Competition STD NMR for the detection of high‐affinity ligands and NMR‐based screening

Abstract

The reported competition STD NMR method combines saturation transfer difference (STD) NMR with competition binding experiments to allow the detection of high-affinity ligands that undergo slow chemical exchange on the NMR time-scale. With this technique, the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappearance or reduction of the STD signals of a low-affinity indicator ligand. This is demonstrated on a BACE1 (b-site amyloid precursor protein cleaving enzyme 1) protein-inhibitor system. This method can also be used to derive an approximate value, or a lower limit, for the dissociation constant of the potential ligand based on the reduction of the signal intensity of the STD indicator, which is illustrated on an HSA (human serum albumin) model system. This leads to important applications of the competition STD NMR method for lead discovery: it can be used (i) for compound library screening against a broad range of drug targets to identify both highand low-affinity ligands and (ii) to rank order analogs rapidly and derive structure-activity relationships, which are used to optimize these NMR hits into viable drug leads.

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

“…Furthermore, the competition STD method was successfully applied to detect the presence of a competing high-affinity ligand, diazepam, in a compound mixture by the reduction of the STD signals of a low-affinity ligand, β-site amyloid precursor protein cleaving enzyme 1 [64].…”

NMR in Pharmacokinetic and Pharmacodynamic Profiling

“…Longitudinal relaxation rates and saturation transfer difference (STD) NMR competition experiments with tryptophan can accurately measure compound binding specifically to the indole-binding site of fulllength albumin. [35,36] As shown in Figure 3, the STD NMR-based experiments monitor the reduction in STD NOEs to a probe compound in the presence of a competing ligand. [36] Because an STD experiment can be collected rapidly (typicallỹ 15 min), the throughput of this assay is sufficient to evaluate hundreds of compounds for their ability to bind to serum albumin.…”

Human Serum Albumin Binding of 2-[(Carboxymethyl)sulfanyl]-4-oxo-4-(4-tert-butylphenyl)butanoic Acid and its Mono-Me Ester

“…Binding of SOM to proteins is studied by different instrumental techniques [8][9][10][11][12]. Fluorescence measurements are often used to study SOM binding to proteins because information on binding mechanism, binding mode, binding constants, binding sites, intermolecular distances, etc., can be easily obtained.…”