Prof Chas Bountra
|Research Area:||Protein Science and Structural Biology|
|Technology Exchange:||Computational biology, Crystallography, Drug discovery, Immunohistochemistry and In vivo imaging|
|Scientific Themes:||Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology|
|Keywords:||target validation, drug discovery, translational science, structural biology, chemical probes and epigenetics|
My interests are in identifying and validating targets for drug discovery. I recognise the limitations of recombinant cell systems and in vitro and in vivo preclinical assays. Recent work has focussed on:
- functional studies in human native cells eg CB2 receptors and TRPV1 channels
- developing new technologies for above eg scanning ion conductance microscopy
- studying changes in expression of proteins in human diseased tissue eg idiopathic rectal hypersensitivity and paroxysmal extreme pain disorder
- establishing new biomarkers for clinical Proof of Concept eg Contact Heat Evoked Potentials (CHEPs) for assessment of small fibre neuropathies, and correlation of skin flare responses with intra epidermal nerve counts, and
- using fMRI to evaluate novel analgesic compounds in patients.
I have used above technologies and strategies to progress clinical candidates into Phase I, II, III and to market, in Neurodegenerative and Gastrointestinal diseases, and Pain disorders.
My current focus is to use knowledge of 3D structure of human protein families, to identify chemical probes, and then use these for target validation (by evaluation in human functional assays, native cells and diseases tissues). I believe that chemical probes have several advantages over other target validation technologies eg knockouts, localisation and RNAi. The department is currently focussed on kinases, metabolic enzymes and human proteins involved in Genome Integrity and Epigenetic control. Our disease focus is primarily oncology, but we recognise potential of our targets/ probes in immunology, metabolic and neurodegenerative diseases.
|Prof Christopher Schofield||Chemistry||Oxford University||GBR|
|Rob Klose||University of Oxford||UK|
|Chris Austin||National Chemical Genomic Centre, NIH, Washington||USA|
|David LLoyd||Trinity College, Dublin||Ireland|
|Luke O'Neill||Trinity College, Dublin||Ireland|
|David Gavaghan||University of Oxford||UK|
Cannabinoid receptor 2 (CB2) agonists provide the potential for treating chronic pain states without CNS effects associated with CB1 receptor activation. Animal models suggest that they act mainly via non-neuronal cells, possibly inhibition of inflammatory cells in the periphery or CNS, or via release of beta-endorphin; however, the clinical relevance and mechanism of analgesic action is uncertain. Here, we demonstrate colocalisation of CB2 with CB1 and the capsaicin receptor TRPV1 in human dorsal root ganglion (DRG) sensory neurons and increased levels of CB2 receptors in human peripheral nerves after injury, particularly painful neuromas. In primary cultures of human DRG neurons, selective CB2 agonists blocked activation of inward cation currents and elevation of cytoplasmic Ca2+ in response to capsaicin. These inhibitory effects were reversed by GW818646X a CB2 antagonist, and 8-bromo cAMP, but not by SR141716 a CB1 antagonist, or naloxone. Thus CB2 receptor agonists functionally inhibited nociceptive signalling in human primary sensory neurons via a mechanism shared with opioids, of adenylyl cyclase inhibition, but not via mu-opioid receptors. We conclude that CB2 agonists deserve imminent clinical trials for nociceptive, inflammatory and neuropathic chronic pain, in which capsaicin or heat-activated responses via TRPV1 may provide a clinical marker. Hide abstract
Handb Clin Neurol, 89 pp. 273-290. | Read more2008. Perspectives of Alzheimer's disease treatments.
Faecal urgency and incontinence with rectal hypersensitivity is a chronic, unexplained condition that is difficult to treat. The aim of this study was to determine if there was an altered level of the voltage gated tetrodotoxin-sensitive (TTX-s) sodium channel Na(v)1.7 in rectal sensory fibres, since this channel has been implicated in clinical nociceptive disorders. Full thickness rectal biopsies from patients with physiologically characterised rectal hypersensitivity (n=7) were compared with control tissues (n=10). Formalin fixed specimens were studied by immunohistochemistry using affinity purified antibodies to Na(v)1.7 and the pan-neuronal structural marker PGP9.5, and the immunoreactive nerve fibres quantified by computerised image analysis. In rectal hypersensitivity, Na(v)1.7 immunoreactive nerve fibres were significantly increased in mucosal (P=0.0004), sub-mucosal (P=0.019), and muscle layers (P=0.0076), while PGP9.5 immunoreactive nerve fibres were increased significantly only in the mucosa (P=0.04); ratios of Na(v)1.7:PGP9.5 showed a significant increase in all layers, suggesting increased expression of Na(v)1.7, and nerve sprouting in the mucosa. The cause of this increase remains uncertain, but may be due to increase of nerve growth factor (NGF), which regulates the expression of both Na(v)1.7 and TRPV1, which we have previously reported to be increased in this condition. In paroxysmal extreme pain disorder (familial rectal pain), where the gene that encodes Na(v)1.7 is mutated, Na(v)1.7 protein was undetectable in the rectum (n=2), which suggests reduced Na(v)1.7 immunoreactivity or expression. Drugs that target Na(v)1.7-expressing nerve terminals may be useful for treating rectal hypersensitivity, and combining these with TRPV1 antagonists may enhance efficacy. Hide abstract
Mechanosensitive ion channels convert external mechanical force into electrical and chemical signals in cells, but their physiological function in different tissues is not clearly understood. One reason for this is that there is as yet no satisfactory physiological method to stimulate these channels in living cells. Using the nanopipette-probe of the Scanning Ion Conductance Microscope (SICM), we have developed a new technique to apply local mechanical stimulus to living cells to an area of about 0.385 microm2, determined by the pipette diameter. Our method prevents any physical contact and damage to the cell membrane by use of a pressure jet applied via the nanopipette. The study used whole-cell patch-clamp recordings and measurements of intracellular Ca2+ concentration to validate the application of the mechanical stimulation protocols in human and rat dorsal root ganglia (DRG) sensory neurons. We were able, for the first time, to produce a non-contact, controlled mechanical stimulation on living neurites of human DRG neurons. Our methods will enable the identification and characterisation of compounds being developed for the treatment of clinical mechanical hypersensitivity states. Hide abstract
Chronic pain is a major problem for the individual and for society. Despite a range of drugs being available to treat chronic pain, only inadequate pain relief can be achieved for many patients. There is therefore a need for the development of new analgesic compounds. The assessment of pain depends to date entirely on the subjective report of the patient, in contrast to many other clinical conditions where biomarkers that help determine the severity and stage of the disease enable the physician to monitor the course of the disease and treatment effects longitudinally. In this article, we illustrate that magnetic resonance-based imaging techniques have the potential to provide sensitive and specific biomarkers of the pain experience, as well as clarifying disease mechanisms. Functional magnetic resonance imaging (FMRI) is particularly suited to investigating the effects of pharmacological agents on pain processing within the human central nervous system. Combination of FMRI and drug administration is termed pharmacological FMRI (phFMRI). In addition to outlining several methodological considerations that have to be taken into account when performing phFMRI, we discuss phFMRI studies that have already used this technique to study the effects of analgesic compounds. These studies provide promising data for the use of phFMRI as sensitive tool in assessing a potential drug effect. Such pharmacodynamic readouts obtained early in the process of drug development would not only save the pharmaceutical industry substantial amounts of money, but would also avoid the unnecessary exposure of patients to molecules with limited or no therapeutic value. We are therefore optimistic that phFMRI will be used as a tool with high sensitivity and specificity for evaluating analgesic agents in early drug development and clinical studies. Hide abstract
Previous human imaging studies have revealed a network of brain regions involved in the processing of allodynic pain; this includes prefrontal areas, insula, cingulate cortex, primary and secondary somatosensory cortices and parietal association areas. In this study, the neural correlates of the perceived intensity of allodynic pain in neuropathic pain patients were investigated. In eight patients, dynamic mechanical allodynia was provoked and brain responses recorded using functional magnetic resonance imaging (fMRI). Voxels in which the magnitude of fMRI signal correlated linearly with the ratings of allodynic pain across the group were determined in a whole brain analysis using a general linear model. To ensure that activation reflected only allodynic pain ratings, a nuisance variable containing ratings of ongoing pain was included in the analysis. We found that the magnitude of activation in the caudal anterior insula (cAI) correlates with the perceived intensity of allodynic pain across subjects, independent of the level of ongoing pain. However, the peak of activation in the allodynic condition was located in the rostral portion (rAI). This matches the representation of other clinical pain syndromes, confirmed by a literature review. In contrast, experimental pain in healthy volunteers resides predominantly in the cAI, as shown by the same literature review. Taken together, our data and the literature review suggest a functional segregation of anterior insular cortex. Hide abstract
We have studied the effect of key neurotrophic factors (NTFs) on morphology, levels of the vanilloid receptor-1 (TRPV1) and responses to capsaicin in adult human sensory neurons in vitro. Avulsed dorsal root ganglia (DRG, n = 5) were cultured with or without a combination of nerve growth factor (NGF), glial cell (line)-derived growth factor (GDNF) and neurotrophin3 (NT3) for 5 days. In the absence of NTFs, the diameter of neurons ranged from 20 to 100 microm (mean 42 +/- 4 microm). Adding NTFs caused a significant increase in neuronal sizes, up to 120 microm (mean diameter 62 +/- 5 microm, P < 0.01, t-test), an overall 35% increase of TRPV1-positive neurons (P < 0.003), and notably of large TRPV1-positive neurons > 80 microm (P < 0.05). Responses to capsaicin were significantly enhanced with calcium ratiometry (P < 0.0001). Short duration (1h) exposure of dissociated sensory neurons to NTFs increased numbers of TRPV1-positive neurons, but not of TRPV3, Nav 1.8 and IK1 and the morphological size-distribution remained similar to intact post-mortem DRG neurons. NTFs thus increase size, elevate TRPV1 levels and enhance capsaicin responses in cultured human DRG neurons; these changes may relate to pathophysiology in disease states, and provide an in vitro model to study novel analgesics. Hide abstract
The P2X(7) purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)-1beta following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1beta, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL-1beta and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1. In addition, we show that this receptor is upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients. We hypothesise that the P2X(7) receptor, via regulation of mature IL-1beta production, plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin. Drugs which block this target may have the potential to deliver broad-spectrum analgesia. Hide abstract