Pharmacological characterisation of small molecule RGD-mimetics targeting αvβ6-mediated activation of TGFβ in idiopathic pulmonary fibrosis

Slack, Robert J.

Thesis

Pharmacological characterisation of small molecule RGD-mimetics targeting αvβ6-mediated activation of TGFβ in idiopathic pulmonary fibrosis

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Creator

Slack, Robert J.

Rights statement

Strathclyde Thesis Copyright

Awarding institution

University of Strathclyde

Date of award

2015

Thesis identifier

T16064

Person Identifier (Local)

201287345

Qualification Level

Doctoral (Postgraduate)

Qualification Name

Doctor of Philosophy (PhD)

Department, School or Faculty

Strathclyde Institute of Pharmacy and Biomedical Sciences

Abstract

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality in disorders that include idiopathic pulmonary fibrosis (IPF). The alpha-v beta-6 (αvβ6) integrin has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesized to be pivotal in the development of IPF. Therefore, the αvβ6 integrin was identified as an attractive therapeutic target for this debilitating disease and a drug discovery programme to identify inhaled small molecule αvβ6 selective arginylglycinyl- aspartic acid (RGD)-mimetics was initiated within GlaxoSmithKline. The primary aim of this study was to pharmacologically characterise the small molecule αvβ6 RGD-mimetics generated in a range of test systems. RGD ligands including peptides, antibodies and small molecule RGD-mimetics were characterised in vitro using methods that included radioligand binding, cell adhesion, flow cytometry, coculture TGFβ bioassays, confocal microscopy and immunohistochemistry. This included the use of soluble recombinant integrin protein, stable and primary cells expressing integrins recombinantly or endogenously and human lung tissue from normal and IPF donors. Compound 3 generated from an internal medicinal chemistry programme was shown to bind to the αvβ6 with high affinity (pM) and displayed fast association with the integrin followed by slow dissociation kinetics in all systems investigated. Using [3H]compound 3 the quantification of the upregulation of αvβ6 from normal to IPF human lung tissue has been determined for the first time with data suggesting αvβ6 exists in a highly activated state within diseased tissue. In human lung primary epithelial cells co-cultured with a TGFβ reporter cell system, compound 3 was shown to inhibit with high potency the activation of TGFβ with a prolonged duration of action. In the same primary lung epithelial cells compound 3 caused a rapid internalisation of αvβ6 (minutes) followed by a slow return of the integrin to the cell surface (hours). It was shown that αvβ6 is degraded in lysosomes post-internalisation by compound 3 that would suggest the slow return of integrin to the surface and sustained duration of action is a consequence of new αvβ6 synthesis. From the chemical series of small molecule RGD-mimetic compounds identified, compound 3 has been shown to display the desirable pharmacological characteristics required for targeting a prolonged inhibition of TGFβ activation in the IPF lung via blockade of the αvβ6 integrin.

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