Concentrations of 100 M and 50M were selected to get LY294002 and PD98059, respectively [12, 13]. GK-2 did not stimulate hyperalgesia, which is one of the primary adverse effects of NGF. By contrast, GK-6 produced a substantial decrease in the pain threshold of rats as based on the tail flick test. == Realization == The information obtained suggest that dimeric dipeptide NGF mimetics are encouraging candidates in the development of pharmacological agents CLEC4M with NGF-like activity that are free of the main side effect of NGF. Keywords: Nerve growth aspect, GK-2, GK-6, PI3K/AKT, MAPK/ERK == History == Nerve growth aspect (NGF), a member of the neurotrophin family, is essential for the development and survival of a number of populations of neurons and a number of nonneural cells. Despite this factors substantial therapeutic potential, the medical application of NGF is limited by its strong side effects, the most important of which are hyperalgesia and weight loss [1]. NGF exerts its main effects by conversation with the TrkA transmembrane receptor. Activation of TrkA by NGF activates signal transduction cascades including phosphatidylinositol 3-kinase/AKT (PI3K/AKT) and mitogen-activated proteins kinase/extracellular-signal-regulated kinases (MAPK/ERK) pathways. The PI3K/AKT pathway is usually involved in the regulation of cell survival but not in the differentiation and formation of neurites [2]. The MAP-kinase pathway is Topiroxostat (FYX 051) associated with neuroprotection and differentiation and appears to be involved with hyperalgesia [3]. The design of small , proteolytically stable NGF mimetics that exert defined biological activities via the selective activation of TrkA-mediated signaling might give a useful strategy for the development of therapeutic providers for several illnesses applications [2, 4]. We created the working hypothesis [5] that by interacting with the same receptor, multiple neurotrophin hairpin loops can stimulate various intracellular signaling cascades and are consequently responsible for numerous neurotrophin effects. Within the platform of this hypothesis, the dimeric dipeptide bis(N-succinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2) was designed based on the NGF loop Topiroxostat (FYX 051) 4 -turn series Asp93Glu94Lys95Gln96, which is the most uncovered fragment and for that reason may play a major role in the conversation of NGF with the receptor. We included the central fragment in the -turn, Glu94Lys95, in the dipeptide composition. The residue Asp93 was substituted by its bioisostere, a succinic acid solution residue, and Gln96 was substituted by an amide group. The purpose of these two substitutions was to stabilize the -turn conformation and to increase the resistance of the substance to peptidases. Because NGF interacts with the TrkA in the homodimer contact form, we linked two -turn mimetics by a hexamethylene diamine spacer. The dimeric dipeptide bis-(N-aminocaproyl-glycyl-L-lysine) hexamethylenediamide (GK-6) was designed analogously to GK-2 based on the NGF loop 1 -turn (RU Patent 2410392, 2010; US Patent Appliction US 2011/0312895 A1). It has been shown in vitro, using both immortalized and primary cell cultures, that GK-2 and GK-6 exert NGF-like neuroprotective activity (10uM-1nM) [5, 6]. Maximal neuroprotective effects were seen at concentrations of 1u (GK-6) and 10n (GK-2); therefore , these concentrations were used for additional in vitro experiments. The neuroprotective activity of GK-2 at doses of 0. 1-1 mg/kg (i. p. ) was also determined in animal models of cerebral ischemia [7, 8] and in a model of rat traumatic brain injury [9]. Herein, we statement a comparative study in the NGF loop 1 and NGF loop Topiroxostat (FYX 051) 4 -turn mimetics, GK-6 and GK-2, respectively. We established that both peptides activate TrkA receptors yet showed diverse patterns of intracellular signal transduction. == Methods == == Drugs and reagents == The dimeric dipeptides GK-6 and GK-2 were synthesized around the base of murine NGF at the Zakusov Institute of Pharmacology (Moscow, Russia). Inhibitors of PI3K (LY294002) and MAPK (PD98059) were purchased from Tocris Bioscience (Bristol, UK). The tetrazolium color 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbeccos Altered Eagles medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OKAY, USA). Glutamine was purchased from ICN Pharmaceuticals. Inc. (Costa Mesa, CA, USA). Poly-D-lysine was purchased coming from BD Biosciences (San Jose, CA, USA). DC proteins assay was purchased.