1. Introduction
The unexpected SARS-CoV-2/COVID-19 outbreak, with over 34 million confirmed cases globally (Oct. 2020) and the struggle for survival in the absence of a proven and efficient treatments, emphasizes the critical need to develop effective, novel, and rapid drug discovery methodologies. Even though the pharmaceutical industry works constantly to discover and develop novel drugs, the process is still slow and expensive. The cost of introducing a new drug has increased steadily, with current cost estimates predicting that a future drug will cost in excess of $2.6 billion. The typical development cost is usually spread out over the course of 14 years [1,2,3], making investment even more difficult (i.e., cost recovery delay). This high investment barrier for drug development is a result of numerous testing phases (Scheme 1), with each phase requiring a statistically significant number of cases. Although there are several other substantial costs to drug development, that discussion of experimental methods to reduce costs is beyond the scope of this review.
The emergence of a pandemic and the emergencies it creates worldwide understandably drive and motivate the rapid development and/or optimization of drugs. However, patient safety and subsequent earned public trust is a primary requirement. Drug redirecting/repurposing (Scheme 1) is an efficient short-cut method in disease treatment that utilizes existing tools, and combines artificial intelligence, machine learning algorithms, and experimental NMR techniques (i.e., “from Bench to Bedside”). This process must be relatively rapid and efficient to have any benefit to patients and the health-care system.
Compared to mass spectrometry and high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) is another powerful technique with several unique advantages [5,6,7,8]. NMR is intrinsically quantitative, and it provides several different approaches that are routinely utilized to identify and structurally elucidate molecules of interest [9,10,11,12,13,14,15,16,17,18]. In contrast to mass spectrometry, NMR is non-destructive, non-invasive, has extremely high reproducibility permitting researchers to acquire measurements under different experimental conditions (e.g., temperature, time points, and concentrations) often while the same sample is inside the magnet [19,20,21,22,23]. NMR can be used in reaction kinetic studies while several consecutive measurements are taken, and while spectral changes (function of the reaction time) are analyzed [24,25,26,27,28]. Moreover, molecules are studied at the atomic level [29,30,31]. Unlike other analytical tools, NMR provides dynamic information, and NMR experiments can be carried out under physiological conditions (e.g., atmospheric pressure, temperature, and different pH values) [32,33]. This is especially important in medical drug design since one must understand the interactions between an enzyme of interest and the ligand(s). NMR provides information on the binding affinity of such ligands, details/location of the binding site, and associated structural changes following binding [32,33,34,35]. These biophysical details are essential when evaluating the potential efficacy of a drug, and during any subsequent optimization. The available literature [32,33,34,36] highlights the practicality of NMR in drug design studies. For these reasons, NMR spectroscopy is highly sought after in drug development [37,38,39,40,41], for both molecule identification [11,13,14,18,42,43,44,45,46] and structural elucidation [15,16,17,45,47,48,49,50,51]. NMR has been successfully applied in stereochemistry [52,53,54,55,56] and isomer determination [57,58,59,60,61], in drug-protein interactions studies [62,63,64], and in the evaluation of drug toxicity [65,66,67,68].
The use of NMR in drug design is not restricted to academic laboratories and gained interest from those in development industries. The use of NMR in drug development increased in the late ‘80s, as seen in both scientific and patent literature (Figure 1). While scientific interest in NMR is still growing, the number of patents has been decreasing since the early 2000s. The top applicants of NMR in pharmaceutical patents are Bristol Myers, AstraZeneca, and Wyeth, with 146, 104, and 67 patent families, respectively.
In addition to the advantages provided by NMR, it is often used with complementary methods such as X-ray crystallography, HPLC, and mass spectrometry [69]. An example of this is found in work by Wyss et al. [36], where they combined X-ray crystallography with NMR fragment-based screening to create the first inhibitor candidate for BACE-1 in Alzheimer’s disease. BACE-1 is a membrane-anchored aspartic acid protease and is responsible for the production of amyloid beta peptides in neurons related to the progression of Alzheimer’s disease [36,70]. Using NMR fragment-based screening, Wyss et al. identified isothiourea as binding to BACE-1 and confirmed this observation with the X-ray crystal structure of the complex of a ligand-efficient isothiourea fragment. Information obtained from these experiments aided in design optimization, resulting in the selection of iminopyrimidinones as BACE-1 inhibitors [36]. This is a perfect example of using different complementary methods to maximize scientific outcome. However, in order to be efficient, one must know the advantages and disadvantages of each method. One of the major issues regarding NMR is the effective size restriction when measuring targets such as proteins above 40kDa. Recent progress has extended this mass limit; an example of this is the resolved structure of chaperone SecB in complex with unstructured proPhoA (PDB ID 5JTL) with a total mass of 119kDa using NMR [71]. In this review, we present practical guideline to use NMR techniques in drug design studies and provide examples of the successful use of NMR in drug-design.