Pharmacological characterisation of GSK3335103, an oral αvß6 integrin small molecule RGD-mimetic inhibitor for the treatment of fibrotic disease.

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality. Activation of the pro-fibrotic cytokine transforming growth factor-ß (TGFß) via the alpha-V beta-6 (αvß6) integrin has been identified as playing a key role in the development of fibrosis. Therefore, a drug discovery programme to identify an orally bioavailable small molecule αvß6 arginyl-glycinyl-aspartic acid (RGD)-mimetic was initiated. As part of a medicinal chemistry programme GSK3335103 was identified and profiled in a range of pre-clinical in vitro and in vivo systems. GSK3335103 was shown to bind to the αvß6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103-induced concentration- and time-dependent internalisation of αvß6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvß6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvß6 integrin and inhibit the activation of TGFß in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvß6 engagement, TGFß signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvß6 inhibitor that attenuates TGFß signalling in vitro and in vivo with a well-defined pharmacokinetic/pharmacodynamic relationship. This translates to a significant reduction of collagen deposition in vivo and therefore GSK3335103 represents a potential novel oral therapy for fibrotic disorders.

Translational pharmacology of an inhaled small molecule αvß6 integrin inhibitor for idiopathic pulmonary fibrosis.

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

Preclinical SPECT/CT imaging of αvß6 integrins for molecular stratification of idiopathic pulmonary fibrosis.

UNLABELLED: Transforming growth factor ß activation by the αvß6 integrin is central to the pathogenesis of idiopathic pulmonary fibrosis. Expression of the αvß6 integrin is increased in fibrotic lung tissue and is a promising therapeutic target for treatment of the disease. Currently, measurement of αvß6 integrin levels in the lung requires immunohistochemical analysis of biopsy samples. This procedure is clinically impractical for many patients with pulmonary fibrosis, and a noninvasive strategy for measuring αvß6 integrin levels in the lungs is urgently required to facilitate monitoring of disease progression and therapeutic responses. METHODS: Using a murine model of bleomycin-induced lung injury, we assessed the binding of intravenously administered (111)In-labeled αvß6-specific (diethylenetriamine pentaacetate-tetra [DTPA]-A20FMDV2) or control (DTPA-A20FMDVran) peptide by nanoSPECT/CT imaging. Development of fibrosis was assessed by lung hydroxyproline content, and αvß6 protein and itgb6 messenger RNA were measured in the lungs. RESULTS: Maximal binding of (111)In-labeled A20FMDV2 peptide to αvß6 integrins was detected in the lungs 1 h after intravenous administration. No significant binding was detected in mice injected with control peptide. Integrin binding was increased in the lungs of bleomycin-, compared with saline-, exposed mice and was attenuated by pretreatment with αvß6-blocking antibodies. Levels of (111)In-labeled A20FMDV2 peptide correlated positively with hydroxyproline, αvß6 protein, and itgb6 messenger RNA levels. CONCLUSION: We have developed a highly sensitive, quantifiable, and noninvasive technique for measuring αvß6 integrin levels within the lung. Measurement of αvß6 integrins by SPECT/CT scanning has the potential for use in stratifying therapy for patients with pulmonary fibrosis.

A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity.

The opportunistic human pathogen, Pseudomonas aeruginosa, is a major cause of infections in chronic wounds, burns and the lungs of cystic fibrosis patients. The P. aeruginosa genome encodes at least three proteins exhibiting the characteristic three domain structure of autotransporters, but much remains to be understood about the functions of these three proteins and their role in pathogenicity. Autotransporters are the largest family of secreted proteins in Gram-negative bacteria, and those characterised are virulence factors. Here, we demonstrate that the PA0328 autotransporter is a cell-surface tethered, arginine-specific aminopeptidase, and have defined its active site by site directed mutagenesis. Hence, we have assigned PA0328 with the name AaaA, for arginine-specific autotransporter of P. aeruginosa. We show that AaaA provides a fitness advantage in environments where the sole source of nitrogen is peptides with an aminoterminal arginine, and that this could be important for establishing an infection, as the lack of AaaA led to attenuation in a mouse chronic wound infection which correlated with lower levels of the cytokines TNFα, IL-1α, KC and COX-2. Consequently AaaA is an important virulence factor playing a significant role in the successful establishment of P. aeruginosa infections.

Properdin deficiency in murine models of nonseptic shock.

Hereditary properdin deficiency is linked to susceptibility to meningococcal disease (Neisseria meningitidis serotypes Y and W-135) with high mortality. Its relative contribution toward the outcome of nonseptic shock has not been investigated. Using properdin-deficient C57BL/6 mice and their littermates, this study examines their survival of zymosan-induced and LPS-induced shock. Properdin-deficient mice were more resistant to zymosan shock compared with wild-type mice, which showed greater impairment of end-organ function 24 h after zymosan injection, higher TNF-alpha production by alveolar and peritoneal macrophages, higher TNF-alpha, and, inversely, lower IL-10 levels in peritoneal lavage and circulation and higher plasma C5a levels. Properdin-deficient mice showed significantly higher mortality in LPS shock, elevated TNF-alpha, and, inversely, reduced IL-10 production by peritoneal macrophages as well as lower plasma C5a levels compared with wild-type littermates. NO production by peritoneal macrophages and plasma alpha1-antitrypsin levels at 24 h after the injection of LPS or zymosan were decreased in properdin-deficient mice in both models, and fewer histopathologic changes in liver were observed in properdin-deficient animals. This study provides evidence that properdin deficiency attenuates zymosan-induced shock and exacerbates LPS-induced shock.

Ex vivo cancer chemoprevention research possibilities.

The concept of cancer prevention with naturally occurring or synthetic compounds is rapidly gaining momentum as a key field in cancer research. The availability of good models for the determination of the molecular mechanisms of these agents, which frequently have multiple sites of action within a cell, is key to the progression of the field. In this review, we concentrate on the emergence of several in vitro techniques that have significant advantages over more traditional monolayer cell culture, and/or in vivo models. In particular, we focus on the potential of 3D multicellular spheroid models as versatile intermediates between monolayer culture and tumours in situ. In these models, cell-cell interactions and cell-extracellular matrix interactions can closely mimic the environment to which tumour cells would be exposed in vivo, while maintaining the advantages of ease of manipulation of an in vitro system. The in vitro tube formation assay for the study of angiogenesis, the availability of human tissues for research, and the sophisticated technology surrounding DNA microarray and proteomics are also briefly discussed.