Application of NMR and Molecular Docking in Structure-Based Drug Discovery - NMR of Proteins and Small Biomolecules - page 5

Fig. 1 An example of the use of a ligand-detect NMR experiment to observe the line broadening
(increase R 2 ) that occurs when one compound, in a mixture of two compounds, binds a protein
target. The 1 H-NOESY spectra of nicotinic acid ( left structure) and 2-phenoxybenzoic acid
( right structure) in a mixture without protein ( top spectrum) and with the protein, p38 MAP
kinase, added ( bottom spectrum). The solid and dashed arrows represent the resonances of
nicotinic acid and 2-phenoxybenzoic acid, respectively. In this case, the resonances corresponding
to 2-phenoxybenzoic acid are broadened, indicating binding of this compound to the protein.
(Reprinted with permission from [ 178 ], copyright 2001 by Academic Press)
NMR ligand affinity screen and protein molecular weight is not a limiting factor
[ 21 ]. In fact, higher molecular-weight proteins enhance the observation of a binding
event in a ligand-based NMR screen. All of these characteristics make ligand-based
NMR screens a routinely used drug discovery technique.
There are several screening techniques created from ligand-based NMR experi-
ments: line broadening [ 56 ], STD NMR [ 57 ], WaterLOGSY [ 58 ], SLAPSTIC
[ 59 ], TINS [ 60 ], transferred NOEs [ 61 ], FAXS [ 62 , 63 ], FABS [ 64 , 65 ], and diffusion
measurements [ 66 , 67 ]. Each of these methods utilizes a specific NMR parameter that
indicates ligand-binding, such as a change in ligand NMR peak width or diffusion,
a saturation transfer from the protein or solvent to the ligand, an NOE transfer between
the free and bound ligand, a spin-label induced paramagnetic relaxation, or fluorine
chemical shift anisotropy. The choice of which method to use typically depends
upon the protein target and the compound library being screened. In addition,
line broadening and STD, among other techniques, can be used to measure dissocia-
tion constants ( K D )[ 68 , 69 ]. Conversely, ligand-based NMR screens don't provide
any structural information about the protein-ligand complex.