Exploring Ligand Affinities for Proteins by NMR of Long-Lived States

The detection of molecules that can bind to active sites of protein targets and the measurement of their affinities is a promising application of NMR. Nowadays, the screening of drug candidates is routinely done by NMR in pharmaceutical industry. We have proposed to use the relaxation of Long-Lived States (LLS) for drug screening by NMR. Long-lived states are nuclear spin states whose decay time constant Tlls can be much longer than the longitudinal relaxation time T1. LLS can be used to screen and determine the dissociation constant KD of molecular fragments that bind weakly to protein targets. The use of LLS for fragment screening leads to a spectacular increase in contrast between free and bound ligands, and thus allows one to characterize binding of fragments with very weak affinities, with KD in the millimolar range, which is difficult to achieve by other methods such as ITC. By exploiting the LLS behavior of a spy molecule, we experimentally demonstrate that it is possible to measure dissociation constants KD as large as 12 mM, corresponding to very weak binding, where most other biophysical techniques fail, including other NMR methods based on the observation of ligands. Furthermore, we have combined LLS for screening for improved contrast with 1H dissolution-DNP to enhance the sensitivity. DNP-enhanced screening for measuring LLS signals of a weak ligand allows one to use very low concentrations of ligands and proteins. We observed dramatic differences between the spectra of the ligand in the presence or absence of a protein, or in the presence of the protein combined with a stronger ligand. Moreover, we have explored LLS involving pairs of 19F nuclei to study binding phenomena. Indeed, fluorine detection is quite interesting because it offers the possibility to perform screening experiments without any problems due to overlapping signals. In a customdesigned fluorinated ligand that binds trypsin, we have observed a promising ratio Tlls / T1 > 4. This fluorinated ligand has been used as spy molecule in competition experiments, which allowed us to rank the affinities and estimate dissociation constants of arbitrary ligands that do not contain any fluorine.

Determining a receptor-ligand interaction useful in e.g. drug screening involves repetitive application of reversibly binding fluorescent ligands with fast association-dissociation kinetics

Marinela Gjoni Boillat, Rudolf Hovius, Christoph Schreiter, Jean-Manuel Segura, Horst Vogel

NOVELTY - Determining a receptor-ligand interaction involves repeatedly contacting a cell comprising a receptor, with a ligand, of the receptor in which the ligand has a fluorescence labeling group and binds reversibly to the receptor under conditions in which the ligand associates with the receptor and then dissociates; and then repeatedly monitoring the fluorescence of the cell. USE - In a screening procedure for the identification and/or characterization of pharmaceutical drug candidate molecules and for determining a receptor-ligand interaction to allow sensitive measurements on single cells carried out in a microfluidic structure or on a chip (claimed) and can be used to characterize single or multiple cells by determining the presence of receptors or other proteins on the surface of the cells e.g. native cells, multiple cells like tissue sections, genetically modified cells or with cells immobilized on a solid surface, or in a matrix like a gel, or with cells in a liquid medium. ADVANTAGE - The method enables pharmacological investigations to be performed on single cells expressing the natural amount of receptor, which usually is very difficult due to photobleaching. The invention performs fluorescence binding assays, the reversible sequential binding assay that solves problems associated with previous measurements of ligand-receptor interactions by applying repetitively-completely reversible and specific fluorescently labeled ligands with fast association-dissociation kinetics. The new approach overcomes problems related to photobleaching so that cells expressing low amounts of receptors down to the single-molecule level can be investigated. The high sensitivity of the method allows the detection of little abundant proteins, thus enabling characterization of native cells. It can be applied even in the case of strong photobleaching opening the possibility to investigate single cells with low receptor concentrations comparable to native conditions in tissue cells. Furthermore, the influence of stimuli, such as ligands, can be rapidly and repetitively investigated using our reversible binding assay. A key advantage of the measurement approach is that pharmacological investigations can be performed on single cells expressing the natural amount of receptor, which usually is very difficult due to photobleaching. Using repetitive measurements, binding parameters can be extracted and investigations can be performed even when photobleaching becomes extremely strong as it is the case at lowest expression levels. Ultimately, single-molecule sensitivity can be achieved as illustrated on the nAChR. This opens the possibility to perform pharmacological investigations on tissue cells expressing minimal amounts of membrane receptors. By performing repetitive measurements, tenth of image sequences can be acquired to collect extensive statistics within a single experiment. High-throughput screening of potential drugs can be performed on a single cell with a rate only depending on the association-dissociation cycle time, consuming only minimal amount of testing components. One can expect that the use of ligands with faster kinetics could reduce this time down to 5 minutes. In addition, the use of more complex microstructures enables complete binding assays on a single cell or increasing statistics by parallel approaches using only some hundred nano liters of cell suspension. This way, screening more than 250 ligands per day using one single cell extracted from an animal tissue or obtained from a body fluid is a realistic goal.

2006

Ligand-Protein Affinity Studies Using Long-Lived States of Fluorine-19 Nuclei

Geoffrey Bodenhausen, Roberto Buratto, Estel Canet Martinez, Daniele Mammoli

The lifetimes T-LLS of long-lived states or T-LLC of long-lived coherences can be used for the accurate determination of dissociation constants of weak protein ligand complexes. The remarkable contrast between signals derived from LLS or LLC in free and bound ligands can be exploited to search for weak binders with large dissociation constants K-D > 1 mM that are important for fragment-based drug discovery but may escape detection by other screening techniques. Alternatively, the high sensitivity of the proposed method can be exploited to work with large ligand-to-protein ratios, with an evident advantage of reduced consumption of precious proteins. The detection of F-19-F-19 long-lived states in suitably designed fluorinated spy molecules allows one to perform competition binding experiments with high sensitivity while avoiding signal overlap that tends to hamper the interpretation of proton spectra of mixtures.

American Chemical Society2016

Determining a receptor-ligand interaction useful in e.g. drug screening involves repetitive application of reversibly binding fluorescent ligands with fast association-dissociation kinetics

Marinela Gjoni Boillat, Rudolf Hovius, Christoph Schreiter, Jean-Manuel Segura, Horst Vogel

2006