Cloning and pharmacological characterization of the dog P2X7 receptor.

BACKGROUND AND PURPOSE: Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues. EXPERIMENTAL APPROACH: A cDNA encoding the dog P2X7 receptor was isolated from a dog heart cDNA library, expressed in U-2 OS cells using the BacMam viral expression system and characterized in electrophysiological, ethidium accumulation and radioligand binding studies. Native P2X7 receptors were examined by measuring ATP-stimulated interleukin-1beta release in dog and human whole blood. KEY RESULTS: The dog P2X7 receptor was 595 amino acids long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. ATP possessed low millimolar potency at dog P2X7 receptors. 2'-&3'-O-(4benzoylbenzoyl) ATP had slightly higher potency but was a partial agonist. Dog P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies on the recombinant receptor although absolute potency was considerably lower. CONCLUSIONS AND IMPLICATIONS: Dog recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic.

Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist.

Vanilloid receptor-1 (TRPV1) is a non-selective cation channel, predominantly expressed by peripheral sensory neurones, which is known to play a key role in the detection of noxious painful stimuli, such as capsaicin, acid and heat. To date, a number of antagonists have been used to study the physiological role of TRPV1; however, antagonists such as capsazepine are somewhat compromised by non-selective actions at other receptors and apparent modality-specific properties. SB-366791 is a novel, potent, and selective, cinnamide TRPV1 antagonist isolated via high-throughput screening of a large chemical library. In a FLIPR-based Ca(2+)-assay, SB-366791 produced a concentration-dependent inhibition of the response to capsaicin with an apparent pK(b) of 7.74 +/- 0.08. Schild analysis indicated a competitive mechanism of action with a pA2 of 7.71. In electrophysiological experiments, SB-366791 was demonstrated to be an effective antagonist of hTRPV1 when activated by different modalities, such as capsaicin, acid or noxious heat (50 degrees C). Unlike capsazepine, SB-366791 was also an effective antagonist vs. the acid-mediated activation of rTRPV1. With the aim of defining a useful tool compound, we also profiled SB-366791 in a wide range of selectivity assays. SB-366791 had a good selectivity profile exhibiting little or no effect in a panel of 47 binding assays (containing a wide range of G-protein-coupled receptors and ion channels) and a number of electrophysiological assays including hippocampal synaptic transmission and action potential firing of locus coeruleus or dorsal raphe neurones. Furthermore, unlike capsazepine, SB-366791 had no effect on either the hyperpolarisation-activated current (I(h)) or Voltage-gated Ca(2+)-channels (VGCC) in cultured rodent sensory neurones. In summary, SB-366791 is a new TRPV1 antagonist with high potency and an improved selectivity profile with respect to other commonly used TRPV1 antagonists. SB-366791 may therefore prove to be a useful tool to further study the biology of TRPV1.