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 development1-5. 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.

A challenge finding P2X1 and P2X4 ligands.

Identifying novel small-molecule P2X1 and P2X4 ligands with sub-type specificity and high-affinity remains a pharmacological challenge. Here we use computational methods, electrophysiology and fluorescent microplate assays to screen for ligand candidates acting at these receptors. Modelling and docking identified 80 compounds for testing at P2X4 receptors, and 20 of these showed >50% inhibition in fluorescence-based assays, making them appealing for further SAR studies. Confirmation of activity by two-electrode voltage clamp, followed by their elaboration resulted in only minor improvements in potency, with the highest IC50 being 295 µM. Testing on P2X1 receptors, resulted in a series of biguanide compounds that yielded a maximum IC50 of 100 µM, but no consistent SAR could be found. Potencies of established antagonists gave expected results, although the measured potencies varied between techniques and no antagonism could be found for compounds such as paroxetine, carbamazepine, 9(10H)-acridanone, acridinol and phenoxazine-type heterocycles. This study highlights the challenge of identifying P2X4 and P2X1 ligands and suggests that a combination of complimentary approaches is needed if we are to be confident of ligand activities at these receptors.

Dihydropyridines Allosterically Modulate Hsp90 Providing a Novel Mechanism for Heat Shock Protein Co-induction and Neuroprotection.

Chaperones play a pivotal role in protein homeostasis, but with age their ability to clear aggregated and damaged protein from cells declines. Tau pathology is a driver of a variety of neurodegenerative disease and in Alzheimer's disease (AD) it appears to be precipitated by the formation of amyloid-ß (Aß) aggregates. Aß-peptide appears to trigger Tau hyperphosphorylation, formation of neurofibrillary tangles and neurotoxicity. Recently, dihydropyridine derivatives were shown to upregulate the heat shock response (HSR) and provide a neuroprotective effect in an APPxPS1 AD mouse model. The HSR response was only seen in diseased cells and consequently these compounds were defined as co-inducers since they upregulate chaperones and co-chaperones only when a pathological state is present. We show for compounds tested herein, that they target predominantly the C-terminal domain of Hsp90, but show some requirement for its middle-domain, and that binding stimulates the chaperones ATPase activity. We identify the site for LA1011 binding and confirm its identification by mutagenesis. We conclude, that binding compromises Hsp90's ability to chaperone, by modulating its ATPase activity, which consequently induces the HSR in diseased cells. Collectively, this represents the mechanism by which the normalization of neurofibrillary tangles, preservation of neurons, reduced tau pathology, reduced amyloid plaque, and increased dendritic spine density in the APPxPS1 Alzheimer's mouse model is initiated. Such dihydropyridine derivatives therefore represent potential pharmaceutical candidates for the therapy of neurodegenerative disease, such as AD.