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-β (Aβ) aggregates. Aβ-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.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development 1 – 5 . This also applies to the PI 3-kinase (PI3K) signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report on the discovery of UCL-TRO-1938 (further referred to as 1938), a small molecule activator of the PI3Kα isoform, a critical effector of growth factor signalling. 1938 allosterically activates PI3Kα through a unique 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, upon local administration, enhances nerve regeneration following nerve crush. This study identifies a unique chemical tool to directly probe PI3Kα signalling and a novel 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.

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MASTL (microtubule-associated serine/threonine kinase-like), commonly known as Greatwall (GWL), is a member of the AGC kinase family and has recently emerged as a novel cancer therapy target. GWL regulates mitotic division via phosphorylation of its known substrates ENSA and ARPP19, which upon phosphorylation act as inhibitors of the PP2A-B55δ phosphatase. Inactivation of PP2A-B55δ induces sustained activity of the CDK1-cyclin B complex, triggering mitotic entry and preventing mitotic collapse during cell division. Towards the end of mitosis, GWL inactivation is required to initiate mitotic exit. Overexpression of GWL in breast cancer cells drives oncogenic properties, such as transformation and invasions. In particular, triple negative breast cancer tumors, which are associated with a poor prognosis, overexpress GWL and are highly sensitive to GWL depletion. In addition, GWL expression has been found to be elevated in oral squamous cell carcinoma and prostate cancer tissues. A number of studies have also indicated that GWL may contribute directly to tumorigenesis, suggesting that modulation of GWL activity with small-molecule inhibitors could afford a potential new anti-cancer therapy. The precise role of GWL in cells remaining largely unexplored, the identification of a pharmacological tool would be useful to further assess the tumor-associated functions of this protein. To that end, we have recently developed and optimized a robust HTRF assay, based on a specific ENSA peptide analogue, and used it to screen kinase focused libraries, made available to us by GlaxoSmithKline and Roche. A 10.2% hit rate was achieved from the screening of the 11,000 compound set at a single concentration of 10 µM. Molecules inducing over 50% inhibition in the single point screen were subsequently tested in concentration-dependent experiments. Analysis of the IC50 generated enabled to identify molecular clusters and potential hits to initiate a medicinal chemistry campaign. A review of the literature on the selectivity of the hits identified indicated that a significant number of these molecules also had affinity for other AGC kinases and that selectivity among the AGC kinase family could be challenging to achieve. Computational studies using an in-house co-crystal structure of GWL in complex with Staurosporine and the published crystal structures of other AGC kinases will be used to design out selectivity and guide the design of inhibitors to obtain a potent chemical probe. Citation Format: Tristan D. Reuillon, Sarah Walker, Darren Le Grand, Simon E. Ward, Ben Wahab, Mohan B. Rajasekaran, Helfrid Hochegger, Antony W. Oliver. Screening of kinase focused libraries for the identification of Greatwall inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4210. doi:10.1158/1538-7445.AM2017-4210