Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR

Abstract

Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained in vitro, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding.

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…This system did not ensure a homogeneous cell survival (Luchinat, personal communication). In their more recent publications, the authors encapsulated 3 × 10 7 cells in agarose threads and delivered fresh medium (supplemented with 2% D 2 O) in the NMR tube without capping the coaxial inlet/outlet tubing with a dialysis membrane. , …”

“…In their more recent publications, the authors encapsulated 3.10 7 cells in agarose threads and delivered fresh medium (supplemented with 2% D2O) in the NMR tube without capping the coaxial inlet/outlet tubing with a dialysis membrane. 269,397 Interestingly, Ito and colleagues also used a flow-probe to maintain insect cells viable in a NMR tube through extended periods of times, but they did not have to encapsulate these cells: they delivered fresh medium at flow rate of 2 mL/hour, which simply kept cells in suspension. 227 Adherent cells have still to be entrapped in hydrogels.…”

Radio Signals from Live Cells: The Coming of Age of In-Cell Solution NMR

“…For protein structure determination in insect cells, the Ito group employed custom-made media supplemented with the desired combination of up to eight different 13 C, 15 N-labeled amino acids . In human cells, the Banci group introduced selectively labeled amino acids in the expressed proteins by using custom-made media supplemented with [ 15 N]­cysteine, [ 13 Cε]­methionine, or [ 15 N]­histidine. , …”

“…The analysis of real-time in-cell NMR spectra greatly benefits from spectral decomposition approaches. The Shekhtman group applied SVD to identify meaningful time-dependent changes in real-time series of spectra. , Later, Luchinat and Banci applied an iterative algorithm, multivariate curve resolution by alternating least-square fitting (MCR-ALS), to reconstruct the pure spectra and the concentration profiles of free and ligand-bound intracellular protein fractions as a function of time from real-time series of 1D and 2D NMR spectra. ,, …”

“…73 In human cells, the Banci group introduced selectively labeled amino acids in the expressed proteins by using custom-made media supplemented with [ 15 N]cysteine, [ 13 Cε]methionine, or [ 15 N]histidine. 85,110 Unlike proteins, nucleic acids are synthesized in vitro and delivered to the desired cells for NMR analysis. Therefore, with the proper synthetic procedure, nucleic acids can be labeled either uniformly or at specific positions.…”