Current Protein & Peptide ScienceCurrent Protein & Peptide ScienceCurrent Protein & Peptide Science1389-20371875-5550Bentham Science64548110.2174/1389203724666230825142558Life SciencesResearch ArticleIn Silico and In vitro Analysis of Phenolic Acids for Identification of Potential DHFR Inhibitors as Antimicrobial and Anticancer AgentsSehrawatRenuinfo@benthamscience.netRatheePriyankainfo@benthamscience.netRatheePoojainfo@benthamscience.netKhatkarSaritainfo@benthamscience.netKüpeli AkkolEsrainfo@benthamscience.netKhatkarAnuraginfo@benthamscience.netSchool of Medical and Allied Sciences, K R Mangalam University, Faculty of Pharmaceutical SciencesDepartment of Pharmaceutical Sciences, Maharshi Dayanand University, Vaish Institute of Pharmaceutical Education and ResearchDepartment of Pharmacognosy, Faculty of Pharmacy, Gazi University01012024251445811012025Copyright ©; 2024, Bentham Science Publishers2024Bentham Science Publishershttps://journals.eco-vector.com/1389-2037/article/view/645481

Background:DHFR is an indispensable enzyme required for the survival of almost all prokaryotic and eukaryotic cells, making it an attractive molecular target for drug design.

Objective:In this study, a combined in silico and in vitro approach was utilized to screen out potential anticancer and antimicrobial agents by using DHFR PDB ID 2W9S (for antimicrobial) and 1U72 (for anticancer).

Methods:Computational work was performed using Maestro Schrodinger Glide software. The DHFR inhibitory activity of the selected compounds was assessed using the DHFR test kit (CS0340-Sigma- Aldrich).

Results:Exhaustive analysis of in-silico results revealed that some natural phenolic acids have a good docking score when compared to standards, i.e., trimethoprim and methotrexate, and have astonishing interactions with crucial amino acid residues available in the binding pocket of DHFR, such as Phe 92, Asp 27, Ser 49, Asn 18, and Tyr 98. In particular, digallic acid and chlorogenic acid have amazing interactions with docking scores of -9.9 kcal/mol and -9.6 kcal/mol, respectively, for the targeted protein 2W9S. Docking scores of -10.3 kcal/mol and -10.2 kcal/mol, respectively, for targeted protein 1U72. The best hits were then tested in vitro to evaluate the DHFR inhibitory activity of the compounds. DHFR inhibition activity results are in correlation with molecular docking results.

Conclusion:In silico and in vitro results confirmed the good binding and inhibitory activity of some phenolic acids to the modeled target proteins. Among all the studied natural phenolic acids, chlorogenic acid, digallic acid, and rosmarinic acid appeared to be the most potential leads for future chemical alteration. This study can provide significant speculative guidance for the design and development of potent DHFR inhibitors in the future by using these compounds as leads.

DHFRmolecular dockingphenolic acidantimicrobialanticancerdrug design.Wang, M.; Yang, J.; Yuan, M.; Xue, L.; Li, H.; Tian, C.; Wang, X.; Liu, J.; Zhang, Z. Synthesis and antiproliferative activity of a series of novel 6-substituted pyrido3,2- dpyrimidines as potential nonclassical lipophilic antifolates targeting dihydrofolate reductase. Eur. J. Med. Chem., 2017, 128, 88-97. doi: 10.1016/j.ejmech.2017.01.033 PMID: 28152430Ducker, G.S.; Rabinowitz, J.D. One-carbon metabolism in health and disease. Cell Metab., 2017, 25(1), 27-42. doi: 10.1016/j.cmet.2016.08.009 PMID: 27641100Brown, P.M.; Pratt, A.G.; Isaacs, J.D. Mechanism of action of methotrexate in rheumatoid arthritis, and the search for biomarkers. Nat. Rev. Rheumatol., 2016, 12(12), 731-742. doi: 10.1038/nrrheum.2016.175 PMID: 27784891Singh, A.; Deshpande, N.; Pramanik, N.; Jhunjhunwala, S.; Rangarajan, A.; Atreya, H.S. Optimized peptide based inhibitors targeting the dihydrofolate reductase pathway in cancer. Sci. Rep., 2018, 8, 3190. doi: 10.1038/s41598-018-21435-5Tonelli, M.; Naesens, L.; Gazzarrini, S.; Santucci, M.; Cichero, E.; Tasso, B.; Moroni, A.; Costi, M.P.; Loddo, R. Host dihydrofolate reductase (DHFR)-directed cycloguanil analogues endowed with activity against influenza virus and respiratory syncytial virus. Eur. J. Med. Chem., 2017, 135, 467-478. doi: 10.1016/j.ejmech.2017.04.070 PMID: 28477572Liu, J.; Bolstad, D.B.; Bolstad, E.S.D.; Wright, D.L.; Anderson, A.C. Towards new antifolates targeting eukaryotic opportunistic infections. Eukaryot. Cell, 2009, 8(4), 483-486. doi: 10.1128/EC.00298-08 PMID: 19168759El-Shershaby, M.H.; El-Gamal, K.M.; Bayoumi, A.H.; El-Adl, K.; Alswah, M.; Ahmed, H.E.A.; Al-Karmalamy, A.A.; Abulkhair, H.S. The antimicrobial potential and pharmacokinetic profiles of novel quinoline-based scaffolds: Synthesis and in silico mechanistic studies as dual DNA gyrase and DHFR inhibitors. New J. Chem., 2021, 45(31), 13986-14004. doi: 10.1039/D1NJ02838CRao, A.S.; Tapale, S.R. A Study on dihydrofolate reductase and its inhibitors: A review. Int. J. Pharm. Sci. Res., 2013, 4(7), 2535-2547. doi: 10.13040/IJPSR.0975-8232.4(7).2535-47Mendelson, M.; Matsoso, M.P. The world health organization global action plan for antimicrobial resistance. S. Afr. Med. J., 2015, 105(5), 325. doi: 10.7196/SAMJ.9644 PMID: 26242647Vijesh, A.M.; Isloor, A.M.; Telkar, S.; Arulmoli, T.; Fun, H.K. Molecular docking studies of some new imidazole derivatives for antimicrobial properties. Arab. J. Chem., 2013, 6(2), 197-204. doi: 10.1016/j.arabjc.2011.10.007Silver, L.L. Challenges of antibacterial discovery. Clin. Microbiol. Rev., 2011, 24(1), 71-109. doi: 10.1128/CMR.00030-10 PMID: 21233508Payne, D.J.; Gwynn, M.N.; Holmes, D.J.; Pompliano, D.L. Drugs for bad bugs: Confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov., 2007, 6(1), 29-40. doi: 10.1038/nrd2201 PMID: 17159923Gwynn, M.N.; Portnoy, A.; Rittenhouse, S.F.; Payne, D.J. Challenges of antibacterial discovery revisited. Ann. N. Y. Acad. Sci., 2010, 1213(1), 5-19. doi: 10.1111/j.1749-6632.2010.05828.x PMID: 21058956Zhang, Y.; Chowdhury, S.; Rodrigues, J.V.; Shakhnovich, E. Development of antibacterial compounds that constrain evolutionary pathways to resistance. eLife, 2021, 10, e64518. doi: 10.7554/eLife.64518 PMID: 34279221Then, R.L. Antimicrobial dihydrofolate reductase inhibitors--achievements and future options: review. J. Chemother., 2004, 16(1), 3-12. doi: 10.1179/joc.2004.16.1.3 PMID: 15077993World Health Organization. Antimicrobial resistance, Available from: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance (Accessed on: December 20, 2022).World Health Organization. Available from: https://www.who.int/news/item/-new-report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis (Accessed on: December 16, 2022).Antibiotic, Resistance Threats In. The United States., Available from: https://www.cdc.gov/drugresistance/biggest-threats.html (Accessed on: December 16, 2022).Hameed, S.; Fatima, Z. Novel regulatory mechanisms of pathogenicity and virulence to combat MDR in Candida albicans. Inter. J. Microbiol., 2013, 2013, 240209. doi: 10.1155/2013/240209Tanwar, J.; Das, S.; Fatima, Z.; Hameed, S. Multidrug resistance: An emerging crisis. Interdisp. Perspect. Infect. Dis., 2014, 2014, 541340. doi: 10.1155/2014/541340Ansari, M.A.; Anurag, A.; Fatima, Z.; Hameed, S. Natural phenolic compounds: A potential antifungal agent. In: Microbial pathogens and strategies for combating them: science, technology and education; A. Méndez-Vilas, 2013.Romanos-Nanclares, A.; Sánchez-Quesada, C.; Gardeazábal, I.; Martínez-González, M.Á.; Gea, A.; Toledo, E. Phenolic acid subclasses, individual compounds, and breast cancer risk in a Mediterranean cohort: The SUN Project. J. Acad. Nutr. Diet., 2020, 120(6), 1002-1015.e5. doi: 10.1016/j.jand.2019.11.007 PMID: 31982373Alanazi, S.; Alenzi, N.; Alenazi, F.; Tabassum, H.; Watson, D. Chemical characterization of Saudi propolis and its antiparasitic and anticancer properties. Sci. Rep., 2021, 11(1), 5390. doi: 10.1038/s41598-021-84717-5 PMID: 33686109Saibabu, V.; Fatima, Z.; Khan, L.A.; Hameed, S. Therapeutic potential of dietary phenolic acids. Adv. Pharmacol. Sci., 2015, 2015, 823539. doi: 10.1155/2015/823539Savych, A.; Marchyshyn, S.; Kyryliv, M.; Bekus, I. Cinnamic acid and its derivatives in the herbal mixtures and their antidiabetic activity. Farmacia, 2021, 69(3), 595-601. doi: 10.31925/farmacia.2021.3.23Hollman, P.C.H.; Katan, M.B. Dietary flavonoids: Intake, health effects and bioavailability. Food Chem. Toxicol., 1999, 37(9-10), 937-942. doi: 10.1016/S0278-6915(99)00079-4 PMID: 10541448Złotek, U.; Lewicki, S.; Markiewicz, A.; Szymanowska, U.; Jakubczyk, A. Effects of drying methods on antioxidant, anti-Inflammatory, and anticancer potentials of phenolic acids in lovage elicited by jasmonic acid and yeast extract. Antioxidants, 2021, 10(5), 662. doi: 10.3390/antiox10050662 PMID: 33923284Merkl, R.; Hrádková, I.; Filip, V.; Šmidrkal, J. Antimicrobial and antioxidant properties of phenolic acids alkyl esters. Czech J. Food Sci., 2010, 28(4), 275-279. doi: 10.17221/132/2010-CJFSWeng, J.R.; Lin, C.S.; Lai, H.C.; Lin, Y.P.; Wang, C.Y.; Tsai, Y.C.; Wu, K.C.; Huang, S.H.; Lin, C.W. Antiviral activity of Sambucus FormosanaNakai ethanol extract and related phenolic acid constituents against human coronavirus NL63. Virus Res., 2019, 273, 197767. doi: 10.1016/j.virusres.2019.197767 PMID: 31560964Asokkumar, K.; Sen, S.; Umamaheswari, M.; Sivashanmugam, A.T.; Subhadradevi, V. Synergistic effect of the combination of gallic acid and famotidine in protection of rat gastric mucosa. Pharmacol. Rep., 2014, 66(4), 594-599. doi: 10.1016/j.pharep.2014.01.006 PMID: 24948059Kassim, M.; Achoui, M.; Mustafa, M.R.; Mohd, M.A.; Yusoff, K.M. Ellagic acid, phenolic acids, and flavonoids in Malaysian honey extracts demonstrate in vitro anti-inflammatory activity. Nutr. Res., 2010, 30(9), 650-659. doi: 10.1016/j.nutres.2010.08.008 PMID: 20934607Francesconi, V.; Giovannini, L.; Santucci, M.; Cichero, E.; Costi, M.P.; Naesens, L.; Giordanetto, F.; Tonelli, M. Synthesis, biological evaluation and molecular modeling of novel azaspiro dihydrotriazines as influenza virus inhibitors targeting the host factor dihydrofolate reductase (DHFR). Eur. J. Med. Chem., 2018, 155, 229-243. doi: 10.1016/j.ejmech.2018.05.059 PMID: 29886325Rashid, U.; Ahmad, W.; Hassan, S.F.; Qureshi, N.A.; Niaz, B.; Muhammad, B.; Imdad, S.; Sajid, M. Design, synthesis, antibacterial activity and docking study of some new trimethoprim derivatives. Bioorg. Med. Chem. Lett., 2016, 26(23), 5749-5753. doi: 10.1016/j.bmcl.2016.10.051 PMID: 28327306Lather, A.; Sharma, S.; Khatkar, A. Virtual screening of novel glucosamine-6-phosphate synthase inhibitors. Comb. Chem. High Throughput Screen., 2018, 21(3), 182-193. doi: 10.2174/1386207321666180330114457 PMID: 29600755Katsila, T.; Spyroulias, G.A.; Patrinos, G.P.; Matsoukas, M.T. Computational approaches in target identification and drug discovery. Comput. Struct. Biotechnol. J., 2016, 14, 177-184. doi: 10.1016/j.csbj.2016.04.004 PMID: 27293534Sharma, K.; Tanwar, O.; Sharma, S.; Ali, S.; Alam, M.M.; Zaman, M.S.; Akhter, M. Structural comparison of Mtb-DHFR and h-DHFR for design, synthesis and evaluation of selective non-pteridine analogues as antitubercular agents. Bioorg. Chem., 2018, 80, 319-333. doi: 10.1016/j.bioorg.2018.04.022 PMID: 29986181Dhiman, P.; Malik, N.; Khatkar, A. 3D-QSAR and in-silico studies of natural products and related derivatives as monoamine oxidase inhibitors. Curr. Neuropharmacol., 2018, 16(6), 881-900. doi: 10.2174/1570159X15666171128143650 PMID: 29189167Cao, H.; Gao, M.; Zhou, H.; Skolnick, J. The crystal structure of a tetrahydrofolate-bound dihydrofolate reductase reveals the origin of slow product release. Commun. Biol., 2018, 1(1), 226. doi: 10.1038/s42003-018-0236-y PMID: 30564747Hawser, S.; Lociuro, S.; Islam, K. Dihydrofolate reductase inhibitors as antibacterial agents. Biochem. Pharmacol., 2006, 71(7), 941-948. doi: 10.1016/j.bcp.2005.10.052 PMID: 16359642RCSB Protein Data Bank https://www.rcsb.org/structure/2W9SMaestro, version 12.6; Schrodinger, LLC: New York, America,. 2020.Halgren, T.A.; Murphy, R.B.; Friesner, R.A.; Beard, H.S.; Frye, L.L.; Pollard, W.T.; Banks, J.L. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J. Med. Chem., 2004, 47(7), 1750-1759. doi: 10.1021/jm030644s PMID: 15027866Friesner, R.A.; Banks, J.L.; Murphy, R.B.; Halgren, T.A.; Klicic, J.J.; Mainz, D.T.; Repasky, M.P.; Knoll, E.H.; Shelley, M.; Perry, J.K.; Shaw, D.E.; Francis, P.; Shenkin, P.S. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem., 2004, 47(7), 1739-1749. doi: 10.1021/jm0306430 PMID: 15027865Friesner, R.A.; Murphy, R.B.; Repasky, M.P.; Frye, L.L.; Greenwood, J.R.; Halgren, T.A.; Sanschagrin, P.C.; Mainz, D.T. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem., 2006, 49(21), 6177-6196. doi: 10.1021/jm051256o PMID: 17034125García-Godoy, M.; López-Camacho, E.; García-Nieto, J.; Nebro, A.; Aldana-Montes, J. Molecular docking optimization in the context of multi-drug resistant and sensitive EGFR mutants. Molecules, 2016, 21(11), 1575. doi: 10.3390/molecules21111575 PMID: 27869781Glide, Version 6.6; Schrödinger, LLC: New York, NY, 2015.Genheden, S.; Ryde, U. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities. Expert Opin. Drug Discov., 2015, 10(5), 449-461. doi: 10.1517/17460441.2015.1032936 PMID: 25835573Can I relate MM-GBSA energies to binding affinity?. Available from: https://www.schrodinger.com/kb/1647 (Accessed on: May 4, 2023).Benet, L.Z.; Hosey, C.M.; Ursu, O.; Oprea, T.I. BDDCS, the Rule of 5 and drugability. Adv. Drug Deliv. Rev., 2016, 101, 89-98. doi: 10.1016/j.addr.2016.05.007 PMID: 27182629Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev., 2012, 64, 4-17. doi: 10.1016/j.addr.2012.09.019 PMID: 11259830Veber, D.F.; Johnson, S.R.; Cheng, H.Y.; Smith, B.R.; Ward, K.W.; Kopple, K.D. Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem., 2002, 45(12), 2615-2623. doi: 10.1021/jm020017n PMID: 12036371Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev., 2001, 46(1-3), 3-26. doi: 10.1016/S0169-409X(00)00129-0 PMID: 11259830Irvine, J.D.; Takahashi, L.; Lockhart, K.; Cheong, J.; Tolan, J.W.; Selick, H.E.; Grove, J.R. MDCK (Madin-Darby canine kidney) cells: A tool for membrane permeability screening. J. Pharm. Sci., 1999, 88(1), 28-33. doi: 10.1021/js9803205 PMID: 9874698Kulkarni, A.; Han, Y.; Hopfinger, A.J. Predicting Caco-2 cell permeation coefficients of organic molecules using membrane-interaction QSAR analysis. J. Chem. Inf. Comput. Sci., 2002, 42(2), 331-342. doi: 10.1021/ci010108d PMID: 11911703Teague, S.J.; Davis, A.M.; Leeson, P.D.; Oprea, T. The design of lead like combinatorial libraries. Angew. Chem. Int. Ed., 1999, 38(24), 3743-3748. doi: 10.1002/(SICI)1521-3773(19991216)38:243.0.CO;2-U PMID: 10649345Azzam, R.A.; Elsayed, R.E.; Elgemeie, G.H. Design, synthesis, and antimicrobial evaluation of a new series of N-sulfonamide 2-pyridones as dual inhibitors of DHPS and DHFR enzymes. ACS Omega, 2020, 5(18), 10401-10414. doi: 10.1021/acsomega.0c00280 PMID: 32426597Dihydrofolate Reductase Assay Kit. Available from: https://www.sigmaaldrich.com/IN/en/product/sigma/cs0340Aslan, E.; Adem, S. Investigation of the effects of some drugs and phenolic compounds on human dihydrofolate reductase activity. J. Biochem. Mol. Toxicol., 2015, 29(3), 135-139. doi: 10.1002/jbt.21677 PMID: 25418905Ogungbe, I.V.; Erwin, W.R.; Setzer, W.N. Antileishmanial phytochemical phenolics: Molecular docking to potential protein targets. J. Mol. Graph. Model., 2014, 48, 105-117. doi: 10.1016/j.jmgm.2013.12.010 PMID: 24463105Sánchez-del-Campo, L.; Sáez-Ayala, M.; Chazarra, S.; Cabezas-Herrera, J.; Rodríguez-López, J.N. Binding of natural and synthetic polyphenols to human dihydrofolate reductase. Int. J. Mol. Sci., 2009, 10(12), 5398-5410. doi: 10.3390/ijms10125398 PMID: 20054477Pandey, K.B.; Rizvi, S.I. Plant polyphenols as dietary antioxidants in human health and disease. Oxid. Med. Cell. Longev., 2009, 2, 270-278. doi: 10.1155/2015/313979 PMID: 20716914Yao, L.H.; Jiang, Y.M.; Shi, J.; Tomás-Barberán, F.A.; Datta, N.; Singanusong, R.; Chen, S.S. Flavonoids in food and their health benefits. Plant Foods Hum. Nutr., 2004, 59(3), 113-122. doi: 10.1007/s11130-004-0049-7 PMID: 15678717Salarbashi, D.; Bahrami, A. Phenolic acids: Structure, classification, and biological activities. J. Food Biochem., 2021, 13639. doi: 10.1111/jfbc.13639González-Sarrías, A.; Giménez-Bastida, J.A.; García-Conesa, M.T.; Gómez-Sánchez, M.B. Phenolic compounds in fruits and beverages consumed as part of the Mediterranean diet: Their role in prevention of chronic diseases. Phytochem. Rev., 2017, 16(4), 555-578. doi: 10.1007/s11101-017-9516-5