Identification, development and in vitro testing of pannexin1 channel inhibitors

Cellular channels consisting of pannexin1 (Panx1) proteins mediate the exchange of ions and small molecules, such as adenosine triphosphate (ATP), between the cytosol and extracellular environment. Given their ability to release different signaling molecules, Panx1 channels are major players in variouscellular communication pathways triggering both inflammation and cell death. Functional inhibition of these channels was put forward as a promising strategy for counteracting the inflammatory response and cell death associated with various diseases, such as coronavirus disease 2019 (COVID-19), non-alcoholic steatohepatitis and multiple sclerosis. Hence, the identification and development of Panx1 channel inhibitors with therapeutic potential could provide a promising generic approach for the treatment of a broad spectrum of diseases. Panx1 channel inhibitors that are eligible for clinical use are scarce. Therefore, the aim of this doctoral project was to identify and/or develop new Panx1 channel inhibitors by following2 strategies namely, the screening of drugs repurposed for the treatment of COVID-19 patients and the development of Panx1 peptide mimetics. Panx1 channels were put forward as a potential therapeutic target in the treatment of COVID-19 patients. Hence, the hypothesis was raised whether the COVID-19 drugs, that were used early on in the pandemic, could exert an effect on Panx1 channels both on afunctional and expression level. This has led to the identification of 3 new potential channel inhibitors, including hydroxychloroquine, favipiravir and lopinavir, which were able to reduce ATP release in a Panx1 channel activity assay without altering Panx1 expression levels. The second approach focused on the development of peptides mimicking amino acid stretches of the different regions of the Panx1 protein. Several of these peptide mimetics with Panx1 channel inhibitory capacity were previously described in literature. However, the unmodified linear character of the peptides impedes their use as potential therapeutics as these compounds are generally characterised by a low in vivo bioavailability due toextensive proteolytic degradation and rapid clearance. Accordingly, a study was set up to uncover important structural features of gold standard peptidic Panx1 channel inhibitor 10Panx1 that could aid in the development of next generation analogues. Gln3and Asp8 of the 10Panx1 sequence were pinpointed as critical residues by means of an alanine scanning method. In parallel, the scissile amide bonds were identified using a plasma stability assay. The design and synthesis of different stabilised analogues based on these results led to fully stable equipotent 10Panx1 analogues. More potent analogues were obtained through the substitutions of the Phe6, Asp8and Tyr10 of the sequence. Next to studying the important structural features and stability of the peptide mimetics, preliminary data were gathered on the molecular mechanism of action of 10Panx1 and 4 novel peptide mimetics. In order to investigate how these peptides interact with the Panx1 channel, localisation and internalisation as well as specificity of the binding was evaluated using fluorescein-labelled analogues of the lead sequences. Due to solubility issues, the fluorescent analogues of lead sequences derived from extracellular regions could not be tested, with the exception of the 10Panx1 analogues. Microscopic imaging showed an interaction of the different peptidesat the cell plasma membrane. In addition, the comparison of the fluorescent signal after exposure of the peptides to a wild-type and Panx1 transduced cell line confirmed the specificity of peptide-protein interaction of 10Panx1 and the peptide derived from the N-terminus. Of note, the peptide mimicking a native segment of the cytoplasmic loop showed distinct results with no clear differences observed between the wild-type and Panx1 transduced cells. The latter also appeared to bind the channel solely in its closed conformation as results showed that preincubation and not incubation of the cells with thepeptide mimetic in a Panx1 channel activity assay was crucial for the inhibitory capacity of the peptide. However, despite current efforts, no-clear cut molecular mechanism of action could be defined yet. In summary, the 2 strategies employed in this doctoral thesis project resulted in a new set of Panx1 channel inhibitors. Further investigation of their therapeutic effect is required to validate their efficiency in different diseases associated with opening of Panx1 channels.