4. In-Cell NMR Approaches
The interactions between targets (proteins) and ligands (small molecules) can be analyzed independently of the biological systems by using ‘cell-based’ NMR drug design approaches. Three basic approaches [409] are as follows: (1) Compound-detected in-cell NMR, (2) Target-detected in-cell NMR, and (3) Reporter-detected in-cell NMR.
These methods, with the exception of compound detected in-cell NMR, differ according to the isotopically labeled structure (protein, cell structure, etc.), which enables NMR detection. A cartoon representation of each of these methods is given in Figure 13.

4.1. Compound-Detected In-Cell NMR
STD NMR is a technique that lies within the compound-detected in-cell NMR method but does not require isotope-labelling of the studied compound. However isotopic labelling of the compound may be used to enhance the quality of the spectra.

4.2. Target-Detected In-Cell NMR
In the target-detected in-cell NMR only the target of interest is isotopically labeled (i.e., 15N labeled protein). For instance, target proteins can be isotopically labeled during cell growth in isotopically enriched (13C, 15N, or both 15N/13C) media [410]. The cell type and the labeling method may vary across experiments. Different cell types, including bacteria [411], oocytes [412], yeast cells [413], mammalian cells [414], HeLa cells [415] and even insect cells [416] have been reported in the literature. The fact that in-cell NMR applies to more than one cell type testifies of the versatility and potential application of this technique.
In terms of labeling, 15N is one of the most commonly used approaches [417] when the targets of interest are proteins. Recently, 19F labeling has been reported as a useful probe for protein-ligand interactions [418]. It was shown that 19F can reveal information about the dynamics of protein-ligand interactions [419]. Methyl groups [420] have also been used as probes for proteins and complexes in vivo [420], proving that labeling specific chemical groups instead of the entire biomolecule (i.e., protein) is feasible.

4.3. Reporter-Detected In-Cell NMR
In-cell NMR extends beyond proteins, and has been applied successfully to DNA [93,421] and RNA molecules [422,423]. Telomeric repeats have also been studied using target detected in-cell NMR [424]. The reporter-detected in-cell NMR technique isotopically labels neither the ligand nor the target, but rather a receptor that indirectly measures the effects of ligand-target binding [409].
The “reporter” varies according to the experimental context. For instance, Dose et al. [425] used acetylation- and deacetylation-based assays to monitor the activity of histone deacetylase and acetyl-transferase. Thongwichian et al. [426] used peptide-based reporters to identify active kinases and phosphatases in cellular conditions. Lastly, Doura et al. [427] designed a 19F probe that operates in biological conditions in order to study the adherence and dynamics of proteins found in human blood.

4.4. “In-Virus” NMR Strategy
In many viruses and phages, scaffolding proteins (SPs) are required to ensure the correct organization of coat proteins (CPs) and other minor capsid proteins into a precursor structure, called a procapsid [428,429]. Although SPs are critical for viral assembly and therefore potential therapeutic targets their structural properties (with only a few exceptions [430,431]) are poorly understood. The size limitation of NMR can be used advantageously as a filter to identify disordered segments even in very large supramolecular protein complexes. In this way, NMR can provide a unique perspective on the dynamic and disordered elements of macromolecules not accessible by other techniques. The procapsid encapsulation experiments described by Whitehead et al. [432] were conceptually analogous to in-cell NMR experiments [433,434,435] in which signals from small proteins, or flexible segments of proteins, can be observed when they are incorporated inside living cells, as long as the isotope-labeled proteins of interest do not interact strongly with other large cellular components [433,434,435]. The so called ‘‘in-virus’’ NMR strategy applied by Whitehead et al. [432] could be more generally used to study the dynamic properties of macromolecules encapsulated into virus particles, including cargo molecules encased in viral capsids for nanotechnology applications. Additionally, such studies could assess the level of interaction of cargo molecules with the virus and probe the release properties of cargo NMR [432].