A small-molecule PI3Kα activator for cardioprotection and neuroregeneration

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1,2,3,4,5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development

Exploring the role of cardiac mast cells in acute myocardial infarction and ischaemia-reperfusion injury

Mast cells are immune cells derived from pluripotent progenitor cells of the bone marrow that mature under the influence of the c-kit ligand and stem cell factor. Cardiac mast cells are known to be involved in many disease processes including plaque rupture, ischaemia post myocardial infarction and myocardial remodelling after infarction. Myocardial infarction (MI) is defined as decreased or complete cessation of blood flow in one or more coronary arteries supplying the myocardium, resulting in irreversible myocardial cell death, usually due to prolonged ischaemia. The deleterious effects of mast cells are thought to be due to a combination of the primary mediators which are found within the secretory granules, and secondary mediators which are generated upon activation. Evidence suggests a clear and important detrimental role of cardiac mast cells in myocardial infarction. However, the extent to which mast cell inhibition confers cardioprotection and the ideal timing to instigate such a therapeutic intervention is unclear. Mast cells release multiple mediators upon activation, one of which is histamine. Histamine is a known vasoconstrictor, and it is yet to be determined whether it has a detrimental impact in relation to no-reflow phenomena. In addition, it is yet to be determined whether cardioprotection due to mast cell inhibition occurs independently of ischaemic preconditioning (IPC), and whether there may be additive protection using a multi-targeted approach. I therefore aimed to characterise the role of cardiac mast cells in MI and ischaemia-reperfusion injury. Using an ex-vivo Langendorff model of global ischaemia, I report that stabilisation of cardiac mast cells prior to ischaemia reduces the myocardial infarct size, independent of IPC. The use of concurrent IPC therapy provided additional protection. When mast cells were degranulated prior to the ischaemic insult, there was reduction in infarct size suggesting that any potential pharmacological therapy needs to be administered prior to reperfusion. When IPC was given to these degranulated mast cells, there was further cardioprotection suggesting that cardioprotection via mast cell inhibition may be due to either an IPC-independent pathway or that IPC pre- 6 treatment does not fully protect against potential damage caused by mast cells. PI3K-regulated signalling events appear to play an important role in mast cell biology such as mast cell growth, differentiation, activation, and survival. I therefore investigated the role of PI3K-α in cardioprotection by using a pharmacological PI3K-α agonist (UCL-TRO-1938). PI3K-α agonism prior to myocardial infarction showed cardioprotective properties. Finally, I describe the use of the endothelial fluorescent dye Thioflavin S to identify regions of no-reflow for the first time in an ex-vivo model. I also described that by stabilising mast cells prior to global ischaemia, the degree of no-reflow may be reduced suggesting a potentially therapeutic target for this process which to date has no definitive therapy. In summary, my thesis describes how mast cells contribute to ischaemic injury after myocardial infarction, and inhibiting mast cell activation confers cardioprotection, which is additive to IPC. Histamine, a major component of mast cell granules appears to play an important role in myocardial damage and no-reflow phenomena. Finally, use of a multi-targeted approach appears to be beneficial, and research is needed to explore this further