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