Investigation of deferiprone binding to different essential metal ions using microscale thermophoresis and electrospray ionization mass spectrometry Mufarreh Asmari a , Lenka Michalcová a,b , Hassan A. Alhazmi c , Zdeněk Glatz b , Sami El Deeb a, ⁎ a Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstrasse 55, 38106 Braunschweig, Germany b Department of Biochemistry, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic c College of Pharmacy, Jazan University, P.O. Box 114, Post code 45142 Jazan, Saudi Arabia abstract article info Article history: Received 6 July 2017 Received in revised form 7 October 2017 Accepted 8 October 2017 Available online 10 October 2017 In this study, the microscale thermophoresis (MST) method was applied to investigate the interaction of deferiprone with Fe 3+ , Cu 2+ , Zn 2+ , Co 2+ , Ni 2+ , Mn 2+ , Mg 2+ , and Ca 2+ . Experiments were performed on a MST (Monolith NT.115 LabelFree®) system. Pretest scanning indicated good fluorescence intensity of deferiprone allowing the use of label-free MST experiments. Different concentrations of the intended metal ions in the range of 0.048 to 100.0 μM were titrated against 100 μM fixed concentration of deferiprone dissolved in 0.1 M Tris buffer pH 7.4. MST measurements were performed in standard capillaries at 50% excitation power and 20% MST power. The results indicated significant interactions of deferiprone with Fe 3+ , Cu 2+ , Zn 2+ , Co 2+ , and Ni 2+ . The data fitted to the Hill model with Hill coefficients of 1.8, 1.5, 3.2, 1.6, and 1.5 for Fe 3+ , Cu 2+ , Zn 2+ , Ni 2+ , Co 2+ , respectively, thus indicating more than 1:1 stoichiometry. EC50 values for the binding of deferiprone to Fe 3+ , Cu 2+ , Zn 2+ , Co 2+ , and Ni 2+ were calculated to be 20.6 ± 3.34, 38.1 ± 3.39, 39.5 ± 4.90, 51.1 ± 6.86, and 101.1 ± 22.70 μM, respectively. No binding was observed for deferiprone with Mn 2+ , Mg 2+ , and Ca 2+ . Electrospray ionization mass spectrometry was used as a complementary technique under similar con- ditions; however, Tris buffer was replaced by ammonium acetate buffer to be compatible with the mass system. Electrospray ionization mass spectrometry confirms the results obtained by MST indicating stable molecular ion peaks for the complexes of deferiprone with either Fe 3+ , Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , and Mn 2+ , but no binding was observed for deferiprone with either Ca 2+ and Mg 2+ under a gas phase state. MST shows a fast and simple ap- proach to study the binding of deferiprone to different essential metal ions. Moreover, the complexes were stable for investigation by mass spectrometry under a soft ionization technique like used electrospray ionization, which aided to confirm binding stoichiometry. © 2017 Published by Elsevier B.V. Keywords: Microscale thermophoresis Mass spectrometry Deferiprone Essential metal ions EC50 Hill coefficient 1. Introduction Essential metals play an important role inside the body and are con- sidered vital for different living systems. They mostly bind with proteins forming catalytic enzymes, or acting as regulators in gene expression and second messenger in signaling pathway or for transportation [1]. The amount of these metals in the living organism varies from kilo- grams, such as calcium (represents about 1–1.2 kg of the body weight), to ultra-trace amounts, such as cobalt (represents about 1–1.5 mg in the body) [2]. Metals toxicity is concomitant to various disease states, such as Wilson disease, diabetes mellitus, Alzheimer disease, and β-thalassemia. Metals accumulation may occur over the years inside body organs and lead to their hypofunctions, fibrosis, mutagenicity, or failure [3,4]. Herein, intervention with chelation therapy is the treatment strategy to protect the organs against metals toxicity. However, this strategy has several threats and needs an intensive monitoring during the treat- ment plan. For example, iron overload is the biggest problem in this context and is frequently occurring in some clinical situations, such as β-thalassemia due to blood transfusion every three to four weeks, which leads to more complications and some organs might be affected, such as the liver, kidney, and heart. Therefore, iron chelators are the most clinically used chelators nowadays either in single or combination therapy [5,6]. Deferiprone (CP20) (3-hydroxy-1,2-dimethylpyridin-4- one) (Fig. 1) is the first used oral iron chelator for patients with β-thal- assemia to remove excess iron from different organ tissues, especially cardiac myocytes [7]. CP20 is bidentate chelator binds to Fe 3+ in a 1:3 metal–CP20 ratio (Fig. 2). Regrettably, the chelation effect of CP20 is not an iron-selective process. There are studies that have described the chelation effect of CP20 on different metal ions, such as Zn 2+ , Microchemical Journal 137 (2018) 98–104 ⁎ Corresponding author. E-mail address: s.eldeeb@tu-bs.de (S. El Deeb). https://doi.org/10.1016/j.microc.2017.10.004 0026-265X/© 2017 Published by Elsevier B.V. Contents lists available at ScienceDirect Microchemical Journal journal homepage: www.elsevier.com/locate/microc