Comparative Assessment of Cytotoxicity of Aliphatic Amino Carboxylic Compounds as Potential Anticoronavirus Agents
DOI:
https://doi.org/10.20535/ibb.2025.9.4.347453Keywords:
cytotoxicity, in vitro, amino carbon compounds, MTT test, image processing, neural networkAbstract
Background. Despite the success in creating vaccines against SARS-CoV-2, the high mutagenicity of coronaviruses, interspecies transmission, and the emergence of new strains require further search for effective antiviral agents. A key step in this process is to evaluate the cytotoxicity of potential compounds to determine their safety and therapeutic potential. Modern IT solutions, such as automated image analysis and artificial intelligence, increase the accuracy and objectivity of assessments.
Objective. To determine the cytotoxicity of compounds with potential anticoronavirus activity and to analyze it using IT tools.
Methods. The study used the grafting cell line BHK-21 of the gerbil hamster, which was incubated with seven aliphatic amino carbon compounds in six concentrations. Cell viability was determined using the MTT assay. Cell monolayer image processing and an exponential dose-response model were used for automated analysis.
Results. The study revealed a pronounced dose- and time-dependent cytotoxicity of most samples, with a maximum decrease in viability at concentrations above 10 mg/ml. The hormesis effect was recorded at low concentrations (up to 5-10 mg/ml), which may indicate the activation of cellular defense mechanisms. The high correlation between measurements at 492 nm and 550 nm (R² > 0.98) confirmed the reliability of the spectrophotometric data. The exponential model allowed us to approximate the toxicity curves, especially in the middle and high concentration ranges. The built neural network based on image data and MTT test showed the ability to predict cell viability even with a limited amount of training data.
Conclusions. The combination of the MTT assay with automated image analysis provides a comprehensive assessment of cytotoxicity. A dose-dependent decrease in cell viability and morphological changes under the influence of the studied compounds were found. Measurements at 550 nm proved to be more sensitive to early changes in cell metabolism. The use of IT algorithms has demonstrated the prospects of an automated approach to the screening of biologically active substances.
References
Galagan R, Andreiev S, Stelmakh N, Rafalska Y, Momot A. Automation of Polycystic Ovary Syndrome Diagnostics Through Machine Learning Algorithms in Ultrasound Imaging. Applied Computer Science. 2024;20(2):194-204. DOI: 10.35784/acs-2024-24
Mulabbi EN, Tweyongyere R, Byarugaba DK. The history of the emergence and transmission of human coronaviruses. Onderstepoort Journal of Veterinary Research. 2021;88(1):e1-e8. DOI: 10.4102/ojvr.v88i1.1872
Lamers MM, Haagmans BL. SARS-CoV-2 pathogenesis. Nature Reviews Microbiology. 2022;20(5):270-84. DOI: 10.1038/s41579-022-00713-0
Yang Y, Peng F, Wang R, Guan K, Jiang T, Xu G, et al. The deadly coronaviruses: The 2003 SARS pandemic and the 2020 novel coronavirus epidemic in China. Journal of Autoimmunity. 2020;109:102434. DOI: 10.1016/j.jaut.2020.102434
Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus ADME, Fouchier RAM. Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia. New England Journal of Medicine. 2012;367(19):1814-20. DOI: 10.1056/NEJMoa1211721
Al-Rohaimi AH, Al Otaibi F. Novel SARS-CoV-2 outbreak and COVID-19 disease; a systematic review on the global pandemic. Genes & Diseases. 2020;7(4):491-501. DOI: 10.1016/j.gendis.2020.06.004
Peng X-L, Cheng J-S-Y, Gong H-L, Yuan M-D, Zhao X-H, Li Z, Wei D-X. Advances in the design and development of SARS-CoV-2 vaccines. Military Medical Research. 2021;8(1):67. DOI: 10.1186/s40779-021-00360-1
Cosar B, Karagulleoglu ZY, Unal S, Ince AT, Uncuoglu DB, Tuncer G, et al. SARS-CoV-2 Mutations and their Viral Variants. Cytokine & Growth Factor Reviews. 2022;63:10-22. DOI: 10.1016/j.cytogfr.2021.06.001
Tan CCS, Lam SD, Richard D, Owen CJ, Berchtold D, Orengo C, et al. Transmission of SARS-CoV-2 from humans to animals and potential host adaptation. Nature Communications. 2022;13(1):2988. DOI: 10.1038/s41467-022-30698-6
Fichtner M, Voigt K, Schuster S. The tip and hidden part of the iceberg: Proteinogenic and non-proteinogenic aliphatic amino acids. Biochimica et Biophysica Acta (BBA) - General Subjects. 2017;1861(1 Pt A):3258-69. DOI: 10.1016/j.bbagen.2016.08.008
Skwarecki AS, Nowak MG, Milewska MJ. Amino Acid and Peptide-Based Antiviral Agents. ChemMedChem. 2021;16(20):3106-35. DOI: 10.1002/cmdc.202100397
Avrahami D, Oren Z, Shai Y. Effect of Multiple Aliphatic Amino Acids Substitutions on the Structure, Function, and Mode of Action of Diastereomeric Membrane Active Peptides. Biochemistry. 2001;40(42):12591-603. DOI: 10.1021/bi0105330
Luiking YC, Deutz NEP. Biomarkers of Arginine and Lysine Excess. The Journal of Nutrition. 2007;137(6):1662S-1668S. DOI: 10.1093/jn/137.6.1662S
Mailoo VJ, Rampes S. Lysine for Herpes Simplex Prophylaxis: A Review of the Evidence. Integrative Medicine. 2017;16(3):42-6.
Dziublyk I, Soloviov S, Trokhimenko O, Dziublyk O, Smetiukh M, Yakovenko O, et al. In vitro Study of the Spectrum Antiviral Activity of Aliphatic Acid toward the Prototype Coronavirus Strain. Biomedical and Biotechnology Research Journal (BBRJ). 2023;7(2):218-24. DOI: 10.4103/bbrj.bbrj_36_23
Parasuraman S. Toxicological screening. Journal of Pharmacology and Pharmacotherapeutics. 2011;2(2):74-9. DOI: 10.4103/0976-500X.81895
Borenfreund E, Babich H. In vitro cytotoxicity of heavy metals, acrylamide, and organotin salts to neural cells and fibroblasts. Cell Biology and Toxicology. 1987;3(1):63-73. DOI: 10.1007/BF00117826
Aslantürk ÖS. In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages. In: Genotoxicity - A Predictable Risk to Our Actual World. InTech; 2018. DOI: 10.5772/intechopen.71923
Edwards V, Markovic E, Matisons J, Young F. Development of an in vitro reproductive screening assay for novel pharmaceutical compounds. Biotechnology and Applied Biochemistry. 2008;51(2):63-71. DOI: 10.1042/BA20070223
Astashkina A, Mann B, Grainger DW. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity. Pharmacology & Therapeutics. 2012;134(1):82-106. DOI: 10.1016/j.pharmthera.2012.01.001
O'Brien P, Haskins JR. In Vitro Cytotoxicity Assessment. In: High Content Screening, vol. 356. New Jersey: Humana Press; 2006, pp. 415-25. DOI: 10.1385/1-59745-217-3:415
Schoonen WGEJ, Westerink WMA, de Roos JADM, Débiton E. Cytotoxic effects of 100 reference compounds on Hep G2 and HeLa cells and of 60 compounds on ECC-1 and CHO cells. I Mechanistic assays on ROS, glutathione depletion and calcein uptake. Toxicology in Vitro. 2005;19(4):505-16. DOI: 10.1016/j.tiv.2005.01.003
Momot A, Zaboluieva M, Galagan R. Automated segmentation of ultrasound medical images using the Attention U-Net model. Norwegian Journal of Development of the International Science. 2024;(128):56-60. DOI: 10.5281/zenodo.10817342
Mirajkar G, Garg L, Alaragisamy M, Shinde S. Image Processing in Toxicology: A Systematic Review. In: Science, Engineering Management and Information Technology: Second International Conference, SEMIT 2023, Ankara, Turkey, September 14–15, 2023, Proceedings, Part-I, vol. 2198. Cham: Springer Nature Switzerland; 2024, pp. 159-75. DOI: 10.1007/978-3-031-72284-4_10
Rajpoot K, Panchal M, Pawar B, Vasdev N, Gupta T, Tekade M, et al. Chapter 15 - High-throughput screening in toxicity assessment. In: Public Health and Toxicology Issues in Drug Research, Volume 2: Toxicity and Toxicodynamics. Elsevier; 2024, pp. 407-49. DOI: 10.1016/B978-0-443-15842-1.00017-X
Prajapati P, Shrivastav P, Prajapati J, Prajapati B. Deep Learning Approaches for Predicting Bioactivity of Natural Compounds. The Natural Products Journal. 2026;16(3):e22103155332267. DOI: 10.2174/0122103155332267241122143118
Naghizadeh A, Tsao W-c, Hyun Cho J, Xu H, Mohamed M, Li D, et al. In vitro machine learning-based CAR T immunological synapse quality measurements correlate with patient clinical outcomes. PLoS Computational Biology. 2022;18(3):e1009883. DOI: 10.1371/journal.pcbi.1009883
4-Aminobutyric Acid, PubChem CID 223130. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/4-Aminobutyric-Acid
5-Aminovaleric acid, PubChem CID 138. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/5-Aminovaleric-acid
6-Aminohexanoic Acid, PubChem CID 564. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/6-Aminohexanoic-Acid
7-Aminoheptanoic acid, PubChem CID 13580. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/7-Aminoheptanoic-acid
Smetiukh MP, Trokhimenko OP, Soloviov SO, Dziublyk IV, Kamatskyi OA, Savchuk IV, Bobyr NA. Biotechnological system for the search of substances with potential activity against coronavirus. Biotechnologia Acta. 2024;17(6):45-55. DOI: 10.15407/biotech17.06.045
Kamiloglu S, Sari G, Ozdal T, Capanoglu E. Guidelines for cell viability assays. Food Frontiers. 2020;1(3):332-49. DOI: 10.1002/fft2.44
Riss T, Niles A, Moravec R, Karassina N, Vidugiriene J. Cytotoxicity Assays: In Vitro Methods to Measure Dead Cells. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004.
Ghasemi M, Liang S, Luu QM, Kempson I. The MTT Assay: A Method for Error Minimization and Interpretation in Measuring Cytotoxicity and Estimating Cell Viability. In: Cell Viability Assays: Methods and Protocols, vol. 2644. New York, NY: Springer US; 2023, pp. 15-33. DOI: 10.1007/978-1-0716-3052-5_2
Tolosa L, Donato MT, Gómez-Lechón MJ. General Cytotoxicity Assessment by Means of the MTT Assay. In: Protocols in In Vitro Hepatocyte Research, vol. 1250. New York, NY: Springer New York; 2015, pp. 333-48. DOI: 10.1007/978-1-4939-2074-7_26
Ghasemi M, Turnbull T, Sebastian S, Kempson I. The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis. International Journal of Molecular Sciences. 2021;22(23):12827. DOI: 10.3390/ijms222312827
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods. 1983;65(1-2):55-63. DOI: 10.1016/0022-1759(83)90303-4
Stone V, Johnston H, Schins RPF. Development of in vitro systems for nanotoxicology: methodological considerations. Critical Reviews in Toxicology. 2009;39(7):613-26. DOI: 10.1080/10408440903120975
Supino R. MTT Assays. In: In Vitro Toxicity Testing Protocols, vol. 43. Totowa, NJ: Humana Press; 1995, pp. 137-49. DOI: 10.1385/0-89603-282-5:137
Berridge MV, Tan AS. Characterization of the Cellular Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): Subcellular Localization, Substrate Dependence, and Involvement of Mitochondrial Electron Transport in MTT Reduction. Archives of Biochemistry and Biophysics. 1993;303(2):474-82. DOI: 10.1006/abbi.1993.1311
Aslantürk Ö, Çelik T, Karabey B, Karabey F. Active Phytochemical Detection, Antioxidant, Cytotoxic, Apoptotic Activities of Ethyl Acetate and Methanol Extracts of Galium aparine L. British Journal of Pharmaceutical Research. 2017;15(6):1-16. DOI: 10.9734/BJPR/2017/32762
Zhang M, Aguilera D, Das C, Vasquez H, Zage P, Gopalakrishnan V, Wolff J. Measuring Cytotoxicity: A New Perspective on LC50. Anticancer Research. 2007;27(1A):35-8.
Hou D, Tang J, Feng Q, Niu Z, Shen Q, Wang L, Zhou S. Gamma-aminobutyric acid (GABA): a comprehensive review of dietary sources, enrichment technologies, processing effects, health benefits, and its applications. Critical Reviews in Food Science and Nutrition. 2024;64(24):8852-74. DOI: 10.1080/10408398.2023.2204373
Cavalcanti-de-Albuquerque JP, De-Souza-Ferreira E, de Carvalho DP, Galina A. Coupling of GABA Metabolism to Mitochondrial Glucose Phosphorylation. Neurochemical Research. 2022;47(2):470-80. DOI: 10.1007/s11064-021-03463-2
Lee XY, Tan JS, Cheng LH. Gamma Aminobutyric Acid (GABA) Enrichment in Plant-Based Food - A Mini Review. Food Reviews International. 2023;39(8):5864-85. DOI: 10.1080/87559129.2022.2097257
Ooka AA, Kuhar KA, Cho N, Garrell RL. Surface interactions of a homologous series of α,ω-amino acids on colloidal silver and gold. Biospectroscopy. 1999;5(1):9-17. DOI: 10.1002/(SICI)1520-6343(1999)5:1<9::AID-BSPY3>3.0.CO;2-T
7-Aminoheptanoic acid hydrochloride, PubChem CID 13257585. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/7-Aminoheptanoic-acid-hydrochloride
8-Aminocaproic acid, PubChem CID 17802677. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/8-Aminocaproic-acid
Nandi N, Gayen K, Ghosh S, Bhunia D, Kirkham S, Sen SK, et al. Amphiphilic Peptide-Based Supramolecular, Noncytotoxic, Stimuli-Responsive Hydrogels with Antibacterial Activity. Biomacromolecules. 2017;18(11):3621-9. DOI: 10.1021/acs.biomac.7b01006
Morimoto J, Sando S. Development and Application of Methodologies for Measuring Passive Membrane Permeability of Peptides toward Understanding the Structure-permeability Relationship of Cyclic Peptides. Journal of Synthetic Organic Chemistry, Japan. 2024;82(6):613-21. DOI: 10.5059/yukigoseikyokaishi.82.613
Steigenberger J, Verleysen Y, Geudens N, Martins JC, Heerklotz H. The Optimal Lipid Chain Length of a Membrane-Permeabilizing Lipopeptide Results From the Balance of Membrane Partitioning and Local Damage. Frontiers in Microbiology. 2021;12:669709. DOI: 10.3389/fmicb.2021.669709
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