Binding copper(II) ion released from azo-amino acid Schiff base complex into lysozyme crystals as models for treatment of Alzheimer’s disease

Abstract

Aim: One of the causes of Alzheimer’s disease (AD) is the structural change and aggregation of target proteins due to the binding of metal ions. In this study, we investigated where copper(II) ions bound to the protein egg white lysozyme crystals in a hydrophilic buffer solution after ions were synthesized from an amino acid Schiff base copper(II) complex with a hydrophobic azobenzene group.

Methods: X-ray crystallographic studies of the complexes and egg white lysozyme were then studied. Molecular docking studies for the binding of copper(II) ion with egg white lysozyme were also carried out.

Results: The results suggest that the hydrophobicity of the introduced complex affected how deeply the resultant copper(II) ion penetrated into the protein. It has been revealed that when metal complexes are soaked into protein crystals, the metal complexes act as carriers, and metal ions tend to dissociate and bind to appropriate functional groups on certain specific residues of the protein. His15 and Glu35 were the more common binding residues of the protein that bound to the metal ion.

Conclusions: An anti-Irving-Williams behaviour was observed for the interaction of the copper(II) complex with the lysozyme. Docking studies revealed various potential binding sites of copper(II) ion with the lysozyme.

Keywords

Alzheimer’s disease / metal ion binding site / copper(II) complex / crystal structure / lysozyme

References

[1] Sohrabi HR, Weinborn M. Cognitive impairments in Alzheimer’s disease and other neurodegenerative diseases. In: Martins RN, Brennan CS, Binosha Fernando WMAD, Brennan MA, Fuller SJ, editors. Neurodegeneration and Alzheimer’s disease: The Role of Diabetes, Genetics, Hormones, and Lifestyle. Wiley Online Library; 2019. pp. 267-90.

[2] Wimo A, Seeher K, Cataldi R, Cyhlarova E, Dielemann JL, Frisell O, et al. The worldwide costs of dementia in 2019. Alzheimers Dement. 2023; 19: 2865-73.

[3] Javaid SF, Giebel C, Khan MA, Hashim MJ. Epidemiology of Alzheimer’s disease and other dementias: Rising global burden and forecasted trends. F1000Research. 2021; 10: 425.

[4] Krishnan A, Asghar MS, Adhiyaman V, Hobson P. 3113 The increasing number of deaths related to Alzheimer's disease (AD) over the last decade. Age Ageing. 2025; 54: afaf133.089.

[5] Sun X, Zhu J, Li R, Peng Y, Gong L. The global research of magnetic resonance imaging in Alzheimer’s disease: a bibliometric analysis from 2004 to 2023. Front Neurol. 2025; 15: 1510522.

[6] Kaur D, Behl T, Sehgal A, Singh S, Sharma N, Bungau S. Multifaceted Alzheimer’s Disease: Building a Roadmap for Advancement of Novel Therapies. Neurochem Res. 2021; 46: 2832—51.

[7] Liu E, Zhang Y, Wang JZ. Updates in Alzheimer’s disease: from basic research to diagnosis and therapies. Transl Neurodegener. 2024; 13: 45.

[8] Jomova K, Vondrakova D, Lawson M, Valko M. Metals, oxidative stress and neurodegenerative disorders. Mol Cell Biochem. 2010; 345: 91-104.

[9] Dellal F, Triaud R, Moyeux A, Gager O, Salerno M. Synthesis of copper complexes for potential use in the diagnosis of Alzheimer’s disease. Med Sci Pulse. 2024; 18: 29-34.

[10] Paterson BM, Donnelly PS. Copper complexes of bis(thiosemicarbazones): from chemotherapeutics to diagnostic and therapeutic radiopharmaceuticals. Chem Soc Rev. 2011; 40: 3005—18.

[11] Mazur T, Malik M, Bieńko DC. The impact of chelating compounds on Cu²⁺, Fe²⁺/³⁺, and Zn²⁺ ions in Alzheimer’s disease treatment. J Inorg Biochem. 2024; 257: 112601.

[12] Leonard E, Takeda C, Akitsu T. Azobenzene—Containing Schiff—Bases—Syntheses and Dyes Applications. Colorants. 2024; 3: 53-72.

[13] Ashok A, Andrabi SS, Mansoor S, Kuang Y, Kwon BK, Labhasetwar V. Antioxidant Therapy in Oxidative Stress—Induced Neurodegenerative Diseases: Role of Nanoparticle—Based Drug Delivery Systems in Clinical Translation. Antioxidants (Basel). 2022; 11: 408.

[14] Santos MA, Chand K, Chaves S. Recent progress in multifunctional metal chelators as potential drugs for Alzheimer’s disease. Coord Chem Rev. 2016; 327: 287-303.

[15] Sharma A, Pachauri V, Flora SJS. Advances in Multi—Functional Ligands and the Need for Metal—Related Pharmacology for the Management of Alzheimer Disease. Front Pharmacol. 2018; 9: 1247.

[16] Burke BP, Seemann J, Archibald SJ. Advanced chelator design for metal complexes in imaging applications: radiopharmaceuticals, protein targeting, and conjugation. In: van Eldik R, Hubbard CD, editors. Advances in Inorganic Chemistry. Academic Press; 2016. pp. 301-39.

[17] Lane AR, Roberts BR, Fahrni CJ, Faundez V. A primer on copper biology in the brain. Neurobiol Dis. 2025; 212: 106974.

[18] Jadhao M, Das C, Rawat A, Kumar H, Joshi R, Maiti S, et al. Development of multifunctional heterocyclic Schiff base as a potential metal chelator: a comprehensive spectroscopic approach towards drug discovery. J Biol Inorg Chem. 2017; 22: 47-59.

[19] Guha R. On Exploring Structure—Activity Relationships. In: Kortagere S, editor. In Silico Models for Drug Discovery. Totowa: Humana Press; 2013. pp. 81-94.

[20] Roy H, Nandi S. In—Silico Modeling in Drug Metabolism and Interaction: Current Strategies of Lead Discovery. Curr Pharm Des. 2019; 25: 3292-305.

[21] Outeiral C, Strahm M, Shi J, Morris GM, Benjamin SC, Deane CM. The prospects of quantum computing in computational molecular biology. Wires Comput Mol Sci. 2020; 11: e1481.

[22] Prieto—Martínez FD, López—López E, Juárez—Mercado KE, Medina—Franco JL. Computational drug design methods—current and future perspectives. In: Kunal P, editor. In silico drug design. Academic Press; 2019. pp. 19-44.

[23] Takahashi K, Miyazaki R, Nakane D, Aiyelabola TO, Akitsu T. Preliminary Investigation of Copper (II) Ion Binding or Complex Coordination in Lysozeme Molecules. J Mater Sci Chem Eng. 2024; 12: 98-103.

[24] Kaneda A, Suzuki S, Nakane D, Kashiwagi Y, Akitsu T. Crystal structure and Hirshfeld surface analysis of (1H—imidazole—κN³)[4—methyl—2—({[2—oxido—5—(2—phenyl—diazen—1—yl)phen—yl]methyl—idene}amino)penta—noate—κ³ O,N,O']copper(II) . Acta Crystallogr E Crystallogr Commun. 2024; 80: 468-71.

[25] Wadayama Y, Kaneda A, Imae T, Nakane D, Akitsu T. Verification of the Inverse Scale Effect Hypothesis on Viscosity and Diffusion by Azo—Amino Acid Schiff Base Copper Complexes. J Compos Sci. 2024; 8: 177.

[26] Lu CH, Lin YF, Lin JJ, Yu CS. Prediction of metal ion—binding sites in proteins using the fragment transformation method. PLoS One. 2012; 7: e39252.

[27] Lin YF, Cheng CW, Shih CS, Hwang JK, Yu CS, Lu CH. MIB: Metal Ion—Binding Site Prediction and Docking Server. J Chem Inf Model. 2016; 56: 2287-91.

[28] Lu CH, Chen CC, Yu CS, Liu YY, Liu JJ, Wei ST, et al. MIB2: metal ion—binding site prediction and modeling server. Bioinformatics. 2022; 38: 4428—9.

[29] Osman D, Martini MA, Foster AW, Chen J, Scott AJP, Morton RJ, et al. Bacterial sensors define intracellular free energies for correct enzyme metalation. Nat Chem Biol. 2019; 15: 241—9.

[30] Young TR, Martini MA, Foster AW, Glasfeld A, Osman D, Morton RJ, et al. Calculating metalation in cells reveals CobW acquires Co^II for vitamin B₁₂ biosynthesis while related proteins prefer Zn^II. Nat Commun. 2021; 12: 1195.

[31] Dudev T, Lim C. Competition among metal ions for protein binding sites: determinants of metal ion selectivity in proteins. Chem Rev. 2014; 114: 538-56.

[32] Barber—Zucker S, Shaanan B, Zarivach R. Transition metal binding selectivity in proteins and its correlation with the phylogenomic classification of the cation diffusion facilitator protein family. Sci Rep. 2017; 7: 16381.

[33] Irving H, Williams RJP. The stability of transition—metal complexes. J Chem Soc. 1953: 3192-210.

[34] Cotruvo JA Jr, Stubbe J. Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study. Metallomics. 2012; 4: 1020—36.

[35] Wienen—Schmidt B, Oebbeke M, Ngo K, Heine A, Klebe G. Two Methods, One Goal: Structural Differences between Cocrystallization and Crystal Soaking to Discover Ligand Binding Poses. ChemMedChem. 2021; 16: 292-300.

[36] Kim JK, Lee C, Lim SW, Adhikari A, Andring JT, McKenna R, et al. Elucidating the role of metal ions in carbonic anhydrase catalysis. Nat Commun. 2020; 11: 4557.

[37] Dai Z. Steric and Stereochemical Modulation in Pyridyl— and Quinolyl—Containing Ligands. Molecules. 2016; 21: 1647.

[38] Lentink S, Salazar Marcano DE, Moussawi MA, Vandebroek L, Van Meervelt L, Parac—Vogt TN. Fine—tuning non—covalent interactions between hybrid metal—oxo clusters and proteins. Faraday Discuss. 2023; 244: 21-38.

[39] Pelosi C, Saitta F, Zerino C, Canil G, Biver T, Pratesi A, et al. Thermodynamic Evaluation of the Interactions between Anticancer Pt(II) Complexes and Model Proteins. Molecules. 2021; 26: 2376.

[40] Kim Y, Chang JY, Kim YY, Lee JW, Kho HS. Effects of Zinc Compounds on the Enzymatic Activities of Lysozyme and Peroxidase and Their Antifungal Activities. Biol Trace Elem Res. 2024; 202: 5850-62.

[41] Choi TS, Tezcan FA. Overcoming universal restrictions on metal selectivity by protein design. Nature. 2022; 603: 522-7.

[42] Robinson NJ, Glasfeld A. Metalation: nature’s challenge in bioinorganic chemistry. J Biol Inorg Chem. 2020; 25: 543—5.

[43] Ferraro G, Paolillo M, Sciortino G, Garribba E, Merlino A. Multiple and Variable Binding of Pharmacologically Active Bis(maltolato)oxidovanadium(IV) to Lysozyme. Inorg Chem. 2022; 61: 16458—67.

[44] Ferraro G, Lyčková T, Massai L, Štarha P, Messori L, Merlino A. Picoplatin binding to proteins: X—ray structures and mass spectrometry data on the adducts with lysozyme and ribonuclease A. Dalton Trans. 2024; 53: 8535—40.

[45] Liu Y, Ma J, Zhang Q, Wang Y, Sun Q. Mechanism of Metal Complexes in Alzheimer’s Disease. Int J Mol Sci. 2024; 25: 11873.

[46] Zhang X, Zhang X, Zhong M, Zhao P, Guo C, Li Y, et al. A Novel Cu(II)—Binding Peptide Identified by Phage Display Inhibits Cu²⁺—Mediated Aβ Aggregation. Int J Mol Sci. 2021; 22: 6842.