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

1. Christiane Riedinger - Nov’04

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. • 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

5. • 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

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

7. • 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!)

8. • 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

9. • 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

10. • 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

11. • 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

12. 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

13. • 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!!!)

15. • 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