A dimeric dipeptide mimetic of nerve growth factor (NGF), bis-(N-monosuccinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2), was previously developed at V. V. Zakusov State Institute of Pharmacology, activated specific TrkA receptors, and exhibited neuroprotective activity in vitro (10–5 – 10–9 M) and in vivo (0.1 – 10 mg/kg i.p. and p.o.). GK-2 was designed based on the beta-turn (-Asp94-Glu95-Lys96-Gln97-) of the NGF 4th loop and preserved the central dipeptide fragment (-Glu95-Lys96-). The Asp94 residue was replaced by its monosuccinyl bioisostere. The dimeric structure of NGF was reproduced using a bivalent hexamethylenediamine spacer. The structure—activity (neuroprotective) relationship for GK-2 was studied in the present work using a glycine scan, i.e., successive replacement of the peptide side groups by H. The bis-(N-acetyl-L-glutamyl-L-lysine) (GK-2Ac), bis-(N-monosuccinylglycyl-L-lysine) (GK-2-Gly1), and bis-(N-monosuccinyl-L-glutamylglycine) hexamethylenediamides (GK-2-Gly2) were less active with neuroprotective activity in vitro under oxidative stress for HT22 cells at concentrations 10 – 100 times greater than GK-2. The conclusion was drawn that each side radical of GK-2 was important for manifestation of the full neuroprotective activity of dimeric dipeptide GK-2, a mimetic of the NGF 4th loop. However, removal of any of the side radicals would probably not change the active structure of the beta-turn so that the two remaining side radicals should retain the ability to bind to their TrkA subsites. This could explain the retention of neuroprotective activity in the GK-2 glycine analogs.

In this work investigated the neuroprotective activity of analogues of substituted glyproline GZK-111 at the N-terminus in vitro . Oxidative stress caused by H 2 O 2 (1.5 mM) led to significant decrease in the hippocampal cells HT-22 viability. The ethyl ester N-phenypropionyl-glycyl-proline (GZK-45) was effective at the concentrations up to 10 –6 M in both experimental schemes. The compounds with N-acethyl (GZK-49) and N-capronoyl (GZK-46) fragments did not protective effect in both experimental schemes. GZK-45 also had protective effect in the 6-hydroxydopamine induced damage of SH-SY5Y neuroblastoma cells.

Resume. The purpose of this study was to compare neuroprotective properties of the dimeric dipeptide mimetics of the 1st, 2nd and 4th Loops of BDNF - GSB-214 (heptamethylenediamide bis-monosuccinyl-methionyl-serine), GTS-201 (hexamethylenediamide bis-hexanoyl-seryl-lysine) and GSB-106 (hexamethylenediamide bis-monosuccinyl-seryl-lysine) in the HT-22 hippocampal cells in conditions of oxidative stress. It has been established that the mimetic of the 4th loop of BDNF shows a neuroprotective effect in lower concentrations compared to mimetics based on the 1st and 2nd loops when applied 24 hours before and immediately after the damages.

Studies at the V. V. Zakusov Science Research Institute of Pharmacology have worked on the basis of the β-pleated structure of loop 4 of brain-derived neurotrophic factor (BDNF) to construct its dimeric peptide mimetic - GSB-106, bis-(N-monosuccinyl-L-serine-L-lysine) hexamethylenediamide. GSB-106 is being developed as an antidepressant with a BDNF-ergic mechanism of action. We report here studies of the physicochemical properties of GSB-106 and development of a method for analysis of GSB-106 substance. The main pharmacopeia quality indicators were determined and IR and NMR spectra were obtained from samples of substance. A method was developed for detecting contaminants in substance using TLC and HPLC.

Resume. Previously in the Zakusov Institute of Pharmacology the linear substituted glyproline GZK-111, N-phenylacetyl-glycyl-L-proline ethyl ester, which is capable to convert into the neuropeptide cyclo-prolylglycine (CPG) in biological media and displaying the nootropic, anxiolytic, antihypoxic and neuroprotective activity, was designed and synthesized. The aim of the work was to study the neuroprotective activity of analogues of substituted glyproline GZK-111 at the C- and N-terminus in vitro on the oxidative stress model in comparison to the GPG. Oxidative stress caused by H2O2 (1.5 mM) led to significant decrease in the hippocampal cells HT-22 viability. L-CPG protected cells against H2O2 in concentrations up to 10-7M when applied both 24 h before and immediately after damage. The compound with an open carboxylic group (GZK-115) like CPG protected HT-22 cells from death in both experimental schemes at concentrations up to 10-7M. The substituted amide GZK-119 added 24 h before cell damage, had a cytoprotective effect at a concentration of 10-7M, and after peroxide damage in concentrations of 10-6 - 10-7M. The D-enantiomer of ethyl ester N-phenylacetyl-glycyl-proline (GZK-121) and the compound with the extension of the N-acyl fragment to the CH2-group (GZK-45) were effective at concentrations up to 10-6M in both experimental schemes. Simultaneous extension of the N-acyl fragment to the CH2 group and replacement of the ester with an amide (GZK-50) resulted to protection of the cells from death only after H2O2. Thus, it was established that all investigated substituted glyprolines possessed neuroprotective activity in experiments in vitro under conditions of oxidative stress in HT-22 cells. The most active compounds were GZK-119 and GZK-115.

Resume. Previously in the Zakusov Institute of Pharmacology the linear substituted glyproline GZK-111, N-phenylacetyl-glycyl-L-proline ethyl ester, which is capable to convert into the neuropeptide cyclo-prolylglycine (CPG) in biological media and displaying the nootropic, anxiolytic, antihypoxic and neuroprotective activity, was designed and synthesized. The aim of the work was to study the neuroprotective activity of analogues of substituted glyproline GZK-111 at the C- and N-terminus in vitro on the oxidative stress model in comparison to the GPG. Oxidative stress caused by H 2 O 2 (1.5 mM) led to significant decrease in the hippocampal cells HT-22 viability. L-CPG protected cells against H 2 O 2 in concentrations up to 10 -7 M when applied both 24 h before and immediately after damage. The compound with an open carboxylic group (GZK-115) like CPG protected HT-22 cells from death in both experimental schemes at concentrations up to 10 -7 M. The substituted amide GZK-119 added 24 h before cell damage, had a cytoprotective effect at a concentration of 10 -7 M, and after peroxide damage in concentrations of 10 -6 - 10 -7 M. The D-enantiomer of ethyl ester N-phenylacetyl-glycyl-proline (GZK-121) and the compound with the extension of the N-acyl fragment to the CH 2 -group (GZK-45) were effective at concentrations up to 10 -6 M in both experimental schemes. Simultaneous extension of the N-acyl fragment to the CH 2 group and replacement of the ester with an amide (GZK-50) resulted to protection of the cells from death only after H 2 O 2 . Thus, it was established that all investigated substituted glyprolines possessed neuroprotective activity in experiments in vitro under conditions of oxidative stress in HT-22 cells. The most active compounds were GZK-119 and GZK-115.

In the present work, the neuroprotective effect stereospecificity of dimeric dipeptide mimetic of the 4th loop of BDNF-GSB-106 is studied. Activity of its diastereomers GT-106DL (hexamethylenediamide bis-monosuccinyl-D-seryl-L-lysine) and GT-106LD (hexamethylenediamide bis-monosuccinyl-L-seryl-D-lysine) in hippocampal HT-22 cells under conditions of oxidative stress is for this purpose studied. Both diastereomers were inactive. Thus, the obtained data indicate dependence of effect on the configuration of amino acid residues in the structure of GSB-106.

We have studied neuroprotective effect of L-cycloprolylglycine (L-CPG) in vitro experiments. It is shown that L-CPG has neuroprotective effect in the conditions an oxidative stress, glutamate and 6-hydroxydopamine toxicity.

On culture of hippocampal HT-22 cells line it is shown that dimeric dipeptide mimetiks of different Loops of NGF realize their action via specific neurotrophic TrkA-receptor and selective activate post-receptor intracellular signal ways. Mimetiks of 1th (GK-6 (10-6M)) and the 3rd Loops (GTS-115 ((10-6M)) evoke activation of two main signal cascades - PI3/Akt and MAP/Erk, while mimetic the 4th loop of NGF (GK-2 (10-8M)) only one - a PI3/Akt-way.

On culture of hippocampal HT-22 cells line it is shown that dimeric dipeptide mimetiks of different Loops of NGF realize their action via specific neurotrophic TrkA-receptor and selective activate post-receptor intracellular signal ways. Mimetiks of 1th (GK-6 (10-6M)) and the 3rd Loops (GTS-115 ((10-6M)) evoke activation of two main signal cascades - PI3/Akt and MAP/Erk, while mimetic the 4th loop of NGF (GK-2 (10-8M)) only one - a PI3/Akt-way.

In previous work we have obtained a dimeric dipeptide mimetic of 4th loop of BDNF - hexamethylenediamide bis-(N-monosuccinil-L-seryl-L-lysine) (GSB-106), having a neuroprotective activity in vitro in a concentration range 10(-5)-10(-8) M and an antidepressant activity in vivo at doses 0.1 and 1 mg/kg i.p. in rats. We have investigated the structural and functional relationships among analogues of GSB-106. Glycine scan was performed and a number of appropriate compounds were synthesized: GT-105 (here lysine is replaced by glycine), GT-107 (here serine is replaced by glycine), GT-106Ac (here monosuccinic radical is replaced by acetyl group). We have studied the dependence of activity of following compounds from the configuration of amino acid residues: GT-107D (D-enantiomer of the GT-107), GT-106DL (L-serine was replaced by D-serine), GT-106LD (L-lysine was replaced by D-lysine). The investigation of these compounds using the HT22 cell culture in conditions of oxidative stress has approved only two analogues of GSB-106 to have a neuroprotective effect: in the case of replacement of serine to glycine and of replacment of succinic radical to acetic group. A disappearance of this effect was observed in event of the replacement of lysine residue to glycine in GT-105, L-lysine residue to D-lysine and also by conversion of serine configuration. These results show that lysine residue is crucial for the neuroprotective activity of GSB-106. L-Configuration of the lysine and serine residues required. Configuration of lysine residue becomes critical in absence of serine side group. Thus, the the following fragment is a minimum pharmacophore of beta-turn of 4 loop of BDNF: HOOC-CH2-CH-CO-NH-(S)-CH(CH2OH)-CO-NH-(S)-CH((CH2)4NHz)-CO-NH-(CH2)3-. Only one (GT-106Ac) out of two analogues of GSB-106 with neuroprotective activity possesses antidepressant activity too. This fact indicates about a necessity of more stringent structural requirements for exposure of antidepressant activity. The results obtained can be useful for designing of new active mimetics of BDNE

Dimeric dipeptide GSB-106, a novel structural analogue of neurotrophin BDNF, in doses from 10−5 to 10−8 M protected cultured immortalized mouse hippocampal HT-22 neurons from H2O2 or glutamate damage. The neuroprotective effect of GSB-106 was also shown on cultured SH-SY5Y human neuroblastoma cells after treatment with neurotoxin 6-hydroxidopamine. These data attest to functional similarity of GSB-106 and neurotrophin BDNF.

The antihypoxic activity of the endogenous cyclic dipeptide cycloprolylglycine (CPG) has been studied on a model of normobaric hypoxia with hypercapnia and its neuroprotective activity has been studied on a model of human neuroblastoma SH-SY5Y cell damage by 6-hydroxydopamine. It is established that CPG exhibits the antihypoxic activity at doses of 0.5 and 1.0 mg/kg (i.p.) on outbred and BALB/c mice, but not on C57Bl/6 mice. The neuroprotective activity of CPG was detected in 10 -5 - 10 -8 M concentration range only when the treatment was carried out 24 h before toxin introduction. The obtained data confirm the hypothesis that piracetam is a mimetic of the endogenous CPG neuropeptide.