The accumulated evidence suggests that varying levels of tyrosine kinase receptor signaling pathway activity may regulate opiate-associated neuroadaptation of noradrenergic system. Neurotrophin-3 (NT-3) interacts with tropomyosin receptor kinases (TRKs), binding mainly to TRKC receptors, which are expressed within noradrenergic neurons in the blue spot (locus coeruleus, LC). Considering the difficulties in delivering full-length neurotrophins to the CNS after systemic administration, low-molecular mimetics of loop 4 in NT-3, hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301), and hexamethylenediamide bis-(N-γ-oxybutyryl-L-glutamyl-L-asparagine) (GTS-302), activating TRKC and TRKB receptors, were synthesized. The aim of the study is comparative examination of the effects of NT-3 dipeptide mimetics on the signs of morphine withdrawal in outbred white rats with opiate dependence, as well as investigation of activation of postreceptor signaling pathways by the mimetics. Dipeptides GTS-301 and GTS-302 after acute administration at doses of 0.1, 1.0, and 10.0 mg/kg (i.p., intraperitoneal) had a dose-dependent effect on the specific morphine withdrawal symptoms with the most effective dose being 1.0 mg/kg. Maximum decrease in the total index of morphine withdrawal syndrome for GTS-301 was 31.3

The pathogenesis of Parkinson's disease (PD) involves abnormalities in the metabolism of catecholamines. The enzyme quinone reductase 2 (NQO2) reduces quinone derivatives of catecholamines, which promotes the formation of reactive oxygen species (ROS), suggesting a role for NQO2 in the development of cellular damage typical of PD. In the present study, we investigated the relationship between 6-hydroxydophamine (6-OHDA) induced cellular damage and NQO2 activity and its levels in SH-SY5Y cell culture to establish an experimental model to evaluate the pharmacological properties of NQO2 inhibitors. Cellular damage was evaluated using the MTT and comet assays. It was shown that oxidative damage of SH-SY5Y cells upon incubation with 6-OHDA for 6, 12 and 24 h was accompanied by an increase in NQO2 activity. The increase in NQO2 protein level in SH-SY5Y cells was observed 24 h after incubation with 6-OHDA at concentrations of 50 and 100 μM. Oxidative damage of SH-SY5Y cells upon 1 h incubation with 6-OHDA is increased in the presence of the selective enzyme co-substrate 1-benzyl-1,4-dihydronicotinamide (BNAH), but is not accompanied by changes in NQO2 activity and protein levels. The data obtained demonstrate the contribution of NQO2 to the cytotoxic mechanism of 6-OHDA action.

A dimeric dipeptide mimetic based on the β-turn of the 4th loop of neurotrophin-3, bis-(N-monosuccinyl-L-asparaginyl-L-asparagine)hexamethylenediamide (GTS-301LL), was recently designed and synthesized by us. GTS-301, like the full-length neurotrophin, activated TrkC and TrkB receptors and exhibited neuroprotective activity on HT22 cells under oxidative stress conditions in the concentration range 10–5 – 10–12 M and antidepressant-like activity in the forced swimming test in mice (10 – 40 mg/kg, intraperitoneally). The stereospecificity of the pharmacological effects of GTS-301LL was revealed by synthesizing the LD, DL, and DD stereoisomers of GTS-301LL and studying their neuroprotective and antidepressant-like properties under the same conditions as for GTS-301LL. Both activities were found to disappear on going from the LL to the DL and DD stereoisomers and were retained on going to the LD stereoisomer. Thus, the stereospecificity of the neuroprotective and antidepressant-like activities of the dipeptide mimetic of neurotrophin-3 GTS-301LL at the N-terminal asparagine residue was proven, which indicated the key role of that amino-acid residue in the interaction with the receptor.

A novel neurotrophin-3 loop 4 dipeptide mimetic, bis(N-γ-hydroxybutyryl-L-glutamyl-L-asparagine) hexamethylenedi-amide (GTS-302), was designed and synthesized. Compound GTS-302 activated the tyrosine kinase receptors TrkC and TrkB, exhibited cytoprotective activity (10-8–10-5 M) in oxidative stress model in HT-22 cells and in 6-hydroxy-dopamine-induced SH-SY5Y cell damage. It also demon- strated an antidepressant-like activity in the forced swim test in mice (0.5–10.0 mg kg–1, intraperitoneally).

Parameters of non-spatial and spatial memory were evaluated in sexually mature offspring of outbred rats (females and males F0) consuming a 10% ethanol solution for 30 weeks before mating. We found a significant increase in the recognition index in F1 males and its decrease in F1 females in the novel object recognition test. During the first days of the experiment in T-maze, a decrease in spatial memory was revealed in F1 males, which remained at the trend level until the end of testing; no significant deviations were detected in F1 females. Memory impairment in F1 females was accompanied by a decrease in BDNF level in the hippocampus, but not in the prefrontal cortex. Thus, ethanol consumption by F0 rats before mating led to impairment of long-term working memory only in female F1 offspring.

Using TrkA or TrkB receptor gene knockout HT-22 cells, the selectivity of the interaction of the low-molecular-weight dipeptide BDNF mimetic GSB-106 (hexamethylenediamide bis(N-monosuccinyl-L-seryl-L-lysine)) with TrkB receptors was shown.

TrkA and TrkB knockout HT-22 cell lines were used to prove that the neuroprotective properties of GK-2 [ bis ( N -monosuccinyl- L -glutamyl- L -lysine hexamethylenediamide], a low-molecular-weight dipeptide mimetic of the NGF 4 th loop, are definitely mediated by selective interaction with TrkA receptor upon oxidative stress-induced cellular damage in vitro .

Background: A dipeptide mimetic of the BDNF loop 4, bis (N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide, GSB-106, was designed and synthesized by V.V. Zakusov Research Institute of Pharmacology. The compound activated in vitro TrkB, MAPK/ERK, PI3K/AKT, and PLCγ, like full-length BDNF. In vivo, GSB-106 exhibited antidepressant-like, neuroprotective and neuroregenerative properties. The aim of this work was to study the effects of GSB-106 on depressive-like behavior, cognitive impairments, as well as on hippocampal neuroplasticity in an experimental model of ischemic stroke. Methods: Male Wistar rats were subjected to 60 minutes of transient middle cerebral artery occlusion (MCAO). Dipeptide GSB-106 was administered intraperitoneally at a dose of 0.1 mg/kg/day for 21 days after surgery. 30-40 days after MCAO, the depressive-like state in the forced swimming test and memory impairment in the novel object recognition test were assessed. Then, the content of CREB, as a neuroplasticity marker, was assessed in the ipsilateral hippocampus. Results: Rats in MCAO group showed depression-like behavior (increase in immobility time in the forced swimming test by 28% compared to sham group), impairments in short-term and long-term memory (decrease in the discrimination index in the novel object recognition test by 70% and 50%, respectively), and a decrease in immunoreactivity to CREB (cAMP response element-binding protein) in the hippocampus by 36% as compared with the sham group. GSB-106 completely prevented the behavior impairments and counteracted the reduction of immunoreactivity to CREB in the hippocampus. Conclusion: The BDNF dipeptide mimetic GSB-106 is promising for further development as a drug for the treatment of poststroke neuropsychiatric disorders.

Background. A dimeric dipeptide mimetic of the brain-derived neurotrophic factor loop 4, bis(N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide (GSB-106), which activates TrkB, PI3K/AKT, MAPK/ERK and PLC-γ1 was created at the V. V. Zakusov Research Institute of Pharmacology. GSB-106 showed neuroprotective activity in vitro and in vivo at systemic administration. Objective. In the present study, we studied the effect of GSB-106 on the brain infarct volume, as well as on neurogenesis and synaptogenesis under conditions of experimental ischemic stroke induced by transient occlusion of the middle cerebral artery in rats, when it was first administered 24 h after ischemia onset. Methods. Dipeptide GSB-106 was administered i.p. in a dose of 0.1 mg/kg 24 h after surgery and then once a day, with the end of administration on theday 6 after surgery. On the day 7 brain samples were collected for morphometric and biochemical (Western-blot) analysis. Results. It was established that GSB-106 reduced the brain damage volume by 24%, restores impaired neurogenesis and/or gliogenesis (by Ki-67) in the hippocampus and in the striatum and completely restored the reduced immunoreactivity to synaptic markers synaptophysin and PSD-95 in the striatum. Conclusions. Thus, the dimeric dipeptide BDNF mimetic GSB-106 exhibits neuroregenerative properties at clinically relevant time window (24 h) in a model of ischemic stroke presumably due to stimulation of neurogenesis (and / or gliogenesis) and synaptogenesis.

The dimeric dipeptide mimetic hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301) was created on the basis of the structure of the exposed region of the neurotrophin-3 4th loop. The new compound, as well as the full-length neurotrophin, activated the TrkC and TrkB receptors. GTS-301 showed neuroprotective activity in experiments on HT-22 mouse hippocampal cells under conditions of oxidative stress and glutamate toxicity at concentrations of 10–12 and 10–8 M, respectively, and antidepressant-like activity in the forced swimming test on mice with 7-day intraperitoneal administration in doses of 10–40 mg/kg.

A mimetic of the BDNF loop 4, bis (N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide, named GSB-106, was designed and synthesized in our scientific group. The compound activated TrkB, MAPK/ERK, PI3K/AKT, and PLCγ in in vitro experiments. In vivo experiments with rodents revealed its antidepressant-like activity in the forced swim and the tail suspension tests at the dose range of 0.1–5.0 mg/kg (i.p., p.o.). However, GSB-106 was not studied in depression models modulating major depression in humans. In the present study, the GSB-106 antidepressant-like activity was revealed in mice at the depression model induced by 28-day social defeat stress with 21-days oral administration (0.1 mg/kg) after stress. At the same time, GSB-106 restored reduced locomotor activity and completely eliminated the anhedonia manifestations. The compound also restored reduced levels of synaptophysin and CREB in the hippocampus. In addition, the Trk receptor antagonist K252A, and the PLC inhibitor U73122, were found to completely block the antidepressant-like activity of GSB-106 in the forced swimming test in mice. Thus, the present results demonstrate the dipeptide BDNF mimetic GSB-106 reversed depressive-like behavior and restored hippocampal neuroplasticity in a rodent depression model. These effects of GSB-106 are probably regulated by TrkB signaling.

The ability of NQO2 to increase the production of free radicals under enhanced generation of quinone derivatives of catecholamines is considered to be a component of neurodegenerative disease pathogenesis. The present study aimed to investigate the neuroprotective mechanisms of original NQO2 inhibitor M-11 (2-[2-(3-oxomorpholin-4-il)-ethylthio]-5-ethoxybenzimidazole hydrochloride) in a cellular damage model using NQO2 endogenous substrate adrenochrome (125 µM) and co-substrate BNAH (100 µM). The effects of M-11 (10–100 µM) on the reactive oxygen species (ROS) generation, apoptosis and lesion of nuclear DNA were evaluated using flow cytometry and single-cell gel electrophoresis assay (comet assay). Results were compared with S29434, the reference inhibitor of NQO2. It was found that treatment of HT-22 cells with M-11 results in a decline of ROS production triggered by incubation of cells with NQO2 substrate and co-substrate. Pre-incubation of HT-22 cells with compounds M-11 or S29434 results in a decrease of DNA damage and late apoptotic cell percentage reduction. The obtained results provide a rationale for further development of the M-11 compound as a potential neuroprotective agent.

Background: A dimeric dipeptide mimetic of the BDNF loop 4, bis(N-monosuccinyl- L-seryl-L-lysine) hexamethylenediamide (GSB-106) activates TrkB, PI3K/AKT, MAPK/ ERK, and PLC-γ1, and was created at the V.V. Zakusov Research Institute of Pharmacology. GSB-106 showed neuroprotective activity in vitro and in vivo at systemic administration. Objective: In this work, we studied the GSB-106 effect on the cerebral infarct volume, as well as on neurogenesis and synaptogenesis under the experimental ischemic stroke induced by intravascular occlusion of the middle cerebral artery in rats. Methods: GSB-106 was administered i.p. in a dose of 0.1 mg/kg, 24 h after the surgery and then once a day, with the end of administration on day 6 after surgery. On day 7, brain samples were collected for morphometric and biochemical (Western-blot) analysis. Results: It was established that GSB-106 reduced the brain damage volume by 24%, restored impaired neurogenesis and/or gliogenesis (by Ki-67) in the hippocampus and the striatum, and completely restored the reduced immunoreactivity to synaptic markers synaptophysin and PSD-95 in the striatum. Conclusions: Thus, the dimer dipeptide BDNF mimetic GSB-106 exhibits neuroregenerative properties at a clinically relevant time window (24 h) in a model of ischemic stroke presumably due to the stimulation of neurogenesis (and/or gliogenesis) and synaptogenesis.

Previous studies at the V. V. Zakusov Science Research Institute of Pharmacology created a dimeric dipeptide mimetic of the most exposed fourth loop of nerve growth factor (NGF), bis-(N-monosuccinyl-L-glutamyl-Llysine) hexamethylenediamide (GK-2), which activates specific TrkA receptors and has neuroprotective activity in vitro (at 10 5 to 10 9 M) and in vivo (0.1 – 10 mg/kg, i.p., or p.o.). The present report describes the construction and synthesis of a mimetic of the first loop of NGF based on the β-turn (-Lys 32 -Gly 33 -Lys 34 -Glu 35 -), bis-(N-aminocaproyl-glycyl-L-lysine) hexamethylenediamide (GK-6). The structure of GK-6 preserves the central dipeptide fragment of the β-turn -Gly 33 -Lys 34 -, the Lys 32 residue being substituted by its bioisostere – a 6-aminocaproic acid residue - and the dimeric structure of NGF being reproduced by dimerization at the C-terminal using a bivalent hexamethylenediamine spacer. Structure-activity relationships of GK-6 were studied by sequential substitution of the side groups of the peptide by hydrogen to produce bis-(N-acetyl-glycyl-Llysine) hexamethylenediamide (GTS-611) and bis-(N-aminocaproyl-glycyl-glycine) hexamethylenediamine (GTS-613). Mimetic GL-6 and its analogs GTS-613, which contains the N-aminocaproyl radical, had neuroprotective effects at concentrations of 10 6 and 10 5 M in conditions of oxidative stress in HT-22 neuron cultures. Dipeptide GTS-611, in which the aminocaproyl fragment was replaced by an acetyl residue, had no neuroprotective activity in these conditions, pointing to the importance of Lys 32 in NGF for this property. In contrast to the loop 4 mimetic GK-2, the loop 1 mimetics GK-6 and GTS-611 showed differentiation-inducing activity on PC12 cells.