Drug Discovery – Small Molecule Binding by NMR
BCMB/CHEM 8190
SAR by NMRShuker, Hajduk, Meadows, Fesik, Science, 274, 1531 (1996)
KA KBKAB
KA = 2 x 103, KB = 5 x 103 , KAB = 1 x 107
ΔGAB = ΔGA + ΔGB, RTln(K) = - ΔGAB , KAB = KAx KB
Chemokine CCL5 Interacting with Chondroitin Sulfate Oligomer – Where is the Oligomer?
Crystal Structure: Murooka et al, JBC 281:25184 (2006)
Titration of CCL5 with Chondroitin Sulfate Pentamer
R47
I24
R47
K45T43
N46
W57
V42
Q48
V49
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0
1H PPM
15N
PP
M13
0.0
125.
0
12
0.0
115.
0
11
0.0
1
05.0
Dissociation constant & Stoichiometry Determined
P/L=1:1 P/L=2:1
Docking Structures Using HADDOCKDe Vries, …, Bonvin, A. (2007) Prot.-Struct. Funct. and Bioinfo. 69, 726-733.
SAR by NMR ExamplesFrom 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))
Protein
Ligand
σijf σij
b
σipb
σjpb
K-1
K1
Transfer NOE
*Cross-relaxation rate α τc, r, ω0
*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
0.0
-1.0
0.5
τc = 1.1(1/ ω0)
Rates of transfer (σ) are proportional to τc for large molecules.
Observe: NOE(obs) = p(bound)•σ(bound) + p(free)•σ(free)
NOE from bound state dominates for p(bound)/p(free) > 0.05
α-Me Mannose Bound to MBP
φ
ωψ
Man I
Man III
Man II
3’
4’1
Transfer NOE studies of Trimannoside with MBP
2
Schematic representation of trimannoside core structure depicting some of the intra and inter-residue NOEs observed
Methyl 3,6-di-O-(α-D-mannopyranosyl)-(α-D-mannopyranoside)
Pulse Sequence for Selective 1D NOE90x 180y 180y 90+/-x 90x
Gradient
x y
z
x y
z
x y
zωa
ωbx y
z
ωa ωbx y
z
Behavior for selected resonance from two different volume elements on +x 90° pulse. Vectors return to +z with –x 90° pulse.
Mix
Selective saturation
NOE
trNOE
a
b
c
H1 H1’ H1’’ H2
H3’
H4’
-OCH3
Tris
Selective saturation NOE difference spectra for trimannoside
0.1 mM MBP, 1 mM Trimannoside
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)
H1
H1H1
Spin Diffusion is Efficient in Macromolecules –Saturation of Protein Spins transfers to Ligands
1/T1,2 = ∑ij Ji (ωi) | Dij|2 , Ji (ωi) = 2τc/(ωi2 τc
2 + 1)
1/T2 ≈ (const) Ji (ωi) | Di(I+I-, I-I+) |2 for large τc
H1
H1H1
H1
H1H1
Saturation Transfer Difference Method (STD) for Ligand Screening
Peptides Used in STD Experiments
STD Results
STD can also be used in screening
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