In silico and in vivo assessment of L-17, a thiadiazine derivative with putative serotonin reuptake properties

: L-17 is a thiadiazine derivative with putative anti-inflammatory, neuroprotective, and antidepressant-like properties. In this study, we applied combined in silico and in vivo electrophysiology techniques to reveal the potential mechanism of action of L-17. PASS 10.4 Professional Extended software suggested that L-17 might have pro-cognitive, antidepressant, and antipsychotic effects. Docking energy assessment with AutoDockVina predicted that the binding affinities of L-17 to the serotonin transporter (SERT) and serotonin receptors 3 and 1A (5-HT 3 and 5HT 1A ) are compatible to the selective serotonin reuptake inhibitor (SSRI) fluoxetine and selective antagonists of 5-HT 3 and 5-HT 1A receptors, granisetron and WAY100135, respectively. However, while the binding mechanisms of L-17 to the SERT and 5-HT 1A receptor were similar to fluoxetine and WAY100135, its interacting with 5-HT 3 receptor might be substantially different from this of granisetron. Acute administration of L-17 led to dose-dependent inhibition of firing activity of 5-HT neurons of the dorsal raphe nucleus. This inhibition was partially reversed by subsequent administration of WAY100135. Based on both in silico and in vivo electrophysiology assessments, we suggest that L-17 is a potent 5-HT reuptake inhibitor and a putative partial agonist of 5-HT 1A receptors. As such, L-17 in particular and thiadiazine derivatives, in general, might be a representative of a new class of antidepressant drugs. Since L-17 also possesses neuro- and cardioprotective properties, it can be useful in affective illness developing due to the general medical condition, such as post-stroke and post-myocardial infarction (MI) depression.


Introduction
L-17 ( In our previous studies, L-17 showed a putative therapeutic effect in animal models of myocardial infarction (MI) [2,3] and pancreatitis [4]. L-17 was also reported to attenuate the immune system response to the immobilization stress in rats, suggesting an immunostimulatory effect of this compound during the stress copying [5]. Neuroprotective [1] and antidepressant-like [6] effects of L-17 in rats were demonstrated as well.
Serotonin (5-HT) is a brain neurotransmitter that is fundamental in the pathophysiology of depression and antidepressant response. The central anti-neuroinflammatory and neuroprotective effects of 5-HT are well established as well. There are also studies reporting the peripheral antiinflammatory effect of 5-HT in MI [7] and pancreatitis [8]. It is therefore likely that the 5-HT system is a target of L-17 [6].
As in our previous study [9], we used a combination of in silico and in vivo experimental techniques to test the hypothesis that the anti-inflammatory, neuroprotective, and antidepressantlike effect of L-17 is mediated, at least in part, via the targeting of the 5-HT system. The 3-D model of L-17 was constructed, and its interaction with different 5-HT targets, such as serotonin transporter (SERT) and 5-HT receptors 3 and 1A (5-HT3/5-HT1A) was examined, used in silico calculation of the minimum binding energy. Finally, the real-time in vivo effect of L-17 administration on the excitability of 5-HT neurons of the rat dorsal raphe nucleus (DRN) was examined using extracellular single-unit electrophysiology under chloral hydrate anesthesia.

In silico predictions
The following pharmacological activities were predicted for L-17 by the PASS 10. While L-17 compound manifests itself as an atypical mild antipsychotic and antidepressant [1], for the subsequent analysis of its multitarget mechanism of action the serotonin transporter (SERT), the serotonin receptor type 3A (5-HT3A), and the serotonin receptor type 1A (5-HT1A) were chosen as target proteins. Table 1 summarizes the docking energy (ΔE), binding affinity (pK), and relative affinity (RA; L-17 th pK as a percent of pK of the corresponding binding compound) of L-17, fluoxetine, granisetron, and WAY100135 with the SERT and 5-HT3 and 5-HT1A receptors, assessed with the

Discussion
In this study, we performed a complex in silico assessment the SERT and an antagonist or a partial agonist of 5-HT1A receptors. Further investigations should be performed to assess the functional character of L-17 interaction with 5-HT3 receptors.
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 were observed with other SSRIs, such as citalopram [13], escitalopram [13,14], Wf-516 [15], or paroxetine [16]. Similar to what was observed with other SSRIs, the L-17-induced inhibition 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 [18]. 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 [19]. The experimental 3D models for 5-HT3 and 5-HT1Areceptors 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 [20]was conducted. Among the available models, the longest ones, with the highest statistical significance, were selected for 5-HT3[21]and 5-HT1A [22]receptors.