Exploring the binding mode of donepezil with calf thymus DNA using spectroscopic and molecular docking methods.

Donepezil (DNP) is one of approved drugs to treat Alzheimer's disease (AD). However, the potential effect of DNP on DNA is still unclear. Therefore, the interaction of DNP with calf thymus DNA (DNA) was studied in vitro using spectroscopic and molecular docking methods. Steady-state and transient fluorescence experiments showed that there was a clear binding interaction between DNP and DNA, resulting from DNP fluorescence being quenched using DNA. DNP and DNA have one binding site between them, and the binding constant (Kb ) was 0.78 × 104 L·mol-1 at 298 K. In this binding process, hydrophobic force was the main interaction force, because enthalpy change (ΔH) and entropy change (ΔS) of DNP-DNA were 67.92 kJ·mol-1 and 302.96 J·mol-1 ·K-1 , respectively. DNP bound to DNA in a groove-binding mode, which was verified using a competition displacement study and other typical spectroscopic methods. Fourier transform infrared (FTIR) spectrum results showed that DNP interacted with guanine (G) and cytosine (C) bases of DNA. The molecular docking results further supported the results of spectroscopic experiments, and suggested that both Pi-Sigma force and Pi-Alkyl force were the major hydrophobic force functioning between DNP and DNA.

Establishment of an RNA-based transient expression system in the green alga Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a unicellular green alga, is a prominent model for green biotechnology and for studying organelles' function and biogenesis, such as chloroplasts and cilia. However, the stable expression of foreign genes from the nuclear genome in C. reinhardtii faces several limitations, including low expression levels and significant differences between clones due to genome position effects, epigenetic silencing, and time-consuming procedures. We developed a robust transient expression system in C. reinhardtii to overcome these limitations. We demonstrated efficient entry of in vitro-transcribed mRNA into wall-less cells and enzymatically dewalled wild-type cells via electroporation. The endogenous or exogenous elements can facilitate efficient transient expression of mRNA in C. reinhardtii, including the 5' UTR of PsaD and the well-characterized Kozak sequence derived from the Chromochloris zofingiensis. In the optimized system, mRNA expression was detectable in 120 h with a peak around 4 h after transformation. Fluorescently tagged proteins were successfully transiently expressed, enabling organelle labeling and real-time determination of protein sub-cellular localization. Remarkably, transiently expressed IFT46 compensated for the ift46-1 mutant phenotype, indicating the correct protein folding and function of IFT46 within the cells. Additionally, we demonstrated the feasibility of our system for studying protein-protein interactions in living cells using bimolecular fluorescence complementation. In summary, the established transient expression system provides a powerful tool for investigating protein localization, function, and interactions in C. reinhardtii within a relatively short timeframe, which will significantly facilitate the study of gene function, genome structure, and green biomanufacturing in C. reinhardtii and potentially in other algae.

Multi-spectroscopic and molecular docking studies of human serum albumin interactions with sulfametoxydiazine and sulfamonomethoxine.

Sulfonamides are a kind of antibiotics which have been widely used as feed additives for livestock and poultry. However, sulfa drugs have raised worldwide concerns because of their adverse impact on human health. In this study, two sulfonamides, sulfametoxydiazine (SMD) and sulfamonomethoxine (SMM), were selected to explore the binding modes with human serum albumin (HSA). The spectroscopic approaches revealed that SMD or SMM could spontaneously enter into the binding site I of HSA through hydrogen bond interactions and van der Waals forces, and that SMD exhibited much stronger binding affinity toward HSA than SMM at different temperatures (p < 0.01, n = 3). The binding constants for SMD-HSA and SMM-HSA were determined to be (8.297 ± 0.010) × 104 L·mol-1 and (1.178 ± 0.008) × 104 L·mol-1 at 298 K, respectively. The interaction of SMD or SMM to HSA induced microenvironmental and conformational changes in HSA, where SMD had a greater effect on the α-helix content of HSA. Results from molecular docking implied that the amino acid residues of HSA, such as Arg222, Ala291 and Leu238, played key roles in the sulfonamide-HSA binding process. Meanwhile, hydrogen bonds might be a key factor contributing to the binding affinity of sulfa drugs and HSA. Additionally, the combined use of SMD and SMM led to an obvious variation in Ka values of binary systems (p < 0.01, n = 3). These findings might be helpful to understand the biological effects of sulfonamides in humans.

Probing the interaction between levamlodipine and hemoglobin based on spectroscopic and molecular docking methods.

In recent years, levamlodipine (LAML) has been widely used as a common drug for the treatment of hypertension. However, no reports exist that focus on the binding process of LAML with the transport proteins present in blood circulation. Here, several spectroscopy techniques, molecular docking and a molecular dynamics simulation were employed to comprehensively analyze the mechanism underlying the interaction between bovine hemoglobin (BHb) and LAML, as well as the effect of other drugs on the BHb-LAML system. The results indicated that a stable BHb-LAML complex was formed and that the binding site for LAML was located at ß-37 tryptophan in the central cavity of BHb. Van der Waals force and hydrogen bonds played major roles in this binding process, and the number of binding sites (n) in the binary system was approximately equal to 1. Multiple spectroscopy experiments (FT-IR and three-dimensional fluorescence spectrometry) and a dynamics simulation revealed that LAML could induce a conformational in BHb and that the microenvironment of Trp/Tyr changed. Interestingly, the values of the binding constant between LAML and BHb significantly increased due to the effect of rofecoxib, propranolol and enalapril. Meanwhile, these drugs did not produce synergistic or negative synergistic effects on the LAML binding with BHb. These results provide new insight into the transport mechanisms for LAML in the human body.