Abstracts

Rationale: Previously, our lab developed a library of 17 fluorinated N-benzamide enaminone analogs. The fluorinated compounds showed good to moderate activity in the 6 Hz 44 mA (the pharmcoresistant animal model) and maximal electroshock seizure rodent models with the para-trifluoromethyl (THA 40), ortho-fluoro, para-trifluoromethyl (GSA 62), and para-trifluoromethoxy (TTA 35) emerging as the lead compounds. Preliminary in-vitro electrophysiology studies on ND7-23 cells containing an assortment of voltage gated ion channels were conducted on the active analogs to determine the plausible mechanism of action. THA 40, GSA 62, and TTA 35 showed activity as potential sodium channel blockers. The current research aims to conduct target-based studies by utilizing two major methods (1) in-silico molecular docking studies on the open-form sodium channel and other seizure related molecular targets from the protein data bank to determine the ligand-target interactions and (2) in-vitro electrophysiology dose response studies to measure the degree of ionotropic blockade of the analogs. Methods: For the in-silico studies, a total of 55 binding sites were found using the site finder module in Molecular Operating Environment (MOE). The 15 largest binding sites were chosen for the ligand binding process. The lowest binding score was used to determine the most possible binding pocket for each ligand with minimal energy. Electrophysiology studies were employed to evaluate the compounds’ ability to inhibit sensory-like ND7/23 cells, which express an assortment of voltage and ligand-gated ion channels. Whole-cell patch-clamp recordings were obtained from the ND7/23 cells. Results: The results from the binding studies produced 4 identical pockets with similar interactions between the enaminone analogs and the amino acid residues in the pocket particularly Lys166 and Tyr169. These residues form strong hydrogen bond interactions with the ligands. With the electrophysiology studies, treatment of ND7/23 cells with 50 µM of THA 40, GSA 62, and TTA 35 generated a significant reduction in the amplitude of whole-cell sodium currents. Similar treatment of ND7/23 cells with these compounds had no effect on T-type calcium currents. Conclusions: This data confirms the preliminary assumption that the potential mechanism of action of the novel fluorinated enaminones is by acting on voltage gated sodium channels since they are responsible for the initial inward current during the depolarization phase of the action potential in excitable cells. The activation and opening of the channel results in the initial phase of an action potential and blockade of this channel reduces the firing of neurons thereby reducing neuronal excitability and suppressing seizures. Funding: The research was supported by a grant from the American Association of College of Pharmacy for which Patrice Jackson-Ayotunde was the principal investigator and the University of Maryland Eastern Shore Department of Pharmaceutical Sciences/Title III. The in silico study was supported, This work was supported by the NSF under Grant 1708959; The Office of Naval Research under Grant N00014-18-1-2145 awarded to Yayin Fang, PhD, Associate Professor of the Department of Biochemistry and Molecular Biology, Howard University, Washington, DC.