Searching for New Leads To Treat Epilepsy: Target-Based Virtual Screening for the Discovery of Anticonvulsant Agents

Abstract

The purpose of this investigation is to contribute to the development of new anticonvulsant drugs to treat patients with refractory epilepsy. We applied a virtual screening protocol that involved the search into molecular databases of new compounds and known drugs to find small molecules that interact with the open conformation of the Nav1.2 pore. As the 3D structure of human Nav1.2 is not available, we first assembled 3D models of the target, in closed and open conformations. After the virtual screening, the resulting candidates were submitted to a second virtual filter, to find compounds with better chances of being effective for the treatment of P-glycoprotein (P-gp) mediated resistant epilepsy. Again, we built a model of the 3D structure of human P-gp, and we validated the docking methodology selected to propose the best candidates, which were experimentally tested on Nav1.2 channels by patch clamp techniques and in vivo by the maximal electroshock seizure (MES) test. Patch clamp studies allowed us to corroborate that our candidates, drugs used for the treatment of other pathologies like Ciprofloxacin, Losartan, and Valsartan, exhibit inhibitory effects on Nav1.2 channel activity. Additionally, a compound synthesized in our lab, N, N'-diphenethylsulfamide, interacts with the target and also triggers significant Na1.2 channel inhibitory action. Finally, in vivo studies confirmed the anticonvulsant action of Valsartan, Ciprofloxacin, and N, N'-diphenethylsulfamide.

Design, synthesis and biological evaluation of N-substituted α-hydroxyimides and 1,2,3-oxathiazolidine-4-one-2,2-dioxides with anticonvulsant activity

“…Initially, the molecules were docked into the Nav1.2 channel with Autodock Vina software 26 . As the 3D structure of human Nav1.2 is not available, we employed a 3D model of this macromolecule previously constructed in our laboratory 24 . It includes the 3D architecture of the α-subunit of the Nav1.2 channel, which is functional on its own and comprises the region of interactions with ACD.…”

“…A docking score of −8.1 kcal/mol was defined as the threshold value to differentiate active from inactive compounds. This cut-off value was previously selected for the screening of a virtual database, since it shows a good balance between specificity and sensibility for the test set that validated the docking model (80% and 88%, respectively) 24 . Table 2 shows the docking scores of the synthesized compounds.…”

“…Docking simulations. The molecules were docked into the model of the 3D structure of the α-subunit of the human Nav1.2 (open-pore conformation) previously constructed by us 24 . The “docking active site” was defined based on the experimental data, and includes residues numbered from Phe1764 to Tyr1771 as important for the interactions of the channel with known ACDs 25 .…”

“…Previous analyses of the performance of different docking protocols allowed us to select Autodock Vina 26 as the best method to discriminate known binders from non-binders of Nav1.2 through the docking score, so the conditions of the docking are already reported. We set the cut-off value as −8.1 Kcal/mol to differentiate active from inactive compounds, which has associated specificity and sensibility rates of 80% and 88%, respectively, in the receiver operating characteristic (ROC) curve of the set of compounds used for the validation of the protocol 24 .…”

Cannabidiol (CBD) Inhibited Rhodamine-123 Efflux in Cultured Vascular Endothelial Cells and Astrocytes Under Hypoxic Conditions

“…Using the docking score as discriminating variable, we analyzed the capacity of different docking protocols to classify them correctly; that is, the non-binders should have higher docking score than binders. We quantified this information through the area under the receiver operating characteristic (ROC) curves (Palestro et al, 2018). This metric also allowed us to decide the threshold score that can be used to discern between the classes.…”