Differential Modulation of Copper(II) Interactions with the 18-22 Coordinating Amylin Fragment by the Geometric Isomers of a New Nicotinoyl Hydrazone: A First Study

Abstract

Type 2 diabetes mellitus is a multifactorial disease associated with insulin resistance and pancreatic β-cell dysfunction. Interestingly, this disease has also been associated with the aggregation of islet amyloid polypeptide (IAPP or amylin). This peptide can bind to physiological metal ions such as copper-(II), which enhances its aggregation and induces oxidative stress. For this reason, the use of moderate chelators constitutes a compelling potential therapeutic strategy. In this work, we synthesized and characterized the geometric isomers of a new compound, 1-methylimidazole-2-carboxaldehyde nicotinoyl hydrazone (X1NIC), aiming to evaluate their differential interactions with copper-(II) and the hIAPP_18-22 peptide fragment. Both compounds demonstrated high aqueous stability and adequate lipophilicity for biological membrane crossing. Coordination studies revealed that both ML and ML₂ complexes can be obtained in solution for the tridentate ligand X1NIC-( E ), with the former being more stable. On the other hand, the bidentate ligand X1NIC-( Z ) only forms ML species. Both isomers effectively set up ternary complexes with Cu²⁺ and hIAPP_18-22, altering the redox behavior of the copper-peptide system. These results, obtained through cyclic voltammetry experiments, suggest a protective effect of the ligands with respect to metal-induced oxidative stress. This study constitutes the first comparative report on the coordination and reactivity of geometric isomers of a bioactive N-acylhydrazone, and the findings described herein highlight this class of compounds as promising chemical tools for the modulation of abnormal metal-peptide interactions implicated in type 2 diabetes pathogenesis.

1. Possible Coordination Patterns for (A) Tridentate X1NIC-( E ), (B) Bidentate X1NIC-( Z ), and (C) Binucleating X1NIC-( Z )

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UV–vis absorption spectra of (A) X1NIC-( E ) and (B) X1NIC-( Z ) along with their precursors (5 × 10^–5 M) in 1% DMSO/Tris (10 mM, pH 7.4), obtained over a period of 12 h at room temperature.

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Thermal stability analysis of X1NIC-( E ) assessed by ¹H NMR between 25 and 65 °C, with spectra taken every 10 °C. A final spectrum was collected after the sample was cooled back to room temperature (25 °C). For the sake of comparison, the spectrum of X1NIC-( Z ) at 25 °C was included as well. Selected spectral windows: (A) 2.0–5.0 ppm; (B) 7.0–9.5 ppm; and (C) 12.0–15.0 ppm.

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Method of continuous variations to evaluate the copper­(II) binding affinity of the two geometric isomers of X1NIC: (A) X1NIC-( E ) and (B) X1NIC-( Z ). Representative electronic spectra of ligand molar fractions ranged from 0.5 (purple) to 1.0 (blue). Insets: absorbance versus molar fraction plots. Conditions: HEPES buffer (50 mM, pH 7.4) at 25 °C.

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Simultaneous X1NIC-( E )/X1NIC-( Z ) interactions with copper­(II) studied through ¹H NMR (400 MHz) spectroscopy at 25 °C. Black: mixture of both X1NIC isomers at an E/Z ratio of 1:1, in DMSO-d ₆. Red: same mixture after addition of 0.05 equiv of copper­(II) in deuterium oxide, resulting in a solution containing 1% D₂O in DMSO-d ₆.

2. Structure of the hIAPP_{18 –22} Peptide Fragment (HSSNN) Employed in This Work

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¹H NMR spectra (700 MHz) of (A) free hIAPP_18–22 and (B) hIAPP_18–22 + 0.1 eq Cu²⁺, both acquired at 25 °C. Samples were prepared in 20 mM Tris-d ₁₁ at pH 7.4, with 8.5% D₂O and 4% DMSO-d ₆. Histidine residues are highlighted in blue, while red was used for serine and green for asparagine. (**) indicates the most affected signals upon copper­(II) addition, while (*) highlights signals moderately modified in this context.

3. Proposed Coordination Sphere for the Neutral Cu²⁺-hIAPP_18‑22 Complex

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Interactions of (A) X1NIC-( E ) and (B) X1NIC-( Z ) with the Cu²⁺-hIAPP_18–22 system. For each isomer, from bottom to top: ¹H NMR spectra (700 MHz) of hIAPP_18–22, hIAPP_18–22 + 0.1 eq Cu²⁺, hIAPP_18–22 + 0.1 eq Cu²⁺ + 0.1 eq compound, hIAPP_18–22 + 0.1 eq Cu²⁺ + 1.0 equiv of compound, and free compound spectrum for comparison purposes. All spectra were acquired at 25 °C. Samples were prepared in 20 mM Tris-d ₁₁ at pH 7.4, with 8.5% D₂O and 4% DMSO-d ₆. Arrows point to relevant spectral changes: red is used for changes in serine-related signals, while blue refers to histidine. Red dotted lines highlight hydrazone signals that remained unchanged. Black dotted lines, on the other hand, were used to point out unaffected peptide signals.

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Cyclic voltammetry of the binary system involving Cu²⁺ and human IAPP coordinating fragment 18–22 (hIAPP_18–22, HSSNN). Top: CVs of Cu²⁺ at 0.1 mM (black), hIAPP_18–22 at 0.1 mM (red), and a 1:1 molar mixture of hIAPP_18–22 and Cu²⁺ (blue). Conditions: 5% DMSO/buffer (50 mM HEPES pH 7.4, 100 mM NaCl) solution, at room temperature. Bottom: CV of Cu²⁺ (0.1 mM) in the absence of DMSO (50 mM HEPES at pH 7.4, containing 100 mM NaCl), at room temperature.

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Cyclic voltammetry of binary systems involving Cu²⁺ and (A) X1NIC-( E ) or (B) X1NIC-( Z ). Conditions: 5% DMSO/buffer (50 mM HEPES pH 7.4, 100 mM NaCl) solution, at room temperature. [Cu²⁺] = 0.1 mM. [hydrazone] = 0.1–0.5 mM. “Free” copper (at a concentration of 0.1 mM) voltammogram is included for the sake of comparison (gray dashed line).

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Titration of the Cu²⁺-hIAPP_18–22 system with increasing amounts of (A) X1NIC-( E ) or (B) X1NIC-( Z ). Conditions: 5% DMSO/buffer (50 mM HEPES pH 7.4, 100 mM NaCl) solution, at room temperature. Red: free hIAPP_18–22 (0.1 mM). Gray: “free” Cu²⁺ (0.1 mM). Blue: Cu²⁺-hIAPP_18–22 at a 1:1 stoichiometry. In (A), from yellow to dark orange: additions of 0.1 eq X1NIC-( E ) (concentration range: 0.01–0.09 mM). Pink: [X1NIC-( E )] = 0.1 mM. Purple: [X1NIC-( E )] = 0.2 mM. In (B), from light to dark green: additions of 0.1 eq X1NIC-( Z ) (concentration range: 0.01–0.09 mM). Pink: [X1NIC-( Z )] = 0.1 mM. Purple: [X1NIC-( Z )] = 0.2 mM.