Christiane Riedinger - Nov’04

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

•  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

•  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

An example of a protein experiencing chemical shift perturbations upon ligand binding.

•  SAR = “Structure-Activity-Relationships” obtained by NMR

•  screen for low-affinity ligands (mM) by chemical shift mapping

•  link ligands obtain high affinity bidentate ligand (nM!)

•  optimise two lead ligands at proximal binding sites

•  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

•  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

•  relaxation enhancement: T2 of ligand decreases as receptor is added

•  acquire spectrum of free ligand and ligand + receptor detect binding!

slow tumbling fast relaxation

fast tumbling slow relaxation

tumbling and relaxation similar to R

•  relaxation also depends on gyromagnetic ratio (γ) of nuclei

•  γ (e- •) = 658 • γ (p+)

•  relaxation rate of nuclei close to paramagnetic centre is increased

•  molecules containing an unpaired electron are paramagnetic

•  this effect is dependent on the distance (p+- e- •), ~ 1/r6

•  Paramagnetic Relaxation Enhancement (PRE)

•  measure distances of up to 20 Å

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:

2. spin-labelled ligand, observe protein resonances

1. spin-labelled protein, observe ligand

•  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!!!)

•  common spin label: TEMPO

•  2,2,6,6-tetramethyl-1-piperidine-N-oxyl

•  residues that can be spin labelled: Lys, Tyr, Cys, His, Met

•  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