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.

African trypanosomiasis: Synthesis & SAR enabling novel drug discovery of ubiquinol mimics for trypanosome alternative oxidase.

African trypanosomiasis is a parasitic disease affecting 5000 humans and millions of livestock animals in sub-Saharan Africa every year. Current treatments are limited, difficult to administer and often toxic causing long term injury or death in many patients. Trypanosome alternative oxidase is a parasite specific enzyme whose inhibition by the natural product ascofuranone (AF) has been shown to be curative in murine models. Until now synthetic methods to AF analogues have been limited, this has restricted both understanding of the key structural features required for binding and also how this chemotype could be developed to an effective therapeutic agent. The development of 3 amenable novel synthetic routes to ascofuranone-like compounds is described. The SAR generated around the AF chemotype is reported with correlation to the inhibition of T. b. brucei growth and corresponding selectivity in cytotoxic assessment in mammalian HepG2 cell lines. These methods allow access to greater synthetic diversification and have enabled the synthesis of compounds that have and will continue to facilitate further optimisation of the AF chemotype into a drug-like lead.

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.

Taurine reduces nitrosative stress and nitric oxide synthase expression in high glucose-exposed human Schwann cells.

The role of taurine in regulating glucose-induced nitrosative stress has been examined in human Schwann cells, a model for understanding the pathogenesis of diabetic neuropathy. Exposure to high glucose increased nitrated proteins (1.56 fold p<0.05), inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS) mRNA expression (1.55 fold and 2.2 fold respectively, p<0.05 both), phospho-p38 MAPK (1.32 fold, p<0.05) abundance and decreased Schwann cell growth (11±2%, p<0.05). Taurine supplementation prevented high-glucose induced iNOS and nNOS mRNA upregulation, reduced nitrated proteins and phospho-p38 MAPK (56±11% and 45±18% (p<0.05 both) respectively) and restored Schwann cell growth to control levels. High glucose and taurine treatment alone reduced phospho-p42/44 MAPK and phospho-AKT to below detectable levels. Treatment of human Schwann cells with donors of nitric oxide and peroxynitrite reduced taurine transporter (TauT) expression (by 35±5% and 29±7% respectively p<0.05 both) as well as the maximum velocity of taurine uptake (TauT Vmax). NOS inhibition prevented glucose-mediated TauT mRNA downregulation, and restored TauT Vmax. These data demonstrate an important role for taurine in the prevention of nitrosative stress in human Schwann cells, which may have important implications for the development and treatment of diabetic neuropathy.

Emerging drugs for diabetic neuropathy.

IMPORTANCE OF THE FIELD: Diabetic neuropathy (DN) is a very common and disabling diabetes-related complication. DN is associated with significant morbidity and mortality. Diabetic peripheral neuropathy (DPN) can be painful in the earlier stages of the disease before becoming painless. Most of the currently available therapies are symptomatic (focusing on pain relief) rather than disease-modifying. With the exception of good glycemic control, there is currently no effective treatment to slow the progression of or reverse DPN. AREAS COVERED IN THIS REVIEW: In this article, we review the epidemiology, pathogenesis, currently available and future treatments for DPN, and the potential development issues/challenges related to such new therapies. Literature search was performed using PubMed, Medline and Pharmaprojects from 1950 onwards. Search terms include a combination of terms such as diabetic neuropathy, pathogenesis, pathophysiology, mechanisms, treatment, therapy, oxidative/nitrosative stress, anti-oxidants, serotonin, nitrotyrosine, protein kinase C, aldose reductase, sodium channels, taurine, lipoic acid and poly (ADP-ribose) polymerase. WHAT THE READER WILL GAIN: The reader will gain an overview of the epidemiology, clinical features and risk factors of DN. In addition, the reader will have a better understanding of the mechanisms that underpin the development of DPN and their relationships to the current and future therapies. The reader will also develop an insight into the limitations of the current approach to DPN treatment and the potential avenues for future research. TAKE HOME MESSAGE: DN is a very common and disabling complication that currently has no effective treatments other than diabetes control. The pathogenesis of DPN is complex and multi-factorial. Several disease-modifying and symptomatic treatments are currently under development. Oxidative and nitrosative stress have been identified as key pathogenic factors in the development of DPN and new treatments target these pathways and/or their downstream consequences. Gene therapy and growth factors have also emerged as potential new therapies that target particular cellular compartments as opposed to being delivered systemically. The recognition of the difficulty in reversing established DN has focused efforts on slowing its progression.

Oxidative stress and dysregulation of the taurine transporter in high-glucose-exposed human Schwann cells: implications for pathogenesis of diabetic neuropathy.

In human Schwann cells, the role of taurine in regulating glucose-induced changes in antioxidant defense systems has been examined. Treatment with high glucose for 7 days induced reactive oxygen species, increased 4-hydroxynoneal adducts (20 +/- 5%, P < 0.05) and poly(ADP-ribosyl)ated proteins (40 +/- 13%, P < 0.05). Increases in these markers of oxidative stress were reversed by simultaneous incubation in 0.25 mM taurine. Both high glucose and taurine independently increased superoxide dismutase and catalase activity and decreased glutathione levels, but their effects were not additive. Glucose reduced taurine transporter (TauT) mRNA and protein in a dose-dependent manner with maximal decreases of 66 +/- 6 and 63 +/- 12%, respectively (P < 0.05 both). The V(max) for taurine uptake was decreased in 30 mM glucose from 61 +/- 5 to 42 +/- 3 pmol x min(-1) x mg protein(-1) (P < 0.001). Glucose-induced TauT downregulation could be reversed by inhibition of aldose reductase, a pathway that depletes NADPH and increases osmotic stress and protein glycation. TauT protein was increased more than threefold, and the V(max) for taurine uptake doubled (P < 0.05 both) by prooxidants. TauT downregulation was reversed both by treatment with the antioxidant alpha-lipoic acid, which increased TauT mRNA by 60% and V(max) by 50% (P < 0.05 both), and by the aldose reductase inhibitor sorbinil, which increased TauT mRNA 380% and V(max) by 98% (P < 0.01 both). These data highlight the potential therapeutic benefits of taurine supplementation in diabetic complications and provide mechanisms whereby taurine restoration could be achieved in order to prevent or reverse diabetic complications.

Mast cells: a unique source of renin.

In addition to the traditional renin-angiotensin system, a great deal of evidence favors the existence of numerous independent tissue-specific renin-angiotensin systems. We report that mast cells are an additional source of renin and constitute a unique extrarenal renin-angiotensin system. We use renin-specific antibodies to demonstrate that cardiac mast cells contain renin. Extending this observation to the human mast cell line HMC-1, we show that these mast cells also express renin. The HMC-1 renin RT-PCR product is 100% homologous to Homo sapiens renin. HMC-1 cells also contain renin protein, as demonstrated both by immunoblot and immunocytochemical analyses. Renin released from HMC-1 cells is active; furthermore, HMC-1 cells are able to synthesize renin. It is known that, in the heart, mast cells are found in the interstitium in close proximity to nerves and myocytes, which both express angiotensin II receptors. Inasmuch as myocardial interstitium contains angiotensinogen and angiotensin-converting enzyme, and because we were able to detect renin only in mast cells, we postulate that the release of renin from cardiac mast cells is the pivotal event triggering local formation of angiotensin II. Because of the ubiquity of mast cells, our results represent a unique paradigm for understanding local renin-angiotensin systems, not just in the heart, but in all tissues. Our findings provide a rationale for targeting mast cells in conjunction with renin-angiotensin system inhibitors in the management of angiotensin II-related dysfunctions.