Design, synthesis and analysis of charged RGD derivatives.

In the present study, negatively charged N-Biotin-RGD and positively charged C-Biotin-RGD were designed, synthesized, and characterized with docking analysis. The fixation of MDA-MB-231 cells with formalin made their cell surface neutrally charged thus removing the electrostatic interactions between charged biotinylated RGD derivatives and MDA-MB-231 cells. The results of the binding affinity of biotinylated RGD derivatives against MDA-MB-231 cells showed that N-Biotin-RGD had higher binding affinity than C-Biotin-RGD. The cytotoxic effect was analyzed by incubating charged biotinylated RGD derivatives with live MDA-MB-231 cells. MDA-MB-231 cell surface is negatively charged due to high hypersialyliation of polyglycans and Warburg effect. The results of their cytotoxic activities against live MDA-MB-231 cells were found to be electrostatic in nature. C-Biotin-RGD had an attractive interaction with the MDA-MB-231 cell surface resulting in a higher cytotoxic effect. In comparison, N-Biotin-RGD had a repulsive interaction with the MDA-MB-231 cell surface resulting in a lower cytotoxic effect. Hence, positively charged C-Biotin-RGD is a better cytotoxic agent than a negatively charged N-Biotin-RGD against MDA-MB-231 cells.

Design of novel anti-quorum sensing peptides targeting LuxO to combat Vibrio cholerae pathogenesis.

Vibrio cholerae, the Gram-negative bacterium abruptly colonizes the human intestine causing diarrhea, employing quorum sensing (QS) system as the major survival technique for mediating biofilm formation, virulence, competence, etc. Two distinct QS systems coordinated by the auto-inducer molecules, cholera-specific CqsA/S system and universal LuxS/PQ system, operate in parallel converging into a common phosphorelay pathway involving LuxU and LuxO. The master regulatory proteins of the QS system, AphA and HapR regulate the biofilm formation based on cell density, whose expression is controlled by the global response regulator LuxO. At low cell density, activated LuxO indirectly represses HapR, favoring the virulence cascade expression. Rather at high cell densities, LuxO represses AphA expression subsequently blocking the expression of virulence factors. Hence, targeting LuxO would be a promising strategy to downregulate the virulence pathway and modulate the QS system. With this insight, the present study has been designed to intrude the interaction between LuxU and LuxO through in silico design of novel peptides and validation of these peptides through molecular simulations. QS peptides were designed using QSPred server by altering the template sequence representing the LuxU-LuxO interaction, among which PEP8 exhibited better interaction and stability with the target protein LuxO. These in silico designed novel peptides would serve as potential target-specific molecules that would inhibit the LuxU-LuxO interaction and modulate the QS system to restrict Vibrio cholerae pathogenesis. However, further in vitro validations would substantiate the efficacy of these novel QS peptides. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00172-2.

Construing recombinant ZFP160 from Aspergillus terreus as pterin deaminase enzyme.

Pterin deaminase stands as a metalloenzyme and exhibits both antitumor and anticancer activities. Therefore, this study aimed to explore the molecular function of zinc finger protein-160 (zfp160) from Aspergillus terreus with its enzyme mechanism in detail. Subsequently, preliminary molecular docking studies on zfp160 from A. terreus were done. Next, the cloning and expression of zfp160 protein were carried out. Following, protein expression was induced and purified through nickel NTA column with imidazole gradient elution. Through the Mascot search engine tool, the expressed protein of MALDI-TOF was confirmed by 32 kDa bands of SDS-PAGE. Furthermore, its enzymatic characterization and biochemical categorization were also explored. The optimum conditions for enzyme were determined to be pH 8, temperature 35°C, km 50 µm with folic acid as substrate, and Vmax of 24.16 (IU/mL). Further, in silico analysis tried to explore the interactions and binding affinity of various substrates to the modeled pterin deaminase from A. terreus. Our results revealed the binding mode of different substrate molecules with pterin deaminase using the approximate scoring functions that possibly correlate with actual experimental binding affinities. Following this, molecular dynamic simulations provided the in-depth knowledge on deciphering functional mechanisms of pterin deaminase over other drugs.

Virtual screening of FOXO3a activators from natural product-like compound library.

FOXO3a is an inevitable transcription factor, which is involved in the regulation of biological processes such as proliferation, DNA damage repair, cell cycle arrest and cell death. Previous studies confirmed that FOXO3a is an excellent tumor suppressor and in cancer cells, it gets phosphorylated followed by proteasomal degradation. FOXO3a is found to be inactivated in cancer cells, whereas in normal cells it gets activated and upregulates its downstream targets, which induces apoptotic pathways. Hence, activation of FOXO3a can be implicated in cancer prevention and treatment. A variety of commercially available FOXO3a activators such as doxorubicin and metformin possess undesirable adverse effects to normal cells and tissues, which are their major limitations. Natural bioactive compounds, eliminating the limitations of such compounds, become an excellent choice for the treatment and prevention of cancer. In this study, a library of natural product-like compounds was screened for their FOXO3a activation potential through in silico approach, which included the use of several bioinformatics tools and processes. Other molecular interaction studies as well as binding and specificity studies were carried out with the help of molecular dynamics simulation. Virtual screening of 7700 small molecules from the Natural Products-like Compound Library revealed the top three FOXO3a activators F3385-6269, F2183-0033 and F3351-0330. Further validation studies are warranted to confirm these findings.

Potent FOXO3a Activators from Biologically Active Compound Library for Cancer Therapeutics: An in silico Approach.

The forkhead transcription factor FOXO3a is a member of the FOXO subfamily, which controls a number of cellular processes including apoptosis, proliferation, cell cycle progression, DNA damage, and carcinogenesis. In addition, it reacts to a number of biological stressors such as oxidative stress and UV radiation. FOXO3a has been predominantly associated with many diseases including cancer. Recent research suggests that FOXO3a suppresses tumor growth in cancer. By cytoplasmic sequestration of the FOXO3a protein or mutation of the FOXO3a gene, FOXO3a is commonly rendered inactive in cancer cells. Furthermore, the onset and development of cancer are linked to its inactivation. In order to reduce and prevent tumorigenesis, FOXO3a needs to be activated. So, it is critical to develop new strategies to enhance FOXO3a expression for cancer therapy. Hence, the present study has been aimed to screen small molecules targeting FOXO3a using bioinformatics tools. Molecular docking and molecular dynamic simulation studies reveal the potent FOXO3a activating small molecules such as F3385-2463, F0856-0033, and F3139-0724. These top three compounds will be subjected to further wet experiments. The findings of this study will lead us to explore the potent FOXO3a activating small molecules for cancer therapeutics.

Scaffold Hopping and Screening for Potent Small Molecule Agonists for GRP94: Implications to Alleviate ER Stress-Associated Pathogenesis.

Disparity in the activity of Endoplasmic reticulum (ER) leads to degenerative diseases, mainly associated with protein misfolding and aggregation leading to cellular dysfunction and damage, ultimately contributing to ER stress. ER stress activates the complex network of Unfolded Protein Response (UPR) signaling pathways mediated by transmembrane proteins IRE1, ATF6, and PERK. In addition to UPR, many ER chaperones have evolved to optimize the output of properly folded secretory and membrane proteins. Glucose-regulated protein 94 (GRP94), an ER chaperone of heat shock protein HSP90 family, directs protein folding through interaction with other components of the ER protein folding machinery and assists in ER-associated degradation (ERAD). Activation of GRP94 would increase the efficacy of protein folding machinery and regulate the UPR pathway toward homeostasis. The present study aims to screen for novel agonists for GRP94 based on Core hopping, pharmacophore hypothesis, 3D-QSAR, and virtual screening with small-molecule compound libraries in order to improve the efficiency of native protein folding by enhancing GRP94 chaperone activity, therefore to reduce protein misfolding and aggregation. In this study, we have employed the strategy of small molecule-dependent ER programming to enhance the chaperone activity of GRP94 through scaffold hopping-based screening approach to identify specific GRP94 agonists. New scaffolds generated by altering the cores of NECA, the known GRP94 agonist, were validated by employing pharmacophore hypothesis testing, 3D-QSAR modeling, and molecular dynamics simulations. This facilitated the identification of small molecules to improve the efficiency of native protein folding by enhancing GRP94 activity. High-throughput virtual screening of the selected pharmacophore hypothesis against Selleckchem and ZINC databases retrieved a total of 2,27,081 compounds. Further analysis on docking and ADMET properties revealed Epimedin A, Narcissoside, Eriocitrin 1,2,3,4,6-O-Pentagalloylglucose, Secoisolariciresinol diglucoside, ZINC92952357, ZINC67650204, and ZINC72457930 as potential lead molecules. The stability and interaction of these small molecules were far better than the known agonist, NECA indicating their efficacy in selectively alleviating ER stress-associated pathogenesis. These results substantiate the fact that small molecule-dependent ER reprogramming would activate the ER chaperones and therefore reduce the protein misfolding as well as aggregation associated with ER stress in order to restore cellular homeostasis.

Inhibition of mecA and blaCTX-M from MRSA and ESBL strains of diabetic foot infection by screening antibiotics compound library: an in silico analysis.

A computational approach was exploited towards new molecule designing to target the inhibition of resistant genes mecA and blaCTX-M in MRSA and ESBL strains cultured from diabetic foot infected patients. The bioinformatic analysis involves the prediction of protein structures for mecA and blaCTX-M employing the Prime module of Schrodinger. The interactions were examined with the control antibiotics using the modelled protein structures, which revealed that Cefixime and Amikacin showed the highest binding affinity with mecA and blaCTX-M, respectively. According to the predictions of pharmacophores, the ADHRN hypothesis for mecA protein and the ADHR hypothesis for blaCTX-M protein were obtained. Subsequently, the antibiotic compound library from Selleckchem was retrieved, and molecular interactions studies were carried out to explore the interaction profiling of mecA with Tobramycin and blaCTX-M with Acyclovir. Further, the stability of protein-ligand interactions was validated through molecular dynamics simulations. Overall, this study suggests that the predicted pharmacophore model provides in-depth knowledge for repurposing an antibiotic drug with effective inhibition to enhance its therapeutic activity in the currently used ones.Communicated by Ramaswamy H. Sarma.

Pharmacological assessment of Ru(II) complex with GidA protein- A novel topoisomerase II inhibitor towards cancer therapeutics.

The interactions of ruthenium(II) complex with Glucose inhibited division protein A (GidA protein) was studied through various spectroscopic techniques with the ultimate goal of preparing adducts with good selectivity for cancer cells. In all the cases, formation of a tight metal-protein conjugate was observed. The influence of pH, reducing agents and chelators on the formation of adduct was analysed by UV- visible spectroscopy. While there was no effect on the addition of sodium ascorbate, some alterations on some selected bands were seen on the UV-visible spectra on the addition of EDTA. The adduct was stable in the pH range of 5-8. Addition of ruthenium(II) complex effectively quenched the intrinsic fluorescence of GidA and it occurred through static quenching. The effect of ruthenium(II) complex on the conformation of GidA has been examined by analyzing CD spectrum. Though, there was some conformational changes observed in the presence of ruthenium(II) complex, α- helix in the secondary structure of GidA retained its identity. Molecular docking of ruthenium(II) complex with GidA also indicated that GidA docks through hydrophobic interaction. The stable semisynthetic complex (ruthenium(II) complex with GidA) was checked for topoisomerase II inhibition. Relaxation and decatenation assay proved topoisomerase II inhibition of semisynthetic complex.Communicated by Ramaswamy H. Sarma.

Pharmacophore based virtual screening, molecular docking and molecular dynamic simulation studies for finding ROS1 kinase inhibitors as potential drug molecules.

Proto-oncogene receptor tyrosine kinase ROS-1 is one of the clinically important biomarker and plays a crucial role in regulation of a number of cellular functions including cell proliferation, migration and angiogenesis. Recently, inhibition of ROS1 kinase has proven to be a promising target of anticancer drugs for non-small cell lung cancer (NSCLC). The very few compounds have been used as potent drug molecules so far and the selective ROS1 inhibitors are relatively rare. Besides the currently available drugs such as Crizotinib and PF-06463922 are becoming sensitive due to mutations in the ROS1 protein. To curtail the problem of the resistant, present study was designed to identify the potent inhibitors against ROS1. Three different screening approaches such as structure based, Atom-based and pharmacophore based screening were carried out against commercially available databases and the retrieved best hits were further evaluated by Lipinski's filter. Thereafter the lead molecule was subjected to pocket specific docking with ROS1. The results show that, total of 9 molecules (3 from each screening) has good docking score (with range of -9.288 to -12.49 Kcal/Mol) and binding interactions within the active site of ROS1. In order to analyze the stability of the ligand- protein complexes, molecular dynamics simulation was performed. Thus, these identified potential lead molecules with good binding score and binding affinity with ROS1 may act as the potent ROS1 inhibitor, and that are worth considering for further experimental studies.Communicated by Ramaswamy H. Sarma.

Macaque trophoblast migration toward RANTES is inhibited by cigarette smoke-conditioned medium.

Trophoblast migration within the endometrium and uterine vasculature is essential for normal placental and fetal development. We previously demonstrated that macaque trophoblasts express the chemokine receptor CCR5 and that this receptor mediates trophoblast migration toward RANTES (regulated upon activation normal T-cell expressed and secreted). In the present paper we have used primary cultures of early gestation macaque trophoblasts to test the hypothesis that tobacco smoke inhibits trophoblast migration as the result of dysregulation of the RANTES/CCR5 chemotactic axis. Early gestation macaque trophoblasts were incubated in the absence or presence of cigarette smoke-conditioned medium (CSM). Cell migration was quantified using migration chambers. CCR5 and G protein receptor kinase 2 (GRK2) expression was measured by immunofluorescence microscopy and Western blotting. cAMP levels were measured by enzyme-linked immunosorbent assay. Trophoblast migration toward RANTES was reduced when cells were incubated in CSM. Trophoblasts also showed reduced expression of CCR5, increased levels of cAMP, and increased expression of GRK2. Finally, the secretion of RANTES by uterine endothelial cells was reduced by exposing the cells to CSM. These results support the idea that cigarette smoke constituents inhibit directional trophoblast migration by causing increased desensitization of trophoblast CCR5 and inhibiting the secretion of RANTES by endothelial cells.

Macaque trophoblast migration is regulated by RANTES.

In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade the endometrium, enter the uterine vasculature, and migrate within the arteries. The mechanisms that regulate this directional migration are unknown. We have used early gestation macaque trophoblasts to test the hypothesis that trophoblast migration is regulated by the chemokine, Regulated on Activation T-Cell Expressed and Secreted (RANTES). Immunohistochemical analysis of cryosections of endometrial tissue showed expression of RANTES by stromal cells and vascular cells. Isolated endothelial cells expressed RANTES as determined by immunocytochemistry and RT-PCR analyses. Immunohistochemical analysis of endometrial cryosections showed that the RANTES receptor, CCR5, was expressed by trophoblasts on anchoring villi and by cells within the trophoblastic shell. Cytokeratin-positive/CCR5-positive cells, consistent with trophoblasts, were also found scattered within the stroma and were often clustered around blood vessels. Isolated trophoblast cells expressed CCR5 as determined by immunocytochemistry and RT-PCR analyses. Isolated trophoblasts migrated towards RANTES when cultured in migration chambers and migration was reduced in the presence of anti-CCR5 antibody. When trophoblasts were cultured on dishes coated with recombinant RANTES, expression of beta1 integrin was increased. The RANTES-induced increase in beta1 integrin expression was inhibited by pertussis toxin. These data suggest a role for RANTES and CCR5 in the regulation of trophoblast migration within the endometrium and within the uterine vasculature.