Characterising the molecular mode of action of connexin therapeutics for the treatment of retinal injury and disease

Abstract

Connexin43 gap junctions are non-selective membrane channels that facilitate cell-to-cell communication. A functional gap junction channel is formed upon docking of two hemichannels, or connexons, but an undocked hemichannel plays a key role in the initial response to cellular injury. In particular Connexin43 hemichannels have been shown to communicate aberrant signals that promote a self-perpetuating cycle involving vascular leak, edema, inflammation and neuronal cell death. Pre-clinical models of central nervous system injuries have demonstrated that systemic delivery of Connexin43 mimetic Peptide5 (VDCFLSRPTEKT, PeptagonTM) significantly improves functional outcomes. Additionally Tonabersat (SB-220453) has been tested in phase II clinical trials to target spreading waves of abnormal activity in cellular injury. Despite the therapeutic potential, the molecular mode of action of these compounds has not yet been described. Therefore, the aim of this thesis is to characterise the molecular mode of action of Peptide5 and Tonabersat to support their clinical progression. An in vitro model of ischemia-reperfusion injury was developed to examine connexin43 hemichannel-mediated ATP release in a human cerebral microvascular endothelial cell (hCMVEC) line. Gap junction channel function was assessed using an established scrapeloading assay and visualised using immunohistochemistry. Modified analogues of Peptide5, including single alanine substituted peptides and truncated, were used to assess the mode of action of Peptide5. The site of action of Peptide5 was tested using extracellular loops in a competition assay. Peptide5 inhibits Connexin43 hemichannel-mediated ATP release in hCMVEC by acting on extracellular loop two of Connexin43. Furthermore, Peptide5 sequence specificity is important for inhibiting hemichannel-mediated ATP release but less so for the uncoupling of gap junctions. The SRPTEKT motif is central to Peptide5 function but on its own is not sufficient to inhibit hemichannels at the concentrations used. Tonabersat exhibits a concentration-dependent response where a low concentration significantly inhibits connexin43 hemichannel-mediated ATP release but relatively higher concentrations uncouple gap junctions, with Connexin43 junction plaques internalised and degraded via the lysosomal pathway. Taken together, this study provides an understanding for the molecular mode of action of Peptide5 and Tonabersat to support their clinical development for the treatment of retinal injury and diseases.