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  • NMDARs are glutamate gated ionotropic receptors that are


    NMDARs are glutamate-gated ionotropic receptors that are involved in excitatory neurotransmission and plasticity (Traynelis et al., 2010). There have been several reports showing that neurosteroids acting as negative modulators of NMDARs exert neuroprotective activity in both in vitro and in vivo models of neurodegeneration (Lapchak, 2004; Rambousek et al., 2011; Vyklicky et al., 2016). The inhibitory GABAA receptors are the major type of receptors that underlie the physiologically and clinically relevant effects of neurosteroids, and they are the most extensively studied type (Belelli and Lambert, 2005). In view of the above, it seems reasonable to continue the evaluation and development of neurosteroids because of their important regulatory activities, neuroprotective properties and multiple mechanisms of action. Therefore, this paper describes the effects of selected neuroactive steroids 1–6 (Fig. 2), which are potent negative modulators of excitatory NMDARs (Borovska et al., 2012; Adla et al., 2017; Weaver et al., 1997; Vyklicky et al., 2016; Chodounska et al., 2016), on inhibitory GABAA and Gly receptors of rat hippocampal neurons. The primary goal of this study was to obtain a structure-activity relationship for neurosteroid actions on GABAARs and GlyRs, and then to compare it with their effect on NMDARs. Such a complex structure-activity relationship study (SAR) has not been reported previously and could be used for the further development of neuroprotective therapeutics, which is a topic of great interest. Pregnanolone glutamate (PA-Glu, Timolol Maleate australia 1, Fig. 2), pregnanolone hemisuccinate (PA-hSuc, compound 2, Fig. 2), and pregnanolone hemipimelate (PA-hPim, compound 3, Fig. 2) were chosen as the main candidates for further screening, as all three compounds were potent inhibitors of NMDA-induced currents, with IC50 values varying from 17 to 51 μM (Borovska et al., 2012; Adla et al., 2017; Weaver et al., 1997; Vyklicky et al., 2016; Chodounska et al., 2016). Moreover, PA-Glu, PA-hSuc, and PA-hPim were shown to exert neuroprotective effects in vivo in several biological models (Rambousek et al., 2011; Holubova et al., 2014; Kleteckova et al., 2014; Lapchak, 2004; Vyklicky et al., 2016). Our previous SAR study showed that non-polar modification of the steroid D-ring in combination with isosteric amide-based C-3 substitution of various lengths and types leads to an increase in the ability of steroids to inhibit the NMDARs current (Kudova et al., 2015, 2016; Adla et al., 2017). In this paper, androstane glutamate (AND-Glu, compound 4, Fig. 2), androstane aspartylamide (N-2′-L-Asp-AND, compound 5, Fig. 2) and 17β-methyl-androstane hemimalonylamide (17β-Me-AND-3β-hMalAmide, compound 6, Fig. 2) were selected for the screening.
    Material and methods
    Discussion Neurosteroids capable of blocking the NMDAR channel attract a lot of attention as possible therapeutic agents for the treatment of neurodegenerative diseases. A large number of new steroids with different structures have been synthesized in order to create an effective antagonist of NMDAR with minimal side effects (Adla et al., 2017; Holubova et al., 2014; Kleteckova et al., 2014; Kudova et al., 2015; Rambousek et al., 2011; Vyklicky et al., 2016). As the neurosteroids can exhibit multi-target action (Korinek et al., 2011; O'Dell et al., 2005), the study of their ability to interact with other receptors, including inhibitory GABAAR and GlyR, is an important task. The effect of steroids on IGABA has been studied by many authors (for a review, see: Belelli and Lambert, 2005; King, 2013; Korinek et al., 2011; Majewska et al., 1988; Wu et al., 1990; Zorumski et al., 2013). It has been shown that progesterone, pregnanolone, and allopregnanolone enhance IGABA while pregnanolone sulfate and pregnenolone sulfate inhibit it. Due to the ability to enhance GABA responses, steroids exhibit anxiolytic, analgesic, anticonvulsant, sedative, hypnotic and anaesthetic properties (for a review see Belelli and Lambert, 2005). In our experiments, the effects of two groups of steroids in the concentration range 0.1–50 μM were studied on the IGABA of rat pyramidal hippocampal neurons: pregnanolone glutamate (PA-Glu) and its derivatives, and androstane glutamate (AND-Glu) and its derivatives. There is an important structural difference between these groups, namely, the presence of a C-17 acetyl moiety. It was found that only analogues of pregnanolone (PA-Glu, PA-hSuc and PA-hPim) and not analogues of androstane could modulate IGABA. These results suggest that a C-17 acetyl moiety is necessary to affect the GABAAR of hippocampal pyramidal neurons. Our data agree with the results of Harrison et al. (1987), who considered that a ketone moiety at C-20 is a structural requirement for the interaction of a steroid with GABAAR. We found that PA-Glu increased IGABA peak amplitude with a maximum potentiation of up to 422%, and an EC50 of 7 μM. Thus, PA-Glu appeared to be a weaker IGABA enhancer than pregnanolone, which effectively potentiated this current at nanomolar concentrations (Belelli and Lambert, 2005; Weir et al., 2004; Wu et al., 1990). In contrast to PA-Glu, its derivates PA-hSuc and PA-hPim decreased IGABA with IC50 values close to 10 μM. These values are close to or lower than those obtained in the study of the effects of pregnenolone sulfate on IGABA: IC50 = 60 μM (Majewska et al., 1988) and 7 μM (Wu et al., 1991). Our study is the first one describing acceleration of desensitization of the IGABA in the presence of steroids. The effect of PA-hSuc on the IGABA desensitization was stronger than on the IGABA peak, and this suggest that tonic inhibition in hippocampus can be suppressed stronger than phasic one with this steroid. In contrast, the effects PA-hPim on the peak and decay of the IGABA were similar, and this means that tonic and fast synaptic inhibition are suppressed in a similar way.