PhD projects

ITP FP7 - SBMPs

Structural Biology of Membrane Proteins

PhD project:

Discovery of High Affinity Small Molecule Ligands for Membrane Proteins

16/07/2007




Principal Investigator:

Gregg Siegal, ZoBio, Marc Baldus, MPI, Jean-Luc Popot, C.N.R.S./Université Paris-7, Jean-Louis Ban ères, CNRS Montpellier

Figure 1
Background:

Membrane proteins form approximately a third of the human genome and represent the target for nearly half of all oral drugs. However, they remain largely refractory to modern, struc­ture-based drug design approaches. ZoBio has developed a ligand-screening approach, cal­led TINS, which can be applied to membrane proteins. At present it is necessary to solubi­li­ze mem­­bra­ne proteins for TINS ligand screening. The first goal of this project will be to assess the suitability of alternatives to detergent micelles for ligand screening studies.


Here we propose a collaborative research project whose goals will be to:

1) Produce a number of target membrane proteins in sufficient quantity for ligand screen­ing and structural studies.

2) Solubilize and immobilize the target and reference proteins in a non-detergent medium amenable to ligand-screening studies. ZoBio can provide as a reference protein micelle-solubilized E. coli OmpA. Once immobilized, the amount of functional membrane protein will be quanti­fied by e.g. radioligand binding studies.

3) Carry out ligand screening using the proprietary TINS ligand screening hardware and compound collection from ZoBio.

4) Validate and further characterize hits from the ligand screening studies using in vitro and cellular assays.

5) Develop/apply novel approaches to characterize the structure of membrane protein-ligand complexes using solid state NMR approaches.

6) Use the structural information to select/design second generation small molecule ligands for the membrane protein.


Target Proteins that can potentially be used for the study:

1) E. coli DsbB has already been screened in micellar form at ZoBio. It could be used to direct­ly compare the screening performance with the non-detergent solubilized form.

2) A chimeric K+ channel (KcsA-Kv1.3) from Marc Baldus’s group. Here, high reso­lu­tion ssNMR studies (using 1H,13C,15N,19F or 31P nuclei) and electrophysiology experiments can be performed. Natural and some synthetic channel blockers are available to test the validi­ty of the approach.

3) Production of one or a couple of GPCRs that can be folded in a non-detergent medium has been developed jointly by the groups of Jean-Louis Banères and Jean-Luc Popot (unpublished data). The two receptors under consideration are currently being routinely folded to their native state with ~50% yield (based on ligand binding), which is more than sufficient for the task at hand.


Points to Investigate for some goals:

1) Best expression system.

2) We have screened immobilized membrane proteins that have been solubilized in detergent micelles. While this works, it is suboptimal due to the requirement to maintain low concen­tra­tions of the detergent in all buffers. Here we will investigate alternative media that will be better suited for TINS ligand screening.

3) With soluble systems we have focused on developing inhibitors. Membrane pro­teins and GPCRs in particular, offer the opportunity to seek co-activators. We could consider for exam­ple, screening for compounds that bind in the presence of an agonist and, therefore, might have the poten­tial to function as a co-agonist.

5-6) Although many membrane proteins produced by members of this RTN have been solu­bi­li­zed (a substantial achievement), few are in a condition appropriate for crystal­lo­gra­phic or high-resolution NMR studies. We therefore propose to develop/implement a new approach which focuses on the receptor bound ligand.


A Ph.D. student working for ZoBio will be formally enrolled in a University from one of the academic participants where that person would receive his/her degree. There is more than suf­ficient work for a coherent and complete thesis.

Relevant literature:

  1. Vanwetswinkel,S., Heetebrij,R.J., van Duynhoven,J., Hollander, J.G., Filippov,D.V., Hajduk,P.J. and Siegal, G.,
    TINS: Target Immobilized NMR Screening – An efficient and sensitive new method for ligand discovery, Chem. Biol., 12, 2005, pp. 207-216.
  2. Marquardsen,T., Hofmann,M., Hollander,J.G., Loch, C.M.P., Kiihne,S.R., Engelke,F. and  Siegal,G.
    Development of a dual cell, flow-injection sample holder and NMR probe for comparative ligand-binding studies, J. Mag. Reson., 2006, 182, pp. 55-65.
  3. Lange et al, J. Recept. Signal Transduc., 2006, 26, p. 379.
  4. Pocanschi, C.L., Dahmane, T., Gohon, Y., Rappaport, F., Apell, H.-J., Kleinschmidt, J.H. & Popot, J.-L. (2006).
    Amphipathic polymers: tools to fold integral membrane proteins to their acti­ve form. Biochemistry 45, 13954-13961.
  5. Popot, J.-L., Berry, E.A., Charvolin, D., Creuzenet, C., Ebel, C., Engelman, D.M., Flöten­meyer, M., Giusti, F., Gohon, Y., Hervé, P., Hong, Q., Lakey, J.H., Leonard, K., Shuman, H.A., Timmins, P., Warschawski, D.E., Zito, F., Zoonens, M., Pucci, B. & Tribet, C. (2003).
    Amphipols: polymeric surfactants for membrane biology research. Cell. Mol. Life Sci. 60, 1559-1574.
  6. Zoonens, M., Catoire, L.J., Giusti, F. & Popot, J.-L. (2005).
    NMR study of a membrane pro­tein in detergent-free aqueous solution. Proc. Natl. Acad. Sci. USA 102, 8893-8898.
  7. Zoonens, M., Giusti, F., Zito, F. & Popot, J.-L. (2007).
    Dynamics of membrane protein/am­phi­pol association studied by Förster resonance energy transfer. Biochemistry, in the press.