Introduction to NMR in Drug Discovery

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					  Introduction to NMR in Drug Discovery




      image from: www.varianinc.com




                                      image from: www.bruker-biospin.com


Dr. Brian Cutting 16.12.09
Although expensive, strong NMR magnets are purchased




 each dome contains a
 800 or 900 MHz

                                            center contains
                                            5 additional 600
                                            MHz magnets
                 Some Recent Applications of NMR

                          Widening the View on Dispersant
                          Pigment Interactions in Colloidal
                          Dispersions with Saturation Transfer
                          Difference NMR Spectroscopy




                                 Quantitative Description of Backbone Conformational Sampling of Unfolded
                                 Proteins at Amino Acid Resolution from NMR Residual Dipolar Couplings




J. Am. Chem. Soc., 131,                                          NMR Structure in a Membrane Environment Reveals
                                                                 Putative Amyloidogenic Regions of the SEVI
   (49), Dec. 16, 2009                                           Precursor Peptide PAP248 86
Introduction to NMR in Drug Discovery



                 Contents

            Some basic concepts
   The NMR chemical shift & ligand binding
           Internuclear distances
              Epitope mapping
       Spin relaxation & ligand binding
               Scalar Coupling
         Others interested in Magnetic Resonance




  Otto Stern       Isidor Rabi         Felix Block Edward Purcell
 Physics 1943     Physics 1944                 Physics 1952




 Richard Ernst   Kurt Wuethrich      Peter Mansfield Paul Lauterbur
Chemistry 1991   Chemistry 2002              Medicine 2003

                        http://nobelprize.org/
                      Intrinsic Spin

1925 Goudsmit and Uhlenbeck suggest electon has an
intrinsic angular momentum, hence a magnetic moment.

1928 Relativistic Quantum Mechanical            n, l, ml, ms
treatment by Dirac suggests need of a
4th quantum number.



    Theory explains Stern-
    Gerlach atomic beam
    experiments




                  Beam is split depending on value of ms
Many isotopes have a nuclear spin



                           I≠ 0
                potentially interesting for NMR




                                                  adapted from
                                                  P.J. Hore OCPpg2
Outside NMR, random dipole orientation




          zero NET magnetic moment
Inside NMR, slight preference in dipole orientation




                                                    net magnetic
                                                      moment




      NON-zero NET magnetic moment from individual nuclei
           NMR Spectrum of H2O




                                 F.I.D.




Spectrum
               High-Resolution NMR



Ethanol 1951                              Ethanol 2009




                                                         CH3
                                              CH2
                                     OH




          Identify compounds through “chemical shift”
Fourier Transform NMR
Measure all signals at once




                  Fourier
                 Transform
   Chemical shifts (roughly) measure electron density


     flow of electrons
      around nucleus

                                Energy




      electron motion
induces a magnetic field that
  opposes that of the NMR
Complicated molecules have complex NMR spectra




                                                             aliphatic side-chain H



                           aromatic H
                                               backbone Hα

                 backbone HN   side-chain HN




                                                                         adapted from
                                                                         P.J. Hore OCPpg8


  complex pattern of peaks represent ≈ 600 hydrogen nuclei
             Chemical Shifts can Indicate Folding





“the
spectrum
of
a
globular
protein
is
more
complex
than
the
sum
of
the
NMR
lines
from
the
cons=tuent
amino
acid
residues
in
the
polypep=de
chain.”


                                        




excerpt
from
Kurt
Wüthrich’s
Nobel
Prize
lecture
                  NMR in Quality Control



    protein
“A”
:

 known
ligands
bind




     protein
“B”
:

known
ligands
do
not
bind

Inhibiting Kinase Phosphorylation
           19F    NMR as a functional assay




                                                                     19F
screening
of
inhibi=on
                                                                    
of
the
Ser/Thr
kinase
AKT1





                                                                        reac=on
quenched
aPer

                                                                        ≈3.5h
with
Staurosporine





Dalvit,
C.,
Ardini,
E.,
FogliaXo,
G.P.,
Mongelli,
N.,
Veronesi,
M.
DDT,
9,
595,
2004
Receptor Observed Methods: “Chemical Shift Mapping”



Kinase
“X”



                 low‐affinity
                   ligand




             1)
chemical
shiP
change
is
propor=onal
to
[ligand]
                   
        result
yields
the
KD

             2)
if
NMR
assignments
are
known,

                            binding
site
is
iden2fied


      Receptor Observed Methods: “SAR by NMR”





add 1st ligand,                   



add 2nd ligand,
identify binding site                 identify binding site




              Shuker,
S.B.,
Hajduk,
P.J.,
Meadows,
R.P.,
Fesik,
S.W.
Science,
274,
1531,
1996
                                    Roberts,
G.C.K.,
DDT,
5,
230,
2000
NMR Distance Restraints from NOESY Spectra



                                                                            
N




                                                                Nuclei
“talk”
to
one
another
                                                              if
they
are
within
5
Angstroms





     Kumar,
A.,
Wagner,
G.,
Ernst,
R.R.,
Wuethrich,
K.
J.
Am.
Chem.
Soc.
103,
3654,
1981
Bigger NOE Signal Indicate Shorter Internuclear Distance




                                                NOE spectrum after 64ms
                                              NOE measurements originating from
                                                not much transfer to other nuclei
                                              the HN(H68) of Ubiquitin




        Chiarparin,
E.,
Pelupessy,
P.,
Cugng,
B.,
Eykyn,
T.R.,
Bodenhausen,
G.
J.
Biomol.
NMR
13,
61,
1999
The Nuclear Overhauser Effect Depends on Tumbling rate



                             small
ligands
                               NOE
≈
0
                          “NO”
NOE
SIGNALS

  NOE efficiency




                                       large
receptors
                                          NOE
≈
-1
                                   STRONG NOE SIGNALS




                                                  adapted
from
                                                  OCPpg64
Application of the Transferred NOESY Experiment




              Becagni,
B.,
Pellecchia,
M.
Chem.
Eur.
J.
12,
2658,
2006
           Li,
D.,
DeRose,
E.F.,
London,
R.E.
J.
Biomol.
NMR
15,
71,
1994
                           Saturation Transfer Difference NMR
                           Determination of Ligand Binding Epitopes

                                                                                    b        c
                                                                             a
                                                                                                       STD
spectrum

                                                                             a
                                                                                    b        c

                                                                                                       reference
spectrum




                                                                STD
=   (





















)
                                                                             Signal
(STD
spectrum)
                                                                         Signal
(reference
spectrum)
                                                                                                        x
(scaling
factor)




                                                                                        STDa
=
25%

                                                                                        STDb
=
50%

                                                                                        STDc
=
100%
M.
Mayer,
B.
Meyer
Angew.
Chem.
Int.
Ed.,
1999,
38,
1784‐1788
B.
Cugng
et
al.
Mag.
Reson.
Chem.,
2007,
45,
720‐724
Saturation Transfer Difference NMR in Practice
           Agreement between traditional SAR




Benzamide moiety removed
   affinity 700x reduced



                           Benzamide AND benzyl moieties removed
                                  affinity >5000x reduced
     Longitudinal relaxation measures -z to +z recovery



         z       1800
                              z            z            z                z
                 pulse

             y                    y            y            y                y
x                        x            x            x            x


                                                                     time



    ΔE                   ΔE           ΔE           ΔE               ΔE
Relaxation rates depends on molecular weight




  Relaxation rate




                                                           molecular weight

                    B.J.
Stockman,
C.
Dalvit
Prog.
NMR.
Spect.,
2002,
41,
1877
     Measuring relaxation to determine binding




  ligand

                  ligand alone




                    ligand with
                      receptor



                                               time(s)


a small ligand binding to a large receptor increases the relaxation rate
                Scalar Coupling
         who has a chemical bond with whom
energy


                                            1J
                                                 CH(formate)   = 195 Hz




                     1H   spectra without scalar coupling



                     1H   spectra with scalar coupling


                                                                     adapted from
                                                                     OCPpg22,23
    Observing Scalar Coupling
electron must penetrate into the nucleus




                   sp3-bond : 1JCH = 125 Hz
                    sp-bond : 1JCH = 250 Hz


                                              adapted from
                                              OCPpg34,19
                     NMR protein structure determination
                         blending magnets with modeling




                                                                           10
conformers
aPer
1
                                                                           “CANDID”
cycle
                          Stimulated Annealing
NMR observables
- chemical shifts
- scalar couplings
- NOEs
- additional data

                                                                         20
conformers
aPer
7
                                                                         “CANDID”

cycles




                                                   Herrmann,
T.,
Guentert,
P.,
Wuethrich.
K.
                                                         J.
Mol.
Biol.
319,
209,
2002


                             no modeling = no structure
Large Protein Structures may be Solved by NMR
Structure of the Membrane-Integral Diacylglycerol Kinase




                             homotrimer MW (42 kD) + DPC micelle (≈100 kD)

                                        = Total MW ≈ 142 kD



                                    van Horn et al., Science, 2009, 324, 1726