Understanding the effect of different assay formats on agonist parameters: a study using the µ-opioid receptor.

The correct interpretation of data is fundamental to the study of G-protein-coupled receptor pharmacology. Often, new assay technologies are assimilated into the drug discovery environment without full consideration of the data generated. In this study, the authors look at µ-opioid receptor agonists in three different assays: (1) [(35)S]GTPγS binding, (2) inhibition of forskolin-stimulated cAMP production, and (3) ß-arrestin recruitment. Agonist-concentration effect curves were performed before and after treatment with the irreversible antagonist ß-funaltrexamine, and where appropriate, these data were fitted to the operational model of agonism. The Z' value was highest in the ß-arrestin assay, followed by the [(35)S]GTPγS and cAMP assays. The cAMP data fitted well to the operational model, as did the [(35)S]GTPγS data, but the [(35)S]GTPγS assay led to an apparent overestimation of K(A) values. However, in the ß-arrestin assay, data did not fit the operational model, as treatment with ß-funaltrexamine reduced the Emax proportionally to receptor number, with no change in EC(50). In addition, the EC(50) values generated correlated well with affinity values. In conclusion, the ß-arrestin recruitment assay does not fit with traditional pharmacological theory but is of great utility as the EC(50) value generated is a good approximation of affinity.

2,4-Diaminopyridine delta-opioid receptor agonists and their associated hERG pharmacology.

A number of libraries were produced to explore the potential of 2,4-diaminopyridine lead 1. The resulting diaminopyridines proved to be potent and selective delta-opioid receptor agonists. Several rounds of lead optimisation using library chemistry identified compound 17 which went on to show efficacy in an electromyography model of neuropathic pain. The structure-activity relationship of the series against the hERG ion channel proved to be a key selectivity hurdle for the series.

The effect of deletion of the orphan G - protein coupled receptor (GPCR) gene MrgE on pain-like behaviours in mice.

BACKGROUND: The orphan GPCR MrgE is one of an extended family of GPCRs that are expressed in dorsal root ganglia (DRG). Based on these expression patterns it has been suggested that GPCRs like MrgE may play a role in nociception however, to date, no direct supporting evidence has emerged. We generated mutant mice lacking MrgE and examined the effects of deletion of this gene in three pain behavioural models. The effect of MrgE gene deletion on expression of Mrgs and genes involved in sensory neurone function was also investigated. RESULTS: The absence of MrgE had no effect on the development of pain responses to a noxious chemical stimulus or an acute thermal stimulus. However, in contrast, the development but not the maintenance of neuropathic pain was affected by deletion of MrgE. The expression of Mrg genes was not significantly affected in the MrgE knockout (KO) mice with the sole exception of MrgF. In addition, the expression of 77 of 84 genes involved in sensory neuron development and function was also unaffected by deletion of MrgE. Of the 7 genes affected by MrgE deletion, 4 have previously been implicated in nociception. CONCLUSION: The data suggests that MrgE may play a role in selective pain behavioural responses in mice.

[3H] pregabalin binding is increased in ipsilateral dorsal horn following chronic constriction injury.

Pregabalin, a 3-substituted analogue of gamma-amino butyric acid has recently been approved for treatment of neuropathic pain. We have investigated the anatomical binding profile of [(3)H] pregabalin following chronic constriction injury (CCI) and compared this with alpha 2 delta 1 subunit expression using in situ hybridisation. We report here that the intensity and distribution pattern of [(3)H] pregabalin binding is altered in the ipsilateral dorsal horn following CCI and this is associated with a corresponding increase in alpha 2 delta 1 mRNA in the ipsilateral dorsal root ganglion (DRG). It is likely that increased DRG mRNA production leads to increased alpha 2 delta 1 protein production and subsequent transport by primary afferents to the dorsal horn. The increased expression of calcium channel subunits and protein in central terminals is interesting, given that abnormal activity within sensory nerves is likely to significantly contribute to the symptomatology of neuropathic pain. The upregulation of pregabalin binding sites in sensory nerve terminals may occur as part of the response to nerve damage in neuropathic pain patients, and therefore, preferential actions of pregabalin at these sites may contribute to its mechanism of action in man.

Identification of the alpha2-delta-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin.

Neuropathic pain is a debilitating condition affecting millions of people around the world and is defined as pain that follows a lesion or dysfunction of the nervous system. This type of pain is difficult to treat, but the novel compounds pregabalin (Lyrica) and gabapentin (Neurontin) have proven clinical efficacy. Unlike traditional analgesics such as nonsteroidal antiinflammatory drugs or narcotics, these agents have no frank antiinflammatory actions and no effect on physiological pain. Although extensive preclinical studies have led to a number of suggestions, until recently their mechanism of action has not been clearly defined. Here, we describe studies on the analgesic effects of pregabalin in a mutant mouse containing a single-point mutation within the gene encoding a specific auxiliary subunit protein (alpha2-delta-1) of voltage-dependent calcium channels. The mice demonstrate normal pain phenotypes and typical responses to other analgesic drugs. We show that the mutation leads to a significant reduction in the binding affinity of pregabalin in the brain and spinal cord and the loss of its analgesic efficacy. These studies show conclusively that the analgesic actions of pregabalin are mediated through the alpha2-delta-1 subunit of voltage-gated calcium channels and establish this subunit as a therapeutic target for pain control.

The human FK506-binding proteins: characterization of human FKBP19.

Analysis of the human repertoire of the FK506-binding protein (FKBP) family of peptidyl-prolyl cis/trans isomerases has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences in tissue distribution and expression levels of each variant. In this article we describe the characterization of human FKBP19 (Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues. The FKBP19 sequence comprises a cleavable N-terminal signal sequence followed by a putative peptidyl-prolyl cis/trans isomerase domain with homology to FKBP12. This domain binds FK506 weakly in vitro. FKBP19 mRNA is abundant in human pancreas and other secretory tissues and high levels of FKBP19 protein are detected in the acinar cells of mouse pancreas.

New targets for neuropathic pain therapeutics.

Neuropathic pain (NeP) is initiated by a lesion or dysfunction in the nervous system. Unlike physiological pain it serves no useful purpose and is usually sustained and chronic. NeP encompasses a wide range of pain syndromes of diverse aetiologies which together account for > 12 million sufferers in the US. Currently, there are a number of therapies available for NeP, including gabapentin, pregabalin, anticonvulsants (tiagabine HCl), tricyclic antidepressants (amitriptyline, nortriptyline) and acetaminophen/opioid combination products (Vicodin, Tylenol #3). However, these products do not provide sufficient pain relief and a significant proportion of sufferers are refractory (60%). Therefore, there is a need for new therapies that provide more predictable efficacy in all patients with improved tolerability. Over the last decade, understanding of the basic mechanisms contributing to the generation of NeP in preclinical animal models has greatly improved. Together with the completion of the various genome sequencing projects and significant advances in microarray and target validation strategies, new therapeutic approaches are being rigourously pursued. This article reviews the rationale behind a number of these mechanism-based approaches, briefly discusses specific challenges that they face, and finally, speculates on the potential of emerging technologies as alternative therapeutic strategies to the traditional 'small-molecule' approach.

Mitogen-activated protein kinase kinase 7 is activated during low potassium-induced apoptosis in rat cerebellar granule neurons.

A stress-activated protein kinase pathway comprising mitogen-activated protein kinase kinases (MKKs), c-Jun N-terminal kinase (JNK) and the transcription factor c-Jun is implicated in neuronal apoptosis. Using an immune-complex kinase assay, we measured the activation of MKK4 and MKK7 in low potassium (LK)-induced apoptosis of rat cerebellar granule neurons (CGN). MKK7, but not MKK4, was activated within the first 4-6 h in four independent sets of 14-h CGN apoptosis time-courses. CEP-1347 (500 nM), an mixed-lineage kinase 3 inhibitor, prevented MKK7 activation and cell death following exposure of CGN cultures to LK-induced apoptosis. Western blot analysis revealed that levels of phosphorylated c-Jun were elevated between 30 min and 10 h of CGN apoptosis, temporally consistent with MKK7 activation. These data suggest that MKK7 is responsible for activating the JNK pathway during LK-induced CGN apoptosis.