Drug Discovery – Small Molecule
Binding by NMR
BCMB/CHEM 8190
SAR by NMR
Shuker, Hajduk, Meadows, Fesik, Science, 274, 1531 (1996)
KA
KB
GAB = GA + GB,
KAB
RTln(K) = - GAB ,
KAB = KAx KB
KA = 2 x 103, KB = 5 x 103 , KAB = 1 x 107
Chemokine CCL5 Interacting with Chondroitin
Sulfate Oligomer – Where is the Oligomer?
Crystal Structure: Murooka et al,
JBC 281:25184 (2006)
110.0
105.0
Titration of CCL5 with Chondroitin Sulfate Pentamer
15N PPM
115.0
120.0
N46
K45
T43
R47
I24
V49
125.0
V42
Q48
130.0
R47
W57
10.5
10.0
9.5
9.0
8.5
8.0
1H PPM
7.5
7.0
6.5
6.0
Dissociation constant & Stoichiometry Determined
P/L=1:1
P/L=2:1
Langmuir isotherm is often mistakenly used: correct binding formula:
FB 
[ P]  [Tb3 ]  K D  ([P]  [Tb 3 ]  K D ) 2  4[ P][Tb3 ]
2[ P]
Docking Structures Using HADDOCK
De Vries, …, Bonvin, A. (2007) Prot.-Struct. Funct. and Bioinfo. 69, 726-733.
SAR by NMR Examples
From Stockman, (1998) Progress in NMR Spec, 33, 109-151
FKBP
Stromelysin
Transferred NOEs in Drug Discovery
• Determine the geometry of a bound ligand
(Sayers and Prestegard, Biophysical J, 81, 0000 (2001))
• Screening of a library of potential ligands
(Meinecke and Meyer, J. Med. Chem, 44, 3059 (2001))
• NMR as a tool for structure-based drug
design
(B. Stockman, Prog. NMR Spec., 33, 109 (1998))
Transfer NOE
sipb
sijf
K-1
sijb
K1
Protein
sjpb
Ligand
*Cross-relaxation rate  c, r, 0
*Chemical exchange fast w.r.t cross-relaxation rate and chemical shift scale
*Observed NOE is weighted average of free and bound states
*Change in sign of NOE upon change in molecular weight
*Spin Diffusion and on, off rates can complicate interpretation
NOEs from large and small molecules
have opposite signs
0.5
0.0
-1.0
c = 1.1(1/0)
Rates of transfer (s) are proportional to c for large molecules.
Observe: NOE(obs) = p(bound)•s(bound) + p(free)•s(free)
NOE from bound state dominates for p(bound)/p(free) > 0.05
-Me Mannose Bound to MBP
Transfer NOE studies of Trimannoside with MBP
Man II
f
4’
1
2
y

Man III
3’
Man I
Methyl 3,6-di-O-(-D-mannopyranosyl)-(-D-mannopyranoside)
Schematic representation of trimannoside core structure
depicting some of the intra and inter-residue NOEs observed
Pulse Sequence for Selective 1D NOE
90x
180y
180y
90x
90+/-x
Mix
Gradient
z
x
z
y x
y x
a b
z
a
b
y
x
z
z
y
x
y
Behavior for selected resonance from two different volume
elements on +x 90° pulse. Vectors return to +z with –x 90° pulse.
c
Tris
0.1 mM MBP, 1 mM Trimannoside
trNOE
b
NOE
-OCH3
Selective
saturation
H3’
H1
H1’
H1’’
H2
a
H4’
Selective saturation NOE difference spectra for trimannoside
STD References
• Saturation Transfer Difference, Mayer, M & Meyer, B.
JACS, 123, 6108-6117 (2001)
• Perspectives on NMR in drug discovery: a technique
comes of age, Pellecchia M; et al., Nature Reviews
Drug Discovery, 7: 738-745 (2008)
• STD-NMR: application to transient interactions
between biomolecules-a quantitative approach,
Angulo J and Nieto PM, European Biophysics J.,
40:1357-1369 (2011)
• NMR Screening and Hit Validation in Fragment Based
Drug Discovery, Campos-Olivas R. Current Topics
Med. Chem., 11:43-67 (2011)
Spin Diffusion is Efficient in Macromolecules –
Saturation of Protein Spins transfers to Ligands
1/T1,2 = ij Ji (i) | Dij|2 ,
Ji (i) = 2c/(i2 c2 + 1)
1/T2  (const) Ji (i) | Di(I+I-, I-I+) |2 for large c
H1
H1
H1
H1
H1
H1
H1
H1
H1
Saturation Transfer Difference Method (STD)
for Ligand Screening
Peptides Used in STD Experiments
STD Results
STD can also be used in screening