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

GAB = GA + GB, RTln(K) = - GAB , 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

K45

T43

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

15

N P

PM

13

0.0

1

25

.0 1

20

.0 1

15

.0 1

10

.0 1

05

.0

Dissociation constant & Stoichiometry Determined

P/L=1:1 P/L=2:1

][2

]][[4)][]([][][ 3233

P

TbPKTbPKTbPF

DD

B

Langmuir isotherm is often mistakenly used: correct binding formula:

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

sijf sij

b

sipb

sjpb

K-1

K1

Transfer NOE

*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

0.0

-1.0

0.5

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

f

y

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-(-D-mannopyranosyl)-(-D-mannopyranoside)

Pulse Sequence for Selective 1D NOE

90x 180y 180y 90+/-x 90x

Gradient

x y

z

x y

z

x y

za

b

x y

z

a bx 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

H1H1’

H1’’ H2

H3’

H4’

-OCH3

Tris

Selective saturation NOE difference spectra for trimannoside

0.1 mM MBP, 1 mM 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)

H1

H1

H1

Spin Diffusion is Efficient in Macromolecules –

Saturation of Protein Spins transfers to Ligands

1/T1,2 = ij Ji (i) | Dij|2 , Ji (i) = 2c/(i

2 c2 + 1)

1/T2 (const) Ji (i) | Di(I+I-, I-I+) |2 for large c

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