Double-modified, thio and methylene ATP analogue facilitates wound healing in vitro and in vivo.

Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue-alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure-activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate SP and RP diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist-AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the SP-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial-mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the SP-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment.

Different aspects in explaining how mutations could affect the binding mechanism of receptor binding domain of SARS-CoV-2 spike protein in interaction with ACE2.

During replication, some mutations occur in SARS-CoV-2, the causal agent of COVID-19, leading to the emergence of different variants of the virus. The mutations that accrue in different variants of the virus, influence the virus' ability to bind to human cell receptors and ability to evade the human immune system, the rate of viral transmission, and effectiveness of vaccines. Some of these mutations occur in the receptor binding domain (RBD) of the spike protein that may change the affinity of the virus for the ACE2 receptor. In this study, several in silico techniques, such as MD and SMD simulations, were used to perform comparative studies to deeply understand the effect of mutation on structural and functional details of the interaction of the spike glycoprotein of SARS-CoV-2, with the ACE2 receptor. According to our results, the mutation in the RBD associated with the Omicron variant increase binding affinity of the virus to ACE2 when compared to wild type and Delta variants. We also observed that the flexibility of the spike protein of the Omicron variant was lower than in comparison to other variants. In summary, different mutations in variants of the virus can have an effect on the binding mechanism of the receptor binding domain of the virus with ACE2.

Designing and generating a single-chain fragment variable (scFv) antibody against IL2Rα (CD25): An in silico and in vitro study.

OBJECTIVES: IL-2Rα plays a critical role in maintaining immune function. However, expression and secretion of CD25 in various malignant disorders and autoimmune diseases are now well established. Thus, CD25 is considered an important target candidate for antibody-based therapy. This study aimed to find the most suitable linker peptide to construct a functional anti-CD25 single-chain fragment variable (scFv) by bioinformatics studies and its production in a bacterial expression system. MATERIALS AND METHODS: Here, the 3D structures of the scFvs with different linkers were predicted and molecular dynamics simulation was performed to compare their structures and dynamics. Then, interactions between five models of scFv and human CD25 were calculated via molecular docking. According to MD and docking results, the anti-CD25 scFvs with (Gly4Ser)3 linker were constructed and cloned into pET-22b(+). Then, recombinant plasmids were transformed into Escherichia coli Bl21 (DE3) for expression using IPTG and lactose as inducers. Anti-CD25 scFv was purified from the periplasm and detected by SDS-PAGE and Western blot. Afterward, functionality was evaluated using ELISA. RESULTS: In silico analysis showed that the model containing (Gly4Ser)3 as a linker has more stability compared with other linkers. The results of SDS-PAGE, Western blot, and ELISA confirmed the accuracy of anti-CD25 scFv production and its ability to bind to the human CD25. CONCLUSION: Conclusively, our work provides a theoretical and experimental basis for production of an anti-CD25 scFv, which may be applied for various malignant disorders and autoimmune diseases.

In silico investigation of critical binding pattern in SARS-CoV-2 spike protein with angiotensin-converting enzyme 2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a newly-discovered coronavirus and responsible for the spread of coronavirus disease 2019 (COVID-19). SARS-CoV-2 infected millions of people in the world and immediately became a pandemic in March 2020. SARS-CoV-2 belongs to the beta-coronavirus genus of the large family of Coronaviridae. It is now known that its surface spike glycoprotein binds to the angiotensin-converting enzyme-2 (ACE2), which is expressed on the lung epithelial cells, mediates the fusion of the cellular and viral membranes, and facilitates the entry of viral genome to the host cell. Therefore, blocking the virus-cell interaction could be a potential target for the prevention of viral infection. The binding of SARS-CoV-2 to ACE2 is a protein-protein interaction, and so, analyzing the structure of the spike glycoprotein of SARS-CoV-2 and its underlying mechanism to bind the host cell receptor would be useful for the management and treatment of COVID-19. In this study, we performed comparative in silico studies to deeply understand the structural and functional details of the interaction between the spike glycoprotein of SARS-CoV-2 and its cognate cellular receptor ACE2. According to our results, the affinity of the ACE2 receptor for SARS-CoV-2 was higher than SARS-CoV. According to the free energy decomposition of the spike glycoprotein-ACE2 complex, we found critical points in three areas which are responsible for the increased binding affinity of SARS-CoV-2 compared with SARS-CoV. These mutations occurred at the receptor-binding domain of the spike glycoprotein that play an essential role in the increasing the affinity of coronavirus to ACE2. For instance, mutations Pro462Ala and Leu472Phe resulted in the altered binding energy from - 2 kcal mol-1 in SARS-COV to - 6 kcal mol-1 in SARS-COV-2. The results demonstrated that some mutations in the receptor-binding motif could be considered as a hot-point for designing potential drugs to inhibit the interaction between the spike glycoprotein and ACE2.

The functional regulatory details of ERK2 in complex with RSK1: an in silico insight.

Protein kinases play a significant role in cellular activation procedures by exhibiting a vivid selection in the target, as well as recognizing and phosphorylating them. Extracellular signal-regulated kinase 2 (ERK2) is one of the main kinases in the mitogen-activated protein kinase (MAPK) signaling cascade and engages in dynamically regulating the activities of signaling proteins and physiological processes, including cell proliferation, differentiation, adhesion, migration, and survival. Predicting collective dynamic and structural motions in biological macromolecules is pivotal to obtain a better understanding of the majority of biological processes. Here, through molecular dynamic simulation and normal mode analysis, we investigated ERK2 conformations, in the forms of active (phosphorylated), inactive (unphosphorylated), and in a complex with its substrate, ribosomal protein S6 kinase alpha-1 (RSK1), to determine functional characteristics. Our finding demonstrated that ERK2 plays a switch role in the regulation of pathways. In the case that this protein kinase is in the active form, all critical regions shift to be prepared to accept the substrate and catalytic action. Meanwhile, inactive ERK2 shows contrasting results in which all motions tend to close the catalytic site and cease the phosphorylation action in the MAPK cascade. These findings are in line with those from other similar studies and provide us with novel molecular target regions and recent details on how this mechanism works.

Novel Peptide Inhibitors for Lactate Dehydrogenase A (LDHA): A Survey to Inhibit LDHA Activity via Disruption of Protein-Protein Interaction.

Lactate dehydrogenase A (LDHA) is a critical metabolic enzyme belonging to a family of 2-hydroxy acid oxidoreductases that plays a key role in anaerobic metabolism in the cells. In hypoxia condition, the overexpression of LDHA shifts the metabolic pathway of ATP synthesis from oxidative phosphorylation to aerobic glycolysis and the hypoxia condition is a common phenomenon occurred in the microenvironment of tumor cells; therefore, the inhibition of LDHA is considered to be an excellent strategy for cancer therapy. In this study, we employed in silico methods to design inhibitory peptides for lactate dehydrogenase through the disturbance in tetramerization of the enzyme. Using peptide as an anti-cancer agent is a novel approach for cancer therapy possessing some advantages with respect to the chemotherapeutic drugs such as low toxicity, ease of synthesis, and high target specificity. So peptides can act as appropriate enzyme inhibitor in parallel to chemical compounds. In this study, several computational techniques such as molecular dynamics (MD) simulation, docking and MM-PBSA calculation have been employed to investigate the structural characteristics of the monomer, dimer, and tetramer forms of the enzyme. Analysis of MD simulation and protein-protein interaction showed that the N-terminal arms of each subunit have an important role in enzyme tetramerization to establish active form of the enzyme. Hence, N-terminal arm can be used as a template for peptide design. Then, peptides were designed and evaluated to obtain best binders based on the affinity and physicochemical properties. Finally, the inhibitory effect of the peptides on subunit association was measured by dynamic light scattering (DLS) technique. Our results showed that the designed peptides which mimic the N-terminal arm of the enzyme can successfully target the C-terminal domain and interrupt the bona fide form of the enzyme subunits. The result of this study makes a new avenue to disrupt the assembly process and thereby oppress the function of the LDHA.