Miconazole mg The ester moiety is a promising structural mot
The ester moiety is a promising structural motif at the C-3 position for the development of drug-like molecules. However, the labile C-3 ester bond impels these derivate to be susceptible to plasmatic degradation by esterases and this is natural steroidal metabolic process [39,40]. Hence, the isosteric replacement of the ester oxygen with a nitrogen or a carbon Miconazole mg is supposed to improve metabolic liability of these analogs while maintain desired biological effect. Here, we demonstrated that the C-3 amide bond is an allowed structural modification while maintaining neuroprotective acitivity. Nevertheless, our results demonstrate that isosteric approach cannot be used in the additive manner. Indeed, compounds 6, 10, 15, and 17 decreased cell viability after co-treatment with glutamate for 24 h, as compared to glutamate alone. This decrease was accompanied by an increase in [Ca2+]i levels, ROS, and caspase-3 activity. This effect is likely caused by the intrinsic cytotoxicity of the metabolites of these compounds (10, 15), since they were also found to decrease neuronal viability in the absence of glutamate after 24 h. This toxicity is clearly caused by ROS generation, as the viability of the neurons was significantly improved after co-treatment with the antioxidant Trolox that has been shown to prevent ROS production as well as cell death [41,42]. Decreasing the concentration of these compounds to 1 μM significantly improved neuronal viability, even in the presence of glutamate. It should be noted that compounds 6, 10, 15 and 17 were recently found to possess adverse toxic effect in HepG2 cells , implicating that the glutamate moiety at C-3 (compound 15) could be responsible for the cytotoxicity in neurons. Conversely, both the aspartate moiety (compound 13) and its Boc-protected analog (compound 14) represent “allowed” structural features, and thus impart neuroprotective effect.
In conclusion, novel neurosteroids presented in this study were able to protect cortical neurons from glutamate-mediated excitotoxicity in vitro through their specific binding to the NMDA receptor. While some of these compounds do have cytotoxic effect, most of them do not show any adverse effects on cell viability. However, it should also be noted that the observed cytotoxicity is highly dependent on both time and concentration. Compound 13 showed the best protection of neuronal cells against glutamate/NMDA-induced excitotoxicity while retaining its neuroprotective properties under the condition of chronic exposure to these stimulants, significantly better than memantine and comparable with that of compound 2 (MK-801).
Conflict of interest
Ethics Animal experiments were approved by the Institutional Animal Ethics Committee of IPHYS CAS, The Ministry of Agriculture of the Czech Republic (http://eagri.cz/public/web/en/mze/) and Prague Veterinary administration (https://www.mevs.cz/), registration number CZ 11760005.
Acknowledgements This work was supported by grant TN01000013, Project PerMed: Personalized Medicine – Diagnostics and Therapy, National Centres of Competence 1, Technology Agency of the Czech Republic; TE01020028Center for Development of Original Drugs from the Technology Agency of the Czech Republic, project InterBioMed LO1302 from the Ministry of Education of the Czech Republic, research project of the RVO67985823, RVO 61388963. The author would like to thank Dr. Martin Horák (IPHYS CAS) for primary cortex tissue donation and Dr. Laurel McGrane for language corrections.
Introduction The amino acid l-glutamate is an essential element of life, and has been very early used as a messenger. During evolution, l-glutamate became one of the major neurotransmitters, being the mediator of the neuro-muscular junction in arthropods and being responsible for transmitting signals at most fast excitatory synapses in the brain (Fonnum, 1984). Such an essential role of glutamate, associated with all the other functions of this amino acid, imposes a tight regulation of its extracellular concentrations. Indeed, an inaccurate control of extracellular glutamate levels can lead to epilepsy and brain damage through excitotoxicity (Meldrum, 2000). Essential mechanisms have then been selected during evolution to monitor extracellular glutamate concentrations, including active uptake mechanisms and a control of glutamate entry at the level of the blood brain barrier. Defects in these mechanisms have been proposed to be responsible for a number of brain diseases, including neurodegenerative disorders as well as psychiatric diseases or addiction (Kalivas, 2009; Lewerenz and Maher, 2015; Miladinovic et al., 2015).