Ligand-Based Nuclear Magnetic Resonance Screening Techniques

Abstract

A critical step in the drug discovery process is the identification of high-affinity ligands for macromolecular targets, and, over the last 10 years, NMR spectroscopy has become a powerful tool in the pharmaceutical industry. Instrumental improvements in recent years have contributed significantly to this development. Digital recording, cryogenic probes, autosamplers, and higher magnetic fields shorten the time for data acquisition and improve the spectral quality. In addition, new experiments and pulse sequences make a vast amount of information available for the drug discovery process. All these techniques take advantage of the fact that upon complex formation between a target molecule and a ligand, significant perturbations can be observed in NMR-sensitive parameters of either the target or the ligand. These perturbations can be used qualitatively to detect ligand binding or quantitatively to assess the strength of the binding interaction. In addition, some of the techniques allow the identification of the ligand-binding site or which part of the ligand is responsible for interacting with the target.In this chapter, we will use examples from our own research to illustrate how NMR experiments to characterize ligand binding may be used to both screen for novel compounds during the process of lead generation, and provide structural information useful for lead optimization during the latter stages of a discovery program.

Current NMR Techniques for Structure-Based Drug Discovery

“…Although the translational diffusion time is less sensitive to the molecular weight of a sample compared to the rotational correlation time [ 33 ], small lined and protein are easily distinguished. The translational diffusion-based approach can be used for pulse schemes to edit coherences, such as COSY-DOSY, TOCSY-DOSY, HSQC-DOSY, NOESY-DOSY and STD-DOSY [ 34 , 35 , 36 ]. These experiments are quite powerful for selective observation of NMR signals from bound-state ligand eliminating signals from free-state ligand and vice versa [ 34 , 35 , 36 ].…”

“…The translational diffusion-based approach can be used for pulse schemes to edit coherences, such as COSY-DOSY, TOCSY-DOSY, HSQC-DOSY, NOESY-DOSY and STD-DOSY [ 34 , 35 , 36 ]. These experiments are quite powerful for selective observation of NMR signals from bound-state ligand eliminating signals from free-state ligand and vice versa [ 34 , 35 , 36 ]. NOE-pumping pulse techniques [ 37 , 38 ] are useful for observing NOE cross-peaks of bound-state ligands, with higher sensitivity and selectivity by filtering signals derived from the free-state ligand before the NOE mixing time.…”

“…0.2 ns) and 30 kDa (τ c = ca. 20 ns), respectively, the intra-ligand NOE becomes 20 times stronger if the ligand interacted with the protein [ 36 ]. Therefore, protein-ligand interactions can be assessed by NOE-based methods, such as saturation-transfer difference (STD) [ 39 , 40 ], SOS-NMR [ 41 ], WaterLOGSY and its related methods [ 42 , 43 ], transferred NOE (trNOE) [ 44 ], INPHARMA [ 45 , 46 ], and inter-ligand NOE (ILOE) [ 47 ].…”

“…The 19 F nucleus, which is a 100% naturally abundant fluorine NMR-visible isotope, is absent in biomolecules, and its NMR sensitivity is comparable to that of 1 H (~83%). 19 F-NMR has a wide chemical shift range and is sensitive in terms of reflecting local chemical environments other than 1 H. Transverse relaxation of the 19 F spin is dominated by chemical shift anisotropy (CSA), even at a lower magnetic field, such as 500 MHz [ 36 ]. The CSA-dominated transverse spin relaxation is sensitive to τ c [ 36 ], so 19 F-NMR is sensitive to τ c of the sample molecules and gives sharp signal for small molecules.…”

“…19 F-NMR has a wide chemical shift range and is sensitive in terms of reflecting local chemical environments other than 1 H. Transverse relaxation of the 19 F spin is dominated by chemical shift anisotropy (CSA), even at a lower magnetic field, such as 500 MHz [ 36 ]. The CSA-dominated transverse spin relaxation is sensitive to τ c [ 36 ], so 19 F-NMR is sensitive to τ c of the sample molecules and gives sharp signal for small molecules. 19 F-NMR is also a sensitive technique used for the examination of interactions between protein and 19 F-containing ligand, and can be applied in cases involving short-lived bound states that include only about 1–3% of the total protein/ligand population [ 36 ].…”