Bivalent Peptidomimetic Ligands of TrkC Are Biased Agonists and Selectively Induce Neuritogenesis or Potentiate Neurotrophin-3 Trophic Signals

Abstract

This study was initiated to find small molecule ligands that would induce a functional response when docked with neurotrophin Trk receptors. "Minimalist" mimics of β-turns were designed for this purpose. These mimics are: (i) rigid, yet easily folded into turn-like conformations, and (ii) readily accessible from amino acids bearing most of the natural side chains. Gram quantities of sixteen of these turn mimics were prepared, then assembled into 152 fluorescein-labeled bivalent peptidomimetics via a solution-phase combinatorial method. Fluorescence-based screening of these molecules using cells transfected with the Trk receptors identified 10 potential ligands of TrkC, the receptor for neurotrophin-3 (NT-3). Analogs of these bivalent peptidomimetics with biotin replacing the fluorescein label were then prepared and tested to confirm that binding was not due to the fluorescein. Several assays were conducted to find the mode of action of these biotinylated compounds. Thus, direct binding, survival and neuritogenic, and biochemical signal transduction assays showed 8 of the original 10 hits were agonistic ligands binding to the ectodomain of TrkC. Remarkably, some peptidomimetics afford discrete signals leading to either cell survival or neuritogenic differentiation. The significance of this work is three fold. First, we succeeded in finding small, selective, proteolytically stable ligands for the TrkC receptor; there are very few of these in the literature. Second, we show that it is possible to activate distinct and biased signaling pathways with ligands binding at the ectodomain of wild type receptors. Third, the discovery that some peptidomimetics initiate different modes of cell signaling increases their potential as pharmacological probes and therapeutic leads.The neurotrophins are dimeric protein growth factors that help regulate the peripheral and central nervous systems and other tissues that express the Trk and p75 neurotrophin receptors.(1,2) Trk receptors are relatively selective for, and bind with high affinity (∼K d 10 -11 M) to the neurotrophin. Nerve growth factor (NGF) docks with TrkA, Brain Derived Neurotrophic Factor (BDNF) interacts with TrkB(3), and Neurotrophin-3 (NT-3) interacts preferentially with TrkC but can also bind TrkA and TrkB ( Figure 1a). Ligand binding induces phosphorylation (pTyr) of Trk receptors and associated signaling partners and activation of the neurotrophic biological signals: cell growth, cell survival under stress (trophic activity), and/or cellular differentiation (neuritogenic or neurogenic activity).(4-6) * Correspondence regarding design and synthesis of the peptidomimetics to burgess@mail.tamu.edu, and on the biology and pharmacology to uri.saragovi@mcgill.ca. There is also a second receptor for the neurotrophins, p75, a member of the tumor necrosis factor (TNF) receptor superfamily. All the neurotrophins also bind the p75 receptor, but with lesser affinities (10 -9 to 10 -11 M) than for the Trk receptors. (7-9) The p75 receptor can transduce signals which ...

Neurotrophic factors and neuroplasticity pathways in the pathophysiology and treatment of depression

“…BMS355349 has been described as a selective potentiator of NT-3 mediated TrkA and TrkB receptor activity and has proven to induce neurogenesis (Chen et al 2009; Lewis et al 2006). …”

Biasing Receptor Tyrosine Kinase Signaling Pathways

“…154 Eight peptidomimetics were identified that bound selectively to TrkC. Five of these, represented by Compound 6E in Figure 5.10B, were shown to potentiate the trophic activity of a submaximal concentration of NT-3 in nnr5-TrkC cells (see above) in a survival assay following serum deprivation.…”

“…Compound 6F, and the other two differentiation-inducing peptidomimetics, were also able to activate TrkC and Akt, but not MAPK, as shown by tyrosine phosphorylation. 154 Akt activation is known to be important for neurotrophin-induced survival and differentiation, while MAPK activation is primarily associated with cell survival. 123 Unfortunately, the ability of Compound 6E and related peptidomimetics to induce phosphorylation of TrkC and downstream signaling intermediaries was not assessed.…”

Selectivity of Cell Signaling in the Neuronal Response Based on NGF Mutations and Peptidomimetics in the Treatment of Alzheimers Disease

“…Good NGF mimetics at this stage appear to be D3 or its derivatives and LM11A-24 or LM11A-31, although each may potentially be useful for a distinct purpose. Also being developed are TrkB (O' Leary & Hughes, 2003;Fletcher, et al, 2008;Fletcher & Hughes, 2009) and TrkC Chen, et al, 2009;Liu, et al, 2010) antagonists/agonists. BDNF mimetics have made progress toward a useful therapeutic reagent with efficacy studies in rodents (Massa et al, 2010).…”