Nmr In Drug Discovery 04

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A presentation I did as a student for a journal club ages ago (2004). No guarantee that everything is correct!!! ;-)

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Nmr In Drug Discovery 04

  1. 1. Christiane Riedinger - Nov’04
  2. 2. 1. NMR in Drug Discovery M. Pellecchia, D.S. Sem and K. Wuethrich Nature Reviews, March 2002 2. Mapping Protein-Protein Interactions in Solution by NMR Spectroscopy E.R.P. Zuiderweg Biochemistry, January 2002 3. Spin Labels as a Tool to Identify and Characterize Protein-Ligand Interactions by NMR Spectroscopy W. Jahnke ChemBioChem, March 2002
  3. 3. • NMR: structure determination and characterisation of molecular dynamics • Drug Discovery: optimisation of lead compounds • Use of NMR to detect and investigate molecular interactions • Advantages :-) : high sensitivity for weak interactions no false positives potential to obtain structural information atomic resolution • Disadvantages :-( : need for large amounts of soluble protein
  4. 4. • using 15N or 13C-labelled protein, acquire HSQC • carry out titration with ligand, monitored by HSQC • ligand alters chemical environment around binding site • this causes perturbation of chemical shift observed in HSQC • if HSQC assigned  mapping of the interface • furthermore: estimation of stoichiometry, affinity, kinetics, specificity
  5. 5. An example of a protein experiencing chemical shift perturbations upon ligand binding.
  6. 6. • SAR = “Structure-Activity-Relationships” obtained by NMR • screen for low-affinity ligands (mM) by chemical shift mapping • optimise two lead ligands at proximal binding sites • link ligands  obtain high affinity bidentate ligand (nM!)
  7. 7. • cross relaxation occurring between nuclei close in space (dipolar coupling) • change of intensity of one resonance when the other is perturbed (saturated) • NOEs can be measured within a 5Å distance between nuclei • measure intra-ligand and ligand-protein distances
  8. 8. • two relaxation mechanisms of perturbed spins: 1.  Magnetisation parallel to the magnetic field (Mz) returns to equilibrium longitudinal relaxation - T1 2.  Magnetisation perpendicular to magnetic field (Mxy) returns to zero transverse relaxation - T2 • relaxation time depends on tumbling rate of molecule in solution • small molecules tumble quickly, large molecules tumble slowly •  large molecules relax much quicker than small molecules
  9. 9. • relaxation enhancement: T2 of ligand decreases as receptor is added • acquire spectrum of free ligand and ligand + receptor  detect binding! slow tumbling fast tumbling tumbling and relaxation fast relaxation slow relaxation similar to R
  10. 10. • relaxation also depends on gyromagnetic ratio (γ) of nuclei • γ (e- •) = 658 • γ (p+) • molecules containing an unpaired electron are paramagnetic •  relaxation rate of nuclei close to paramagnetic centre is increased • Paramagnetic Relaxation Enhancement (PRE) • this effect is dependent on the distance (p+- e- •), ~ 1/r6 • measure distances of up to 20 Å
  11. 11. Different Effects of Paramagnetics: • some cause chemical shift changes, but no peak broadening (e.g. Eu3+) • some cause no chemical shift changes, but significant broadening (e.g. Mn2+, Cu2+) Two Possibilities: 1. spin-labelled protein, observe ligand 2. spin-labelled ligand, observe protein resonances
  12. 12. • common spin label: TEMPO • 2,2,6,6-tetramethyl-1-piperidine-N-oxyl • residues that can be spin labelled: Lys, Tyr, Cys, His, Met • difference in relaxation rate of ligand upon binding largely enhanced • advantage :-) : amounts of protein needed are much smaller • disadvantage :-( : exchange between bound/unbound state must be fast (in case of tight binder with slow exchange, you don’t detect anything!!!)
  13. 13. • if ligand contains Mg(II), exchange for Mn(II) • if ligand small organic inhibitor, add NO• - substituent • map the changes observed in HSQC onto structure • use degree of broadening to measure distance to paramagnetic site

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