Latest ArticlesThe Dionex CaboPacTM PA10 BioLCTM Analyical 2 mm × 250 mm column was used with a protective column (Dionex CaboPacTM PA10 BioLCTM Guard 2 mm × 50 mm). 100 mmol·L-1 sodium hydroxide solution was used as eluent; the flow rate was 0.25 mL·min-1. Sample tray temperature: 35 ℃. The pulse amperometric detector was adopted, and the waveform was Gold CWE, Ag-AgCl RE, Carbo, Quad. The samples were cultured with 8 concentrations of glycogen substrates (0.31, 1.25, 2.5, 5, 10, 20, 30, and 40 mg·mL-1). D-Glucose concentrations were measured at 5 different time points (T0, T1, T2, T3 and T4). The glucose concentration from T1 to T4 minus the glucose concentration at T0. The reaction rate was calculated at different glycogen substrate concentrations. These reaction rates are plotted against substrate concentrations using Michaelis-Menten equation. The kinetic parameters were expressed as Vmax (nmol·mg-1·min-1) and Km (mg·mL-1). The RSD of glucose standard curve R2 (n = 6, linear range: 1.25-500 μmol·L-1) was 0.1% and the RSD (n = 6) of the slope of the standard curve was 2.2%. The mean limit of quantitation was 0.14 μmol·L-1, and the mean limit of detection was 0.05 μmol·L-1. The RSD of Km and Vmax were 4.4% and 4.6% respectively in three separate experiments. The durability of the method was good. The method was developed for the on-line automatic determination of the hydrolysis kinetics of acid α-glucosidase (GAA) for injection by ion chromatography. The method has good precision, repeatability and durability, and can be used for the determination of glycogen hydrolysis kinetics of GAA for injection, and could reference value for the enzyme kinetics evaluation of recombinant enzyme replacement therapy.
According to the requirements of the regulatory authorities, degree of modification (DP) should be included in the characterisation of the PEGylated protein drug substance, and is one of the critical quality attributes for quality control. In this study, based on the fundamental assumption that the refractive index (RI) signal and the ultraviolet (UV) signal of PEGylated protein are equal to the sum of the corresponding signal produced by the polyethylene glycol (PEG) and protein parts of the conjugates in their uncoupled state, we developed a method to determine the DP of PEGylated recombinant human growth hormone (inpegsomatropin). In this method, 20 μL of 1 mg·mL-1 human growth hormone (hGH) standard, 2 mg·mL-1 PEG reference substance and 1 mg·mL-1 drug substance solution were each injected to size exclusion chromatographic (SEC) column for separation, detected with ultraviolet and refractive index (UV-RI) detectors in series. Finally, the DP was calculated as the formula derived from the fundamental assumption. The developed SEC-UV-RI method showed good specificity, repeatability (RSD = 0.63%, n = 9) and accuracy, with a recovery of 100.0%, compared with the result obtained from the simultaneously established classical acid hydrolysis method, demonstrating pretty handleability and accessibility, and is appropriate for quality control test of DP for this drug substance.
In order to improve the poor photostability of nifedipine, this study designed a cocrystal based on the principles of crystal engineering and prepared nifedipine-imidazole cocrystal by suspension method. The new cocrystal was characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TG) and infrared spectroscopy (IR) to confirm the formation of the cocrystal. The photostability of nifedipine and its cocrystal was measured by powder X-ray diffraction and high-performance liquid chromatography (HPLC). The results showed that the nifedipine-imidazole cocrystal improved the photostability of nifedipine to a certain extent. This study provides guidance for the development of nifedipine cocrystals and the improvement of its druggability.
The limitations of antifungal drugs and severe drug resistance make the treatment of invasive fungal infections (IFIs) a great challenge. Itraconazole (ITZ), as a clinical first-line drug, has a wide range of antifungal activity, but it is still limited by adverse reactions such as liver and kidney toxicity, headache and abdominal pain due to its poor water solubility and easy to cause drug accumulation by injection. In this study, the amphiphilic polymer gallic acid-chitosan-cinnamaldehyde (GA-CS-CN) was prepared by amide reaction and Schiff-base reaction. The drug-loaded nanoparticles (GA-CS-CN/ITZ) were prepared by ultrasonic method. The properties of nanoparticles formulations and its in vitro antifungal activity were investigated in the study. Studies have shown that GA-CS-CN/ITZ is spherical, homogeneous and stable, and has good biological safety. The average particle size was 239.57 ± 31.37 nm, ITZ encapsulation efficiency was (93.41 ± 1.12)%, and the cumulative drug release was (62.25 ± 1.88)% at 48 h in vitro. The antifungal activity results of Candida albicans ATCC 10231 (C. albicans) showed that it had the optimal antifungal effect and could significantly enhance the antifungal activity of free drugs. GA-CS-CN/ITZ prepared in this study has excellent biological safety and anti-C. albicans performance, which provides a new choice for the treatment of C. albicans infection and has good application potential in the treatment of this fungal infection.
Intracellular neurofibrillary tangles resulting from abnormal hyperphosphorylation of Tau protein constitute one of the principal pathological markers of Alzheimer′s disease. Existing studies have indicated that BSc3094 is an efficacious inhibitor of Tau protein aggregation, capable of binding to Tau protein, inhibiting Tau protein phosphorylation, and enhancing cell viability concurrently, holding significant potential in treating Alzheimer′s disease. Nevertheless, due to the presence of the blood-brain barrier, it is challenging for drugs to penetrate the brain and exert their effects, and whether BSc3094 can treat Alzheimer′s disease by inhibiting Tau protein aggregation has not been profoundly investigated. Hence, in this study, small-sized (PLGA) nanoparticles were fabricated through the stirring method. BSc3094 was loaded into the nanoparticles (PLGA@BSc). To further enhance the brain entry efficiency of PLGA nanoparticles, a pathological BBB-targeting peptide was modified on the surface to obtain PLGA@BSc@K. In this study, the stability, cytotoxicity, and pathological targeting of the nanosystem were characterized. The particle size of the nanosystem was about 90 nm, which was negatively charged. The results demonstrated that the particle size of the nanoparticles did not fluctuate conspicuously within 168 h, and the stability was favorable. PLGA and BSc3094 had no notable impact on cell viability and displayed low cytotoxicity. At 1 and 4 h, it was observed that the uptake of targeted modified nanoparticles by cells in pathological states augmented, suggesting that PLGA@BSc@K had an excellent pathological blood-brain barrier targeting effect. This study provides a novel concept for the targeting of BSc3094 nanoparticles in the brain and the treatment of Alzheimer′s disease.
The objective of this study was to optimise the extraction process of peptide of Poecilobdella manillensis by the Box-Behnken design-response surface methodology, and to investigate its whitening and anti-aging effects. Based on single factor experiments, NaCl solution concentration, extracting time and extracting times were taken as influencing factors, and peptide yield was used as the response value. The response surface model was designed and used to obtain the optimal extraction conditions of peptides of Poecilobdella manillensis: NaCl solution concentration of 4.3%, ultrasonic time of 4 h, and ultrasonic times of 2 times (2 + 2 h), which was significant and well-fitted to the actual experiment. The tyrosinase inhibition activity of peptide of Poecilobdella manillensis was evaluated by measuring the oxidation rate of levodopa (L-DOPA) catalyzed by tyrosinase. Moreover, using Caenorhabditis elegans as a model organism, the effects of peptide of Poecilobdella manillensis on body length, locomotion, reproductive capacity, reactive oxygen species (ROS), and lipofuscin levels were determined. The results showed that peptide of Poecilobdella manillensis exhibited a significant inhibitory effect on tyrosinase, with an IC50 of 0.58 mg·mL-1, which was stronger than that of the positive control arbutin (2.24 mg·mL-1). Compared to the control group, 0.1, 0.5 and 1.0 mg·mL-1 peptide of Poecilobdella manillensis showed no significant differences in the body length, eggs, and body bending of Caenorhabditis elegans. However, 0.5 mg·mL-1 peptide of Poecilobdella manillensis significantly reduced ROS levels in Caenorhabditis elegans; 0.5 and 1.0 mg·mL-1 peptide of Poecilobdella manillensis significantly reduced lipofuscin levels in Caenorhabditis elegans. Peptide of Poecilobdella manillensis exhibits effective whitening and anti-aging activities, potentially mediated by its inhibition of tyrosinase activity and antioxidant effects.
Arginase 1 deficiency (ARG1-D) is a rare genetic metabolic disorder that leads to progressive spastic paralysis, cognitive impairment, and seizures. Recombinant human arginase 1 (rhArg1) is a potential therapeutic agent for this condition, but its clinical application is limited by low activity and short half-life. In this study, we employed directed evolution to address these issues. A random mutation library of rhArg1 was constructed using error-prone PCR, and high-throughput screening was used to identify mutants with enhanced activity. Site-saturation mutagenesis was also performed to investigate the effects of residues R21 and V182 on enzyme activity. Our findings revealed that under reaction conditions devoid of Mn2+, the kcat values of the mutants V182D, V182S, V182H, and R21N increased by 2.0, 1.9, 1.7, and 1.3 times respectively, compared to rhArg1. The kcat/Km values of mutants V182D, V182S, R21D, and R21N were 2.1, 1.7, 1.4, and 1.4 times higher than those of rhArg1, respectively. Additionally, mutants R21D and V182L showed enhanced substrate affinity. Through directed evolution and site-saturation mutagenesis, we successfully obtained rhArg1 mutants with improved activity, thereby enhancing its potential for clinical application.
Folding and post-translational modification of proteins are vital for their proper functionality, with various functional regulatory systems playing significant roles, including molecular chaperone systems, ubiquitination systems, phosphorylation systems, acetylation systems, etc. Precise regulations of these systems have emerged as an important trend in drug development. This review systematically summarizes the molecular control strategies related to protein folding and post-translational modification, with a specific focus on the molecular chaperone system and the strategy of heterobifunctional molecules. On one hand, based on the similarities and differences in molecular mechanisms and design strategies, we summarize the drug development process targeting the molecular chaperone system. On the other hand, we discuss the design principles and characteristics of dual-functional molecules, and summarize their applications and developments in the precise control of post-translational modifications, aiming to provide new insights for future design.
Protein phosphorylation modification is an important mechanism of physiological regulation that is closely related to protein biological functions. In particular, protein kinases are responsible for catalyzing the phosphorylation process of proteins, and phosphatases are responsible for catalyzing the dephosphorylation process of phosphorylation-modified proteins, which together mediate the achievement of dynamic and reversible phosphorylation modifications of proteins. Abnormal phosphorylation levels of proteins contribute to the development of many diseases, such as cancer, neurodegenerative diseases, and chronic diseases. Therefore, rational design of small molecules to regulate protein phosphorylation is an important approach for disease treatment. Based on the mechanism of protein phosphorylation regulation, small molecule drug design strategies can be classified into three types, protein kinase modulators, phosphatase modulators, and bifunctional molecules with proximity-mediated mechanism. This review emphasizes the above three small molecule design strategies for targeting protein phosphorylation regulation, including molecular design ideas, research progress and current challenges, and provides an outlook on small molecule modulators targeting protein phosphorylation modification.
The ubiquitin-proteasome system (UPS) is responsible for protein degradation in both normal and pathological states. E3 ligases selectively attach ubiquitin to specific substrates, which is essential for regulating cellular homeostasis. The function of E3 ligases has been associated with a variety of diseases, such as cancer and cardiovascular disease. The discovery of E3 ligands can help regulate E3 ligases, thus expanding new ideas for disease treatment. Targeted protein degradation (TPD) drugs, including proteolysis targeting chimera (PROTAC), have become increasingly popular in recent years due to their dependence on E3 ligands. In this paper, we review the discovery techniques of E3 ligands, including activity-based protein mapping, fragment-based drug discovery, and library-based methods, and briefly introduce the protein interaction detection techniques involved in the ligand discovery techniques, in the hope of providing certain ideas for the future discovery of E3 ligands as well as the treatment of diseases.
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