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Modelling Antagonism and Dye Permeation in P2X7 Receptors

Principal Supervisor: Dr Ralf Schmid, Department of Molecular and Cell Biology

Co-supervisor: Prof Richard Evans

PhD project title: Modelling Antagonism and Dye Permeation in P2X7 Receptors

University of Registration: University of Leicester

Project outline:

P2X receptors (P2XR) form a family of ligand-gated ion channels activated upon binding of extracellular ATP. In humans, there are seven P2XR homologs (P2XR1-P2XR7) that have intracellular N- and C-termini, two transmembrane domains and an extracellular ligand binding domain. P2XR assemble as trimeric ion channels which after channel opening allow the influx of small cations [1]. P2X7R are expressed in a variety of cell types e.g. neurons, oligodendrocytes and macrophages, but have little activity under normal physiological conditions [2]. Extracellular ATP levels increase upon necrosis, cell damage and inflammation leading to activation of P2X7R. Activation of P2X7R can trigger a series of biochemical signalling pathways leading to inflammatory and immune response activation [3], due to this role P2X7R as seen as an emerging target in CNS and other diseases [4]. RS and RE labs have a long-standing successful collaboration working on P2X receptors (2 BBSRC grants, 5 joint publications). The proposed project will focus on two major questions:

Allosteric antagonism. We recently characterised an allosteric binding site in the P2X7R by site directed mutagenesis and molecular modelling [5]. This site was confirmed by X-ray structures of the panda P2X7R with allosteric antagonists bound [6], which allowed us to confirm the accuracy of our modelling approach [7]. The project aims to characterise the energetics of P2X7R allosteric binding by taking advantage of recent progress in free energy perturbation calculations [8] and the characterisation of the chemical inhibitor space with descriptors based on molecular dynamics [9]. Establishing accurate free energy calculations and quantitative pharmacore descriptors for allosteric P2X7R inhibitors are extremely useful for lead optimization, for classification of known antagonists and filtering candidates from virtual screening. Binding modes and pharmacore descriptors for selected antagonists can also be validated experimentally based on voltage clamp of a set of P2X7R mutants that serve as ‘finger prints’ for specific binding modes [7]. Dye permeant state. An intriguing feature of the P2X7R is that the open channel can increase in diameter to allow permeation of large fluorescent dyes (dye permeant state) [10]. This concept is supported by experiments showing time-dependent changes in the Nernst potential and permeability of N-methyl-d-glucamine (NMDG). The structural rearrangements from the currently identified ATP bound open state to the dye permeant state remain to be determined, but further expansion of the open pore of P2X7R is required to allow dye permeation. The project will model in atomistic detail how NMDG is transferred through the ion permeable pore of the P2X7R receptor by using steered MD simulations. NMDG is placed into the extracellular lateral portal (a cavity with access to the pore) and an artificial force perpendicular to the membrane is applied to “pull” NMDG through the transmembrane domains into the intracellular region (alternatively this force is generated by applying an artificial electrostatic potential across the membrane). The conformational changes accomplished by force correspond to the path of minimum energy for the transition. This can be quantified by the calculation of the potential mean force. Typical controls are simulations with P2XR that do not form the dye permeant state. Mechanistic models will be validated by electrophysiological characterisation of P2X7R chimeras and mutants.

References:

  1. North & Jarvis. Mol. Pharm. (2013), 83, 759.
  2. Bartlett et al. Pharm. Rev. (2014), 66, 638.
  3. Orioli et al. Curr. Med. Chem. (2017), 24, 2261.
  4. Sperlágh B & Illes P. Trends Pharm. (2014), 35, 537.
  5. Allsopp et al. Sci. Rep. (2017), 7, 725.
  6. Karasawa & Kawate. Elife (2016), 5, e22153.
  7. Allsopp et al. JBC submitted.
  8. Wang et al. JACS (2015), 137, 2695.
  9. Ash & Fourches. J. Chem. Inf. Mod. (2017).
  10. Wei et al., Front. Pharm. (2016), 7, 5.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

Computational

  • Molecular dynamics simulations of membrane proteins
  • Free energy calculations
  • Ligand docking with receptor
  • Ligand flexibility

Experimental

  • Site directed mutagenesis
  • Voltage clamp

 Contact: Dr Ralf Schmid, University of Leicester