Abstract This study was performed in order to reveal the effect of Noopept (ethyl ester of N-phenylacetyl-Lprolylglycine, GVS-111) on the DNA-binding activity of transcriptional factors (TF) in HEK293 cells transiently transfected with luciferase reporter constructs containing sequences for CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Noopept (10 μM) was shown to increase the DNA-binding activity of HIF-1 only, while lacking the ability to affect that of CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, and HSF1. Noopept provoked an additional increase in the DNA-binding activity of HIF-1 when applied in conditions of CoCl2-induced HIF- 1 stabilization. The degree of this HIF-positive effect of Noopept was shown to be concentration-dependent. Piracetam (1 mM) failed to affect significantly any of the TF under study. The results of molecular docking showed that Noopept (L-isomer), as well as its metabolite, L-isomer of N-phenyl-acetylprolyl, unlike its pharmacologically ineffective D-isomer, is able to bind to the active site of prolyl hydroxylase 2. Taking into account the important role of the genes activated by HIF-1 in the formation of an adaptive response to hypoxia, data on the ability of Noopept to provoke a selective increase in the DNA-binding activity of HIF-1 explain the wide spectrum of neurochemical and pharmacological effects of Noopept revealed before. The obtained data allow one to propose the HIF-positive effect as the primary mechanism of the activity of this Pro-Gly-containing dipeptide. Keywords: Noopept, transcriptional factors, HIF-1, hypoxia, HIF-prolyl hydroxylase 2, docking, neuroprotection

INTRODUCTION Noopept (ethyl ester of N-phenylacetyl-L-prolylglycine) was designed as a drug at State Zakusov Institute of Pharmacology. The synthesis of the drug is based on the original hypothesis of peptide design, according to which structures similar to known psychotropic agents are reproduced using appropriate amino acids [1]. The non-peptide prototype of Noopept is the nootropic drug Piracetam. The pharmacological activity of the new compound turned out to be generally similar to the activity of Piracetam; but, it manifestes itself at doses 1,000 times lower than those for Piracetam [2, 3]. Moreover, Noopept has more pronounced anxiolytic [4] and neuroprotective properties [5-7]. A clinical study of Noopept (registration number 015770) confirmed the nootropic effects established experimentally. In patients with mild cognitive impairment of cerebrovascular and post-traumatic origin, the drug decreased cognitive impairment, showed an anxiolytic effect, and vegetostabilizing activity (www. noopept.ru). The mechanism of Noopept action has been studied since its synthesis. It has been established that the drug increases the expression of NGF and BDNF in the hippocampus [8], exhibits choline-positive properties at behavioral and neuronal levels [9], reduces oxidative stress and enhances the activity of antioxidant systems [7, 10], and represses kinases pSAPK/JNK and pERK1 induced by stress [11]. However, the study of the primary interactions of Noopept with more than 100 known receptors conducted according to our protocol by the CEREP company (France) did not lead to the expected identification of the primary targets. At the same time, the wide range of the neurochemical and pharmacological effects of Noopept prompted the further search for its targets. In order to obtain more exhaustive information on the targets of Noopept, we analyzed in vitro the influence of the drug on the DNA-binding activity of pharmacologically significant biological targets, the transcription factors (TF) CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Having identified the selective influence of Noopept on HIF-1, we examined the effect of the drug on the activity of this transcription factor under conditions mimicking the hypoxia in vitro.

CONCLUSIONS The data obtained on the ability of the effective nootropic and neuroprotective drug Noopept to cause an increase in the DNA-binding activity of HIF-1 allow one to advance a novel interpretation of the wide spectrum of its action: namely, assume that the HIF-1-positive effect of the drug can be considered as the primary mechanism of its action. Clarification of the molecular mechanisms underlying the HIF-1-positive action of Noopept certainly requires further investigation; but the presence of this effect definitely has a significant value, since it allows one to explain almost all known to date effects of Noopept and, probably, the effects of other biologically active Pro-Gly peptides. These data provide additional evidence for current concepts of the importance of the components of the HIF-1-dependent signaling pathway and the compensation processes activated by this transcription factor in the mechanisms of neuroprotection.

Acknowledgments The studies were performed using equipment from Biomika (Department of Biochemical Methods of Research and Nanobiotechnology Agidel, Ufa) and KODINK. This work was supported by a Russian President grant for leading scientific schools (5923.2014.4).