3.6. Other Methods Used to Determine the Drug-Target Complexes
Substantial progress has been made in the NMR field over the past 5–10 years, and various methods were established to determine the drug-target complexes. Most of them utilize either NOE or chemical shift perturbations (CSP) although in silico models/programs, using NMR-derivate data also exist.

3.6.1. DIRECTION
One of the methods called difference of inversion recovery rate with and without target irradiation (DIRECTION) is used to map pharmacophores and can be an alternative to STD experiments. This method uses the difference between longitudinal relaxation rates of ligand protons with- and with-out irradiation of the protons of the target protein. The DIRECTION approach, however cannot be used for slowly exchanging (strong binding) ligands. The practical approach of this method was demonstrated on the experiment when analyzed the interactions between p38 MAPK (p38 a mitogen-activated protein kinase) and its inhibitor-SB203580 [389,390]. The results from this experiment showed that protons H1, H4, H5, and H6 of SB203580, are in close neighborhood with the protons of p38 MAPK when compared with H2, H3, and methyl protons. It indicates that two aromatic rings (a pyridine ring and fluorophenyl ring) of SB203580 interact tightly with p38 MAPK. The results were later confirmed with proton density map of each ligand’s proton, based on the crystal structure of SB203580–p38 MAPK complex [391]. Moreover, the same authors already created a new and improved protein–ligand docking method by combining the DIRECTION obtained NMR data with docking software. [392].

3.6.2. ILOE
A second method that can be used to map pharmacophores is called inter-ligand nuclear Overhauser effect (ILOE). This 2D NMR experiment detects when two ligands bind simultaneously to adjacent sites on a protein surface although both of the ligands do not have to bind to the same binding pocket (opposite to INPHARMA, see above) [5,393]. A negative ligand−ligand NOE signal will be created when ligands bind in close proximity to each other whereas ligands that do not bind will show no NOEs, or at most very weak positive ones [372,394]. ILOE also enables determination of the ligand orientations with respect to one another [393]. As in the case of INPHARMA, ILOE can be utilized even in the absence of a 3D protein structure and used with large proteins. Additionally, ILOE differs from INPHARMA in mixing times—for ILOE the mixing times are typically in the range of 600–800 ms [345]. Application of ILOE was first shown on glycolate+NAD+ in the presence of porcine heart lactatedehydrogenase, and by glucose-6-phosphate+NADPH in the presence of L. mesenteroides glucose-6-phosphatedehydrogenase and from that time it has been widely used [393,395,396].

3.6.3. SOS-NMR
A third method called structural information using Overhauser effects and selective labeling (SOS-NMR), relies of STD experiments performed on ligand complexes with different protein samples that have been fully deuterated excluding a specific type of amino acid. In other words, the data obtained by SOS-NMR gives insight into the ligand-binding amino acid composition and when taken into consideration the 3D structure of targeted protein can be used to establish the structure of protein-ligand complex. This approach has been demonstrated using two complexes—FKBP complexed to 2-(3′-pyridyl)-benzimidazole and MurA complexed to uridine diphosphateN-acetylglucosamine (UDP-GlcNAc). The results showed that for FKBP and MurA, only four and three amino acids (FKBP: Ile, Val, Leu, Met; MurA: Trp, Phe, His) were needed to be selectively protonated in perdeuterated samples to establish the ligand-binding site. Additionally, on average only 6 amino acids were required for accurate identification of ligand-binding surface. According to authors SOS-NMR can greatly improve the early stages of the drug discovery process [397]. Moreover, combining SOS-NMR with other methods can even further increase chances for a positive outcome of an experiment [398].

3.6.4. Tert-butyl Labelling
A completely different approach to this topic was taken by Chen et al. [399,400]. Instead of using isotope labeling, Chen’s group decided to use a tert-butyl group contained within ligand-1 to obtain structural information about the protein-ligand complex [400]. The tert-butyl group formed an intense singlet in 1.0 to 1.5 ppm range thanks to rapid methyl rotation and methyl reorientation within that group. When compared with the protein’s 1H-NMR signal, the tert-butyl signal tended to be much narrower and resulted in easy detection without the need for isotopic enrichment even in protein complexes of high molecular mass such as Bacillus stearothermophilus DnaB hexamer (320 kDa) [399]. Additionally, the tert-butyl group produces intense NOESY cross peaks that can be observed even in the situations where normally cross-peaks of the proteins are barely detectable. This is partially because the signal corresponded to nine protons within tert-butyl group. Those aspects enable measurements of pseudo-contact shifts generated by paramagnetic tags attached to the protein. As a result, it allows positioning of the ligand on the protein. An example of this approach, is dengue virus NS2B-NS3 protease from serotype 2 (referred as DENpro) in complexed with ligand containing a tert-butyl group. The result of this experiment showed NOEs between the tert-butyl group of ligand-1 and residue Val155 from DENpro [400].

3.6.5. SALMON
Solvent accessibility, ligand binding, and mapping of ligand orientation by NMR spectroscopy (SALMON) is another method based on the data obtained via nuclear Overhauser effect. This method utilizes WaterLOGSY [401] to probe for solvent accessibility to the ligand and determine the orientation of the ligand by analyzing signal changes in WaterLOGSY spectra (positive signal from unbound ligand vs. negative for protein-bound ligands). This method was first used to determine the orientation of prodrug called tretazicar ((5-(aziridin-1-yl)-2,4-dinitrobenzamide) known as CB1954 in NQO2 (quinone oxidoreductase 2) binding site. Previous attempts had been made to obtain the orientation of tretazicar bounded to NQO2, however the results obtained from X-ray crystallography were inconclusive as two orientations of tretazicar could be possible. The information obtained via SALMON showed that the side chain of asparagine at position 161 formed a hydrogen bond with 2-nitrogroup of tretazicar, and that the aziridine moiety of tretazicar pointed toward the solvent [401].

3.6.6. LOGSY Titration
Another variant of WaterLOGSY method called LOGSY utilizes the titration slopes as a measure of solvent accessibility. The titration slopes are created by a constant increase of protein concentrations. This method also provides more insight into the process of ligand solvation by checking the influence of protein concentration onto the process. This approach was used on the bromodomain 1 of protein 4 (Brd4-BD1) by mapping epitopes of two ligands interacting with Brd4-BD1 and predicting ligands position. The results showed that the triazolopyridazine moiety of both ligands was implanted into the binding pocket of the Brd4. Additionally, the results from LOGSY titration showed that methyl-group 1 of ligand 1, aromatic proton 8 of ligand 2 and aromatic proton 8 of ligand 1 exhibit strong water NOE. This information enabled researchers to utilize a chemical replacement strategy (substitute bound water molecules by suitable functional groups) for aromatic proton 8 in a series of ligands containing the triazolopyridazine ring. Those protons were replaced with an amino or aminomethyl groups and as a result, the binding affinity of those ligands increased 100-fold. Finally, the results obtained from X-ray crystallography for ligands with such modifications allowed to find the binding mode of the triazolopyridazine ring of ligand 1 (with methyl group pointing internally) and the substituted amino group was found to create hydrogen bond to the side chain of Asn140 of Brd4-BD1 [402].

3.6.7. Nuclear Magnetic Resonance Molecular Replacement (NMR2)
The most recent approach called Nuclear Magnetic Resonance Molecular Replacement (NMR2) utilizes spatial data obtained through solution-state NMR in order to locate the binding pocket of a complex structure. For that, it uses a receptor model, e.g., a X-ray structure of a homolog, to conduct an analysis and at the same time excluding the need for protein resonance assignment. To conduct an experiment using such an approach requires a few steps. First, either the protein or ligand used in the complex must be uniformly 13C and 15N labeled. Then, an experiment to assign the ligand is needed such as 2D 13C,1H-HMQC or 13C,1H-HMBC. The next step is the evaluation of ligand intra- and ligand–protein intermolecular distances through NOE cross peaks obtained from F1-15N,13C-filtered 1H,1H-NOESY. Lastly, choosing a proper input structure is required which can be either X-ray or NMR structures in apo form, with another bound ligand, or a homolog to the protein of interest. Then the NMR2 program analyzes for all possible partial assignments (such as methyl groups of a protein) and calculates the complex structures for all options [403,404]. This method was already successfully used to resolve complex structures in case of slow and fast exchange ligands [403,404,405,406].

3.6.8. HECSP
In silico methods combined with NMR derived information can also be used to determine accurate drug-target complexes. 1H empirical chemical shift perturbation (HECSP) is an empirical model that is based on chemical shift perturbation (CSP) of a protein. CSP represents the change in chemical shifts in a protein due to alteration of its chemical environment (which can happen upon ligand binding). The CSP of a target protein is obtained by a series of 2D HSQC experiments with a set of ligand titrations involving samples that contain 15N-labelled protein. The calculation of 1H-CSPs inside the protein are based on four contributors: 1) ring current, 2) electric field, 3) hydrogen bonding, and last 4) magnetic anisotropy. To show the value of the HECSP model two CSP examples were used: apo-neocarzinostatin (apoNCS)-naphthoate ester complex, and human intestinal fatty acid binding protein (hIFABP)-ketorolac-ANS complex. The results from the experiment showed that HECSP model can distinguish native ligand from decoys and more clearly define protein-ligand complex structures with NMR derived information [407].

3.6.9. SAMPLEX
Another program that can utilize CSP called Smoothed Automatic Mapping of Protein from Listed Extremes (SAMPLEX) can help to determine the interaction surface of proteins complexes. SAMPLEX takes the chemical shifts of the protein of interests in both the free and bound state and corresponding 3D structure of a protein in the free state. The programs returns a confidence value for each residue to be in a perturbed or unperturbed state (0.05 as being in a perturbed state, −0.05 as remaining in their unperturbed state). This approach was tested on five examples, one of which was Subtilisin BPN’ (serine protease) complexed with its inhibitor–chymotrypsin inhibitor 2. The results showed that residue 2, and residues 56–62 of chymotrypsin inhibitor-2 were perturbed and residue 63 was in an ambiguous state. To compare, the X-ray crystallography data showed residues 50 and 54–61 to be involved in the interaction. For subtilisin BPN’ the program predicted residues 33, 97, 99–109, 126-128, 141, 154–156, 167–171 and 218–219 to perturbed and residues 65, 98 and 220 to be in ambiguous state. That information was also confronted with the X-ray crystallography data which shown residues 99–104, 125–128, 154–157, 167, 218–221 to be perturbed [408].