The student will be trained in expertise available both in the School of Biological Sciences (Dr Samuel Fountain) and the School of Pharmacy (Professor Mark Searcey), chiefly biological assays of ion channel function, molecular biology, combinatorial chemistry and in silico drug design.  Cell biology will include maintenance and growth of human cells, use of lentivirus to generate stable gene knockdown cell lines, and ELISA and qRT-PCR to quantify markers of inflammation.

The project:

P2X receptors (P2X1-P2X7) comprise a family of ligand-gated ion channels activated by extracellular ATP.  Mammalian cells release ATP constitutively and in response to physiology and pathophysiological cues, and consequently P2X receptor activation is associated with both physiological (breathing, taste and blood pressure) and pathological processes.  The P2X4 receptor is a calcium permeable ATP-activated channel associated with signalling during pain, inflammation and cardiovascular disease.  Research into the contribution of P2X receptors in cellular function during wellbeing and disease has been severely hampered by poor selective pharmacology, particularly pertinent for the P2X4 receptor subtype which lack selective agonists, antagonists or allosteric modulators.  Discovery of small molecules that modulate P2X4 function would greatly accelerate basic research into the role P2X4 plays in wellbeing and disease, and may also provide building blocks and lead compounds for future therapies for the treatment of pain, inflammation and cardiovascular disease.  Recently the crystal structure of the P2X4 receptor in an open and ATP bound state was resolved, paving the way for in silico drug design and molecular docking experiments.

The studentship will employ in silico screening, molecular docking, compound library screening, and rational drug design in combination with biological assays to discovery new small molecules which antagonise and modulate the activity of the human P2X4 receptor.  Biological assays of ion channel activity will include high throughput calcium influx to identify lead compounds and patch clamp electrophysiology on recombinant human P2X4 to determine modes of action and drug kinetics.  Site-directed mutagenesis will be employed to determine the structural requirements for drug action at the receptor level, and combinatorial chemistry to determine chemical moieties important for drug function.  The combinatorial chemistry approach will start with several newly identified lead compounds discovered by the group.

Later experiments will determine the ability of identified compounds to modulate the inflammatory function of human monocytes and microglia.  The dependency of drug action on native P2X4 receptors will be determined by stably suppressing P2X4 gene expression by lentivirus.  Currently experiments in P2X4 knockout mice identify a role for P2X4 in the development of chronic and neuropathic pain though the molecular mechanisms are unclear, again hampered by poor pharmacology.  The project therefore has the potential to define novel mechanisms of pain and inflammation, major factors influencing human wellbeing.

Sivaramakrishnan V, Bidula S, Campwala H, Katikaneni D, Fountain SJ, 2012.  Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes.  J Cell Sci. (In Press).

Li J & Fountain SJ, 2012.  Fluvastatin suppresses native and recombinant human P2X4 receptor function.  Purinergic Signal. 8(2):311-6.

Fountain SJ, North RA, 2006.  A C-terminal lysine that controls human P2X4 receptor desensitization.  J Biol Chem. 281(22):15044-9.

Steel RA, Cowan J, Payerne E, O'Connell MA, Searcey M, 2012. Anti-inflammatory effect of a cell penetrating peptide targeting the Nrf2/Keap1 interaction. ACS Med. Chem. Lett.  3: 407-410.

Shaginian A, Whitby LR, Hong S, Hwang I, Farooqi B, Searcey M, Chen J, Vogt PK, Boger DL, 2009. Design, synthesis and evaluation of an a-helic mimetic library targeting protein-protein interactions. J. Am. Chem. Soc. 131, 5564-5572