Pharmacogenetic Analysis of the Interaction of the Low-Molecular-Weight BDNF Mimetic Dipeptide GSB-106 with TRK Receptors.

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.

Dimeric mimetic of BDNF loop 4 promotes survival of serum-deprived cell through TrkB-dependent apoptosis suppression.

Brain-derived neurotrophic factor (BDNF) is involved in the regulation of neuronal cell growth, differentiation, neuroprotection and synaptic plasticity. Although aberrant BDNF/TrkB signaling is implicated in several neurological, neurodegenerative and psychiatric disorders, neurotrophin-based therapy is challenging and is limited by improper pharmacokinetic properties of BDNF. Dimeric dipeptide compound GSB-106 (bis-(N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide) has earlier been designed to mimic the TrkB-interaction 4 loop of BDNF. It displayed protective effect in various cell-damaging models in vitro. Animal studies uncovered antidepressive and neuroprotective properties upon GSB-106 per os administration. Current study shows that GSB-106 acts similarly to BDNF, promoting survival of serum-deprived neuronal-like SH-SY5Y cells. 100 nmol concentration of GSB-106 provided maximum neurotrophic effect, which corresponds to about 37% of the maximum effect provided by BDNF. Protective properties of GSB-106 arise from its ability to counteract cell apoptosis via activation of TrkB-dependent pro-survival mechanisms, including inactivation of pro-apoptotic BAD protein and suppression of caspases 9 and 3/7. Thus, our study has characterized neurotrophic activity of small dimeric compound GSB-106, which mimics certain biological functions of BDNF and neurotrophin-specific protective mechanisms. GSB-106 also displays similarities to some known low weight peptide and non-peptide TrkB ligands.

Peptide Mimetic of BDNF Loop 4 Blocks Behavioral Signs of Morphine Withdrawal Syndrome and Prevents the Increase in ΔFosB Level in the Striatum of Rats.

Activity of compound GSB-106, a low-molecular mimetic of loop 4 of the brain neurotrophic factor (BDNF), was studied in experimental morphine withdrawal syndrome simulated in outbred rats. Single and subchronic (5 intraperitoneal injections) administration of GSB-106 in a dose of 0.1 mg/kg significantly reduced the total index of morphine withdrawal syndrome by 55.2 and 45.6%, respectively. GSB-106 reduced the severity of some behavioral signs (piloerection, gnashing of teeth, wet-dog shaking, and runaway attempts), but had no effect on mechanical allodynia formed in the rats with dependence. Subchronic treatment with GSB-106 prevented the increase in the content of ΔFosB (product of early response gene) in the striatum induced by morphine withdrawal. The results confirmed the concept on the involvement of neurotrophins, specifically BDNF and its analogs, in the mechanisms associated with the formation of opiate dependence.

The PI3K/Akt Cascade Is Involved in the Antidiabetic Effect of Compound GSB-214, a Low-Molecular-Weight BDNF Mimetic.

In our previous studies on the streptozotocin model of diabetes we hypothesized that activation of the PI3K/Akt signaling pathway is essential for the realization of the antidiabetic effect of low-molecular-weight NGF and BDNF mimetics. Here we analyze the effect of a specific PI3K/Akt pathway inhibitor (LY 294002) on the antidiabetic effect of the BDNF loop 1 mimetic GSB-214. The experiments on C57BL/6 mice with streptozotocin-induced diabetes showed that GSB-214 attenuated the hyperglycemic effect of streptozotocin and prevented weight loss typical of diabetes, while LY 294002 eliminated these effects of GSB-214. These findings clearly demonstrate the involvement of PI3K/Akt pathway in the implementation of the effects of this low-molecular-weight BDNF mimetic.

Dipeptide Mimetics of Different NGF and BDNF Loops Activate PLC-γ1.

Previously, we designed and synthesized dipeptide mimetics of individual loops of the nerve growth factor (NGF) and the brain-derived neurotrophic factor (BDNF). It was shown that these mimetics activate the corresponding tyrosine kinase (Trk) receptors and have different patterns of activation of the PI3K/AKT and MAPK/ERK postreceptor signaling pathways in vitro. In the present study, it was shown on HT-22 cells that all these compounds activate the phospholipase C-γ1 (PLC-γ1) cascade.

Cognitive Enhancer Noopept Activates Transcription Factor HIF-1.

The effect of noopept (N-phenylacetyl-L-prolyl-glycine ethyl ester) on the DNA-binding activity of HIF-1 in SH-SH5Y cells and the mechanisms of stabilization of this transcription factor were studied in vitro. Noopept was shown to increase both the basal DNA-binding activity of HIF-1 and the activity induced by various hypoxia mimetics. The mechanism of stabilization of the oxygen-sensitive HIF1α subunit by noopept involves the inhibition of HIF-1 prolyl hydroxylase, which is indirectly indicated by the data obtained using the ODD-Luc reporter, and the positive effect on the level of the HIF1α protein. It was revealed that the effect of noopept is accompanied by changes in gene expression, which belong to different metabolic pathways and are controlled by the transcription factor HIF-1.

A Nerve Growth Factor Dipeptide Mimetic Stimulates Neurogenesis and Synaptogenesis in the Hippocampus and Striatum of Adult Rats with Focal Cerebral Ischemia.

The nerve growth factor (NGF) and its mimetics, which have neuroprotective and neuroregenerative properties, are attractive candidates for developing new drugs for brain injury therapy. A dipeptide mimetic of NGF loop 4, bis(N-succinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2), developed at the Zakusov Research Institute of Pharmacology, has the NGF-like ability to activate TrkA receptors, but unlike NGF, GK-2 activates mainly the PI3K/AKT pathway associated with neuroprotection and has no effect on the MAPK cascade associated with hyperalgesia, the main side effect of NGF. That GK-2 possesses neuroprotective activity has been observed in various models of cerebral ischemia. GK-2 was found to statistically significantly reduce the cerebral infarct volume in experimental stroke, even at treatment onset 24 h after injury. This suggests that GK-2 possesses neuroregenerative properties, which may be associated with the activation of neurogenesis and/or synaptogenesis. We studied the effect of GK-2 on neurogenesis and synaptogenesis in experimental ischemic stroke caused by transient occlusion of the middle cerebral artery in rats. GK-2 was administered 6 or 24 h after surgery and then once a day for 7 days. One day after the last administration, proliferative activity in the hippocampus and striatum of the affected hemisphere was assessed using Ki67 and synaptogenesis in the striatum was evaluated using synaptophysin and PSD-95. Ki67 immunoreactivity, both in the striatum and in the hippocampus of the ischemic rats, was found to have dropped by approximately 30% compared to that in the sham-operated controls. Synaptic markers - synaptophysin and PSD-95 - were also statistically significantly reduced, by 14 and 29%, respectively. GK-2 in both administration schedules completely restored the level of Ki67 immunoreactivity in the hippocampus and promoted its increase in the striatum. In addition, GK-2 restored the level of the postsynaptic marker PSD-95, with the therapeutic effect amounting to 70% at the start of its administration after 6 h, and promoted restoration of the level of this marker at the start of administration 24 h after an experimental stroke. GK-2 had no effect on the synaptophysin level. These findings suggest that the neurotrophin mimetic GK-2, which mainly activates one of the main Trk receptor signaling pathways PI3K/ AKT, has a stimulating effect on neurogenesis (and, probably, gliogenesis) and synaptogenesis in experimental cerebral ischemia. This effect may explain the protective effect observed at the start of dipeptide administration 24 h after stroke simulation.

A Novel Dipeptide NGF Mimetic GK-2 Selectively Activating the PI3K/AKT Signaling Pathway Promotes the Survival of Pancreatic ß-Cells in a Rat Model of Diabetes.

We investigated the cytoprotective effect of a novel low-molecular-weight NGF mimetic, GK-2 (hexamethylenediamide bis-N-monosuccinyl-L-glutamyl-L-lysine), on pancreatic ß-cells. The neuroprotective effect of GK-2 had been previously shown to be associated with selective activation of the PI3K/Akt signaling pathway. In this study, rats with streptozotocin (STZ)-induced type 2 diabetes mellitus were used. Metformin was used as a reference drug. STZ was immunohistochemically demonstrated to reduce the number of ß-cells and affect their morphological structure. Treatment of diabetic animals with GK-2 (at a dose of 0.5 mg/kg intraperitoneally or 5 mg/kg orally) or metformin (300 mg/kg orally) for 28 days reduced the damaging effect of STZ. The effect of GK-2 on manifestations of STZ-induced diabetes, such as hyperglycemia, weight loss, polyphagia, and polydipsia, was comparable to that of metformin, while the cytoprotective activity of GK-2 was slightly stronger than that of metformin. A strong positive correlation between morphometric parameters and the blood glucose level was revealed. The GK-2 cytoprotective effect on ß-cells is supposed to manifest through the PI3K/Akt signaling pathway.

Drug with Neuroprotective Properties Noopept Does Not Stimulate Cell Proliferation.

Effects of Noopept (N-phenylacetyl-L-prolylglycine ethyl ester) on the relative level of proliferation marker Ki-67 and cell cycle parameters were studied in HEK293 and SH-SY5Y cell lines. The previously established multifactorial mechanism of action of the drug includes enhancement of neurotrophin NGF and BDNF expression and increase in HIF-1 activity. The possible mitogenic action of Noopept was estimated by its effect on cell proliferation. Noopept did not affect cell distribution over G1, S, G2 cell cycle phases and the relative level of proliferation marker Ki-67 in the cell lines under study. These data suggest that Noopept does not stimulate cell growth.

Low-Molecular-Weight Compound GSB-106 Mimics the Cellular Effects of BDNF after Serum Deprivation.

The in vitro model of serum deprivation shows that the survival of SH-SY5Y neuronal cells is ensured by the intrinsic trophic activity of BDNF loop 4 mimetic GSB-106 (10-7 М), which is comparable to that of endogenous neurotrophin (10-9 М). The analysis of the cell cycle and S-phase showed that GSB-106, similarly to BDNF, induces the cell-cycle arrest in the G1 phase, diminishes the number of cells in the S-phase, reduces the number of apoptotic cells, and does not stimulate proliferation.

Antidiabetic Activity of Afobazole in Wistar Rats.

Using the streptozotocin model of type 2 diabetes mellitus in Wistar rats, we compared antidiabetic activity of anxiolytic Afobazole with that of metformin. Afobazole in a dose of 10 mg/kg reduced streptozotocin-induced hyperglycemia and polyphagia and prevented accumulation of malonic dialdehyde, being not inferior to metformin in a dose of 300 mg/kg, and was even more effective than metformin in body weight recovery, elimination of polydipsia, and preservation of these effects after treatment withdrawal.

[An experimental evaluation of the therapeutic window of the neuroprotective activity of a low-molecular nerve growth factor mimetic GK-2]. / Éksperimental'naia otsenka terapevticheskogo okna neiroprotektivnoi aktivnosti preparata GK-2, nizkomolekuliarnogo mimetika faktora rosta nervov.

AIM: To identify the time interval for the preservation of the effect of GK-2 depending on the start of administration after modeling ischemic stroke by the transient occlusion of the middle cerebral artery in rats. MATERIAL AND METHODS: The experiments were performed on 33 wild-type male rats and 81 male Wistar rats. Animals were kept in standard conditions. Ischemic stroke was modelled by thread occlusion of the middle cerebral artery. RESULTS AND CONCLUSION: It was found that GK-2 at a daily dose of 1 mg/kg, intraperitoneally, during 7 days statistically significantly reduces brain infarct volume by 20-60% at the first injection from 4 to 24h, with the highest effect 6-8 hours after surgery. Thus, the 'therapeutic window' of GK-2 detected in the experiment is no less than 24 hours, which exceeds the existing neuroprotective agents.

Antidiabetic Properties of Low-Molecular-Weight BDNF Mimetics Depend on the Type of Activation of Post-Receptor Signaling Pathways.

Reduced proliferation and enhanced apoptosis of ß cells in diabetes mellitus are associated with a deficiency of brain-derived neurotrophic factor (BDNF). Low-molecular weight compounds similar to different BDNF loops were synthesized at the V. V. Zakusov Research Institute of Pharmacology. They produce a potentiating effect on TrkB phosphorylation, but differently activate post-receptor signaling pathways. We compared their effects on the severity of streptozotocin-induced diabetes mellitus in C57Bl/6 mice. The antidiabetic effect (estimated from the degree of hyperglycemia and dynamics of body weight) was typical of GSB-214 compound that selectively activates PI3K/Akt. This activity was not revealed in GTS-201, selective activator of MAPK/Erk. GSB-106 compound activating both signaling pathways exhibited weak antidiabetic activity. Our results indicate that the antidiabetic effect is mainly related to activation of the PI3K/Akt signaling pathway.

Comparison of the Pharmacological Effects of Dimeric Dipeptide Nerve Growth Factor Mimetic GK-2 and Mexidol on the Model of Ischemic Stroke in Rats.

We compared the effects of GK-2 (dimeric dipeptide mimetic of nerve growth factor) and Mexidol (standard preparation for the therapy of stroke) on rat model of transient occlusion of the middle cerebral artery. GK-2 and Mexidol were administered intraperitoneally in the most active doses (1 and 100 mg/kg, respectively) 6 h after surgery and then once a day for 6 days. The preparations reduced the volume of cerebral infarction (by 60 and 30%, respectively). At the same time, GK-2 had a pronounced and statistically more reliable effect in a dose that is lower by two orders of magnitude. In addition, GK-2 significantly reduced the neurological deficit in the limb placement test, while Mexidol was ineffective in this test.

Antiangiogenic Effects of Nerve Growth Factor Loop 4 Monomeric Dipeptide Mimetic.

The effects of GK-1, a monomeric dipeptide mimetic of nerve growth factor (NGF) loop 4, on angiogenesis were studied in vitro and in vivo. Experiments on human umbilical vein endothelial cells HUVEC showed that the test compound did not affect tubulogenesis (initial stage of angiogenesis) and prevented realization of the angiogenic effect of NGF and its dimeric dipeptide mimetic GK-2. Experiments on rat hind limb ischemia model demonstrated that GK-1 (1 mg/kg/day intraperitoneally over 14 days) significantly reduced the density of the capillary network in ischemic tissue and increased the number and area of Zenker necrosis in comparison with the control. These data suggest that GK-1 exhibits a pronounced antiangiogenic activity.

Angiogenic Effects of Dimeric Dipeptide Mimetic of Loop 4 of Nerve Growth Factor.

Angiogenic action of compound GK-2, a dimeric dipeptide mimetic of loop 4 of nerve growth factor (NGF), was studied in in vitro and in vivo experiments. Experiments on human endothelial cell culture HUVEC showed that compound GK-2 significantly (p<0.05) stimulated the initial stage of angiogenesis, and its angiogenic activity was not inferior to the reference neurotrophin NGF. In experiments with hindlimb ischemia modeled in rats, GK-2 (1 mg/kg intraperitoneally for 14 days) significantly increased the total length of capillary vessels (p<0.003) and the number of vessels per 1 mm2 ischemic tissue (p<0.001) in comparison with the control. Our findings indicate that under experimental conditions compound GK-2 exhibits not only angiogenic, but also anti-ischemic activity.

Effect of Afobazole and Betaine on DNA Damage in Placental and Embryonic Tissues of Rats with Experimental Streptozocin Diabetes.

DNA comet assay showed that the level of DNA damage in the placental and embryonic tissues of rats with streptozotocin-induced diabetes mellitus increased on gestation days 14 and 20. Afobazole and betaine administered per os effectively decreased the level of genotoxic damage; afobazole was most efficient in doses of 10 and 50 mg/kg and betaine in a dose of 100 mg/kg.

[The development of a pharmacologically active low-molecular mimetic of the nerve growth factor].

Authors present an overview of theirs author's works on the design of low-molecular mimetic of the nerve growth factor and studies of mechanisms of action and pharmacological properties of the compound. The original working hypothesis, underlying the design of the compound, posited that different neurotrophin hairpin loops could activate different signaling cascades by interaction with the receptor and so be responsible for different effects. The mimetic bis(N-succinyl-L-glutamyl-L-lysine)hexametylendiamide (GK-2), that was designed on the basis of NGF loop 4 ß-turn sequence, activated TrkA and PI3K/Akt, but not MAPK/Erk. GK-2 showed neuroprotective activity in concentrations up to 10-9М against H(2)O(2) or glutamate or MPTP-induced neurotoxicity in РС12, НТ22 cells and primary rat hippocampal neurons. At that, GK-2 has no differentiating activity. In in vivo experiments, GK-2 exhibited significant anti-ischemic, anti-parkinsonic effect, reversed impaired cognitive functions in models of Alzheimer's disease in doses 0.01 - 5 mg/kg intraperitoneally and 5-10 mg/kg orally, but does not induce side effects accompanying the full-length neurotrophin treatment, which are hyperalgesia and weight loss. It was shown that GK-2 was a low-toxicity compound (LD50=700 mg/kg, intraperitoneally, mice) and capable of crossing the blood-brain barrier. The agent GK-2 is promising for development as a neuroprotective agent and is currently in preclinical studies.

Cytoprotective Effect of Afobazole and Its Main Metabolite M-11.

Cell damage depending on activity of quinone reductase 2 (MT3 receptor) was simulated in experiments on bone marrow cell suspension and assessed by menadione-induced DNA breaks measured by comet assay. We analyzed the protective effect of afobazole interacting with MT1, MT3, σ1 receptors, and monoamine oxidase A and its main metabolite M11 that specifi cally binds to MT3 receptors. Both compounds reduced the level of menadione-induced DNA damage (afobazole was effective in lower concentrations in comparison with M-11). Conclusion was made on the contribution of MT3 receptors to the protective effect of afobazole, but the observed concentration differences indicate possible contribution of other targets of anxiolytic drug to the protective mechanisms.