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.

Supramolecular self-associating amphiphiles (SSAs) as nanoscale enhancers of cisplatin anticancer activity.

Many chemotherapeutic drugs have a narrow therapeutic window due to inefficient tumour cell permeation. Supramolecular self-associating amphiphilic salts (SSAs) are a unique class of small molecules that offer potential as next generation cancer drugs and/or therapeutic enhancement agents. Herein, we demonstrate the cytotoxicity of seven SSAs towards both ovarian and glioblastoma cancer cells. We also utilize the intrinsic fluorescent properties of one of these lead SSAs to provide evidence for this class of compound to both bind to the exterior cancer cell surface and permeate the cell membrane, to become internalized. Furthermore, we demonstrate synergistic effects of two lead SSAs on cisplatin-mediated cytotoxicity of ovarian cancer cells and show that this correlates with increased DNA damage and apoptosis versus either agent alone. This work provides the first evidence that SSAs interact with and permeate cancer cell membranes and enhance the cytotoxic activity of a chemotherapeutic drug in human cancer cells.

Structural insights and activating mutations in diverse pathologies define mechanisms of deregulation for phospholipase C gamma enzymes.

BACKGROUND: PLCγ enzymes are key nodes in cellular signal transduction and their mutated and rare variants have been recently implicated in development of a range of diseases with unmet need including cancer, complex immune disorders, inflammation and neurodegenerative diseases. However, molecular nature of activation and the impact and dysregulation mechanisms by mutations, remain unclear; both are critically dependent on comprehensive characterization of the intact PLCγ enzymes. METHODS: For structural studies we applied cryo-EM, cross-linking mass spectrometry and hydrogen-deuterium exchange mass spectrometry. In parallel, we compiled mutations linked to main pathologies, established their distribution and assessed their impact in cells and in vitro. FINDINGS: We define structure of a complex containing an intact, autoinhibited PLCγ1 and the intracellular part of FGFR1 and show that the interaction is centred on the nSH2 domain of PLCγ1. We define the architecture of PLCγ1 where an autoinhibitory interface involves the cSH2, spPH, TIM-barrel and C2 domains; this relative orientation occludes PLCγ1 access to its substrate. Based on this framework and functional characterization, the mechanism leading to an increase in PLCγ1 activity for the largest group of mutations is consistent with the major, direct impact on the autoinhibitory interface. INTERPRETATION: We reveal features of PLCγ enzymes that are important for determining their activation status. Targeting such features, as an alternative to targeting the PLC active site that has so far not been achieved for any PLC, could provide new routes for clinical interventions related to various pathologies driven by PLCγ deregulation. FUND: CR UK, MRC and AstaZeneca.

Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a 5'-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II (TOP2). TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for high-throughput screen (HTS)-screening. We have gone on to determine crystal structures of these compounds bound to a 'humanized' form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2.

Development of an oligonucleotide-based fluorescence assay for the identification of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors.

Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3'-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.