Structure-Based Design and Synthesis of Stapled 10Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases.

Following a rational design, a series of macrocyclic ("stapled") peptidomimetics of 10Panx1, the most established peptide inhibitor of Pannexin1 (Panx1) channels, were developed and synthesized. Two macrocyclic analogues SBL-PX1-42 and SBL-PX1-44 outperformed the linear native peptide. During in vitro adenosine triphosphate (ATP) release and Yo-Pro-1 uptake assays in a Panx1-expressing tumor cell line, both compounds were revealed to be promising bidirectional inhibitors of Panx1 channel function, able to induce a two-fold inhibition, as compared to the native 10Panx1 sequence. The introduction of triazole-based cross-links within the peptide backbones increased helical content and enhanced in vitro proteolytic stability in human plasma (>30-fold longer half-lives, compared to 10Panx1). In adhesion assays, a "double-stapled" peptide, SBL-PX1-206 inhibited ATP release from endothelial cells, thereby efficiently reducing THP-1 monocyte adhesion to a TNF-α-activated endothelial monolayer and making it a promising candidate for future in vivo investigations in animal models of cardiovascular inflammatory disease.

Mitochondrial pannexin1 controls cardiac sensitivity to ischaemia/reperfusion injury.

AIMS: No effective therapy is available in clinics to protect the heart from ischaemia/reperfusion (I/R) injury. Endothelial cells are activated after I/R, which may drive the inflammatory response by releasing ATP through pannexin1 (Panx1) channels. Here, we investigated the role of Panx1 in cardiac I/R. METHODS AND RESULTS: Panx1 was found in cardiac endothelial cells, neutrophils, and cardiomyocytes. After in vivo I/R, serum Troponin-I, and infarct size were less pronounced in Panx1-/- mice, but leukocyte infiltration in the infarct area was similar between Panx1-/- and wild-type mice. Serum Troponin-I and infarct size were not different between mice with neutrophil-specific deletion of Panx1 and Panx1fl/fl mice, suggesting that cardioprotection by Panx1 deletion rather involved cardiomyocytes than the inflammatory response. Physiological cardiac function in wild-type and Panx1-/- hearts was similar. The time to onset of contracture and time to maximal contracture were delayed in Panx1-/- hearts, suggesting reduced sensitivity of these hearts to ischaemic injury. Moreover, Panx1-/- hearts showed better recovery of left ventricle developed pressure, cardiac contractility, and relaxation after I/R. Ischaemic preconditioning failed to confer further protection in Panx1-/- hearts. Panx1 was found in subsarcolemmal mitochondria (SSM). SSM in WT or Panx1-/- hearts showed no differences in morphology. The function of the mitochondrial permeability transition pore and production of reactive oxygen species in SSM was not affected, but mitochondrial respiration was reduced in Panx1-/- SSM. Finally, Panx1-/- cardiomyocytes had a decreased mitochondrial membrane potential and an increased mitochondrial ATP content. CONCLUSION: Panx1-/- mice display decreased sensitivity to cardiac I/R injury, resulting in smaller infarcts and improved recovery of left ventricular function. This cardioprotective effect of Panx1 deletion seems to involve cardiac mitochondria rather than a reduced inflammatory response. Thus, Panx1 may represent a new target for controlling cardiac reperfusion damage.

Pannexin1 channels-a potential therapeutic target in inflammation.

An exaggerated inflammatory response is the hallmark of a plethora of disorders. ATP is a central signaling molecule that orchestrates the initiation and resolution of the inflammatory response by enhancing activation of the inflammasome, leukocyte recruitment and activation of T cells. ATP can be released from cells through pannexin (Panx) channels, a family of glycoproteins consisting of three members, Panx1, Panx2, and Panx3. Panx1 is ubiquitously expressed and forms heptameric channels in the plasma membrane mediating paracrine and autocrine signaling. Besides their involvement in the inflammatory response, Panx1 channels have been shown to contribute to different modes of cell death (i.e., pyroptosis, necrosis and apoptosis). Both genetic ablation and pharmacological inhibition of Panx1 channels decrease inflammation in vivo and contribute to a better outcome in several animal models of inflammatory disease involving various organs, including the brain, lung, kidney and heart. Up to date, several molecules have been identified to inhibit Panx1 channels, for instance probenecid (Pbn), mefloquine (Mfq), flufenamic acid (FFA), carbenoxolone (Cbx) or mimetic peptides like 10Panx1. Unfortunately, the vast majority of these compounds lack specificity and/or serum stability, which limits their application. The recent availability of detailed structural information on the Panx1 channel from cryo-electron microscopy studies may open up innovative approaches to acquire new classes of synthetic Panx1 channel blockers with high target specificity. Selective inhibition of Panx1 channels may not only limit acute inflammatory responses but may also prove useful in chronic inflammatory diseases, thereby improving human health. Here, we reviewed the current knowledge on the role of Panx1 in the initiation and resolution of the inflammatory response, we summarized the effects of Panx1 inhibition in inflammatory pathologies and recapitulate current Panx1 channel pharmacology with an outlook towards future approaches.

Canonical and Non-Canonical Roles of Connexin43 in Cardioprotection.

Since the mid-20th century, ischemic heart disease has been the world's leading cause of death. Developing effective clinical cardioprotection strategies would make a significant impact in improving both quality of life and longevity in the worldwide population. Both ex vivo and in vivo animal models of cardiac ischemia/reperfusion (I/R) injury are robustly used in research. Connexin43 (Cx43), the predominant gap junction channel-forming protein in cardiomyocytes, has emerged as a cardioprotective target. Cx43 posttranslational modifications as well as cellular distribution are altered during cardiac reperfusion injury, inducing phosphorylation states and localization detrimental to maintaining intercellular communication and cardiac conduction. Pre- (before ischemia) and post- (after ischemia but before reperfusion) conditioning can abrogate this injury process, preserving Cx43 and reducing cell death. Pre-/post-conditioning has been shown to largely rely on the presence of Cx43, including mitochondrial Cx43, which is implicated to play a major role in pre-conditioning. Posttranslational modifications of Cx43 after injury alter the protein interactome, inducing negative protein cascades and altering protein trafficking, which then causes further damage post-I/R injury. Recently, several peptides based on the Cx43 sequence have been found to successfully diminish cardiac injury in pre-clinical studies.

Non-canonical roles of connexins.

Gap junctions mediate cellular communication and homeostasis by controlling the intercellular exchange of small and hydrophilic molecules and ions. Gap junction channels are formed by the docking of 2 hemichannels of adjacent cells, which in turn are composed of 6 connexin subunits. Connexin proteins as such can also control the cellular life cycle independent of their channel activities. This has been most demonstrated in the context of cell growth and cell death. Different mechanisms are involved mainly related to direct interaction with cell growth or cell death regulators, but also implying effects on the expression of cell growth and cell death regulators. The present paper focuses on these atypical roles of connexin proteins.