Previously, we prepared dimeric dipeptide mimetics of the first and the fourth loops of the nerve growth factor (NGF): hexamethylenediamides of bis(N-aminocaproyl-glycyl-L-lysine) (GK-6) and bis(N-monosuccinyl-L-glutamyl-L-lysine) (GK-2). Both mimetics activated TrkA-receptors, but induced different postreceptor signal pathways. GK-2 selectively activated PI3K/AKT, whereas GK-6 activated both PI3K/AKT and MAPK/ERK. Both mimetics exhibited a neuroprotective activity. In this study, we continued the investigation of a contribution of separate loop-like structures in the NGF functions and created and studied dimeric dipeptide mimetics based on a beta-turn of the NGF third loop: hexamethylenediamides of bis(N-gamma-hydroxybutyryl-L-lysyl-L-histidine) (GTS-115) and bis(N-acetyl-L-lysyl-L-histidine) (GTS-113). GTS-115 was shown to exhibit the neuroprotective activity in the concentration range from 10–5 to 10–7 М towards the HT-22 cell culture under the conditions of oxidative stress. The acetyl-containing GTS-113 mimetic proved to be inactive. GTS-115 (1 mg/kg/day intraperitoneally, for 7 days, the administration was started 4 h after the operation) exhibited the neuroprotective properties and decreased the infarction volume by 25% on the model of a stroke that was induced by a transient occlusion of the medial cerebral artery of rats. The action mechanism of GTS-115 was studied by Western-blot analysis and this mimetic in a concentration of 10–6 М was shown to activate the TrkA-receptor and both MAPK/ERK and PI3K/AKT basic postreceptor signal pathways. The inhibitory analysis revealed different contributions of these pathways into the GTS-115 neuroprotective effect. The LY294002 selective inhibitor of PI3K completely blocked the neuroprotective effect of GTS-115 in vitro, whereas the PD98059 specific inhibitor of MEK1 and MEK2 decreased this effect only by 10–15%. GTS-115 peptide stimulated a differentiation of the PC12 cells and caused a hyperalgesia in rats. These facts were in a good agreement with the literature data on the participation of the MAP-kinase pathway in these effects. Thus, the third NGF loop and the neighboring first NGF loop activated the postreceptor pathways in a similar way and exhibited the similar activities.

В НИИ фармакологии им. В. В. Закусова РАМН на основе структуры бета-изгиба 4-й петли BDNF-Asp 93 -Ser 94 -Lys 95 -Lys 96 - был создан димерный дипептидный миметик гексаметилендиамид бис-( N -моносукцинил- L -серил- L -лизина) (ГСБ-106). Он обладал нейропротективной активностью in vitro в концентрациях 10 –6 – 10 –8 М и антидепрессивной активностью in vivo при внутрибрюшинном введении в дозах 0,1 – 1 мг/кг. В настоящей работе для выбора оптимальной схемы синтеза мы получили пептид ГСБ-106 по 3 схемам. Первая схема основана на Boc/Z-стратегии защитных групп с применением метода пентафторфениловых эфиров; вторая также на Boc/Z-стратегии с использованием метода N -оксисукцинимидных эфиров; третья — на Z/Boc-стратегии с использованием азидного метода. Проведено сравнение этих способов получения ГСБ-106 по выходу и оптической чистоте. Оптимальной схемой синтеза оказалась третья, использующая Z/Boc стратегию защитных групп и азидный метод образования пептидной связи.

The BDNF-mimetic dimeric dipeptide bis-(N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide (GSB-106) based on the structure of beta-turn loop 4 (BDNF-Asp93-Ser94-Lys95-Lys96-) was synthesized. GSB-106 showed neuroprotective activity in vitro at concentrations 10–6-10–8 M and antidepressant activity in vivo in rats at i.p. injected doses of 0.1-1 mg/kg. The target peptide GSB-106 was obtained using three schemes in order to select the optimum synthetic pathway. The first scheme was based on the strategy of Boc/Z protecting groups using the method of pentafluorophenyl esters. The second scheme also used the Boc/Z strategy and the N-hydroxysuccinimide ester method. The third scheme used the Z/Boc strategy and the azide method. These three methods for synthesizing GSB-106 were compared with respect to yield and optical purity. The optimum result was obtained by using the third scheme that involved Z/Boc protecting groups and the azide method of peptide bond formation.

Low-molecular-weight mimetics of loops 1 and 4 of the brain-derived neurotrophic factor (BDNF) have been designed and synthesized. The compounds represent monomeric and dimeric amides of N -acyldipeptides. Their dipeptide fragments coincide in sequence with the central regions of beta-turns of the corresponding neurotrophin loops, and acyl groups are the bioesosteres of preceding amino acid residues. Hexa- or heptamethylenediamines were used as spacers to link the C-terminal regions of dipeptides in dimeric mimetics of BDNF. These compounds were synthesized by classical methods of peptide synthesis in solution and received the laboratory codes GSB-104 (HO-Suc-Ser-Lys-NH 2 ), GSB-106 {[HO-Suc-Ser-Lys-NH-(CH 2 ) 3 −] 2 }, GSB-207 (HO-Suc-Met-Ser-NH 2 ), and GSB-214 ([HO-Suc-Met-Ser-NH-(CH 2 ) 7/2 -] 2 ). It was shown using immortalized hippocampal cells of the HT22 line under conditions of oxidative stress that the dimeric mimetics of both loops at concentrations of 10 −5 −10 −8 M possess a neuroprotective activity. The monomeric loop 1 mimetic GSB-207 in the same concentration range is inactive, and the monomeric loop 4 mimetic GSB-104 at a concentration of 10 −7 impairs the survival of neurons. The finding that only dimeric mimetics possess the neuroprotective activity is consistent with the data indicating that BDNF is active in the homodimeric form. As opposed to the dimeric loop 1 mimetic GSB-214, the dimeric loop 4 mimetic GSB-106 exhibits the antidepressant activity typical for BDNF in the Porsolt test on rats at doses of 0.1 and 1 mg/kg injected intraperitoneally. This suggests that the antidepressant activity of BDNF is related to its 4th loop. We believe that the compounds obtained will be useful in studies of the mechanism of action of BDNF and may form the basis for the design of a novel group of drugs with antidepressant and neuroprotective activities.