Anticonvulsant Evaluation of 2-Amino Pyrimidine-Based Mannich Base Derivatives Targeting the GABA-A Receptor: In Silico, In Vitro and In Vivo Studies
DOI:
https://doi.org/10.63682/jns.v14i2S.7056Keywords:
2-Amino Pyrimidine, Mannich bases, Anticonvulsant activity, GABA-A receptor, PDB ID, 6X3D, Maximal electroshock seizure (MES) modelAbstract
Epilepsy affects over 50 million individuals worldwide and is characterized by recurrent seizures that are sometimes unresponsive to current therapies and induce negative effects, underscoring the urgent need for new anticonvulsant medications. This study examines the anticonvulsant effects of 2-Amino Pyrimidine-based Mannich base derivatives that target the GABA-A receptor, a critical inhibitory neurotransmitter site for seizure regulation. Fifteen new derivatives were produced and subjected to molecular docking using Auto Dock Tools 1.5.7 against the GABA-A receptor (PDB ID: 6X3D), indicating strong binding affinities for compounds SSN6, SSN15, SSN10 and SSN12, with docking scores of 14.0, 12.8, 9.2 and 9.0 kcal/mol, respectively. These compounds showed important contacts with the receptor active site residues. In vitro MTT testing on SHSY5Y neuroblastoma cells revealed more than 75% cell viability at 40 µg, suggesting excellent neuroprotection and low cytotoxicity. In vivo confirmation using the maximal electroshock seizure (MES) model in mice revealed that both SSN6 and SSN15 substantially reduced seizure duration (p < 0.05), similar to the standard drug Phenobarbitone, with no toxicity or mortality reported. The combined in silico, in vitro and in vivo findings indicate that 2-Amino Pyrimidine Mannich base derivatives, notably SSN6 and SSN15, are promising candidates for developing effective and safer anticonvulsant drugs
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Thurman DJ, Beghi E, Begley CE, Berg AT, Buchhalter JR, Ding D, et al. Standards for epidemiologic studies and surveillance of epilepsy. Epilepsia. 2011;52(Suppl 7):2–26. doi:10.1111/j.1528-1167.2011.03121.x
Olsen RW, Sieghart W. GABAA receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology. 2009;56(1):141–148. doi:10.1016/j.neuropharm.2008.07.045.
Kharb R, Sharma PC, Yar MS. Pharmacological significance of synthetic heterocyclic Mannich bases: a review. Eur J Med Chem. 2011;46(7):2327–2346. doi:10.1016/j.ejmech.2011.03.032.
Rangaswamy CR, Dastagiri Reddy N, Basavanag U, Goudanavar PS, Yaragatti C, Ramesh C. In silico docking and in vivo evaluation of novel 1,2,4-triazole derivatives as potential anticonvulsant agents. Eur J Pharm Sci. 2019;132: 96-106. doi:10.1016/j.ejps.2019.02.021.
Iwata H, Mizutani H, Yamamoto M. Structure drawing programs for chemical structure database: ChemDraw and ChemSketch. J Chem Inf Comput Sci. 2003;43(4):1269–1275.
DeLano WL. The PyMOL Molecular Graphics System, Version 2.5. Schrödinger, LLC; 2020. Available from: https://pymol.org
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem. 2009; 30(16): 2785–2791. https://doi.org/10.1002/jcc.21256.
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The Protein Data Bank. Nucleic Acids Res. 2000; 28(1):235–242. https://doi.org/10.1093/nar/28.1.235
Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem. 1998; 19(14):1639–1662.
Kitchen DB, Decornez H, Furr JR, Bajorath J. Docking and scoring in virtual screening for drug discovery: Methods and applications. Nat Rev Drug Discov. 2004;3(11):935–949. https://doi.org/10.1038/nrd1549.
BIOVIA Discovery Studio Visualizer, version 2024. San Diego: Dassault Systèmes; 2024.
Xicoy H, Wieringa B, Martens GJM. The SH-SY5Y cell line in Parkinson’s disease research: A systematic review. Mol Neurodegener. 2017;12(1):10. https://doi.org/10.1186/s13024-017-0149-0
Riss TL, Moravec RA, Niles AL, et al. Cell Viability Assays. In: Markossian S, Grossman A, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004.
Kandasamy M, Lehner B, Kraus S, et al. Neuroprotective effect of resveratrol against Aβ-induced oxidative stress and apoptosis in SH-SY5Y cells. Neurochem Int. 2010;57(6):651–658. https://doi.org/10.1016/j.neuint.2010.07.003
Freshney RI. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. 7th ed. Hoboken, NJ: Wiley-Blackwell; 2016.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods.1983;65(1-2):55–63. https://doi.org/10.1016/0022-1759(83)90303-4
Stepanenko AA, Dmitrenko VV. HEK293 in cell biology and cancer research: Phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene. 2015;569(2):182–190. https://doi.org/10.1016/j.gene.2015.05.065
Vohora D, Pillai KK, Khanam R. Evaluation of CNS activity of newer antiepileptic drugs using different animal models. Indian J Pharmacol. 2001;33(4):268–276.
Löscher W, Schmidt D. Which animal models should be used in the search for new antiepileptic drugs? A proposal based on experimental and clinical considerations. Epilepsy Res. 1988;2(3):145–181. https://doi.org/10.1016/0920-1211(88)90054-X
Löscher W. Animal models of seizures and epilepsy: Past, present, and future role for the discovery of antiseizure drugs. Neurochem Res. 2020;45(1):187–203. https://doi.org/10.1007/s11064-019-02820-2
Dixit S, Goyal A, Jadhav HR. Evaluation of anticonvulsant activity of novel 1,3,4-oxadiazole derivatives using MES and PTZ models in mice. Eur J Pharm Sci. 2016;89:108–116. https://doi.org/10.1016/j.ejps.2016.03.004
CPCSEA Guidelines for Laboratory Animal Facility. Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment & Forests, Government of India. 2003.
Bhutada P, Mundhada Y, Bansod K, Ubgade A, Patil S, Mundhada D. Anticonvulsant and anxiolytic activity of hydroalcoholic extract of Delphinium denudatum root. Indian J Pharmacol. 2010;42(3):171–175. https://doi.org/10.4103/0253-7613.68424
LoPachin RM, Lehning EJ. Mechanism of action of the anticonvulsant valproic acid. DrugMetab Rev. 1997;29(1-2):243–267. https://doi.org/10.3109/03602539709037578
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