• Share
  • Email
  • Embed
  • Like
  • Save
  • Private Content
Nmr In Drug Discovery 04

Nmr In Drug Discovery 04



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

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



Total Views
Views on SlideShare
Embed Views



5 Embeds 256

http://www.dr-cee.net 153
http://new.dr-cee.net 75
http://dr-cee.net 25
http://www.slideshare.net 2
http://localhost 1



Upload Details

Uploaded via as Adobe PDF

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
Post Comment
Edit your comment

    Nmr In Drug Discovery 04 Nmr In Drug Discovery 04 Presentation Transcript

    • 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 • optimise two lead ligands at proximal binding sites • link ligands  obtain high affinity bidentate ligand (nM!)
    • • 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 tumbling tumbling and relaxation fast relaxation slow relaxation similar to R
    • • 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
    • 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
    • • 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!!!)
    • • 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