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 IТ 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 ІТ 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. Appl Comput Sci. 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 J Vet Res. 2021 Feb 10;88(1). DOI:10.4102/ojvr.v88i1.1872
Lamers MM, Haagmans BL. SARS-CoV-2 pathogenesis. Nat Rev Microbiol. 2022 May 30;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. J Autoimmun. 2020 May; 10(9):1024-34. 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. N Engl J Med. 2012 Nov 8;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 Dis. 2020 Dec;7(4):491-501. DOI:10.1016/j.gendis.2020.06.004
Peng XL, Cheng JSY, Gong HL, Yuan MD, Zhao XH, Li Z, et al. Advances in the design and development of SARS-CoV-2 vaccines. Mil Med Res. 2021 Dec 16;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 Rev. 2022 Feb; 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. Nat Commun. 2022 May 27;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. Biochim Biophys Acta - Gen Subj. 2017 Jan;1861(1):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 Oct 15;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 Oct 1;40(42):12591-603. DOI:10.1021/bi0105330
Luiking YC, Deutz NEP. Biomarkers of Arginine and Lysine Excess. J Nutr. 2007 Jun;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. Integr Med (Encinitas). 2017 Jun;16(3):42-6. PMID: 30881246 PMCID: PMC6419779
Dziublyk I, Soloviov S, Trokhimenko O, Dziublyk O, Smetiukh M, Yakovenko O, et al. In vitro study of the spectrum of antiviral activity of aliphatic acid against the prototype coronavirus strain. Biomed Biotechnol Res J. 2023;7(2):218. DOI:10.4103/bbrj.bbrj_36_23
S. P. Toxicological screening. J Pharmacol Pharmacother. 2011 Jun 1;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 Biol Toxicol. 1987 Mar;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. Biotechnol Appl Biochem. 2008 Oct 23;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. Pharmacol Ther. 2012 Apr;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. New Jersey: Humana Press; p. 415-426. 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. Toxicol Vitr. 2005 Jun;19(4):505-16. DOI:10.1016/j.tiv.2005.01.003
A. Momot, M. Zaboluieva R. Galagan Automated segmentation of ultrasound medical images using the Attention U-Net model. Nor J Dev Int Sci. 2024;№128.:56-60. DOI: 10.5281/zenodo.10817342
Mirajkar G, Garg L, Alaragisamy M, Shinde S. Image Processing in Toxicology: A Systematic Review. 2024.: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. High-throughput screening in toxicity assessment. In: Public Health and Toxicology Issues Drug Research, Volume 2. Elsevier; 2024.: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. Nat Prod J. 2025 Jan 10;15. DOI:10.2174/0122103155332267241122143118
Naghizadeh A, Tsao W chung, 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. Faeder JR, editor. PLOS Comput Biol. 2022 Mar 18;18(3):e1009883. DOI:10.1371/journal.pcbi.1009883
PubChem. 4-Aminobutyric Acid. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/4-Aminobutyric-Acid
PubChem. 5-Aminovaleric acid . Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/5-Aminovaleric-acid
PubChem. 6-Aminohexanoic Acid. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/6-Aminohexanoic-Acid
PubChem. 7-Aminoheptanoic acid. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/7-Aminoheptanoic-acid
Smetiukh MP. BIOTECHNOLOGICAL SYSTEM FOR THE SEARCH OF SUBSTANCES WITH POTENTIAL ACTIVITY AGAINST CORONAVIRUS. Biotechnol Acta. 2024 Dec 16;17(6):45-55. DOI:10.15407/biotech17.06.045
Kamiloglu S, Sari G, Ozdal T, Capanoglu E. Guidelines for cell viability assays. Food Front. 2020 Sep 16;1(3):332-49. DOI: 10.1002/fft2.44
Terry Riss, PhD, Andrew Niles, MS, Rich Moravec, BS, Natashia Karassina, MS, and Jolanta Vidugiriene P. Cytotoxicity Assays: In Vitro Methods to Measure Dead Cells 2019. PMID: 31070879
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. 2023. 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 2015. p. 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. Int J Mol Sci. 2021 Nov 26;22(23):12827. DOI:10.3390/ijms222312827
Mosmann T. Rapid colourimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec;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. Crit Rev Toxicol. 2009 Aug 3;39(7):613-26. DOI:10.1080/10408440903120975
Supino, R. (1995). MTT Assays. In: O’Hare, S., Atterwill, C.K. (eds) In Vitro Toxicity Testing Protocols. Methods in Molecular Biology™, vol 43. Humana Press. 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. Arch Biochem Biophys. 1993 Jun;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. Br J Pharm Res. 2017 Jan 10;15(6):1-16. DOI:10.9734/BJPR/2017/32762
Zhang M, Aguilera D, Das C, Vasquez H, Zage P, Gopalakrishnan V, et al. Measuring cytotoxicity: a new perspective on LC50. Anticancer Res. 2007;27(1A):35-8. PMID: 17352213
Hou D, Tang J, Feng Q, Niu Z, Shen Q, Wang L, et al. Gamma-aminobutyric acid (GABA): A comprehensive review of dietary sources, enrichment technologies, processing effects, health benefits, and its applications. Crit Rev Food Sci Nutr. 2024 Sep 20;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. Neurochem Res. 2022 Feb 8;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 Rev Int. 2023 Sep 8;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 alpha,omega-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
National Center for Biotechnology Information. 7-Aminoheptanoic acid hydrochloride. 2025. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/7-Aminoheptanoic-acid-hydrochloride
PubChem. 8-Aminocaproic acid. Bethesda (MD): National Library of Medicine (US). 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 Nov 13;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. J Synth Org Chem Japan. 2024 Jun 1;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. Front Microbiol. 2021 Sep 14;12. DOI:10.3389/fmicb.2021.669709
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