Direct Interaction of Minocycline to p47phox Contributes to its Attenuation of TNF-α-Mediated Neuronal PC12 Cell Death: Experimental and Simulation Validation.

Minocycline, a repurposed approved medication, shows promise in treating neurodegeneration. However, the specific pathways targeted by minocycline remain unclear despite the identification of molecular targets. This study explores minocycline's potential protective effects against TNF-α-mediated neuronal death in PC12 cells, with a focus on unraveling its interactions with key molecular targets. The study begins by exploring minocycline's protective role against TNF-α-mediated neuronal death in PC12 cells, showcasing a substantial reduction in cleaved caspase-3 expression, DNA fragmentation, and intracellular ROS levels following minocycline pretreatment. Subsequently, a comprehensive analysis utilizing pull-down assays, computational docking, mutation analysis, molecular dynamics simulations, and free energy calculations is conducted to elucidate the direct interaction between minocycline and p47phox-the organizer subunit of NADPH oxidase-2 (NOX2) complex. Computational insights, including a literature survey and analysis of key amino acid residues, reveal a potential binding site for minocycline around Trp193 and Cys196. In silico substitutions of Trp193 and Cys196 further confirm their importance in binding with minocycline. These integrated findings underscore minocycline's protective mechanisms, linking its direct interaction with p47phox to the modulation of NOX2 activity and attenuation of NOX-derived ROS generation. Minocycline demonstrates protective effects against TNF-α-induced PC12 cell death, potentially linked to its direct interaction with p47phox. This interaction leads to a reduction in NOX2 complex assembly, ultimately attenuating NOX-derived ROS generation. These findings hold significance for researchers exploring neuroprotection and the development of p47phox inhibitors.

Insights Into The Effects of Amino Acid Substitutions on The Stability of Reteplase Structure: A Molecular Dynamics Simulation Study.

Background: Reteplase (recombinant plasminogen activator, r-PA) is a recombinant protein designed to imitate the endogenous tissue plasminogen activator and catalyze the plasmin production. It is known that the application of reteplase is limited by the complex production processes and protein's stability challenges. Computational redesign of proteins has gained momentum in recent years, particularly as a powerful tool for improving protein stability and consequently its production efficiency. Hence, in the current study, we implemented computational approaches to improve r-PA conformational stability, which fairly correlates with protein's resistance to proteolysis. Objectives: The current study was developed in order to evaluate the effect of amino acid substitutions on the stability of reteplase structure using molecular dynamic simulations and computational predictions. Materials and Methods: Several web servers designed for mutation analysis were utilized to select appropriate mutations. Additionally, the experimentally reported mutation, R103S, converting wild type r-PA into non-cleavable form, was also employed. Firstly, mutant collection, consisting of 15 structures, was constructed based on the combinations of four designated mutations. Then, 3D structures were generated using MODELLER. Finally, 17 independent 20-ns molecular dynamics (MD) simulations were conducted and different analysis were performed like root-mean-square deviation (RMSD), root-mean-square fluctuations (RMSF), secondary structure analysis, number of hydrogen bonds, principal components analysis (PCA), eigenvector projection, and density analysis. Results: Predicted mutations successfully compensated the more flexible conformation caused by R103S substitution, so, improved conformational stability was analyzed from MD simulations. In particular, R103S/A286I/G322I indicated the best results and remarkably enhanced the protein stability. Conclusion: The conformational stability conferred by these mutations will probably lead to more protection of r-PA in protease-rich environments in various recombinant systems and potentially enhance its production and expression level.

An in-silico insight into the substrate binding characteristics of the active site of amorpha-4, 11-diene synthase, a key enzyme in artemisinin biosynthesis.

The enzyme amorphadiene synthase (ADS) conducts the first committed step in the biosynthetic conversion of the substrate farnesyl pyrophosphate (FPP) to artemisinin, which is a highly effective natural product against multidrug-resistant strains of malaria. Due to the either low abundance or low turn-over rate of the enzyme, obtaining artemisinin from both natural and synthetic sources is costly and laborious. In this in silico study, we strived to elucidate the substrate binding site specificities of the ADS, with the rational that unraveling enzyme features paves the way for enzyme engineering to increase synthesis rate. A homology model of the ADS from Artemisia annua L. was constructed based on the available crystal structure of the 5-epiaristolochene synthase (TEAS) and further analyzed with molecular dynamic simulations to determine residues forming the substrate recognition pocket. We also investigated the structural aspects of Mg2+ binding. Results revealed DDYTD and NDLMT as metal-binding motifs in the putative active site gorge, which is composed of the D and H helixes and one loop region (aa519-532). Moreover, several representative residues including Tyr519, Asp444, Trp271, Asn443, Thr399, Arg262, Val292, Gly400 and Leu405, determine the FPP binding mode and its fate in terms of stereochemistry as well as the enzyme fidelity for the specific end product. These findings lead to inferences concerning key components of the ADS catalytic cavity, and provide evidence for the spatial localization of the FPP and Mg2+. Such detailed understanding will probably help to design an improved enzyme.

Identification of novel bacterial DNA gyrase inhibitors: An in silico study.

Owing to essential role in bacterial survival, DNA gyrase has been exploited as a validated drug target. However, rapidly emerging resistance to gyrase-targeted drugs such as widely utilized fluoroquinolones reveals the necessity to develop novel compounds with new mechanism of actions against this enzyme. Here, an attempt has been made to identify new drug-like molecules for Shigella flexneri DNA gyrase inhibition through in silico approaches. The structural similarity search was carried out using the natural product simocyclinone D8, a unique gyrase inhibitor, to virtually screen ZINC database. A total of 11830 retrieved hits were further screened for selection of high-affinity compounds by implementing molecular docking followed by investigation of druggability according to Lipinski's rule, biological activity and physiochemical properties. Among the hits initially identified, three molecules were then confirmed to have reasonable gyrase-binding affinity and to follow Lipinski's rule. Based on these in silico findings, three compounds with different chemical structures from previously identified gyrase inhibitors were proposed as potential candidates for the treatment of fluoroquinolone-resistant strains and deserve further investigations.

Protective effect of telmisartan against oxidative damage induced by high glucose in neuronal PC12 cell.

Telmisartan is an angiotensin II type 1 receptor blocker and partial agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ). Here, we investigated the protective capacity of telmisartan against high glucose (HG)-elicited oxidative damage in PC12 cells. The activity of lactate dehydrogenase (LDH), NADPH oxidase (NOX), superoxide dismutase (SOD), catalase (CAT) as well as the levels of malondialdehyde (MDA), glutathione (GSH), intracellular reactive oxygen species (ROS), cell viability and DNA fragmentation were measured in HG-treated PC12 cells with and without telmisartan co-treatment. Moreover, the direct antioxidant effect of telmisartan was determined by 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay and protein expression of Bax, Bcl-2, cleaved caspase-3 and NOX subunit p47phox by western blotting. Telmisartan exhibited antioxidant activity in the ABTS assay with the IC50 value of 37.5 µM. Pretreatment of PC12 cells with telmisartan, prior to HG exposure, was associated with a marked diminution in cleaved caspase-3 expression, DNA fragmentation, Bax/Bcl-2 ratio, intracellular ROS and MDA levels. Additionally, the cell viability, GSH level, SOD and CAT activity were notably elevated by telmisartan, whereas the activity and the protein expression of NADPH oxidase subunit p47phox were attenuated. Interestingly, co-treatment with GW9662, a PPAR-γ antagonist, partially inhibited the beneficial effects of telmisartan. These findings suggest that telmisartan has protective effects on HG-induced neurotoxicity in PC12 cells, which may be related to its antioxidant action and inhibition of NADPH oxidase. Furthermore, the results show that PPAR-γ activation is involved in the neuroprotective effects of telmisartan.

Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells.

Hyperglycemia, which occurs under the diabetic condition, is widely recognized as the causal link between diabetes and its serious complications. Diabetic neuropathies, which are among the most frequent complications of diabetes, affect sensory, motor, and autonomic nerves. The exact molecular mechanisms of high glucose-induced toxicity on neuronal cells, is still unclear. We previously reported that high glucose can induce apoptosis in PC12 cells, as evidenced by DNA fragmentation and high Bax/Bcl-2 ratio. The present study examined the involvement of caspase-3, the executioner, and two initiators of apoptosis, caspase-8 and caspase-9, during high glucose-induced apoptosis in PC12 cells, a neuronal cell line. Cells were exposed to high glucose with or without z-VAD-fmk, a pan-caspase inhibitor. Cell viability was measured by MTT assay. Caspase activity was determined spectrophotometrically using enzyme specific substrates. To correlate and confirm the caspase activity with changes in protein expression, procaspase-8, -9, and -3 were evaluated by Western blot analysis. The DNA-fragmentation was determined by DNA ladder using gel electrophoresis. The PC12 cell viability on high glucose exposure was decreased compared to controls, which was reversed by z-VAD-fmk. The activities of caspase-8, -9, and -3 were significantly increased in treated cells compared to controls. Moreover, high glucose exposure induced a significant decrease in protein levels of procaspases, indicating conversion of pro-form into the mature caspases. Finally, DNA fragmentation (Ladder) was shown in treated cells by high glucose. Based on the current data, it could be concluded that high glucose-induced apoptosis in PC12 cells is mediated, in part, by activation of caspase-8, -9, and -3 dependent pathways.