Fluorine-NMR Competition Binding Experiments for High-Throughput Screening of Large Compound Mixtures

Abstract

High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection. Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target. A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands. Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits. Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened. Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.

NMR methods in fragment based drug discovery

“…In our K-Ras project, we had a trouble with a biochemical guanine nucleotide exchange assay due to a narrow dynamic range of read-out signals and to a compound precipitation issue. Fluorine-NMR competition binding experiments 12 , exploiting fluorine's ultrahigh sensitive line-broadening feature upon target biding events, could be an effective solution to overcome the aforementioned problems. In our fluorine-NMR competition binding experiments, we were able to successfully measure the dissociation binding constants of tested compounds, minimizing the precipitation issue by lowering the concentrations of K-Ras binding 'spy' molecule and synthesized compounds to 20 µM and 200 µM, respectively, which are one-fifth of 1 mM used in our biochemical assay.…”

Theoretical analysis of the competition ligand‐based NMR experiments and selected applications to fragment screening and binding constant measurements

“…It is evident that in the slow motion regime, the D F R 2 is dominated by Hi R 1,s so that fluorine anti-phase magnetization decays faster than in-phase magnetization. In a CPMG pulse sequence with long t CP , typically used for the fluorine chemical shift anisotropy and exchange for screening (FAXS) experiments (25)(26)(27), the transverse relaxation can be approximated to…”

“…Water suppression is required in these experiments, whereas water presaturation is recommended as discussed in the text. [25] where I 0 corresponds to the Zeeman magnetization transferred to the xy plane via a p/2 pulse, 2t CP is the CPMG pulse separation period, n is the number of cycles of the spin-echo scheme filter, t is the separation period between the selective p pulse and the hard p/2 pulse used in the R 1,s filter experiment (as described in Fig. 13), and o 0 is the apparent precession frequency of the spy resonance.…”

SEAL by NMR: Glyco‐Based Selenium‐Labeled Affinity Ligands Detected by NMR Spectroscopy

“…We foresee that SEAL by NMR will be useful for the screening of selenium-containing ligands in general, the selenium atom acting as an NMR-handle, reporting on binding. The technique which is operationally straightforward could be extended with an R 2 relaxation-filter [36] or be used in a competition-based approach with a 77 Se-labeled weak ligand acting as a spy molecule [7,37,38] . It should be particularly suitable for ligand screening with a large number of compounds and it is also tolerant to various conditions, otherwise challenging for 1 H NMR-based experiments, such as the presence of H 2 O, buffers, detergents, additives or even whole cell systems.…”