Deciphering anti-biofilm property of Arthrospira platensis-origin peptides against Staphylococcusaureus.

Arthrospira platensis is a valuable natural health supplement consisting of various types of vitamins, dietary minerals, and antioxidants. Although different studies have been conducted to explore the hidden benefits of this bacterium, its antimicrobial property has been poorly understood. To decipher this important feature, here, we extended our recently introduced optimization algorithm (Trader) for aligning amino acid sequences associated with the antimicrobial peptides (AMPs) of Staphylococcus aureus and A.platensis. As a result, similar amino acid sequences were identified, and several candidate peptides were generated accordingly. The obtained peptides were then filtered based on their potential biochemical and biophysical properties, and their 3D structures were simulated based on homology modeling techniques. Next, to investigate how the generated peptides can interact with S. aureus proteins (i.e., heptameric state of the hly and homodimeric form of the arsB), molecular docking approaches were used. The results indicated that four peptides included better molecular interactions relative to the other generated ones in terms of the number/average length of hydrogen bonds and hydrophobic interactions. Based on the outcomes, it can be concluded that the antimicrobial property of A.platensis might be associated with its capability in disturbing the membrane of pathogens and their functions.

Recombinant production and characterization of L-glutaminase (glsA) as a promiscuity therapeutic enzyme.

Because of the therapeutical impacts of hydrolytic enzymes in different diseases, in particular malignancies, we aimed to produce a recombinant putative L-glutaminase (GLS ASL-1) from a recently characterized halo-thermotolerant Bacillus sp. SL-1. For this purpose, the glsA gene was identified and efficiently overexpressed in the Origami™ B (DE3) strain. The yield of the purified GLS ASL-1 was ~ 20 mg/L, indicating a significant expression of recombinant enzyme in the Origami. The enzyme activity assay revealed a significant hydrolytic effect of the recombinant GLS ASL-1 on L-asparagine (Asn) (i.e., Km 39.8 µM, kcat 19.9 S-1) with a minimal affinity for L-glutamine (Gln). The GLS ASL-1 significantly suppressed the growth of leukemic Jurkat cells through apoptosis induction (47.5%) in the IC50 dosage of the enzyme. The GLS ASL-1 could also change the Bax/Bcl2 expression ratio, indicating its apoptotic effect on cancer cells. The in silico analysis was conducted to predict structural features related to the histidine-tag exposure in the N- or C-terminal of the recombinant GLS ASL-1. In addition, molecular docking simulation for substrate specificity revealed a greater binding affinity of Asn to the enzyme binding-site residues than Gln, which was confirmed in experimental procedures as well. In conclusion, the current study introduced a recombinant GLS ASL-1 with unique functional and structural features, highlighting its potential pharmaceutical and medical importance. GLS ASL-1 represents the first annotated enzyme from Bacillus with prominent asparaginase activity, which can be considered for developing alternative enzymes in therapeutic applications. KEY POINTS: • Hydrolytic enzymes have critical applications in different types of human malignancies. • A recombinant L-glutaminase (GLS ASL-1) was produced from halo-thermotolerant Bacillus sp. SL-1. • GLS ASL-1 displayed a marked hydrolytic activity on L-asparagine compared to the L-glutamine. • GLS ASL-1 with significant substrate promiscuity may be an alternative for developing novel pharmaceuticals.

Pharmacoinformatics-based phytochemical screening for anticancer impacts of yellow sweet clover, Melilotus officinalis (Linn.) Pall.

To date, much attention has been paid to phytochemicals because of their diverse pharmacological effects on a variety of diseases such as cancer. In this regard, computer-aided drug design, as a cost- and time-effective approach, is primarily applied to investigate the drug candidates before their further costly in vitro and in vivo experimental evaluations. Accordingly, different signaling pathways and proteins can be targeted using such strategies. As a key protein for the initiation of eukaryotic DNA replication, mini-chromosome maintenance complex component 7 (MCM7) overexpression is related to the initiation and progression of aggressive malignancies. The current study was conducted to identify new potential natural compounds from the yellow sweet clover, Melilotus officinalis (Linn.) Pall, by examining the potential of 40 isolated phytochemicals against MCM7 protein. A structure-based pharmacophore model to the protein active site cavity was generated and followed by virtual screening and molecular docking. Overall, four compounds were selected for further evaluation based on their binding affinities. Our analyses revealed that two novel compounds, namely rosmarinic acid (PubChem CID:5281792) and melilotigenin (PubChem CID:14059499) might be druggable and offer safe usage in human. The stability of these two protein-ligand complex structures was confirmed through molecular dynamics simulation. The findings of this study reveal the potential of these two phytochemicals to serve as anticancer agents, while further pharmacological experiments are required to confirm their effectiveness against human cancers.

A novel multi-objective metaheuristic algorithm for protein-peptide docking and benchmarking on the LEADS-PEP dataset.

Protein-peptide interactions have attracted the attention of many drug discovery scientists due to their possible druggability features on most key biological activities such as regulating disease-related signaling pathways and enhancing the immune system's responses. Different studies have utilized some protein-peptide-specific docking algorithms/methods to predict protein-peptide interactions. However, the existing algorithms/methods suffer from two serious limitations which make them unsuitable for protein-peptide docking problems. First, it seems that the prevalent approaches require to be modified and remodeled for weighting the unbounded forces between a protein and a peptide. Second, they do not employ state-of-the-art search algorithms for detecting the 3D pose of a peptide relative to a protein. To address these restrictions, the present study aims to introduce a novel multi-objective algorithm, which first generates some potential 3D poses of a peptide, and then, improves them through its operators. The candidate solutions are further evaluated using Multi-Objective Pareto Front (MOPF) optimization concepts. To this end, van der Waals, electrostatic, solvation, and hydrogen bond energies between the atoms of a protein and designated peptide are computed. To evaluate the algorithm, it is first applied to the LEADS-PEP dataset containing 53 protein-peptide complexes with up to 53 rotatable branches/bonds and then compared with three popular/efficient algorithms. The obtained results indicate that the MOPF-based approaches which reduce the backbone RMSD between the original and predicted states, achieve significantly better results in terms of the success rate in predicting the near-native conditions. Besides, a comparison between the different types of search algorithms reveals that efficient ones like the multi-objective Trader/differential evolution algorithm can predict protein-peptide interactions better than the popular algorithms such as the multi-objective genetic/particle swarm optimization algorithms.

Vaccination with rEGVac elicits immunoprotection against different stages of Echinococcus granulosus life cycle: A pilot study.

Vaccination against dog-sheep transmission cycle is necessary to control cystic echinococcosis (CE) infection. A multi-epitope multi-antigenic recombinant vaccine was developed-comprising the three putative vaccine antigens EG95, Eg14-3-3 and EgEnolase-was cloned and expressed. In a pilot experiment, the multi-antigen vaccine was assessed in 15 dogs and 15 sheep (five experimental groups and three animals in each group) by two subcutaneous doses 28 days apart. To evaluate the efficacy of the vaccine candidate first immunological analysis were done comprising IgG and IgE antibodies and the cytokine IL-4 in sera of the immunized dogs and sheep. Serum IgG, IgE, and IL-4, in particular in the dogs, were increased after the two rounds of vaccine candidate injection, while the total number of hydatid cysts was reduced (~85.43%). This pilot trial indicated significant immune protection efficacy against E. granulosus especially in dogs, while its efficacy in sheep was not as high as dogs. The multi-antigenic candidate vaccine is proposed as a protective vaccine modality in both dogs and sheep.

Nanobody-based therapeutics against colorectal cancer: Precision therapies based on the personal mutanome profile and tumor neoantigens.

Monoclonal antibodies and vaccines have widely been studied for the immunotherapy of cancer, while their large size appears to limit their functionality in solid tumors, in large part due to unique properties of tumor microenvironment such as high pressure of tumor interstitial fluid. To tackle such limitations, smaller formats of antibodies have been developed, including antigen-binding fragments, single-chain variable fragments, single variable domain of camelid antibody (so-called nanobody (Nb) or VHH). Of these, Nbs offer great immunotherapy potentials because of their advantageous physicochemical and pharmacological features, including small size, high stability, and excellent tissue penetration. Besides, the therapeutic impacts of Nbs can be improved by their modifications and functionalizations (e.g., PEGylation and conjugation to the Fc domain, peptide tags, drugs, toxins, aptamers, and radionuclides). This review aims to provide comprehensive insights into key signaling networks of colorectal cancer and discuss Nb-based precision immunotherapy of colorectal cancer.

Functional expression and impact of testis-specific gene antigen 10 in breast cancer: a combined in vitro and in silico analysis.

Introduction: Testis-specific gene antigen 10 (TSGA10) is a less-known gene, which is involved in the vague biological paths of different cancers. Here, we investigated the TSGA10 expression using different concentrations of glucose under hypoxia and also its interaction with the hypoxia-inducible factor 1 (HIF-1). Methods: The breast cancer MDA-MB-231 and MCF-7 cells were cultured with different concentrations of glucose (5.5, 11.0 and 25.0 mM) under normoxia/hypoxia for 24, 48, and 72 hours and examined for the HIF-1α expression and cell migration by Western blotting and scratch assays. The qPCR was employed to analyze the expression of TSGA10. Three-dimensional (3D) structure and the energy minimization of the interacting domain of TSGA10 were performed by MODELLER v9.17 and Swiss-PDB viewer v4.1.0/UCSF Chimera v1.11. The UCSF Chimera v1.13.1 and Hex 6.0 were used for the molecular docking simulation. The Cytoscape v3.7.1 and STRING v11.0 were used for protein-protein interaction (PPI) network analysis. The HIF-1a related hypoxia pathways were obtained from BioModels database and reconstructed in CellDesigner v4.4.2. Results: The increased expression of TSGA10 was found to be significantly associated with the reduced metastasis in the MDA-MB-231 cells, while an inverse relationship was seen between the TSGA10 mRNA level and cellular migration but not in the MCF-7 cells. The C-terminal domain of TSGA10 interacted with HIF-1α with high affinity, resulting in PPI network with 10 key nodes (HIF-1α, VEGFA, HSP90AA1, AKT1, ARNT, TP53, TSGA10, VHL, JUN, and EGFR). Conclusions: Collectively, TSGA10 functional expression alters under the hyper-/hypo-glycemia and hypoxia, which indicates its importance as a candidate bio-target for the cancer therapy.

Peptide-mediated drug delivery across the blood-brain barrier for targeting brain tumors.

INTRODUCTION: Transportation of the nutrients and other substances from the blood to the brain is selectively controlled by the brain capillary endothelial cells that form a restrictive barrier, so-called blood-brain barrier (BBB). Currently, there is no unimpeachable approach to overcome the BBB obstructiveness because the existing options are either invasive or ineffective. AREAS COVERED: This review delineates the biological impacts of BBB on brain drug delivery and targeting. The nanoscaled multifunctional shuttles armed with the targeting entities (e.g., antibodies and peptides) are discussed. Important insights are remarked into the combinatorial screening methodologies used for the identification of de novo peptides capable of crossing BBB and targeting the brain. EXPERT OPINION: Depending on the physicochemical properties of small molecules and macromolecules, they may cross the BBB and get into the brain either through passive diffusion or active/facilitated transportation and transcytosis in a very selectively controlled manner. Phage-derived shuttle peptides can specifically be selected against BBB endocytic machinery and used in engineering novel peptide-drug conjugates (PDCs). Nanoscaled multitargeting delivery systems encompassing PDCs can overcome the BBB obstructiveness and deliver drugs specifically to diseased cells in the brain with trivial side effects.

In silico design of a triple-negative breast cancer vaccine by targeting cancer testis antigens.

Introduction: Triple-negative breast cancer (TNBC) is an important subtype of breast cancer, which occurs in the absence of estrogen, progesterone and HER-2 receptors. According to the recent studies, TNBC may be a cancer testis antigen (CTA)-positive tumor, indicating that the CTA-based cancer vaccine can be a treatment option for the patients bearing such tumors. Of these antigens (Ags), the MAGE-A family and NY-ESO-1 as the most immunogenic CTAs are the potentially relevant targets for the development of an immunotherapeutic way of the breast cancer treatment. Methods: In the present study, immunoinformatics approach was used to design a multi-epitope peptide vaccine to combat the TNBC. The vaccine peptide was constructed by the fusion of three crucial components, including the CD8+ cytotoxic T lymphocytes (CTLs) epitopes, helper epitopes and adjuvant. The epitopes were predicted from the MAGE-A and NY-ESO-1 Ags. In addition, the granulocyte-macrophage-colony-stimulating factor (GM-CSF) was used as an adjuvant to promote the CD4+ T cells towards the T-helper for more strong induction of CTL responses. The components were conjugated by proper linkers. Results: The vaccine peptide was examined for different physiochemical characteristics to confirm the safety and immunogenic behavior. Furthermore, the 3D-structure of the vaccine peptide was predicted based on the homology modeling approach using the MODELLER v9.17 program. The vaccine structure was also subjected to the molecular dynamics simulation study for structure refinement. The results verified the immunogenicity and safety profile of the constructed vaccine as well as its capability for stimulating both the cellular and humoral immune responses. Conclusion: Based on our in-silico analyses, the proposed vaccine may be considered for the immunotherapy of TNBC.

Phage antibody library screening for the selection of novel high-affinity human single-chain variable fragment against gastrin receptor: an in silico and in vitro study.

BACKGROUND: As a membrane G protein coupled receptors (GPCRs) family, gastrin/cholecystokinin-2 receptor (CCK2R) plays a key role in the initiation and development of gastric cancer. OBJECTIVES: Targeting CCK2R by immunotherapeutics such as single-chain variable fragments (scFvs) may provide an effective treatment modality against gastric cancer. Thus, the main objective of this study was to isolate scFvs specific to CCK2R. METHODS: To isolate scFvs specific to the CCK2R, we capitalized on a semi-synthetic diverse phage antibody library (PAL) and a solution-phase biopanning process. The library was panned against a biotinylated peptide of the second extracellular loop (ECL2) of CCK2R. After four rounds of biopanning, the selected soluble scFv clones were screened by enzyme-linked immunosorbent assay (ELISA) and examined for specific binding to the peptide. The selected scFvs were purified using immobilized metal affinity chromatography (IMAC). The binding affinity and specificity of the scFvs were examined by the surface plasmon resonance (SPR), immunoblotting and flow cytometry assays and molecular docking using ZDOCK v3.0.2. RESULTS: Ten different scFvs were isolated, which displayed binding affinity ranging from 0.68 to 8.0 (nM). Immunoblotting and molecular docking analysis revealed that eight scFvs were able to detect the denatured form of CCK2R protein. Of the isolated scFvs, two scFvs showed high-binding affinity to the human gastric adenocarcinoma AGS cells. CONCLUSIONS: Based on our findings, a couple of the selected scFvs showed markedly high-binding affinity to immobilized CCK2R peptide and CCK2R-overexpressing AGS cells. Therefore, these scFvs are proposed to serve as targeting and/or treatment agents in the diagnosis and immunotherapy of CCK2R-positive tumors. Graphical abstract ᅟ.

A novel in silico minigene vaccine based on CD4+ T-helper and B-cell epitopes of EG95 isolates for vaccination against cystic echinococcosis.

EG95 oncospheral antigen plays a crucial role in Echinococcus granulosus pathogenicity. Considering the diversity of antigen among different EG95 isolates, it seems to be an ideal antigen for designing a universal multivalent minigene vaccine, so-called multi-epitope vaccine. This is the first in silico study to design a construct for the development of global EG95-based hydatid vaccine against E. granulosus in intermediate hosts. After antigen sequence selection, the three-dimensional structure of EG95 was modeled and multilaterally validated. The preliminary parameters for B-cell epitope prediction were implemented such as the possible transmembrane helix, signal peptide, post-translational modifications and allergenicity. The high ranked linear and conformational B-cell epitopes derived from several online web-servers (e.g., ElliPro, BepiPred v1.0, BcePred, ABCpred, SVMTrip, IEDB algorithms, SEPPA v2.0 and Discotope v2.0) were utilized for multiple sequence alignment and then for engineering the vaccine construct. T-helper based epitopes were predicted by molecular docking between the high frequent ovar class II allele (Ovar-DRB1*1202) and hexadecamer fragments of the EG95 protein. Having used the immune-informatics tools, we formulated the first EG95-based minigene vaccine based on T-helper epitope with high-binding affinity to the ovar MHC allele. This designed construct was analyzed for different physicochemical properties. It was also codon-optimized for high-level expression in Escherichia coli k12. Taken all, we propose the present in silico vaccine constructs as a promising platform for the generation of broadly protective vaccines for species and genus-specific immunization of the natural hosts of the parasite.

Molecular machineries of pH dysregulation in tumor microenvironment: potential targets for cancer therapy.

Introduction: Cancer is an intricate disorder/dysfunction of cells that can be defined as a genetic heterogeneity in human disease. Therefore, it is characterized by several adaptive complex hallmarks. Among them, the pH dysregulation appears as a symbol of aberrant functions within the tumor microenvironment (TME). In comparison with normal tissues, in the solid tumors, we face with an irregular acidification and alkalinization of the extracellular and intracellular fluids. Methods: In this study, we comprehensively discussed the most recent reports on the hallmarks of solid tumors to provide deep insights upon the molecular machineries involved in the pH dysregulation of solid tumors and their impacts on the initiation and progression of cancer. Results: The dysregulation of pH in solid tumors is fundamentally related to the Warburg effect and hypoxia, leading to expression of a number of molecular machineries, including: NHE1, H+ pump V-ATPase, CA-9, CA-12, MCT-1, GLUT-1. Activation of proton exchangers and transporters (PETs) gives rise to formation of TME. This condition favors the cancer cells to evade from the anoikis and apoptosis, granting them aggressive and metastasis phenotype, as well as resistance to chemotherapy and radiation therapy. This review aimed to discuss the key molecular changes of tumor cells in terms of bio-energetics and cancer metabolism in relation with pH dysregulation. During this phenomenon, the intra- and extracellular metabolites are altered and/or disrupted. Such molecular alterations provide molecular hallmarks for direct targeting of the PETs by potent relevant inhibitors in combination with conventional cancer therapies as ultimate therapy against solid tumors. Conclusion: Taken all, along with other treatment strategies, targeting the key molecular machineries related to intra- and extracellular metabolisms within the TME is proposed as a novel strategy to inhibit or block PETs that are involved in the pH dysregulation of solid tumors.