Novel tetranuclear grid-like Zn(II) complexes derived from dihydrazone pyrimidine derivatives as antitumor agents.

Due to the antitumor properties, Zn(II) complexes have attracted more and more attention. Herein, three novel tetranuclear Zn(II) complexes 1-3 based on dihydrazone pyrimidine derivatives H2L1-H2L3 were synthesized and characterized using IR spectroscopy, 1H NMR spectroscopy, single crystal X-ray diffraction analysis, XRD, TG and elemental analysis. Single crystal X-ray diffraction analysis revealed that 1-3 all displayed a [2 × 2] grid-like topology. The stability in solution, lipophilicity, confocal imaging and antitumor activities were investigated. Complexes 1-3 displayed high structural stability, membrane permeability and different lipophilicities. They can target mitochondria due to the cation charge. The MTT assay indicated that all of them exhibited stronger antiproliferative activity than the corresponding derivatives H2L1-H2L3 and the well-known cisplatin against all the selected tumor cells (BGC-823, BEL-7402, MCF-7 and A549), with IC50 values ranging from 2.83 µM to 7.97 µM. AO/EB double staining, flow cytometry and ROS detection suggested that complexes 1 and 2 could induce BGC-823 apoptosis in a dose-dependent manner. UV-Vis spectra, CD spectra, viscosity analysis and molecular docking revealed that complexes 1 and 2 interact with DNA mainly via partial intercalation and groove binding. Tetranuclear [2 × 2] grid-like Zn(II) complexes have the potential to be promising antitumor agents in the future.

Synthesis, Biological Evaluation, DNA Binding, and Molecular Docking of Hybrid 4,6-Dihydrazone Pyrimidine Derivatives as Antitumor Agents.

In the present paper, on the basis of molecular hybridization, a series of 4,6-dihydrazone pyrimidine derivatives containing the pyridine moiety were synthesized, structurally characterized, and evaluated in vitro for their antitumor activity. According to the results, all the tested compounds demonstrated broad-spectrum antitumor activity against selected tumor cell lines (MCF-7, BGC-823, A549, and BEL-7402) and no obvious toxicity toward normal cells HL-7702. In particular, compounds 10a and 10f were found to be the most promising antitumor agents among the tested compounds against BGC-823 cells (IC50 = 9.00 µM and 7.89 µM) and BEL-7402 cells (IC50 = 6.70 µM and 7.66 µM), respectively. Compounds 10a and 10f exhibited higher potency against BGC-823 and BEL-7402 than the positive control 5-FU (IC50 = 15.18 µM and 15.81 µM). Further mechanism investigations demonstrated that compounds 10a and 10f could significantly increase the level of cellular ROS and induce early apoptosis of BGC-823 cells in a dose-dependent manner. Moreover, the DNA binding results from UV/Vis, CD spectroscopy, and molecular docking studies indicated that 10a and 10f bind with DNA via groove binding and partial intercalation. These results demonstrated that 10a and 10f may serve as novel lead compounds for the discovery of more dihydrazone pyrimidine derivatives with improved antitumor potency and selectivity.

The Role of Surface Properties on Protein Aggregation Behavior in Aqueous Solution of Different pH Values.

This study aimed to investigate the effect of pH-mediated surface properties of bovine serum albumin (BSA) on protein aggregation and the changes of protein structure and colloidal stability at different solution pH levels. The hydrophobicity of BSA surface was characterized by endogenous fluorescence spectroscopy, fluorescence quenching of acrylamide, and fluorescence probe. The results showed that the hydrophobicity of BSA surface was similar at pH 5, 6, 7.4, followed by pH 4, 8, 9, 10, and finally by pH 3 and 11 with strong acidity and alkalinity. The positive charge on the BSA surface was increased gradually with the decrease of solution pH, while the negative charge on protein surface was increased gradually with the increase of solution pH. The degree of protein aggregation was examined by turbidimetry, flow cytometry, and SDS-PAGE. The results showed that the oscillating aggregation of BSA did not change with the solution pH, but was partially dependent on the relative contribution of electrostatic and hydrophobic interactions between the protein molecules. In addition, the secondary structure, conformational stability, unfolding degree, and colloidal stability of proteins were investigated by circular dichroism, fluorescence spectroscopy, protein pulse hydrolysis, and dynamic light scattering, respectively. The results suggested that the solution pH could change the structure and stability of the protein at different levels. Solution pH has distinct effects on the structural stability of protein at different levels. The change of protein surface properties mediated by solution pH is related to protein aggregation.

Protein Aggregation and Performance Optimization Based on Microconformational Changes of Aromatic Hydrophobic Regions.

Protein aggregation is a key concern in biopharmaceutical development and manufacturing. There is growing interest in understanding how the changes in protein microconformation affect the aggregation behavior. This study selected several representative proteins and first manipulated microconformational changes of the aromatic hydrophobic regions of proteins, especially tryptophan residues, by using amine or guanidine additives. The effects of the interactions between the additives and proteins on the aromatic hydrophobic regions could be grouped into three categories: exposure to solvent, burial into core, and no change. The microconformational parameters of the tryptophan residue, including fluorescence peak position (λm), degree of hydrolysis, solvent accessible surface area ( SAS), and packing density ( Den), were obtained by steady-state fluorescence spectroscopy, proteolysis coupled with electrophoresis, and molecular dynamics simulation. Furthermore, the aggregation degrees of globular proteins with distinct surface aromatic hydrophobilities under mechanical stress were investigated. A strong correlation was observed between protein aggregation and the microconformational changes of the aromatic hydrophobic regions incurred by amine or guanidine additives. Protein aggregation was enhanced when the aromatic hydrophobic regions were exposed to the solvent but suppressed when the additives led to burial of the aromatic hydrophobic regions with lower-polarity microenvironment. These findings shed light on the relationship between protein aggregation and molecular conformation and paved way for future preformulation studies of therapeutic proteins.

Bilateral Effects of Excipients on Protein Stability: Preferential Interaction Type of Excipient and Surface Aromatic Hydrophobicity of Protein.

PURPOSE: Understanding the mechanism of protein-excipient interaction and illuminating the influencing factors on protein stability are key steps in the rational design of protein formulations. The objective of this study was to assess effects of preferential interaction type of excipient and surface aromatic hydrophobicity of protein on protein solution stability. METHODS: The preferential interaction between excipient and aromatic hydrophobic area of protein was investigated by solubility and fluorescence studies of amino acid derivatives in excipient solutions. We examined conformational, colloidal and mechanical stabilities of model proteins with different surface aromatic hydrophobicities, including bovine serum albumin (BSA) and ovalbumin (OVA), and then stability data were visualized by three-index empirical phase diagram. RESULTS: The result showed that preferentially excluded excipients (trehalose, sucrose and sorbitol) protected protein conformation against damage, but they could accelerate mechanical stress-induced aggregation. Preferentially bound excipients (propanediol and arginine) suppressed BSA aggregation, but arginine failed to inhibit OVA aggregation, which might be attributed to the disparate conformational perturbing effects of arginine on aromatic hydrophobic regions of BSA and OVA. CONCLUSIONS: These findings provided strong evidence that excipient possessed bilateral effects, and its application should be determined on different preferential interaction behaviors of excipients with protein, especially with the aromatic hydrophobic region.