Acknowledgments
The authors would like to thank King Abdullah University of Science and Technology (KAUST) for financial support.

Author Contributions
A.-H.E. designed the outline of the manuscript, and offered numerous suggestions for each part. B.G.P. contributed to Section 3 and Section 4 of this paper, and provided English editing services. K.S. arranged and organized the references. K.S. and K.C. designed the figures representing common NMR mechanisms and concepts, as well as the NMR pulse sequences. R.T.M., M.J., and L.J. contributed their NMR expertise to make “An Introduction to NMR Spectroscopy” (Section 2) scientifically correct and reader accessible. M.D. and F.A. created the figures in the Introduction and made valuable revisions for the final remarks section (Section 5). J.I.L. did extensive research and added the information regarding NMR as it applies to studying viruses. M.D. and J.I.L. developed and contributed to all parts, especially the final remarks section. All authors have read and agreed to the published version of the manuscript.

Funding
This research received no external funding.

Conflicts of Interest
The authors declare no conflict of interest.

Figures, Schemes and Tables
Scheme 1  Schematic representation of drug discovery [4]. Numerical data were reported from Meigs et al. [3].
Figure 1  Data search results for: “NMR” and “Drug design” in Scopus (blue) (www.scopus.com) and Orbit patent (orange) (www.orbit.com) databases.
Figure 2  The most simplified layout of an NMR experiment. The NMR spectrum of ethyl acetate Table 1. H chemical shift assignments is shown as an example. The chemical structure and simulated 1H-NMR spectrum were created using ChemDraw 18.1.
Figure 3  The pulse sequence of HSQC. I and S represent two heteronuclear spins. The τ is (1/4J) where J is the coupling constant. The thick and thin bars represent the 180° and 90° pulse respectively [142].
Figure 4  RF (radiofrequency) pulse causes the bulk magnetization to move to the transverse plane. Over time (ms to seconds, and in extreme cases, minutes [155]), the bulk magnetization will decrease in the transverse plane, and increase in the longitudinal axis, returning to its original, equilibrium value. (A) represents T1 relaxation and (B) represents T2 relaxation.
Figure 5  CPMG pulse sequence. First, a 90° RF pulse is applied and results in transverse magnetization in the xy plane. Then a 180°y pulse is applied to re-phase the magnetization vectors. After 180°y, the vectors who were faster during the dephasing are overtaken by the slower vectors, which results in re-phasing and generation of a spin-echo signal. This process is repeated several times.
Figure 6  Underlying mechanisms of (A) Traditional High-throughput Screening, and (B) Fragment Based Drug Discovery.
Figure 7  Overlaid 1H-15N-HSQC for FKBP in the absence (black contours) and presence (red contours) of 2-phenylimidazole. Adapted with permission from Hajduk et al. [249].
Figure 8  Basic layout of the different NOE effect on NMR signals.
Figure 9  Illustration of STD NMR (a) and WaterLOGSY (b) in drug design. Copied with permission from Robson-Tull [209].
Figure 10  Schematic representation of the principle of the INPHARMA. The inter-ligand NOEs observed between two competitive ligands A (in green) and B (in blue), which bind consecutively to the same target receptor T. The figure is adapted from [284].
Figure 11  Visual representation of how molecules diffuse in solution from a high concentration to a low concentration across an arbitrarily defined point (x = 0) after some period of time (t = ∞) [289]. The red arrow indicates the direction of translational motion as the molecules (green) move from an area of higher concentration (left) to an area of lower concentration (right) [287].
Figure 12  Spin echo (SE) sequence, as discovered by Stejskal et al. [293]. G1 is the first gradient pulse applied after the first 90° pulse, and G2 is the second gradient pulse applied after the first 180° pulse. δ and ∆ are the gradient length, and diffusion time, respectively.
Figure 13  Representation of different in-cell NMR techniques. (A) Target-detected in-cell NMR, (B) compound-detected in-cell NMR, (C) Reporter-detected in-cell NMR. The red color is the isotopically labeled component of the system. Adapted with permission from [409].
Scheme 2  Analysis of the cost and time between different NMR methods used in drug design. Meaning of some of the acronyms are listed below: SOFAST—Band-Selective Optimized Flip Angle Short Transient; NUS—Non-uniform sampling; MDD—Multidimensional Decomposition; CS—Compressed Signaling; MAX ENT—Maximum Entropy; IST—Iterative Soft Threshold; STD—Saturation Transfer Difference; PNMR—Paramagnetic NMR; ALARM—A La Assay to detect Reactive Molecules; ssNMR—Solid State NMR; FBDD—Fragment Based Drug Design; SAR—Structure Activity Relationship.
Table 1  Recent pharmacological studies complemented with NMR approaches for evaluating the efficacy/safety of new or existing drugs.
Tested Substance  Evaluated Effect  NMR Experiments Used in the Study  Ref.
Isoniazid (INH)  INH induces oxidative stress, disturbs energy metabolism, and causes disorders in neurotransmission and neuromodulation processes in Sprague Dawley rats.  1H-NMR CPMG1H,1H-TOCSY1H,13C-HSQC  [178]
Naproxen  Naproxen induces a disturbance in energy and choline metabolism, and promotes catabolism of tryptophan in Sprague Dawley rats.  1H-NOESYPRESAT-1D  [179]
Cisplatin (CP)  Identification of six serum (alanine, betaine, glucose, glutamine, lactate, and leucine) and eight urinary (alanine, acetate, citrate, glucose, glycine, guanidinoacetate, hippurate, and lactate) metabolites that can be used as biomarkers for cisplatin nephrotoxicity.  1H-NMR  [180]
Shell of Herpetospermum caudigerum Wall (SHCW)  A high dosage of SHCW causes the disturbance of energy and amino acid metabolism and induces oxidative stress in Sprague-Dawley rats.  1H-NMR CPMG1H,13C-HSQC1H,13C-TOCSY  [86]
Ampicillin, Maculatin 1.1  Both antibiotics cause destabilization of membrane integrity and increase breakdown of nucleic acids in E. coli.  1H-31P CP  [94]
Emodin  Emodin can affect the immune response and interrupt energy metabolism (citric acid cycle) along with glutathione synthesis, which can lead to oxidative stress.  1H-NMR CPMG1H,1H-COSY1H,13C-HMBC1H,13C-HSQC  [65]
“RenqingMangjue” pill (RMP)  RMP can disturb the citric acid cycle in cells, and decreases levels of glutamate, glutamine and BCAAs in the plasma of Wistar rats.  1H-NMR CPMG1H,1H-COSY1H,13C-HSQC1H-13C HMBC  [66]
Ptac2S  Ptac2S limits cancer cell (Skov-3) proliferation by reducing the efficiency of the citric acid cycle and induces changes in cell membranes in a shorter period of time (6h) compared to cisplatin (24h). Additionally, Ptac2S may inhibit lactate dehydrogenase.  1H-NMR CPMG1H,1H-COSY1H,13C-HSQC1H,13C-HMBC  [145]
Gemcitabine-carboplatin (GC)  Identification of two biomarkers (formate and acetate) that can predict a positive response in MBC (metastatic breast cancer) patients treated with GC chemotherapy.  1H-NMR CPMG1H JRES1H,1H-COSY1H,1H-TOCSY1H,13C-HSQC1H,13C-HMBC  [137]
Doxorubicin (DOX)/dexrazoxane (DEX)  DOX decreases ATP production and induces oxidative stress in H9C2 cells. DEX counteracts those changes, having a cardioprotective effect on H9C2 cell lines.  1H-NOESYPRESAT-1D  [67]
Curcumin  Curcumin shows antihyperlipidemic effects on C57BL/6Slac mice by partially restoring metabolic defects induced by a high-fat diet. Affected metabolic pathways include the citric acid cycle, glycolysis and gluconeogenesis, ketogenesis of BCAA, synthesis of ketone bodies and cholesterol, and choline and fatty acid metabolism.  1H-NOESYPRESAT-1D1H-1H TOCSY1H,13C-HSQC  [136]
Formosanin C (FC)  Formosanin C shows the ability to inhibit synthesis and methylation of DNA as well as reducing the activity of the citric acid cycle and energy metabolism in the mitochondria of HepG2 cells.  1H-NMR  [181]
Melamine  Melamine disrupts metabolism of glucose, nitrogen, and protein in the liver of Wistar rat.  1H-NMR CPMG  [182]
Aristolochic acid (AA)  Aristolochic acid causes renal lesions and a disorder in tubular reabsorption in Wistar rats.  1H-NMR  [68]
Rituximab  Evaluating response outcome for patients with rheumatoid arthritis, treated with rituximab. Identification of metabolites changes between responders and non-responders such as succinate, taurine, lactate, pyruvate and aspartate.  1H-NMR  [183]
Levetiracetam, Lamotrigine, Topiramate  No distinction between metabolite profiles of serum from patients treated with levetiracetam, lamotrigine and topiramate. Could not evaluate response of initial treatment of epilepsy.  1H-NOESYPRESAT-1D  [184]
Hexacationic Ruthenium Metallaprism  Metallaprism mainly affects lipid metabolism in A2780 (human ovarian cancer) and HEK-293 (human embryonic kidney) cells, and increases GSH levels in all cell lines. In A2780cisR (cisplatin resistant A2780) cells, lipid biogenesis and glycosylation are affected by treatment with metallaprism.  HR-MAS:a)1D-1H NOESY-1H CPMGb)2D-1H,1H-TOCSY-1H J-resolved  [138]
Centella asiatica extract  Extract from Centella asiatica promotes glycolysis, boosts the citric acid cycle and decreases gluconeogenesis and lipid metabolism in T2DM Sprague–Dawley rats.  1H-NMR CPMG  [185]
VR24, VR27 (1,3,4-thiadiazoles)  VR24 and VR27 improve glycerol metabolism, decrease betaine levels, and normalize the altered level of myoinositol in serum of DMH-induced CRC (colorectal cancer) Wistar rats.  1H-NMR CPMG  [186]
Xiaoyaosan  Xiaoyaosan regulates energy metabolism, can play an important role in the regulation of the nervous system, and might restore the balance in gut microbiota of depressed patients.  1H-NMR CPMG1H,1H-COSY1H,13C-HMQC  [187]
Sini decoction (SND)  SND may restore balance in myocardial energy metabolism, and regulate the citric acid cycle and amino acid metabolism. Identification of 10 biomarkers showing potential efficiency of SND administration in Sprague-Dawley rats.  1H-NMR  [188]
Fu Fang Jin Jing Oral Liquid (FJJOL) with Herba Rhodiol  FJJOL regulates energy metabolism of brain tissue, can affect the function of neurons abundant in GABA and glycine receptors, and may help to maintain the membrane integrity of the cells in Kunming-strain mice exposed to hypobaric hypoxia.  1H-NOESYPRESAT-1D1H,1H-gCOSY1H,1H-TOCSY  [87]
Acyclovir, Pyrazinamide, Isoniazid, Sulfamethoxazole  Evaluating the efficacy (determined by the concentration of a drug able to reach the therapeutic site) of four drugs in cerebrospinal fluid of tuberculous meningitis patients.  1H-NOESYPRESAT-1D1H,1H-COSY  [189]
Genipin  Genipin can recover energy metabolism to normal levels, and regulate methylamine and amino acid metabolisms of diabetic Sprague Dawley rats.  1H-NOESY-1D  [190]
Adriamycin (ADR)  Identification of seven biomarkers: trimethylamine oxide (TMAO), taurine, trimethylamine (TMA), hippurate, trigonelline, citrate and 2-oxoglutarate that can predict tumor’s (gastric adenocarcinoma) response to ADR treatment in BALB/c-nu/nu mice.  1H-NOESYPRESAT-1D  [191]
Danggui/European Danggui  Comparison between Danggui and European Danggui showed that Danggui has a different chemical composition and provides a better enriching effect on blood than European Danggui. Identification of 18 metabolites affected by Danggui treatment.  1H-NMR CPMG1H-NOESYPRESAT-1D1H,1H-COSY1H,13C-HSQC  [88]
Erythromycin  Erythromycin decreases citric acid cycle activity, enhances fatty acid oxidation, causes dysfunction in amino acid metabolism, and creates oxidative stress in livers of Wistar rats.  1H-NMR CPMG1H-NMR BPPLED  [192]
Fuzi/Gancao  Fuzi causes a shift in energy metabolism (from aerobic respiration to anaerobic), induces membrane toxicity, and disrupts the balance of gut microbiota of Wistar rats. Administrating Fuzi with Gancao diminishes the toxic effects of Fuzi.  1H-NOESY-1D  [193]
Kijitsu, Tohi, Chimpi,  Kippi, Seihi  1H-NMR spectra enabled the identification of three compounds (naringin, sucrose, and β-glucose), and 13C-NMR enabled the identification of eight compounds (naringin, neohesperidin, ɑ- and β-glucose, sucrose, limonene, narirutin, and synephrine).  1H-NMR13C-NMR  [112]
Table 2  The names, structures, targets, FBDD optimization strategy used, biophysical techniques used, and status in clinical trials of select drugs derived from the FBDD approach.
Drug (Company)  Target  Original Fragment(s) *  Advanced Molecule and Progress in Clinical Trials  Techniques Used
Vemurafenib (Plexxikon) [224,226]  BRAF-V600E   Approved  high-concentration biochemical fragment screening, X-ray crystallography
Venetoclax (AbbVie, Genetech) [227,228,229,230]  BCL-2   Approved  NMR, X-ray crystallography
Ribociclib (Novartis Europharm Limited) [231]  CDK4 and 6  Information Not Available  Approved  Information not Available
PLX3397 (Plexxikon) [232,233,234]  FMS, KIT, and FLT3-ITD   Phase 3  X-ray crystallography, Structure Confirmed by NMR, MS, and HPLC
Verubecestat (Merck) [235,236]  BACE1   Phase 3  NMR, X-ray crystallography, inhibition of cathepsin D
Onalespib (Astex) [237,238]  HSP90   Phase 2  X-ray crystallography, isothermal titration calorimetry, NMR
AZD5099 [239,240]  Topoisomerase II   Phase 1  NMR, Surface Plasmon Resonance, isothermal calorimetry, X-ray cystallography
AT7519 [241,242,243,244]  CDK 1, 2, 4, and 5   Phase 2  NMR, MS, X-ray crystallography
* Structures for some of the fragments are taken directly from Dan Erlanson’s blog at [245].
Table 3  Advantages and limitations of different NMR techniques developed in recent years.
NMR Technique  Advantages  Disadvantages
1D-NMR  Ability to identify simple chemical compounds.High quality resolution and sensitivity for many 1D experiments.Less time consuming compared to 2D NMR.  Compared to 2D NMR less details can be obtained for more complex molecules.For nuclei other than 1H and 19F, relative sensitivity is fairly low—requires extra labeling to obtain better spectra.
2D NMR  Ability to identify complex molecules and observe different interactions between the nuclei, e.g., correlations between all spins in one spin system using TOCSY experiment.  Requires long times to obtain a proper spectra (up to days).
Ultrafast 2D NMR  Greatly reduces time to obtain 2D spectra.  Reduced sensitivity.
SOFAST  Significantly reduces acquisition time of HMQC.  Relatively low resolution
NUS  Lowers the time of measurement while keeping the same level of resolution.  Requires use of reconstruction algorithms since missing data points can lead to artifacts in spectra.
MDD  Multidimensional data are “broken” to one dimensional, which are easier to analyze.Ability to resolve overlapping resonances.   Data must be (approximately) symmetrical (Lorentzian shape) to obtain good spectra.
CS  Good reconstruction of weaker peaks.  Large computational costs, low performance on noisy data.
MAX ENT  Significant reduction in acquisition time.  Nominally Lorentzian peak shapes may be distorted, and peak intensities may be altered.
IST  Greatly reduced time to obtain NMR spectra.  Requires a grid of uniformly sampled data points.
FBDD  Often makes stronger binding ligands from weakly binding fragments.Less time and resource intensive.  Can be used only for small fragments of compound of interest.
SAR  Direct observation of target binding to ligand. Several types of NMR experiments are possible.  Inability to distinguish binding modes, difficult to gage the “true” binding site of ligand to protein.
STD  Only requires a small amount of sample.Highly reproducible.Allows direct observations of ligand binding.  Only works for ligands with low binding affinity (fast chemical exchange). Inability to distinguish binding modes.
In cell NMR  In vivo studies are possible, can focus on specific cell parts.  Special labeling techniques may be required. Spectra may be more challenging to interpret.
In silico + NMR  Can model protein drug interactions, helps speed up and reduce cost of drug delivery  Protein models need to be validated through experimental approach.
PNMR  Can observe proteins interacting with metal ions, long observation distance (10-25 angstroms) between paramagnetic left and nearby atoms.  Paramagnetic left required in the system.
ALARM NMR  Elimination of false positives from HTS methods  It requires synthesis of human La antigen protein.
ssNMR  Enables the characterization of a chemical compound in a solid-state form such as a tablet/pill.Provides insight into the physical properties of a compound.  Significant broadening of the spectral lineshapes due to anisotropic spin interactions.
Relaxation editing  Noticeable difference in spectra of binding and non-binding ligands.  Sets the lower limit of time for which experiments can be performed.