Combined in silico, ex vivo, and in vivo assessment of L-17, a thiadiazine derivative with putative neuroprotective and antidepressant-like effects

: L-17 is a thiadiazine derivative with putative anti-inflammatory, neuroprotective, and antidepressant-like


Introduction
L-17 (2-morpholino-5-phenyl-6H-1,3,4-thiadiazine, hydrobromide; Fig. 1) is a thiadiazine derivative, synthesized by cyclocondensation of α-bromoacetophenone with the original morpholine-4-carbothionic acid hydrazide [1]: In our previous studies, L-17 we have shown a putative therapeutic effect of myocardial infarction (MI) [2][3][4] and pancreatitis [5] in animal models. L-17 has also been reported to attenuate the immune system response to the immobilization stress in rats, suggesting an immunostimulatory effect of this compound during the stress copying [4]. Neuroprotective [1] and antidepressant-like [6] effects of L-17 in rats have been demonstrated, as well. In this study, we aimed to investigate the mechanism of the putative beneficial effect of L-17 on the central nervous system (CNS), using a combination of in silico, ex vivo, and in vivo methods [7].
For in silico assessments, we constructed three-dimensional (3D) model of L-17, and examined its interaction with different targets, such as serotonin (5-HT) transporter (SERT) and 5-HT receptors 3 and 1A (5-HT3/5-HT1A), used the calculation of the minimum binding energy. SERT and 5-HT3 were chosen because they play a primary role in the pathophysiology and treatment of CNS disorders.
Immunohistochemical assessment of proto-oncogene Fos across the brain is an ex vivo experimental technique used for the investigation of mechanism of action of novel CNS drugs. The activation of the proto-oncogene Fos, resulting in an increased expression of its protein product c-Fos, is a well-established marker for the neuronal excitability within the specific brain area [8][9][10][11].
Increased proto-oncogene Fos expression in the amygdala, indicating increased neural excitability in this brain area, has been observed after administration of various CNS drugs, such as typical [12] and atypical antipsychotics [12-14], tricyclic antidepressants [13], and selective 5-HT reuptake inhibitors (SSRIs) [13,15,16]. The SSRI citalopram-induced increase in c-Fos immunoreactivity has been shown to be potentiated by the selective antagonist of 5-HT1A receptors WAY100638 [17], suggesting a key role of 5-HT system in the modulation of amygdaloid excitability by CNS drugs. With regards to the prefrontal cortex (PFC), some SSRIs, such as fluvoxamine [16], and some atypical antipsychotics, such as olanzapine [18,19], increased local c-Fos immunoreactivity. On the other hand, 5-HT depletion led to an increased c-Fos immunoreactivity in the PFC and hippocampus [20], suggesting that extracellular 5-HT suppresses neural excitability and Fos proto-oncogene expression in these brain areas.
Finally, we used single-unit extracellular electrophysiology in vivo to examine the effect of L-17 on the excitability of 5-HT neurons in the rat dorsal raphe nucleus (DRN). Multiple antidepressant drugs, such as tricyclic antidepressants, SSRIs, dual 5-HT/norepinephrine, and triple 5-HT/ norepinephrine/dopamine reuptake inhibitors, pyridoindoles (experimental drugs with putative triple reuptake inhibition property), and some atypical antipsychotics, excerpt potent acute inhibitory effect on the excitability of 5-HT neurons of the DRN. This inhibition is usually reversed by the selective 5-HT1A receptors antagonist, suggesting the involvement of the extracellular 5-HT. The assessment of the effect of an experimental CNS drug on the excitability of 5-HT neurons is a key marker for the preclinical assessment of its efficacy.

In silico predictions
The following pharmacological activities were predicted for L-17 by the PASS 10.

Discussion
In this study, we performed a complex in silico assessment of pharmacotherapeutic properties  Two WAY100135 molecule fragments participate in   three nonspecific hydrophobic interactions with ALA69, TYR72, TRP78, ILE89, VAL93, ILE165, ALA179, PHE338, LEU357 of the binding site of the 5-HT3 receptor.
It has been demonstrated that the pre-treatment with L-17 robustly increased c-Fos immunoreactivity in the amygdala. Amygdala is a fundamental structure in the emotional responses, including fear and anxiety [24]. Thus, it is possible that the modulation of amygdaloid neural excitability is involved in the therapeutic response to antidepressant, anxiolytic, and mood stabilizing medicines. It has indeed been reported that various CNS drugs, such typical [12] and atypical antipsychotics [12][13][14], tricyclic antidepressants [13], and selective 5-HT reuptake inhibitors (SSRIs) [13,15,16], increased c-Fos immunoreactivity in the amygdala, indicating an increased neural excitability in this brain area. In our previous studies, we have found that pyridoindole derivate SMe1EC2M3, a molecule with putative 5-HT reuptake inhibition properties and antidepressant-like behavioral effect [7], stimulated amygdaloid c-Fos immunoreactivity, as well [11]. The fact that SSRI- With regards to proto-oncogene Fos expression in other brain areas, we found that acute administration of L-17 significantly decreased c-Fos immunoreactivity in the hippocampus and tended to decrease it also in the PFC. It has been previously reported that 5-HT depletion led to an increased c-Fos expression in these brain areas [20]. Thus, it is possible that the L-17-induced decrease in forebrain c-Fos immunoreactivity might be explained by the ability of this drug to elevate extracellular 5-HT concentrations. Further studies should be, however, performed to test this hypothesis.
We found that acute intravenous administration of L-17 significantly and dose-dependently inhibited the firing activity of 5-HT neurons of the DRN. Similar inhibitory effects on 5-HT neuronal firing activity has been observed with other SSRIs, such as citalopram [25], escitalopram [25,26], Wf-516 [27] or paroxetine [28]. Similar to that observed in other SSRIs, the L-17-induced inhibition of 5-HT neuronal firing activity was reversed by WAY100135. It is likely that L-17 is acting as a potent Further, for a comparative evaluation of the L-17 compound's affinity to the selected biotargets, the docking of the compound to the specific binding sites of these proteins was performed. Five experimental X-ray 3D models of SERT were obtained from Protein Data Bank in Europe [30]. Among these five models, the longest one (PDB code 5I6X), with the maximum resolution including an inhibitor, was chosen to allow the unambiguous determination of the binding site position [31]. The experimental 3D models for 5-HT3 and 5-HT1A receptors were not available in Protein Data Bank in Europe, therefore a search for the best theoretical 3D models from the Database of Comparative Protein Structure Models [32]was conducted. Among the available models, the longest ones, with the highest statistical significance, were selected for 5-HT3 [33] and 5-HT1A [34] receptors.
The 3D models of L-17 and reference molecules were constructed using the molecular mechanics' methods in the MarvinSketch15.6.15software [35], followed by optimization with the semi-empirical quantum chemical method PM7 in the MOPAC2016 software [36]. The docking was performed using AutoDockVina 1.1.2 [23], five times for each compound into each target and then the spectrum of energies were used to determine the minimum binding energies. To reveal the mechanisms of serotoninergic action of the compound L-17, an analysis of its affinity spectrum in comparison with the affinity spectra of reference compounds was performed. The primary information about reference compounds for target proteins was obtained from the UniProtKB base [37]. For each reference compound found in the UniProtKB, the mechanism of its action was clarified using the DrugBank[38]. In the case of the information in UniProtKB and DrugBank datasets was not sufficient, the search for references was performed in the database of pharmacologically relevant proteins and their ligands IUPHAR [39]. The SSRI fluoxetine and the selective antagonists of 5-HT3(granisetron) and 5-HT1A(WAY100135) receptors were used as reference molecules. Using the obtained energy spectra of ΔE, the pK values were calculated using the formula: where R is a universal gas constant (8.314 J×mol -1 ×K -1 ) and T is a temperature, set as 300 K. The molecular binging mechanism of L-17, granisetron, and WAT100135 with SERT and with 5-HT3 and 5-HT1Areceptors was furtherly evaluated using the LigandScout 4.2.1 software [22]. The data on binding sites were found in the available literature. For SERT, the key amino acids of the binding site were Gly94, Ala96, Val97, Asn101, Ser336, Asn368, Leu434, Asp437, Ser438 [31]. For 5-HT3receptor, the key amino acids of the binding site were Tyr229, Phe221, Asn123, Trp85, Trp178, Tyr148, Arg87, Gln146, Tyr138 [33]. For 5HT1Areceptor, the key amino acids of the binding site were Tyr56, Gln57, Asp76, Val77, Ser159, Trp318, Phe321, Phe322, Thr339, Gly342, Ala343, Ile345, Asn346 [34].

Assessment of c-Fos immunoreactivity
For the assessment of the proto-oncogene c-Fos expression, rats were pre-treated with L-17 (60 mg/kg, intraperitoneally, i.p.) twenty-four and one hour before immunoreactivity measurements. C-Fos expression was analyzed, as we have reported previously [11,40]. The topographic mapping of the c-Fos protein immunolabeled cells was performed in three brain areas (PFC, hippocampus, and central nucleus of the amygdala) identified based on the rat brain atlas [11,41]. The individual structures investigated were captured unilaterally from 6-10 representative sections using an Axio-Imager A1 light microscope (Carl Zeiss, Jena) coupled to a video camera and monitor. Since the staining of each individual Fos-immunoreactive cell varied from very intense to very light, c-Fos profiles counting was performed on pictures inverted by the Adobe