Covalent EGFR family inhibitors bind to and induce the degradation of the pseudokinase TRIB2 to kill cancer cells.

The opportunistic pathogen Pseudomonas aeruginosa employs the type III secretion system (T3SS) and four effector proteins, ExoS, ExoT, ExoU, and ExoY, to disrupt cellular physiology and subvert the host’s innate immune response. Of the effector proteins delivered by the T3SS, ExoU is the most toxic. In P. aeruginosa infections, where the ExoU gene is expressed, disease severity is increased with poorer prognoses. This is considered to be due to the rapid and irreversible damage exerted by the phospholipase activity of ExoU, which cannot be halted before conventional antibiotics can successfully eliminate the pathogen. This review will discuss what is currently known about ExoU and explore its potential as a therapeutic target, highlighting some of the small molecule ExoU inhibitors that have been discovered from screening approaches.

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system (T3SS) toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration (MIC) for 24 h, but, when used in combination with either PSA, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa, as well as the means to identify more potent ExoU inhibitors for future therapeutics.

The opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments. ExoU is a phospholipase which causes destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosain vitro than other antimicrobial classes. Their overuse over the past decade, however, has resulted in the emergence of antibiotic resistance. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered that tobramycin disrupted T3SS expression and reduced both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-t cells at concentrations below the minimal inhibitory concentration (MIC)_. The fluoroquinolones moxifloxacin and ciprofloxacin, however, upregulated the T3SS and did not inhibit and may have increased the cytotoxic effects of ExoS and ExoU.

Objectives: Activation of neutrophils and release of neutrophil extracellular traps (NETs), proteases and reactive oxygen species (ROS) is pathogenic in immune-mediated inflammatory diseases like rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), driving inflammation and damaging host tissues. The aim of this research was to identify small molecule inhibitors of NET production using a highly curated panel of narrow-spectrum small molecule kinase inhibitors termed the kinase chemogenomic set (KCGS). Methods: Neutrophils were isolated from healthy controls (HC) and people with RA or SLE. Over 220 small molecules were screened for their ability to inhibit NET production and decrease ROS production without impairing neutrophil apoptosis or killing of Staphylococcus aureus bacteria. Results: Fifty compounds, nominally targeting 29 individual protein kinases, were found to inhibit NADPH oxidase 2 (NOX2) -dependent (phorbol 12-myristate 13-acetate-stimulated) and NOX2-independent (A23187-stimulated) NET production (P < 0.05, n = 5 HC, RA and SLE). Of these, seven compounds did not significantly impair ROS production or apoptosis. The deconvoluted targets of these small molecules inhibit kinases that operate in three cellular pathways: autophagy, cell cycle checkpoint and epidermal growth factor tyrosine kinase signalling. Of these, only inhibitors of ULK1, JNK and ROCK1/2, broadly implicated in the regulation of autophagy, did not significantly impair bacterial killing (n = 5 HC, P > 0.05). Autophagy inhibitors were also able to inhibit immune-complex driven NET production (P < 0.05, n = 5 HC, RA and SLE). Conclusion: We propose that autophagy signalling pathways represent novel and exciting targets for the development of small molecule therapeutics to block unwanted neutrophil activation and NET release in immune-mediated inflammatory disease.