Systematic study of halide-induced ring opening of 2-substituted aziridinium salts and theoretical rationalization of the reaction pathways

The ring-opening reactions of 2-alkyl-substituted 1,1-bis(arylmethyl)- and 1-methyl-1-(1-phenylethyl)aziridinium salts with fluoride, chloride, bromide and iodide in acetonitrile have been evaluated for the first time in a systematic way. The reactions with fluoride afforded regioisomeric mixtures of primary and secondary fluorides, whereas secondary β-chloro, β-bromo and β-iodo amines were obtained as the sole reaction products from the corresponding halides by regiospecific ring opening at the substituted position. Both experimental and computational results revealed that the reaction outcomes in the cases of chloride, bromide and iodide were dictated by product stability through thermodynamic control involving rearrangement of the initially formed primary halides to the more stable secondary halides. The ring opening of the same aziridinium salts with fluoride, however, was shown to be mediated by steric interactions (kinetic control), with the corresponding primary β-fluoro amines being obtained as the main reaction products. Only for 2-acylaziridinium ions was the reaction outcome shown to be under full substrate control, affording secondary β-fluoro, β-chloro, β-bromo and β-iodo amines through exclusive attack at the activated α-carbonyl carbon atom

A "target class" screen to identify activators of two-pore domain potassium (K2P) channels

Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a key role in regulating resting membrane potential and cellular excitability. Accumulating evidence points to a role for K2Ps in human pathophysiologies, most notably in pain and migraine, making them attractive targets for therapeutic intervention. However, there remains a lack of selective pharmacological tools. The aim of this work was to apply a "target class" approach to investigate the K2P superfamily and identify novel activators across all the described subclasses of K2P channels. Target class drug discovery allows for the leveraging of accumulated knowledge and maximizing synergies across a family of targets and serves as an additional approach to standard target-based screening. A common assay platform using baculovirus (BacMam) to transiently express K2P channels in mammalian cells and a thallium flux assay to determine channel activity was developed, allowing the simultaneous screening of multiple targets. Importantly, this system, by allowing precise titration of channel function, allows optimization to facilitate the identification of activators. A representative set of channels (THIK-1, TWIK-1, TREK-2, TASK-3, and TASK-2) were screened against a library of Food and Drug Administration (FDA)-approved compounds and the LifeArc Index Set. Activators were then analyzed in concentration-response format across all channels to assess selectivity. Using the target class approach to investigate the K2P channels has enabled us to determine which of the K2Ps are amenable to small-molecule activation, de-risk multiple channels from a technical point of view, and identify a diverse range of previously undescribed pharmacology

Structural analysis of the PATZ1 BTB domain homodimer

PATZ1 is a ubiquitously expressed transcriptional repressor belonging to the ZBTB family that is functionally expressed in T lymphocytes. PATZ1 targets the CD8 gene in lymphocyte development and interacts with the p53 protein to control genes that are important in proliferation and in the DNA-damage response. PATZ1 exerts its activity through an N-terminal BTB domain that mediates dimerization and co-repressor interactions and a C-terminal zinc-finger motif-containing domain that mediates DNA binding. Here, the crystal structures of the murine and zebrafish PATZ1 BTB domains are reported at 2.3 and 1.8 Å resolution, respectively. The structures revealed that the PATZ1 BTB domain forms a stable homodimer with a lateral surface groove, as in other ZBTB structures. Analysis of the lateral groove revealed a large acidic patch in this region, which contrasts with the previously resolved basic co-repressor binding interface of BCL6. A large 30-amino-acid glycine- and alanine-rich central loop, which is unique to mammalian PATZ1 amongst all ZBTB proteins, could not be resolved, probably owing to its flexibility. Molecular-dynamics simulations suggest a contribution of this loop to modulation of the mammalian BTB dimerization interface

Aprepitant is a novel, selective activator of the K2P channel TRAAK

TRAAK (KCNK4, K2P4.1) is a mechanosensitive two-pore domain potassium (K2P) channel. Due to its expression within sensory neurons and genetic link to neuropathic pain it represents a promising potential target for novel analgesics. In common with many other channels in the wider K2P sub-family, there remains a paucity of small molecule pharmacological tools. Specifically, there is a lack of molecules selective for TRAAK over the other members of the TREK subfamily of K2P channels. We developed a thallium flux assay to allow high throughput screening of compounds and facilitate the identification of novel TRAAK activators. Using a library of ∼1200 drug like molecules we identified Aprepitant as a small molecule activator of TRAAK. Aprepitant is an NK-1 antagonist used to treat nausea and vomiting. Close structural analogues of Aprepitant and a range of NK-1 antagonists were also selected or designed for purchase or brief chemical synthesis and screened for their ability to activate TRAAK. Electrophysiology experiments confirmed that Aprepitant activates both the 'long' and 'short' transcript variants of TRAAK. We also demonstrated that Aprepitant is selective and does not activate other members of the K2P superfamily. This work describes the development of a high throughput assay to identify potential TRAAK activators and subsequent identification and confirmation of the novel TRAAK activator Aprepitant. This discovery identifies a useful tool compound which can be used to further probe the function of TRAAK K2P channels

Pharmacological inhibition of MERTK induces in vivo retinal degeneration: a multimodal imaging ocular safety assessment

The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes

Terbinafine is a novel and selective activator of the two-pore domain potassium channel TASK3

Two-pore domain potassium channels (K2Ps) are characterized by their four transmembrane domain and two-pore topology. They carry background (or leak) potassium current in a variety of cell types. Despite a number of important roles there is currently a lack of pharmacological tools with which to further probe K2P function. We have developed a cell-based thallium flux assay, using baculovirus delivered TASK3 (TWIK-related acid-sensitive K+ channel 3, KCNK9, K2P9.1) with the aim of identifying novel, selective TASK3 activators. After screening a library of 1000 compounds, including drug-like and FDA approved molecules, we identified Terbinafine as an activator of TASK3. In a thallium flux assay a pEC50 of 6.2 ( ±0.12) was observed. When Terbinafine was screened against TASK2, TREK2, THIK1, TWIK1 and TRESK no activation was observed in thallium flux assays. Several analogues of Terbinafine were also purchased and structure activity relationships examined. To confirm Terbinafine's activation of TASK3 whole cell patch clamp electrophysiology was carried out and clear potentiation observed in both the wild type channel and the pathophysiological, Birk-Barel syndrome associated, G236R TASK3 mutant. No activity at TASK1 was observed in electrophysiology studies. In conclusion, we have identified the first selective activator of the two-pore domain potassium channel TASK3

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