Bromodomain inhibitor i-BET858 triggers a unique transcriptional response coupled to enhanced DNA damage, cell cycle arrest and apoptosis in high-grade ovarian carcinoma cells.

BACKGROUND: Ovarian cancer has a specific unmet clinical need, with a persistently poor 5-year survival rate observed in women with advanced stage disease warranting continued efforts to develop new treatment options. The amplification of BRD4 in a significant subset of high-grade serous ovarian carcinomas (HGSC) has led to the development of BET inhibitors (BETi) as promising antitumour agents that have subsequently been evaluated in phase I/II clinical trials. Here, we describe the molecular effects and ex vivo preclinical activities of i-BET858, a bivalent pan-BET inhibitor with proven in vivo BRD inhibitory activity. RESULTS: i-BET858 demonstrates enhanced cytotoxic activity compared with earlier generation BETis both in cell lines and primary cells derived from clinical samples of HGSC. At molecular level, i-BET858 triggered a bipartite transcriptional response, comprised of a 'core' network of genes commonly associated with BET inhibition in solid tumours, together with a unique i-BET858 gene signature. Mechanistically, i-BET858 elicited enhanced DNA damage, cell cycle arrest and apoptotic cell death compared to its predecessor i-BET151. CONCLUSIONS: Overall, our ex vivo and in vitro studies indicate that i-BET858 represents an optimal candidate to pursue further clinical validation for the treatment of HGSC.

Alginate oligosaccharides enhance the antifungal activity of nystatin against candidal biofilms.

Background: The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required. Methods: The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. auris, C. tropicalis and C. dubliniensis), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM). Results: MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces (p < 0.001), with increased cell death (p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment (p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control (p < 0.001). Conclusions: Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents.

Highly glycosylated MUC1 mediates high affinity L-selectin binding at the human endometrial surface.

BACKGROUND: Sialyl-Lewis X/L-selectin high affinity binding interactions between transmembrane O-glycosylated mucins proteins and the embryo have been implicated in implantation processes within the human reproductive system. However, the adhesive properties of these mucins at the endometrial cell surface are difficult to resolve due to known discrepancies between in vivo models and the human reproductive system and a lack of sensitivity in current in vitro models. To overcome these limitations, an in vitro model of the human endometrial epithelial was interrogated with single molecule force spectroscopy (SMFS) to delineate the molecular configurations of mucin proteins that mediate the high affinity L-selectin binding required for human embryo implantation. RESULTS: This study reveals that MUC1 contributes to both the intrinsic and extrinsic adhesive properties of the HEC-1 cellular surface. High expression of MUC1 on the cell surface led to a significantly increased intrinsic adhesion force (148 pN vs. 271 pN, p < 0.001), whereas this adhesion force was significantly reduced (271 pN vs. 118 pN, p < 0.001) following siRNA mediated MUC1 ablation. Whilst high expression of MUC1 displaying elevated glycosylation led to strong extrinsic (> 400 pN) L-selectin binding at the cell surface, low expression of MUC1 with reduced glycosylation resulted in significantly less (≤200 pN) binding events. CONCLUSIONS: An optimal level of MUC1 together with highly glycosylated decoration of the protein is critical for high affinity L-selectin binding. This study demonstrates that MUC1 contributes to cellular adhesive properties which may function to facilitate trophoblast binding to the endometrial cell surface through the L-selectin/sialyl-Lewis x adhesion system subsequent to implantation.

Phenotypic and Genotypic Adaptations in Pseudomonas aeruginosa Biofilms following Long-Term Exposure to an Alginate Oligomer Therapy.

Chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) evolve to generate environmentally adapted biofilm communities, leading to increased patient morbidity and mortality. OligoG CF-5/20, a low-molecular-weight inhaled alginate oligomer therapy, is currently in phase IIb/III clinical trials in CF patients. Experimental evolution of P. aeruginosa in response to OligoG CF-5/20 was assessed using a bead biofilm model allowing continuous passage (45 days; ∼245 generations). Mutants isolated after OligoG CF-5/20 treatment typically had a reduced biofilm-forming ability and altered motility profile. Genotypically, OligoG CF-5/20 provided no selective pressure on genomic mutations within morphotypes. Chronic exposure to azithromycin, a commonly prescribed antibiotic in CF patients, with or without OligoG CF-5/20 in the biofilm evolution model also had no effect on rates of resistance acquisition. Interestingly, however, cross-resistance to other antibiotics (e.g., aztreonam) was reduced in the presence of OligoG CF-5/20. Collectively, these findings show no apparent adverse effects from long-term exposure to OligoG CF-5/20, instead resulting in both fewer colonies with multidrug resistance (MDR)-associated phenotypes and improved antibiotic susceptibility of P. aeruginosaIMPORTANCE The emergence of multidrug-resistant (MDR) pathogens within biofilms in the cystic fibrosis lung results in increased morbidity. An inhalation therapy derived from alginate, OligoG CF-5/20, is currently in clinical trials for cystic fibrosis patients. OligoG CF-5/20 has been shown to alter sputum viscoelasticity, disrupt mucin polymer networks, and disrupt MDR pseudomonal biofilms. Long-term exposure to inhaled therapeutics may induce selective evolutionary pressures on bacteria within the lung biofilm. Here, a bead biofilm model with repeated exposure of P. aeruginosa to OligoG CF-5/20 (alone and in combination with azithromycin) was conducted to study these long-term effects and characterize the phenotypic and genotypic adaptations which result. These findings, over 6 weeks, show that long-term use of OligoG CF-5/20 does not lead to extensive mutational changes and may potentially decrease the pathogenicity of the bacterial biofilm and improve the susceptibility of P. aeruginosa to other classes of antibiotics.

Bi-Functional Alginate Oligosaccharide-Polymyxin Conjugates for Improved Treatment of Multidrug-Resistant Gram-Negative Bacterial Infections.

The recent emergence of resistance to colistin, an antibiotic of last resort with dose-limiting toxicity, has highlighted the need for alternative approaches to combat infection. This study aimed to generate and characterise alginate oligosaccharide ("OligoG")-polymyxin (polymyxin B and E (colistin)) conjugates to improve the effectiveness of these antibiotics. OligoG-polymyxin conjugates (amide- or ester-linked), with molecular weights of 5200-12,800 g/mol and antibiotic loading of 6.1-12.9% w/w, were reproducibly synthesised. In vitro inflammatory cytokine production (tumour necrosis factor alpha (TNFα) ELISA) and cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) of colistin (2.2-9.3-fold) and polymyxin B (2.9-27.2-fold) were significantly decreased by OligoG conjugation. Antimicrobial susceptibility tests (minimum inhibitory concentration (MIC), growth curves) demonstrated similar antimicrobial efficacy of ester- and amide-linked conjugates to that of the parent antibiotic but with more sustained inhibition of bacterial growth. OligoG-polymyxin conjugates exhibited improved selectivity for Gram-negative bacteria in comparison to mammalian cells (approximately 2-4-fold). Both OligoG-colistin conjugates caused significant disruption of Pseudomonas aeruginosa biofilm formation and induced bacterial death (confocal laser scanning microscopy). When conjugates were tested in an in vitro "time-to-kill" (TTK) model using Acinetobacter baumannii, only ester-linked conjugates reduced viable bacterial counts (~2-fold) after 4 h. Bi-functional OligoG-polymyxin conjugates have potential therapeutic benefits in the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, directly reducing toxicity whilst retaining antimicrobial and antibiofilm activities.

Anti-pseudomonad Activity of Manuka Honey and Antibiotics in a Specialized ex vivo Model Simulating Cystic Fibrosis Lung Infection.

Pseudomonas aeruginosa causes problematic chronic lung infections in those suffering from cystic fibrosis. This is due to its antimicrobial resistance mechanisms and its ability to form robust biofilm communities with increased antimicrobial tolerances. Using novel antimicrobials or repurposing current ones is required in order to overcome these problems. Manuka honey is a natural antimicrobial agent that has been used for many decades in the treatment of chronic surface wounds with great success, particularly those infected with P. aeruginosa. Here we aim to determine whether the antimicrobial activity of manuka honey could potentially be repurposed to inhibit pulmonary P. aeruginosa infections using two ex vivo models. P. aeruginosa isolates (n = 28) from an international panel were tested for their susceptibility to manuka honey and clinically relevant antibiotics (ciprofloxacin, ceftazidime, and tobramycin), alone and in combination, using conventional antimicrobial susceptibility testing (AST). To increase clinical applicability, two ex vivo porcine lung (EVPL) models (using alveolar and bronchiolar tissue) were used to determine the anti-biofilm effects of manuka honey alone and in combination with antibiotics. All P. aeruginosa isolates were susceptible to manuka honey, however, varying incidences of resistance were seen against antibiotics. The combination of sub-inhibitory manuka honey and antibiotics using conventional AST had no effect on activity against the majority of isolates tested. Using the two ex vivo models, 64% (w/v) manuka honey inhibited many of the isolates where abnormally high concentrations of antibiotics could not. Typically, combinations of both manuka honey and antibiotics had increased antimicrobial activity. These results highlight the potential of manuka honey as a future antimicrobial for the treatment of pulmonary P. aeruginosa isolates, clearing potential infection reservoirs within the upper airway.

Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides.

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn = 3200 g mol-1) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca2+ and DNA were studied using molecular dynamics (MD) simulations, Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (≥0.5%) inhibited biofilm formation, revealing a significant reduction in both biomass and biofilm height (P < 0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of EPS polysaccharides, and extracellular (e)DNA (P < 0.05) with a corresponding increase in nanoparticle diffusion (P < 0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca2+ evident in FTIR and MD modelling. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca2+-DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections.

A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness.

In chronic respiratory disease, the formation of dense, 3-dimensional "microcolonies" by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates (n = 3) and reference nonmucoid and mucoid multidrug-resistant (MDR) CF isolates (n = 7). Bacterial growth and biofilm development and disruption were studied using cell viability assays and image analysis with scanning electron and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production (P < 0.05) and the formation (at 48 h) of discrete (>10-µm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 µg/ml) in AS medium compared to Mueller-Hinton (MH) medium. In contrast, in an established-biofilm model in AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment, however, induced dose-dependent biofilm disruption (P < 0.05) and led to colistin retaining its antimicrobial activity (P < 0.05). While circular dichroism indicated that OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, mass spectrometry demonstrated that the oligomers induced dose-dependent (>0.2%; P < 0.05) reductions in pseudomonal quorum-sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung and highlight a novel approach to treat MDR pseudomonal infections.

A New Class of Safe Oligosaccharide Polymer Therapy To Modify the Mucus Barrier of Chronic Respiratory Disease.

The host- and bacteria-derived extracellular polysaccharide coating of the lung is a considerable challenge in chronic respiratory disease and is a powerful barrier to effective drug delivery. A low molecular weight 12-15-mer alginate oligosaccharide (OligoG CF-5/20), derived from plant biopolymers, was shown to modulate the polyanionic components of this coating. Molecular modeling and Fourier transform infrared spectroscopy demonstrated binding between OligoG CF-5/20 and respiratory mucins. Ex vivo studies showed binding induced alterations in mucin surface charge and porosity of the three-dimensional mucin networks in cystic fibrosis (CF) sputum. Human studies showed that OligoG CF-5/20 is safe for inhalation in CF patients with effective lung deposition and modifies the viscoelasticity of CF-sputum. OligoG CF-5/20 is the first inhaled polymer therapy, represents a novel mechanism of action and therapeutic approach for the treatment of chronic respiratory disease, and is currently in Phase IIb clinical trials for the treatment of CF.