In addition to Surface Plasmon Resonance (SPR) and X-ray crystallography, Ligand observed NMR is one of the most popular techniques used in Fragment Based Drug Discovery (FBDD). In recent years methodological and technical advancements have enabled NMR-based fragment screening to be performed in full automation and with significantly reduced consumption of fragments and unlabeled target protein. Ligand observed NMR can not only be used as a primary screening tool but also to confirm hits from other screening methods such as Thermal Shift, which results in high quality hits. Furthermore, all experiments can be performed as competition experiments in which a known binder is displaced and the Kd is determined by titration.
Here we present practical aspects on how to setup ligand observed NMR screening experiments in an automated fashion for the three basic experiments: Saturation Transfer Difference (STD), Water-Ligand Observation with Gradient SpectroscopY (waterLOGSY) and relaxation based methods (T1r and T2).
Ligand Observed NMR Screening Experiments:
T2/T1r – Relaxation based methods
T2 and T1r relaxation times of large biomolecules are much shorter than those of fragments. Usually two spectra are measured and compared, one with 10 ms relaxation delay and one with 200 ms relaxation delay. Fragments that bind can be identified from a comparison of the spectra:
In WLOGSY experiments polarization is transferred from water molecules to small molecules by NOE. Binding ligands interact with water that is bound to the protein, while non-binders only “see” the solvent water. The NOE effect, however, from bound water has the opposite sign than the one from free water. As a result, the signal from binding fragments is opposite to signals from non-binders.
Saturation Transfer Difference (STD)
In STD experiments, two spectra are recorded, (1) a spectrum with selective radio frequency (RF) irradiation on the biomolecular target, and (2) a spectrum with off-resonance irradiation.
Saturation of the target due to RF irradiation is transferred to binding ligands via NOE, resulting in a reduced intensity in the on-resonance spectrum. In a difference spectrum of (1) and (2) only signals from binders can be observed.
Key Advantages of NMR Screening
In the new TopSpin release 3.5 patch level 6 parameter sets for WLOGSY and STD are included with optimized experimental settings and routines for automatic acquisition and processing (rpar WLOGSY_PREP, WLOGSY and STDDIFFESGP.3). Parameter sets for T2 and T1r are available on request (firstname.lastname@example.org).
In the new Topspin release 3.5 patch level 6 parameter sets for WLOGSY and STD are included with optimized experimental settings and routines for automatic acquisition and processing (rpar WLOGSY_PREP, WLOGSY and STDDIFFESGP.3). Parameter sets for T2 and T1r are available on request (email@example.com).
Ligand observed NMR is an automated fragment screening technique that yields high quality hits in primary and secondary screens. Nowadays, parameter sets with optimized measurement conditions are available which significantly facilitates the setup of NMR-based screening experiments, even for novice users. In addition, a key advantage is the QC of fragments from 1D 1H spectra which enables the identification of compound degradation and aggregation, avoiding false positives. This is especially useful for confirmation of hits from primary screens performed by using SPR or Thermal Shift.
A.D. Gossert and W. Jahnke, Prog. NMR Spect. 2016
Stefan Jehle1, Helena Kovacs1, Till Kuehn1, and Alvar Gossert2
1 Bruker BioSpin AG, Industriestrasse 26, 8117 Faellanden, Switzerland, 2Novartis Institutes for BioMedical Research, Basel, Switzerland.