Multiscale Computational Investigations of Keap1 Inhibitors for Kidney Disease Treatments: Integrating <i>in silico</i> and <i>in vivo</i> Approaches
DOI:
https://doi.org/10.26538/tjpps/v4i3.6Keywords:
Molecular docking, Molecular dynamic simulations, Geophila obvallata, Quercetin-3-rhamnoside, Kidney injuryAbstract
Kidney injury, a growing global health concern, lacks effective therapeutic interventions. This study aimed to identify natural compounds from Geophila obvallata (Gob) that modulate the Keap1/Nrf2 pathway, a key mediator of cadmium-induced nephrotoxicity. Eighty-five bioactive compounds from Gob were screened via molecular docking, quantitative structure-activity relationship (QSAR) modeling, and absorption, distribution, metabolism, excretion, toxicity (ADMET) profiling against Keap1. The top candidates, Quercetin-3-rhamnoside (Q3R) and Narcissin, along with the reference compound Resveratrol, were further evaluated using 100-ns molecular dynamics (MD) simulations. The Q3R-Keap1 complex demonstrated superior conformational stability (RMSD: 1–3 Å), outperforming Resveratrol. To validate these computational insights, an in vivo study was conducted with 28 male mice divided into four groups: control, Cd-exposed (0.3 mg/kg), Cd + Q3R (0.3 mg/kg Cd + 50 mg/kg Q3R), and Q3R alone (50 mg/kg). Treatments were administered via oral gavage for 28 days. Cadmium exposure significantly increased blood urea nitrogen (BUN) and creatinine levels, elevated reactive oxygen species (ROS: 9.93 U/mg tissue) and malondialdehyde (MDA: 5.03 nmol/mg protein), and suppressed antioxidant enzymatic activities (GPx, GSH, GST, CAT, SOD). Q3R co-administration significantly attenuated cadmium-induced renal dysfunction BUN (46.78 mg/dL vs. 72.24 mg/dL; creatinine: 1.03 vs. 2.48 mg/dL), reversed oxidative stress markers, and restored antioxidant enzyme activity. These findings demonstrate the potential of integrated computational and natural product approaches in developing novel therapies for kidney diseases.
References
Singh A, Sharma A, Verma RK, Chopade RL, Pandit P, Nagar V. Heavy metal contamination of water and their toxic effect on living organisms. IntechOpen. 2022. https://doi.org/10.5772/intechopen.105075.
Genchi G, Sinicropi MS, Lauria G, Carocci A, Catalano A. The effects of cadmium toxicity. Int J Environ Res Public Health. 2020; 17(11):3782. https://doi.org/10.3390/ijerph17113782.
Kowalczyk P, Sulejczak D, Kleczkowska P, Bukowska-Ośko I, Kucia M, Popiel M, Kaczyńska K. Mitochondrial oxidative stress: A causative factor and therapeutic target in many diseases. Int J Mol Sci. 2021; 22(24):13384. https://doi.org/10.3390/ijms222413384.
Chen PY, Qin L, Simons M. TGFβ signaling pathways in human health and disease. Front Mol Biosci. 2023; 10. https://doi.org/10.3389/fmolb.2023.1113061.
Gui T, Sun Y, Shimokado A, Muragaki Y. The roles of mitogen-activated protein kinase pathways in TGF-β-induced epithelial-mesenchymal transition. J Signal Transduct. 2012; https://doi.org/10.1155/2012/289243.
Falodun A, Igbinosa E. EDITORIAL: Prospects of Microbial Natural Products and Their Biological Entities in Drug Discovery: http://www.doi.org/10.26538/tjpps/v1i1.1. Trop J Phyto and Pharm Sci. 2022; 1(1): 1. Retrieved from https://tjpps.org/index.php/home/article/view/7
Iserhienrhien LO, Okolie PN. Phytochemical screening and in vitro antioxidant properties of methanol and aqueous leaf extracts of Geophila obvallata. AJRB. 2018; 3(2):1-11. https://doi.org/10.9734/AJRB/2018/4505.
Iserhienrhien LO, Okolie PN. Protective effect of Geophila obvallata (Shumach) Didr leaf extract and its fractions against cadmium-induced nephrotoxicity in male Wistar rats. Toxicol Rep. 2022; 87-93. https://doi.org/10.1016/j.toxrep.2021.12.008.
Iserhienrhien LO, Okolie PN. Acute and subacute toxicity profile of Geophila obvallata (Shumach.) Didr methanol leaf extract on renal and hepatic indices in Wistar rats. Cogent Food Agric. 2020; 6(1):7-14.
Iserhienrhien LO, Iyoha A, Memudu A. Renoprotective effect of hyperin against CdCl2 prompted renal damage by activation of Nrf-2/Keap-1 ARE pathway in male mice. Toxicol Mech Methods. 2024. https://doi.org/10.1080/15376516.2024.2329655.
Gan F, Lou J, Duan H, Qin Q, Teng Z, Zhou X, Zhou X. Advances in oral drug delivery systems: Challenges and opportunities. Pharm. 2023; 15(2):484. https://doi.org/10.3390/pharmaceutics15020484.
Challapa-Mamani MR, Tomás-Alvarado E, Espinoza-Baigorria A, León-Figueroa DA, Sah R, Rodriguez-Morales AJ, Barboza JJ. Molecular docking and molecular dynamics simulations in related to Leishmania donovani: An update and literature review. Trop Med Infect Dis. 2023; 8(10):457. https://doi.org/10.3390/tropicalmed8100457.
Bodun DS, Omoboyowa DA, Omotuyi OI, Olugbogi EA, Balogun TA, Ezeh CJ. QSAR-based virtual screening of traditional Chinese medicine for the identification of mitotic kinesin Eg5 inhibitors. Comput Biol Chem. 2023; 104:107865. https://doi.org/10.1016/j.compbiolchem.2023.107865.
Schrödinger L. Release 2017–1: Lig Prep. Schrödinger, LLC. 2017.
Iserhienrhien LO, Enoyoze GE. Comparative evaluation of antioxidant activity in extracts and fractions from leaves and stem of Geophila obvallata (Schumach)Didr. Adv Clin Toxicol. 2024; 9(2):001-009. https://doi.org/10.23880/act-16000309.
Lipinski CA. Lead- and drug-like compounds: The rule-of-five revolution. Drug Discov Today Technol. 2004; 1(4):337-341. https://doi.org/10.1016/j.ddtec.2004.11.007.
Asagba S, Obi F. Effects of oral cadmium exposure on renal glomerular and tubular functions in the rat. J Appl Sci Environ Manag. 2004; 8(1):29–32. https://doi.org/10.4314/jasem.v8i1.17222.
Chen C, Narayanapillai S, Zhang W, Sham YY, Xing C. Rapid identification of Keap1-Nrf2 small-molecule inhibitors through structure-based virtual screening and hit-based substructure search. J Med Chem. 2022; 57(3):1121–1126. https://doi.org/10.1021/jm4017174.
Aebi HU. Methods of enzymatic analysis. 3rd ed. Academic Press; 1984.
Samira B, Menaceur F, Gasmi S, Lidoughi A, Rais T, Gattel H. Oxidative stress assessment and its relationship with the prevalence of atherogenic risk in patients with type 2 diabetes. J Diabetes Metab Disord. 2021; 20(1):583-590.
Hayashi I, Morishita Y, Imai K, Nakamura M, Nakachi K, Hayashi T. High-throughput spectrophotometric assay of reactive oxygen species in serum. Mutat Res Genet Toxicol Environ Mutagen. 2007; 631(1):55-61. https://doi.org/10.1016/j.mrgentox.2007.04.006.
Daniele D, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as a toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis. 2005; 15(4):316-328.
Hosten AO. BUN and creatinine. In: Walker HK, Hall WD, Hurst JW, editors. Clinical methods: The history, physical, and laboratory examinations. 3rd ed. Butterworths; 1990.
Wang Y, Sun Q, Liu J, Jin F, Dai Z. Cadmium-induced mitochondrial dysfunction in kidney injury. Redox Biol. 2020; 33:101540. https://doi.org/10.1016/j.redox.2020.101540.
Nordberg GF, Nordberg BA, Friberg LM. Handbook on the toxicology of metals. Academic Press; 2007.
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. https://doi.org/10.1517/17460441.2015.1032936.
Alzain AA, Mukhtar RM, Abdelmoniem N, Shoaib TH, Osman W, Alsulaimany M, Aljohani AKB. Modulation of NRF2/KEAP1-mediated oxidative stress for cancer treatment by natural products using pharmacophore-based screening, molecular docking, and molecular dynamics studies. Mol. 2023; 28(16):6003. https://doi.org/10.3390/molecules28166003.
Alves FS, Rodrigues Do Rego JDA, Da Costa ML, Lobato Da Silva LF, Da Costa RA, Cruz JN. Spectroscopic methods and in silico analyses using density functional theory to characterize and identify piperine alkaloid crystals isolated from pepper (Piper nigrum L.). J Biol Struct Dyn. 2020; 38(9):2792-2799. https://doi.org/10.1080/07391102.2020.1747546.
Hagar M, Ahmed HA, Aljohani G, Alhaddad OA. Investigation of some antiviral N-heterocycles as COVID-19 drug: Molecular docking and DFT calculations. Int J Mol Sci. 2020; 21(11):3922. https://doi.org/10.3390/ijms21113922.
Salo-Ahen OMH, Alanko I, Bhadane R, Bonvin AMJJ, Honorato RV, Hossain S, Juffer AH, Kabedev A, Lahtela-Kakkonen M, Larsen AS. Molecular dynamics simulations in drug discovery and pharmaceutical development. Processes. 2021; 9(1):71. https://doi.org/10.3390/pr9010071.
Liu S, Stanley A, Yadav L, Sudhakar A. JAK2/STAT3 signaling pathway mediates cadmium-induced inflammatory response and renal fibrosis. Ecotoxicol Environ Saf. 2023; 262:115280. https://doi.org/10.1016/j.ecoenv.2023.115280.
Liu J, Liu J, Klaassen CD. Cadmium toxicity and its mechanisms in animals. Recent Pat Toxicol. 2010; 4(1):1–12. https://doi.org/10.2174/1877652101004010001.
Salehi B, Mishra AP, Nigam M, Sener B, Kilic M, Sharifi-Rad M, Fokou PVT, Martins N, Sharifi-Rad J. Resveratrol: A double-edged sword in health benefits. Biomed. 2018; 6(3):91. https://doi.org/10.3390/biomedicines6030091.
Zhang DD. Keap1-Nrf2 signaling pathway: An emerging target for the treatment of oxidative stress-related disorders. Antioxid Redox Signal. 2015; 22(1):29–48. https://doi.org/10.1089/ars.2014.6013.
Chi F, Cheng C, Zhang M, Su B, Hou Y, Bai G. Resveratrol targeting NRF2 disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage in mice pulmonary infection. J Ethnopharmacol. 2024; 332:118353. https://doi.org/10.1016/j.jep.2024.118353.
Patil R, Das S, Stanley A, Yadav L, Sudhakar A, Varma AK. Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-designing. PLoS One. 2010; 5(8):e12029. https://doi.org/10.1371/journal.pone.0012029.
Cui J, Feng Y, Yang T, Wang X, Tang H. Computer-aided designing peptide inhibitors of human hematopoietic prostaglandin D2 synthase combined molecular docking and molecular dynamics simulation. Mol. 2023; 28(15):5933. https://doi.org/10.3390/molecules28155933.
Velichkova M, Hasson T. Keap1 regulates the oxidation-sensitive shuttling of Nrf2 into and out of the nucleus via a Crm1-dependent nuclear export mechanism. Mol and Cell Bio. 2005; 25(11): 4501–4513. doi: 10.1128/MCB.25.11.4501-4513.2005
Kołacz K, Wójcik M, Balcerzyk M. Resveratrol targeting NRF2 disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage in mice pulmonary infection. Int J Environ Res Public Health. 2017; 14(12):1501. https://doi.org/10.3390/ijerph14121501.
Dinkova-Kostova AT, Talalay P. Direct and indirect antioxidant activities of isothiocyanates: Their potential in prevention and therapy of human diseases. J Nutr. 2010; 140(1):75–81. https://doi.org/10.3945/jn.109.111336.
Grunenwald A, Roumenina LT, Frimat M. Heme oxygenase 1: A defensive mediator in kidney diseases. Int J Mol Sci. 2021; 22(4):2009. https://doi.org/10.3390/ijms22042009.
Nath KA, Grande JP, Haggard JJ, Croatt AJ, Katusic ZS, Solovey A, Hebbel RP. Oxidative stress and induction of heme oxygenase-1 in the kidney in sickle cell disease. Am J Pathol. 2001; 158(3):893–903. https://doi.org/10.1016/S0002-9440(10)64037-4.
Xiong L, Xie J, Song C, Liu J, Zheng J, Liu C. The activation of Nrf2 and its downstream regulated genes mediates the antioxidative activities of Xueshuan Xinmaining Tablet in human umbilical vein endothelial cells. Evid Based Complement Alternat Med. 2015; 2015:187265. https://doi.org/10.1155/2015/187265.
Hammad M, Raftari M, Cesário R, Salma R, Godoy P, Emami SN, Haghdoost S. Roles of oxidative stress and Nrf2 signaling in pathogenic and non-pathogenic cells: A possible general mechanism of resistance to therapy. Antioxidants. 2023; 12(7):1371. https://doi.org/10.3390/antiox12071371.
Yan X, Allen D. Chemical Reviews. 2019; 119(18):10520–10594. https://doi.org/10.1021/acs.chemrev.8b00728.
Lee JM, Surh YJ. Targeting Keap1-Nrf2 system with natural compounds for cancer chemoprevention and treatment. Mutat Res. 2013; 751(1-2):116–126. https://doi.org/10.1016/j.mrfmmm.2013.06.003.

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