Chemokine Receptors-Structure-Based Virtual Screening Assisted by Machine Learning.

Chemokines modulate the immune response by regulating the migration of immune cells. They are also known to participate in such processes as cell-cell adhesion, allograft rejection, and angiogenesis. Chemokines interact with two different subfamilies of G protein-coupled receptors: conventional chemokine receptors and atypical chemokine receptors. Here, we focused on the former one which has been linked to many inflammatory diseases, including: multiple sclerosis, asthma, nephritis, and rheumatoid arthritis. Available crystal and cryo-EM structures and homology models of six chemokine receptors (CCR1 to CCR6) were described and tested in terms of their usefulness in structure-based drug design. As a result of structure-based virtual screening for CCR2 and CCR3, several new active compounds were proposed. Known inhibitors of CCR1 to CCR6, acquired from ChEMBL, were used as training sets for two machine learning algorithms in ligand-based drug design. Performance of LightGBM was compared with a sequential Keras/TensorFlow model of neural network for these diverse datasets. A combination of structure-based virtual screening with machine learning allowed to propose several active ligands for CCR2 and CCR3 with two distinct compounds predicted as CCR3 actives by all three tested methods: Glide, Keras/TensorFlow NN, and LightGBM. In addition, the performance of these three methods in the prediction of the CCR2/CCR3 receptor subtype selectivity was assessed.

Accessing the In Vivo Efficiency of Clinically Isolated Phages against Uropathogenic and Invasive Biofilm-Forming Escherichia coli Strains for Phage Therapy.

Escherichia coli is one of the most common members of the intestinal microbiota. Many of its strains are associated with various inflammatory infections, including urinary or gut infections, especially when displaying antibiotic resistance or in patients with suppressed immune systems. According to recent reports, the biofilm-forming potential of E. coli is a crucial factor for its increased resistance against antibiotics. To overcome the limitations of using antibiotics against resistant E. coli strains, the world is turning once more towards bacteriophage therapy, which is becoming a promising candidate amongst the current personalized approaches to target different bacterial infections. Although matured and persistent biofilms pose a serious challenge to phage therapy, they can still become an effective alternative to antibiotic treatment. Here, we assess the efficiency of clinically isolated phages in phage therapy against representative clinical uropathogenic and invasive biofilm-forming E. coli strains. Our results demonstrate that irrespective of host specificity, bacteriophages producing clear plaques with a high burst size, and exhibiting depolymerizing activity, are good candidates against biofilm-producing E. coli pathogens as verified from our in vitro and in vivo experiments using Galleria mellonella where survival was significantly increased for phage-therapy-treated larvae.

Bacterial RNA virus MS2 exposure increases the expression of cancer progression genes in the LNCaP prostate cancer cell line.

Bacteriophages effectively counteract diverse bacterial infections, and their ability to treat most types of cancer has been explored using phage engineering or phage-virus hybrid platforms. In the present study, it was demonstrated that the bacteriophage MS2 can affect the expression of genes associated with the proliferation and survival of LNCaP prostate epithelial cells. LNCaP cells were exposed to bacteriophage MS2 at a concentration of 1×107 plaque forming units/ml for 24-48 h. After exposure, various cellular parameters, including cell viability, morphology, and changes in gene expression, were examined. MS2 affected cell viability adversely, reducing viability by 25% in the first 4 h of treatment; however, cell viability recovered within 24-48 h. Similarly, the AKT, androgen receptor, integrin α5, integrin ß1, MAPK1, MAPK3, STAT3, and peroxisome proliferator-activated receptor-γ coactivator 1α genes, which are involved in various normal cellular processes and tumor progression, were significantly upregulated, whereas the expression levels of HSP90, ITGB5, ITGB3, HSP27, ITGAV, and PI3K genes were unchanged. Therefore, based on viability and gene expression changes, bacteriophage MS2 severely impaired LNCaP cells by reducing anchorage-dependent survival and androgen signaling. A caveolin-mediated endocytosis mechanism for MS2-mediated signaling in prostate cancer cells was proposed based on reports involving bacteriophages T4, M13, and MS2, and their interactions with LNCaP and PC3 cell lines.

Computational and experimental approaches to probe GPCR activation and signaling.

G protein-coupled receptors (GPCRs) regulate different physiological functions, e.g., sensation, growth, digestion, reproductivity, nervous and immune systems response, and many others. In eukaryotes, they are also responsible for intercellular communication in response to pathogens. The major primary messengers binding to these cell-surface receptors constitute small-molecule or peptide hormones and neurotransmitters, nucleotides, lipids as well as small proteins. The simplicity of the way how GPCR signaling can be regulated by their endogenous agonists prompted the usage of GPCRs as major drug targets in modern pharmacology. Drugs targeting GPCRs inhibit pathological processes at the very beginning. This enables to significantly reduce the occurrence of morphological changes caused by diseases. Until recently, X-ray crystallography was the method of the first choice to obtain high-resolution structural information about GPCRs. Following X-ray crystallography, cryo-EM gained attention in GPCR studies as a quick and low-cost alternative. FRET microscopy is also widely used for GPCRs in the analysis of protein-protein interactions (PPIs) in intact cells as well as for screening purposes. Regarding computational methods, molecular dynamics (MD) for many years has proven its usefulness in studying the GPCR activation. MODELLER and Rosetta were widely used to generate preliminary homology models of GPCRs for MD simulation systems. Apart from the conventional all-atom approach with explicitly defined solvent, also other techniques have been applied to GPCRs, e.g., MARTINI or hybrid methods involving the coarse-grained representation, less demanding regarding computational resources, and thus offering much larger simulation timescales.

Bacteriophages M13 and T4 Increase the Expression of Anchorage-Dependent Survival Pathway Genes and Down Regulate Androgen Receptor Expression in LNCaP Prostate Cell Line.

Wild-type or engineered bacteriophages have been reported as therapeutic agents in the treatment of several types of diseases, including cancer. They might be used either as naked phages or as carriers of antitumor molecules. Here, we evaluate the role of bacteriophages M13 and T4 in modulating the expression of genes related to cell adhesion, growth, and survival in the androgen-responsive LNCaP prostatic adenocarcinoma-derived epithelial cell line. LNCaP cells were exposed to either bacteriophage M13 or T4 at a concentration of 1 × 105 pfu/mL, 1 × 106 pfu/mL, and 1 × 107 pfu/mL for 24, 48, and 72 h. After exposure, cells were processed for general morphology, cell viability assay, and gene expression analyses. Neither M13 nor T4 exposure altered cellular morphology, but both decreased the MTT reduction capacity of LNCaP cells at different times of treatment. In addition, genes AKT, ITGA5, ITGB1, ITGB3, ITGB5, MAPK3, and PI3K were significantly up-regulated, whilst the genes AR, HSPB1, ITGAV, and PGC1A were down-regulated. Our results show that bacteriophage M13 and T4 interact with LNCaP cells and effectively promote gene expression changes related to anchorage-dependent survival and androgen signaling. In conclusion, phage therapy may increase the response of PCa treatment with PI3K/AKT pathway inhibitors.

Exposure to Bacteriophages T4 and M13 Increases Integrin Gene Expression and Impairs Migration of Human PC-3 Prostate Cancer Cells.

The interaction between bacteriophages and integrins has been reported in different cancer cell lines, and efforts have been undertaken to understand these interactions in tumor cells along with their possible role in gene alterations, with the aim to develop new cancer therapies. Here, we report that the non-specific interaction of T4 and M13 bacteriophages with human PC-3 cells results in differential migration and varied expression of different integrins. PC-3 tumor cells (at 70% confluence) were exposed to 1 × 107 pfu/mL of either lytic T4 bacteriophage or filamentous M13 bacteriophage. After 24 h of exposure, cells were processed for a histochemical analysis, wound-healing migration assay, and gene expression profile using quantitative real-time PCR (qPCR). qPCR was performed to analyze the expression profiles of integrins ITGAV, ITGA5, ITGB1, ITGB3, and ITGB5. Our findings revealed that PC-3 cells interacted with T4 and M13 bacteriophages, with significant upregulation of ITGAV, ITGA5, ITGB3, ITGB5 genes after phage exposure. PC-3 cells also exhibited reduced migration activity when exposed to either T4 or M13 phages. These results suggest that wildtype bacteriophages interact non-specifically with PC-3 cells, thereby modulating the expression of integrin genes and affecting cell migration. Therefore, bacteriophages have future potential applications in anticancer therapies.

Development of pipette tip gap closure migration assay (s-ARU method) for studying semi-adherent cell lines.

This work presents a pipette tip gap closure migration assay prototype tool (semi-adherent relative upsurge-s-ARU-method) to study cell migration or wound healing in semi-adherent cell lines, such as lymph node carcinoma of the prostate (LNCaP). Basically, it consists of a 6-well cover plate modification, where pipette tips with the filter are shortened and fixed vertically to the inner surface of the cover plate, with their heights adjusted to touch the bottom of the well center. This provides a barrier for the inoculated cells to grow on, creating a cell-free gap. Such a uniform gap formed can be used to study migration assay for both adherent as well as semi-adherent cells. After performing time studies, effective measurement of gap area can be carried out conveniently through image analysis software. Here, the prototype was tested for LNCaP cells, treated with testosterone and flutamide as well as with bacteriophages T4 and M13. A scratch assay using PC3 adherent cells was also performed for comparison. It was observed that s-ARU method is suitable for studying LNCaP cells migration assay, as observed from our results with testosterone, flutamide, and bacteriophages (T4 and M13). Our method is a low-cost handmade prototype, which can be an alternative to the other migration assay protocol(s) for both adherent and semi-adherent cell cultures in oncological research along with other biological research applications.