Targeting fatty acid amide hydrolase as a therapeutic strategy for antitussive therapy.

Cough is the most common reason to visit a primary care physician, yet it remains an unmet medical need. Fatty acid amide hydrolase (FAAH) is an enzyme that breaks down endocannabinoids, and inhibition of FAAH produces analgesic and anti-inflammatory effects. Cannabinoids inhibit vagal sensory nerve activation and the cough reflex, so it was hypothesised that FAAH inhibition would produce antitussive activity via elevation of endocannabinoids.Primary vagal ganglia neurons, tissue bioassay, in vivo electrophysiology and a conscious guinea pig cough model were utilised to investigate a role for fatty acid amides in modulating sensory nerve activation in vagal afferents.FAAH inhibition produced antitussive activity in guinea pigs with concomitant plasma elevation of the fatty acid amides N-arachidonoylethanolamide (anandamide), palmitoylethanolamide, N-oleoylethanolamide and linoleoylethanolamide. Palmitoylethanolamide inhibited tussive stimulus-induced activation of guinea pig airway innervating vagal ganglia neurons, depolarisation of guinea pig and human vagus, and firing of C-fibre afferents. These effects were mediated via a cannabinoid CB2/Gi/o-coupled pathway and activation of protein phosphatase 2A, resulting in increased calcium sensitivity of calcium-activated potassium channels.These findings identify FAAH inhibition as a target for the development of novel, antitussive agents without the undesirable side-effects of direct cannabinoid receptor agonists.

The chimeric approach reveals that differences in the TRPV1 pore domain determine species-specific sensitivity to block of heat activation.

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

The biophysical and molecular basis of TRPV1 proton gating.

The capsaicin receptor TRPV1, a member of the transient receptor potential family of non-selective cation channels is a polymodal nociceptor. Noxious thermal stimuli, protons, and the alkaloid irritant capsaicin open the channel. The mechanisms of heat and capsaicin activation have been linked to voltage-dependent gating in TRPV1. However, until now it was unclear whether proton activation or potentiation or both are linked to a similar voltage-dependent mechanism and which molecular determinants underlie the proton gating. Using the whole-cell patch-clamp technique, we show that protons activate and potentiate TRPV1 by shifting the voltage dependence of the activation curves towards more physiological membrane potentials. We further identified a key residue within the pore region of TRPV1, F660, to be critical for voltage-dependent proton activation and potentiation. We conclude that proton activation and potentiation of TRPV1 are both voltage dependent and that amino acid 660 is essential for proton-mediated gating of TRPV1.

A novel series of positive modulators of the AMPA receptor: discovery and structure based hit-to-lead studies.

Starting from an HTS derived hit 1, application of biostructural data facilitated rapid optimization to lead 22, a novel AMPA receptor modulator. This is the first demonstration of how structure based drug design can be exploited in an optimization program for a glutamate receptor.

Development of a novel high-throughput assay for the investigation of GlyT-1b neurotransmitter transporter function.

The glycine transporter (GlyT-1b) is a Na(+)/Cl(-)-dependent electrogenic transporter which mediates the rapid re-uptake of glycine from the synaptic cleft. Based on its tissue distribution, GlyT-1 has been suggested to co-localise with the NMDA receptor where it may modulate the concentration of glycine at its co-agonist binding site. This data has led to GlyT-1 inhibitors being proposed as targets for disorders such as schizophrenia and cognitive dysfunction. Radiolabelled uptake assays (e.g. [(3)H]glycine) have been traditionally used in compound screening to identify glycine transporter inhibitors. While such an assay format is useful for testing limited numbers of compounds, the identification of novel glycine uptake inhibitors requires a functional assay compatible with high-throughput screening (HTS) of large compound libraries. Here, the authors present the development of a novel homogenous cell-based assay using the FLIPR membrane potential blue dye (Molecular Devices) and FLEXstation. Pharmacological data for the GlyT-1 inhibitors Org 24598 and ALX 5407 obtained using this novel electrogenic assay correlated well with the conventional [(3)H]-glycine uptake assay format. Furthermore, the assay has been successfully miniaturised using FLIPR(3) and therefore has the potential to be used for high-throughput screening.

Voltage-gated K+ channel from mammalian brain: 3D structure at 18A of the complete (alpha)4(beta)4 complex.

Voltage-sensitive K(+) channels (Kv) serve numerous important roles, e.g. in the control of neuron excitability and the patterns of synaptic activity. Here, we use electron microscopy (EM) and single particle analysis to obtain the first, complete structure of Kv1 channels, purified from rat brain, which contain four transmembrane channel-forming alpha-subunits and four cytoplasmically-associated beta-subunits. The 18A resolution structure reveals an asymmetric, dumb-bell-shaped complex with 4-fold symmetry, a length of 140A and variable width. By fitting published X-ray data for recombinant components to our EM map, the modulatory (beta)(4) was assigned to the innermost 105A end, the N-terminal (T1)(4) domain of the alpha-subunit to the central 50A moiety and the pore-containing portion to the 125A membrane part. At this resolution, the selectivity filter could not be localised. Direct contact of the membrane component with the central (T1)(4) domain occurs only via peripheral connectors, permitting communication between the channel and beta-subunits for coupling of responses to changes in excitability and metabolic status of neurons.

Characteristics of brain Kv1 channels tailored to mimic native counterparts by tandem linkage of alpha subunits: implications for K+ channelopathies.

Most neuronal Kv1 channels contain Kv1.1, Kv1.2 alpha, and Kvbeta2.1 subunits, yet the influences of their stoichiometries on properties of the (alpha)(4)(beta)(4) variants remain undefined. cDNAs were engineered to contain 0, 1, 2, or 4 copies of Kv1.1 with the requisite number of Kv1.2 and co-expressed in mammalian cells with Kvbeta2.1 to achieve "native-like" hetero-oligomers. The monomeric (Kv1.1 or 1.2), dimeric (Kv1.1-1.2 or 1.2-1.2), and tetrameric (Kv1.1-(1.2)(3)) constructs produced proteins of M(r) approximately 62,000, 120,000, and 240,000, which assembled into (alpha)(4)(beta)(4) complexes. Each alpha cRNA yielded a distinct K(+) current in oocytes, with voltage dependence of activation being shifted negatively as the Kv1.1 content in tetramers was increased. Channels containing 1, 2, or 4 copies of Kv1.1 were blocked by dendrotoxin k (DTX)(k) with similarly high potencies, whereas Kv(1.2)(4) proved nonsusceptible. Accordingly, Kv1.2/beta2.1 expressed in baby hamster kidney cells failed to bind DTX(k); in contrast, oligomers containing only one Kv1.1 subunit in a tetramer exhibited high affinity, with additional copies causing modest increases. Thus, one Kv1.1 subunit largely confers high affinity for DTX(k), whereas channel electrophysiological properties are tailored by the content of Kv1.1 relative to Kv1.2. This notable advance could explain the diversity of symptoms of human episodic ataxia I, which is often accompanied by myokymia, due to mutated Kv1.1 being assembled in different combinations with wild-type and Kv1.2.