Pranlukast is a novel small molecule activator of the two-pore domain potassium channel TREK2.

TREK2 (KCNK10, K2P10.1) is a two-pore domain potassium (K2P) channel and a potential target for the treatment of pain. Like the majority of the K2P superfamily, there is currently a lack of useful pharmacological tools to study TREK2. Here we present a strategy for identifying novel TREK2 activators. A cell-based thallium flux assay was developed and used to screen a library of drug-like molecules, from which we identified the CysLT1 antagonist Pranlukast as a novel activator of TREK2. This compound was selective for TREK2 versus TREK1 and showed no activity at TRAAK. Pranlukast was also screened against other members of the K2P superfamily. Several close analogues of Pranlukast and other CysLT1 antagonists were also tested for their ability to activate K2P channels. Consistent with previous work, structure activity relationships showed that subtle structural changes to these analogues completely attenuated the activation of TREK2, whereas for TREK1, analogues moved from activators to inhibitors. Pranlukast's activity was also confirmed using whole-cell patch clamp electrophysiology. Studies using mutant forms of TREK2 suggest Pranlukast does not bind in the K2P modulator pocket or the BL-1249 binding site. Pranlukast therefore represents a novel tool by which to study the mechanism of TREK2 activation.

Transcription factors NRF2 and HSF1 have opposing functions in autophagy.

Autophagy plays a critical role in the maintenance of cellular homeostasis by degrading proteins, lipids and organelles. Autophagy is activated in response to stress, but its regulation in the context of other stress response pathways, such as those mediated by heat shock factor 1 (HSF1) and nuclear factor-erythroid 2 p45-related factor 2 (NRF2), is not well understood. We found that the Michael acceptor bis(2-hydoxybenzylidene)acetone (HBB2), a dual activator of NRF2 and HSF1, protects against the development of UV irradiation-mediated cutaneous squamous cell carcinoma in mice. We further show that HBB2 is an inducer of autophagy. In cells, HBB2 increases the levels of the autophagy-cargo protein p62/sequestosome 1, and the lipidated form of microtubule-associated protein light chain 3 isoform B. Activation of autophagy by HBB2 is impaired in NRF2-deficient cells, which have reduced autophagic flux and low basal and induced levels of p62. Conversely, HSF1-deficient cells have increased autophagic flux under both basal as well as HBB2-induced conditions, accompanied by increased p62 levels. Our findings suggest that NRF2 and HSF1 have opposing roles during autophagy, and illustrate the existence of tight mechanistic links between the cellular stress responses.

Dimethylarginine dimethylaminohydrolase 1 is involved in spinal nociceptive plasticity.

Activation of neuronal nitric oxide synthase, and consequent production of nitric oxide (NO), contributes to spinal hyperexcitability and enhanced pain sensation. All NOS isoforms are inhibited endogenously by asymmetric dimethylarginine, which itself is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Inhibition of DDAH can indirectly attenuate NO production by elevating asymmetric dimethylarginine concentrations. Here, we show that the DDAH-1 isoform is constitutively active in the nervous system, specifically in the spinal dorsal horn. DDAH-1 was found to be expressed in sensory neurons within both the dorsal root ganglia and spinal dorsal horn; L-291 (NG-[2-Methoxyethyl]-L-arginine methyl ester), a DDAH-1 inhibitor, reduced NO synthesis in cultured dorsal root ganglia neurons. Spinal application of L-291 decreased N-methyl-D-aspartate-dependent postdischarge and windup of dorsal horn sensory neurons--2 measures of spinal hyperexcitability. Finally, spinal application of L-291 reduced both neuronal and behavioral measures of formalin-induced central sensitization. Thus, DDAH-1 may be a potential therapeutic target in neuronal disorders, such as chronic pain, where elevated NO is a contributing factor.