Exploring the Role of Nuclear Magnetic Resonance Spectroscopy in Drug Discovery

Nuclear magnetic resonance spectroscopy (NMR) is becoming increasingly prevalent in drug discovery. The technique has demonstrated a number of advantages in drug design, and has proven to be a useful tool in fragment-based approaches especially. While this technique continues to evolve, improving in sensitivity, it is becoming evident that NMR is an important tool to better understand target-ligand interactions. 

Introduction 

NMR spectroscopy is an analytical chemistry technique used in quality control and research which uses a magnetic field for determining the content and purity of a sample and molecular structure. 

NMR can be used to define the structure and purity of a compound in a solid or liquid state, pure and mixed, and if the sample weight is less than a milligram. When combined with infrared spectroscopy, it provides sufficient information to define the molecular structure. 

Both mass spectrometry (MS) and NMR are popular techniques in research and development, metabolomic studies especially, each with its own advantages and limitations. In comparison with MS, NMR is quantitative and does not require extra steps for sample preparation. 

This is a particular advantage for pharmaceutical companies, saving time and resources that would otherwise be allocated to separation or derivatisation. In addition, NMR gives rise to less false positive hits and a mixture of fragments can be screened.

Despite the significant improvement in sensitivity, this area remains a weak point for NMR spectroscopy compared with MS. The potential for this technique to be widely used in drug discovery however, remains promising and an area of focus for a number of companies across the industry. 

Fragment-based drug discovery (FBDD) in particular has exploited the advancement of NMR techniques, with a number of drug candidates and FDA-approved products have been developed from FBDD with the support of NMR techniques. 

NMR in Fragment-Based Drug Design 

FBDD is a target-based method which identifies chemical fragments and optimises them towards drug-like leads and further to clinical candidates. According to a review in Cell, the process of FBDD runs as follows: FBDD begins by screening libraries of low-molecular weight compounds (fragments) against the target of interest to identify hits.

These initial hits usually have only very weak affinities, often in the millimolar or high micromolar range. However, when robust biophysical methods like X-ray crystallography have been employed for their detection and validation, they can be readily optimised to higher affinity with the help of structural information.

FBDD is a popular technique due the low experimental cost, offering a diverse range of hits, and exhibiting a number of ways to develop novel compounds. NMR has been utilised by FBDD in a number of experiments to identify various hits that are binding to a specific site on targets. 

Structure-activity relationship (SAR) by NMR is a technique developed in 1996 and is the “first experimental demonstration of the fragment-based approach to drug discovery”. In this method, a small structurally diverse chemical library is screened by NMR with the aim of identifying ligands that bind proximal to each other in the protein’s active site.

Experiments utilising NMR can be summarised as two approaches: one monitors the signal changes from fragments (ligand-observed NMR) and the other monitors signal changes from targets (target-observed NMR). SAR by NMR is considered a protein/target approach to NMR drug discovery. 

The main advantages of ligand-observed NMR is that there is no molecular weight limitation for targets, and the technique is capable of detecting weak binders. Unfortunately, false positives can arise due to the aggregation of compounds. Target-observed NMR is also capable of providing information about the binding site and detecting weak binders, but requires isotope labelling for compounds which is a lengthy process. In addition, the technique is typically low throughput and the molecular weight is a limitation for targets. 

Hetero-nuclear single quantum coherence (HSQC) spectroscopy is a frequently used method in probing protein-ligand interactions. In this experiment, the chemical shifts of amino acids are compared in the absence and presence of a ligand. The chemical shift refers to the resonant frequency of a nucleus relative to a standard in a magnetic field. 

This method is used to confirm the target-ligand interactions, determine the binding affinity as well as mapping the ligand binding site. HSQC is a popular technique for a number of reasons – firstly, it is more sensitive than other techniques and less-time consuming to acquire the desired data. Secondly, it is richer in information about molecular structure and interactions.  

NMR in Structure-Based Drug Design

Structure-based drug design (SBDD) is rapidly becoming a fundamental part of drug discovery projects and academic research. In comparison with the conventional rational drug design, SBDD is a more specific, efficient, and rapid process for lead discovery and optimisation. 

SBDD is a computational technique widely used by pharmaceutical companies and scientists. The basic step in a typical SBDD process is target protein identification and validation. The 3D structures of therapeutically important proteins are determined by structural biology techniques like NMR and X-ray crystallography. 

The use of NMR in SBDD is similar to that of FBDD, focusing on target-observed and ligand-observed approaches. The SOS-NMR method is an example of a technique in SBDD, and is characterised by the use of a target protein whose binding site is labelled with a hydrogen isotope. Ligands bound to the H1-labeled site of the target protein can be selectively detected using this method, while excluding ligands bound to undesired target sites. 

The method is called SOS-NMR for “structural information using Overhauser effects and selective labeling and is validated on two protein-ligand complexes”.

The benefit of this method is that it provides structural information about the relative orientation of the ligand with the target protein in a bound state. This enables chemists to better understand the dynamics of the molecular interaction between target and ligand – determining the structure alone limits the drug design, as the successful binding may be determined by a specific orientation. 

Charlotte Di Salvo, Lead Medical Writer
PharmaFeatures