Novel Dinuclear Gold Complex Demonstrates Selective Anticancer Activity and Internal Apoptotic Induction

A newly synthesized dinuclear gold(III) coordination compound utilizing 1,5-naphthyridine as a bridging scaffold has demonstrated selective cytotoxicity against human colon and breast carcinoma cells while sparing non-cancerous keratinocytes, according to laboratory evaluations and computational docking models. Metal-based anticancer therapeutics have historically centered on platinum-based agents such as cisplatin, but research into gold(III) chemistry has expanded as investigators seek alternative therapeutic complexes. Because gold(III) shares an isoelectronic low-spin configuration with platinum(II) and adopts a four-coordinate square-planar geometry, researchers have investigated its capacity to bypass cisplatin-associated pathways. In this newly reported study, chemists designed a bimetallic framework where a rigid 1,5-naphthyridine ligand directs two gold(III) centers away from each other. Across comprehensive spectroscopic characterizations, stability assays, DNA-interaction titrations, serum albumin binding experiments, and in vitro cellular assays, the dinuclear architecture demonstrated stable physiological compatibility, minor groove DNA recognition, carrier protein affinity at defined subdomains, and cell death directed selectively through the internal, caspase-9-mediated mitochondrial pathway.

Synthesizing polynuclear gold complexes requires careful ligand selection because gold(III) ions are inherently prone to rapid chemical reduction in physiological environments, particularly in the presence of thiols or disulfides, which can yield gold(I) species or metallic gold precipitates. To enforce structural stability across the coordination sphere, nitrogen-donor aromatic heterocycles serve as stabilizing ligands. The research team synthesized the dinuclear complex, formulated as [Au2(1,5-naph)Cl6] where 1,5-naph represents 1,5-naphthyridine, by reacting potassium tetrachloridoaurate(III) with 1,5-naphthyridine in absolute methanol under light-protected conditions at room temperature for three hours. This reaction achieved an isolated yield of 73.9 percent. Structural verification performed through proton nuclear magnetic resonance spectroscopy resolved six distinct aromatic proton signals corresponding to the symmetrical naphthyridine backbone, while cryospray-ionization quadrupole time-of-flight mass spectrometry detected the intact cationic fragment [C8H6Au2Cl5N2]+ at an experimental mass-to-charge ratio of 700.8246 following the dissociation of a single chloride counter-ligand. Molar conductivity measurements in acetone yielded a value of 36.2 reciprocal ohms square centimeters per mole, confirming that the isolated coordination complex exists as a neutral non-electrolyte in solution.

Evaluating physiological persistence is a necessary prerequisite for any prospective metal-based therapeutic candidate, prompting the researchers to record electronic absorption profiles across twenty-four hours in both pure aqueous media and buffered saline. In pure water, ultraviolet-visible spectrophotometry demonstrated preserved spectral shapes but an observable reduction in characteristic band intensities, an effect attributed to the potential spontaneous hydrolysis of gold(III)-halide coordination bonds. Conversely, when monitored in physiological phosphate-buffered saline, the complex exhibited exceptional stability over the entire twenty-four-hour observation period, showing only minor variations in its principal absorption maxima. Electronic absorption spectroscopy revealed characteristic intra-ligand pi-to-pi-star transitions at 265 nanometers alongside ligand-to-metal charge transfer bands located between 300 and 315 nanometers. This persistent stability in physiological saline confirmed that the bimetallic assembly retains its structural integrity under ionic and buffering conditions representative of biological fluids, enabling valid evaluations of macromolecular binding interactions and cellular biological response.

Because cellular deoxyribonucleic acid is often targeted by cytotoxic metal complexes, the researchers systematically analyzed the interaction between the dinuclear gold(III) compound and calf thymus DNA using electronic absorption titrations, thermodynamic profiling, and hydrodynamic viscosity measurements. During spectrophotometric titrations conducted in phosphate buffer with a fixed complex concentration of 13.5 micromolar and DNA concentrations extending up to 65.5 micromolar, addition of DNA produced hyperchromism at 258 nanometers alongside hypochromism at 310 nanometers without any positional shifts in absorption maxima. This spectral profile indicated non-covalent groove surface binding rather than classical base intercalation. Thermodynamic variables derived from van ‘t Hoff equations revealed negative standard Gibbs free energy values across varying experimental temperatures, verifying that complex association with the duplex is an inherently spontaneous thermodynamic process. Concurrently, hydrodynamic measurements showed that the relative viscosity of DNA solutions increased only slightly upon titration with the gold complex, contrasting sharply with the extensive lengthening and pronounced viscosity increases that accompany classic intercalators.

To rigorously differentiate whether the dinuclear gold complex binds DNA through groove association or base intercalation, the investigators conducted competitive fluorescence displacement assays utilizing two established fluorescent probes: ethidium bromide and the synthetic derivative 2-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl)-benzimidazo-2-yl]-benzimidazole, designated as HOE. Ethidium bromide functions as an archetypal intercalating agent possessing an established DNA binding constant of 1.4 multiplied by 10 to the sixth reciprocal molar. While titrating the gold(III) complex into ethidium bromide-bound DNA reduced fluorescence emission at 608 nanometers, the resulting Stern-Volmer quenching constant was limited to the order of 10 to the fourth reciprocal molar, indicating incomplete displacement that failed to displace the intercalator entirely. In contrasting experiments employing HOE, which binds specifically within the minor groove, titration of the dinuclear complex produced marked quenching of HOE fluorescence at 512 nanometers, accompanied by emission growth at 409 nanometers reflecting complex luminescence. The Stern-Volmer quenching constant calculated for HOE displacement reached the order of 10 to the fifth reciprocal molar, an order of magnitude higher than that observed for ethidium bromide, providing corroborating evidence that minor groove association represents the predominant binding mode.

The pharmacological behavior, tissue distribution, and systemic lifetime of therapeutic coordination compounds depend directly on interactions with circulating blood plasma carrier proteins, leading the research team to investigate binding kinetics against bovine serum albumin and human serum albumin. Steady-state fluorescence quenching spectroscopy was conducted at an excitation wavelength of 295 nanometers to selectively monitor emission from intrinsic tryptophan fluorophores located near 360 nanometers. Titrating the gold(III) complex into albumin solutions caused concentration-dependent fluorescence quenching alongside discernible blue shifts, shifting the emission maximum from 362 to 360 nanometers in human serum albumin and from 366 to 363 nanometers in bovine serum albumin. These blue shifts confirmed direct alterations in the microenvironment surrounding tryptophan residues. Stern-Volmer quenching constants exceeded 10 to the tenth reciprocal molar per second, demonstrating that dynamic collision was not responsible and that quenching operated via a static complexation mechanism. Binding constants calculated using Hill formulations reached the magnitude of 10 to the fifth reciprocal molar for both carrier proteins, with calculated binding site numbers indicating a single primary interaction locus per albumin macromolecule.

To locate the exact biological domain responsible for accommodating the dinuclear structure within serum albumin, competitive binding assays were performed using site-specific fluorescent markers alongside molecular docking simulations. Eosin Y was employed as a diagnostic probe targeting subdomain IIA, designated as site I, while ibuprofen was utilized to interrogate binding within subdomain IIIA, recognized as site II. Spectrofluorometric titrations in the presence of these markers demonstrated that binding constants remained elevated across both sites but were quantitatively higher in the presence of ibuprofen, indicating direct competitive displacement within subdomain IIIA. Molecular docking modeling against crystallographic albumin structures validated these empirical findings, revealing that while the dinuclear framework docks into hydrophobic pockets at both subdomains, subdomain IIIA yields superior docking scores. The docking simulations further revealed that the nitrogen donor atoms of the bridging ligand are fully coordinated to the gold centers, precluding the formation of direct hydrogen bonds and indicating that association is governed predominantly by shape compatibility and hydrophobic interactions within the carrier cavity.

Following biomacromolecular binding characterization, the cytotoxic potential of the synthesized dinuclear gold(III) complex was evaluated against two human malignant lines alongside non-tumorigenic control cells using standardized MTT cell viability assays. The cancer models comprised HCT116 colorectal carcinoma and MDA-MB-231 breast adenocarcinoma, while non-cancerous HaCaT human skin keratinocytes served as healthy tissue benchmarks. Cell viability was measured following twenty-four and seventy-two hours of exposure across complex concentrations spanning 1 to 200 micromolar. In colorectal HCT116 cultures, the complex demonstrated time-dependent cytotoxicity, whereas in breast cancer MDA-MB-231 cells, pronounced cytotoxic activity was established within twenty-four hours. Significantly, half-maximal inhibitory concentration values determined in non-cancerous HaCaT keratinocytes were notably higher than those calculated for both carcinoma lines, demonstrating that the dinuclear compound exhibits distinct selective toxicity toward malignant cells while exerting substantially lower cytotoxic stress against healthy human epithelial cells.

To clarify whether the observed reduction in carcinoma viability resulted from passive necrosis or targeted programmed cell death, the investigators examined cellular morphological changes and activated biochemical pathways following twenty-four hours of treatment at half-maximal inhibitory concentrations. Double staining with acridine orange and ethidium bromide, visualized under fluorescence microscopy across more than six hundred counted cells per sample, showed that the complex markedly reduced the fraction of viable cells while predominantly inducing apoptosis. Microscopic micrographs displayed characteristic hallmarks of programmed apoptotic death, including pronounced membrane blebbing, cytoplasmic volume reduction, cellular condensation, and nuclear chromatin consolidation, while necrotic red-fluorescing cells remained at consistently minimal percentages. Colorimetric evaluation of caspase 8, the principal initiator enzyme governing extrinsic death-receptor pathways, revealed no statistically significant variation relative to untreated controls. In sharp contrast, immunocytochemical detection of caspase 9, the initiator caspase that triggers the intrinsic mitochondria-dependent cascade, demonstrated statistically significant elevation in relative cellular fluorescence across both treated cancer cell lines, verifying internal apoptotic activation.

Computational molecular docking simulations were carried out to corroborate empirical observations regarding DNA groove recognition, albumin distribution, and internal apoptotic pathway triggering. Docking calculations utilized a B3LYP functional paired with a def2-SVP basis set to optimize the geometry of the neutral bimetallic complex prior to rigid docking against crystallographic targets from the Protein Data Bank. When docked against canonical B-DNA and an intercalation gap model, MolDock scoring functions indicated superior geometric compatibility with the canonical minor groove, matching spectroscopic displacement findings. Similarly, comparative docking into the binding groove of the anti-apoptotic regulator Bcl-2 demonstrated favorable structural accommodation, supporting observations that the complex impairs mitochondrial membrane integrity and triggers caspase 9 expression. While these comprehensive physical, spectroscopic, and cellular findings demonstrate that the 1,5-naphthyridine-bridged dinuclear gold(III) assembly achieves selective anticancer potency and favorable buffer stability, the authors emphasize that current investigations remain confined to in vitro cell cultures and in silico models, underscoring that broader preclinical evaluations and in vivo animal testing are necessary to determine clinical viability.

Subject of Research: Synthesis, macromolecular binding, and selective in vitro anticancer activity of a dinuclear gold(III) complex

Article Title: New dinuclear gold(III) complex with 1,5-naphthyridine as bridging ligand: synthesis, characterization, DNA/BSA binding studies, and anticancer activity

Article References: Radisavljević, S., Ćoćić, D., Petrović, B., Kellner, I., Ivanović-Burmazović, I., Radenković, N., Nikodijević, D., & Milutinović, M. (2024). New dinuclear gold(III) complex with 1,5-naphthyridine as bridging ligand: synthesis, characterization, DNA/BSA binding studies, and anticancer activity. Gold Bulletin, 57(1-2), 9-25. https://doi.org/10.1007/s13404-024-00344-8

Image Credits: AI Generated

DOI: 10.1007/s13404-024-00344-8

Keywords: gold(III) complex, anticancer activity, 1,5-naphthyridine, DNA binding, bovine serum albumin, caspase 9, apoptosis, molecular docking, dinuclear, gold, complex, naphthyridine