Development of Pleiotropic TrkB and 5-HT4 Receptor Ligands as Neuroprotective Agents

Abstract

One common event that is the most detrimental in neurodegenerative disorders, even though they have a complex pathogenesis, is the increased rate of neuronal death. Endogenous neurotrophins consist of the major neuroprotective factors, while brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine kinase receptor TrkB are described in a number of studies for their important neuronal effects. Normal function of this receptor is crucial for neuronal survival, differentiation, and synaptic function. However, studies have shown that besides direct activation, the TrkB receptor can be transactivated via GPCRs. It has been proven that activation of the 5-HT₄ receptor and transactivation of the TrkB receptor have a positive influence on neuronal differentiation (total dendritic length, number of primary dendrites, and branching index). Because of that and based on the main structural characteristics of LM22A-4, a known activator of the TrkB receptor, and RS67333, a partial 5-HT₄ receptor agonist, we have designed and synthesized a small data set of novel compounds with potential dual activities in order to not only prevent neuronal death, but also to induce neuronal differentiation in neurodegenerative disorders.

Keywords: 5-HT4 receptor; TrkB receptor; neurite differentiation; neurodegeneration; neuronal survival.

Figure 1

Design of TrkB/5-HT₄ receptor ligands.

Scheme 1

Synthesis of ENT-C199 (8) (a) CDI, THF, rt; (b) KO₂CCH₂CO₂Et, MgCl₂, 40 °C; (c) K₂CO₃, DMF, rt; (d) KOH, EtOH/H₂O, reflux; (e) TFA, DCM, rt; (f) tert-butyl-N-(2-bromoethyl)carbamate, K₂CO₃, DMF, 110 °C; (g) 6, Et₃N, EDC, HOBt, THF, 0 °C—rt; (h) ethanolamine, MeOH, 170 °C.

Scheme 2

Synthesis of ENT-232 (13) (a) TFA, DCM, rt; (b) (Boc)₂O, Et₃N, DCM, 0 °C—rt; (c) K₂CO₃, DMF, 110 °C; (d) TFA, DCM, rt; (e) 6, Et₃N, EDC, HOBt, THF, 0 °C—rt; (f) ethanolamine, MeOH, 170 °C.

Scheme 3

Synthesis of ENT-236 (19) (a) (Boc)₂O, Et₃N, DCM, 0 °C—rt; (b) p-TsCl, Et₃N, DCM, 0 °C—rt; (c) TFA, DCM, rt; (d) K₂CO₃, ACN, 80 °C; (e) 6, Et₃N, EDC, HOBt, THF, 0 °C—rt; (f) ethanolamine, MeOH, 170 °C.

Figure 2

Celltox cell toxicity assay in the NIH-3T3 TrkB expressing cell line. (A) Representative images of NIH-3T3 cells untreated and treated with BDNF or LM22A-4 and ENT-C199. (B) Quantification of the toxicity assay (n = 3–5 independent experiments). Cells were treated with compounds (final concentration 1 μM) or BDNF (500 ng/mL) for 24 h after 24 h of serum starvation. Error bars represent SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 [18].

Figure 3

Celltox cell toxicity assay in the NIH-3T3 TrkB expressing cell line. (A) Representative images of NIH-3T3 cells untreated and treated with BDNF and ENT-C232. (B) Quantification of the toxicity assay (n = 3–5 independent experiments). Cells were treated with compounds (final concentration 1 μM) or BDNF (500 ng/mL) for 24 h after 24 h of serum starvation. Error bars represent SEM. * p < 0.05 [18].

Figure 4

Phosphorylation assay of NIH-3T3 TrkB cell line. Quantification of the toxicity assay (n = 3 independent experiments). Cells were treated with compounds (final concentration 1 μM) or BDNF (500 ng/mL) (represented as light green colored bar) for 20 min after 6 h of serum starvation. Error bars represent SEM. * p < 0.05; ** p < 0.01; *** p < 0.001. Representative images of Western blot for TrkB and Akt of compounds ENT-C199 (represented as teal colored bar) and ENT-C232 (represented as lavender colored bar) and BDNF [18].

Figure 5

Determination of the 5-HT₄ pharmacological profile for compound ENT-C199 by quantification of cAMP production (with serotonin and the partial agonist RS67333 as references); n = 3 independent experiments.

Figure 6

Results of the efficacy assay—neurite outgrowth assay of ENT-C compounds ¹. ¹ Control line (yellow interrupted line), RA control (light grey), BDNF (light green), LM22A-4 (dark blue), RS67333 (dark grey), and ENT-C199 (teal), ENT-C232 (lavender), ENT-C236 (light blue). N = 3; error bars represent S.E.M.; * p ≤ 0.05; *** p ≤ 0.001.

Figure 7

Representation of the effect on neurite outgrowth of human SH-SY5 cells: (A) control—SH-SY5 cells cultured 3 days in DMEMF12 with 1% FBS, followed by 2 days in DMEMF12 FBS-free; (B) control FBS—SH-SY5 cells treated with DMEMF12 with 10% FBS; (C) RA—SH-SY5 cells treated with DMEMF12 with 1% FBS in the presence of RA 10 µM, followed by 2 days in DMEMF12 FBS-free; (D) BDNF—SH-SY5 cells treated with BDNF (20 ng/mL); (E) ENT-C225—SH-SY5 cells treated with ENT-C199 (10 nM) [18].