated with solvent (negative control), optimal concentrations of growth factor alone (positive control) or growth factor plus 2Cl at 50 µM as indicated. Detergent lysates were resolved in SDS-PAGE and analyzed by Western blotting with anti-phosphotyrosine (pTyr) antibody 4G10 (Upstate Biotechnology, Lake Placid, NY), or anti-phospho-MAPK (New EnglandBiolabs), or anti-phospho-Akt (Ser473) antibody (New EnglandBiolabs). After stripping, membrane was rebloted with anti-actin (Sigma) to gauge protein loading. Quantification was done by densitometric analysis . For receptor pTyr assays the stimulation was for 10 min. For phospho-Akt assays stimulation was for 5 or 15 min as indicated. For phospho-MAPK assays ligand stimulation was for 15 min.
We designed a library of 12 peptidomimetics based on the β-turn structure of NT-3 (Figure 1). Their synthesis [8, 9] and the codes  used to name these compounds were based on our previous literature. The code contains a number, and two letters.
The number of the code corresponds to the ring closure (containing an S or an O), the first letter of the code corresponds to the appendix (-NHSO2 or -NH2), and the second letter of the code corresponds to the dipeptide side chains displayed at the β-turn. For an abbreviated summary of the dipeptide see Table 1. The compounds were purified, and characterized by 1D-NMR and LC-MS (data not shown).
12 peptidomimetics were tested in assays of cell survival, using the quantitative MTT method. Cells undergo apoptotic death when cultured in serum-free medium (SFM). NGF protects TrkA-expressing cells, IGF-1 protects IGF-1R-expressing cells, while NT-3 protects TrkC-expressing cells from apoptotic death in SFM. Growth-factor protection in SFM is dose-dependent and time-dependent. Optimal concentrations of growth factor afford maximal protection (2 nM, 100%). Suboptimal concentrations of growth factor (0.2 nM) protect at ~30% of maximal (in a 3 day bioassay) or at ~60 % of maximal (in a 2 day bioassay).
First, the peptidomimetics were tested for their effect on NGF or NT-3-mediated survival. From the 12 compounds, 2Cl significantly lowered the survival induced by NT-3 (Figure 2A). In selectivity controls, 2Cl had no effect on the survival of TrkA-expressing cells responding to 2 nM NGF, and did not even affect sub-optimal doses of NGF either (Figure 2B).
Further survival assays were performed to test dose-dependent effects of 2Cl (5, 10, 20 µM) on the metabolism of NIH-TrkC, NIH-TrkA or NIH-IGF-1R (Figure 3). These assays were performed in the presence of growth factor to test for antagonism of ligand-dependent signals (as above), and also in the absence of growth factor to test the effects of the compounds on ligand-independent receptor activity.
In the absence of neurotrophins 2Cl at 10 µM or at 20 µM significantly reduced the metabolism of NIH-TrkC cells (p < 0.05 versus untreated control); but had no effect on NIH-TrkA or NIH-IGF-1R (Figure 3). These data suggest that 2Cl antagonizes ligand-independent TrkC activity selectively. Lack of effects on the growth of NIH-TrkA or NIH-IGF-1R, and lack of general toxicity when cells are grown in normal serum conditions (data not shown) suggest that the reduced metabolic activity of NIH-TrkC may be due to reduced TrkC signaling.
The same dose-dependent assays were performed in the presence of neurotrophins; to further test the effects of the compounds on ligand-dependent receptor activity. The NT-3 induced survival of NIH-TrkC cells was also blocked significantly by 2Cl at 10 µM or at 20 µM (p < 0.05 versus untreated control). These doses of 2Cl had no effect on the NGF-induced survival of NIH-TrkA or on the IGF-1-induced survival of NIH-IGF-1R (Figure 3). These data suggest that 2Cl antagonizes ligand-dependent TrkC signaling selectively.
To confirm the antagonistic activity of 2Cl we analyzed signal transduction biochemically (Figure 4). The tyrosine phosphorylation of the receptors (Figure 4A), the phosphorylation of Akt (Figure 4B), and the phosphorylation of MAPK (Figure 4C) were studied in western blots of cell lysates, after stimulation of cells with the appropriate growth factor ± 2Cl.
In NIH-TrkC cells NT-3 induces strong TrkC-pTyr, and the phosphorylation of Akt and MAPK; indicating activation. 2Cl decreases these signals. Inhibition was significant for all treatments (p < 0.05 versus NT-3 stimulated control). The only exception was that the inhibition of phospho-Akt was significant versus control for the 15 minute of stimulation with NT-3, but not for the time point of 5 minute of stimulation with NT-3. In selectivity control assays, 2Cl does not reduce the activating signals that NGF affords through TrkA, or the activating signals that IGF-1 affords through IGF-1R. These data indicate that an NT-3 derived peptidomimetic 2Cl can inhibit NT-3-dependent activation and tyrosine phosphorylation of TrkC and of signals downstream of TrkC; and these data are consistent with the effects reported on ligand-dependent survival bioassays.
As well, the inhibition of ligand-independent receptor pTyr by 2Cl was tested in biochemical assays (Figure 4D). A fraction of the TrkC or the TrkA receptors in transfected NIH-3T3 cells are tyrosine phosphorylated in the absence of ligand activation. Treatment of the cells with 2Cl for 15 minutes at 37°C reduced the pre-existing levels TrkC-pTyr, but did not affect the pre-existing levels of TrkA-pTyr; relative to actin control. These data were reproduced in three independent assays. However, these biochemical data are not quantified by densitometry because to reveal the ligand-independent Trk-pTyr the X-ray films are generally over-exposed and outside the linear range. Nevertheless, these data does show qualitatively that 2Cl selectively antagonizes ligand-independent TrkC activation; and the data are consistent with the effects reported on ligand-independent survival bioassays.
Here we report on the biological characterization of a library of twelve small molecule peptidomimetics designed to mimic NT-3, one of which termed 2Cl, acts as a selective TrkC antagonist in bioassays and in biochemical assays. Antagonism of TrkC by 2Cl was evident in inhibiting ligand-independent activity (which is often oncogenic) as well as a more conventional antagonism of NT-3–dependent activity.
The easiest interpretation is that 2Cl antagonizes receptor signals by binding to TrkC selectively. However, we have no direct binding data to TrkC, as we failed to obtain labeled ligands (either 2Cl or NT-3) for binding studies. Thus, we can not formally rule out that 2Cl could potentially bind to NT-3 itself and neutralize it, therefore causing antagonism. Against that possibility, however, we show that 2Cl selectively antagonizes ligand-independent TrkC activity (e.g. in the absence of growth factor); thus at least some of the activity takes place at the TrkC receptor. Antagonism of ligand-dependent activation can be most easily explained in terms of competitive antagonism, meaning that the small molecule prevents the binding of the natural ligand to the receptor. This could be anticipated from an NT-3 mimetic.
However, in previous work we made NT-3 mimetics with agonistic activity [8, 9, 24]. These mimetics have a different pharmacophore than 2Cl, and can bind to and activate both TrkC and to TrkA. While this is consistent with the ability of NT-3 to bind to and activate both receptors , the selectivity of 2Cl suggests that it interacts functionally only with TrkC. Hence, it seems that the unique 2Cl pharmacophore is more restricted compared to the other mimetics. In addition, 2Cl is an antagonist, whereas the other agents are partial agonists.
The mechanism by which small molecule 2Cl may antagonize the ligand-independent oncogenic activation of a RTK is intriguing. It is speculated that over-expressed RTKs, including TrkC, may require lateral or rotational mobility within the membrane to become activated [10, 17, 18, 22]. It is possible that 2Cl restricts this mobility thereby causing antagonism. It is also conceivable that 2Cl prevents events that are required for receptor activation such as receptor-receptor dimerization, or dimer stabilization, or receptor conformational changes [6, 7].
This compound is further proof that small molecule mimetics can be developed from neurotrophin β-turns. While previous work using this strategy resulted in peptidomimetics with partial agonistic activity, the present work is the first example of a TrkC antagonist.
Grants form the Canadian Institutes of Health Research (to HUS) and the National Institutes of Health (MH070040, GM076261, to KB) funded this work. TAMU/LBMS-Applications Laboratory headed by Dr Shane Tichy provided mass spectrometric support. The NMR instrumentation in the Biomolecular NMR Laboratory at Texas A&M University was supported by a grant from the National Science Foundation (DBI-9970232) and the Texas A&M University System.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.