3.4. Solid State NMR in Drug Discovery
Since the late 1970s solid state NMR (ssNMR) has demonstrated its usefulness in complex biomolecular systems such as collagen or lipid bilayers [354]. However, over the past years ssNMR has gained attention in the field of drug design and is slowly becoming a commonly used technique as its proving to be a powerful tool for structural analysis of membrane proteins and amyloid fibrils [354,355,356].
ssNMR is becoming a more attractive alternative for several different reasons. One of them is the fact that it enables the characterization of a chemical compound in a solid-state form such as in a tablet/pill [356,357,358]. Moreover, ssNMR is not only restricted to analyzing the chemical structure but it can also provide insight into the physical properties of a compound such as polymorphism (different crystalline structures of the same compound), disorder (crystal defects and amorphous solids in the compound) or the presence of cocrystals (multicomponent crystal made of a compound and one or more small organic molecules) [356,357]. ssNMR can also be used to quantify the amount of crystalline against the amount of amorphous material in the sample to establish phase purity (the amount of desired phase separated from other, undesirable phase) [356,357,358].
ssNMR differs from liquid state NMR by the presence of anisotropic interactions. In liquids NMR these effects are averaged to zero as a consequence of rapid molecular tumbling. In solid state however, the molecules are not tumbling rapidly and the residual effects of anisotropic (orientation depended) interactions such as anisotropic chemical shift, magnetic dipolar coupling, and quadrupolar coupling could be observed in the form of broad peaks, with could be much wider than the chemical shift range of the nucleus [355,358,359]. As a results, there has been a constant effort to improve the sensitivity and resolution of solid state NMR spectra, which increased the potential of ssNMR in future applications [360]. One of the methods that works for nuclei with spin value of I = 1/2 is called magic-angle spinning (MAS). It increases the resolution by rapidly rotating the sample around a fixed (or so-called magic) angle of 54.736° [360]. This method can be combined with decoupling, to remove the dipolar couplings between spins. This is done by applying radiofrequency pulses or cross-polarization (CP) transfer of magnetization from abundant and sensitive nuclei such as 1H to less sensitive such as 13C [328,333]. A broader comparison between ssNMR and liquid state NMR is provided in [361].
As mentioned before, ssNMR can provide information about membranes and membrane proteins. For this reason, ssNMR can be used to detect interactions of ligands with receptors embedded to the membrane which enables the mapping of binding site of a receptor by utilizing CP-MAS (cross-polarization magic-angle spinning) NMR and site specific mutagenesis [355]. ssNMR can provide the conformation of ligands bound to the receptor which can then be used to optimize future drug in terms of better affinity and efficiency [355]. Since ssNMR is also applicable to amyloid research, it can be used for probing polypeptide structures of amyloid and intermolecular contacts between fibrils. The potential is for the design a drug that will inhibit the process of aggregation of proteins and peptides [355]. Lastly, since ssNMR gains insight into physical properties of a chemical compound it can be used for control of the process of formulation and processing of a drug to help assess the purity of a compound [358].
An example of ssNMR application related to drug design is the work of Callari et al., who monitored the effect of drug loading on the properties of micelles [362]. Polymer micelles are widely used as nano-carries for drug delivery, but so far the effects of drug loading on the morphology of a drug carrier had not been thoroughly investigated [362]. They created a model consisting of a fructose hydrophilic block and a PMAA block (micelle), to which a different amount of platinum complex was anchored. The results from this experiment showed that micelles loaded with a higher amount of platinum complex had reduced cellular uptake, release, and cytotoxicity. The micelles with a lower load (LL) of platinum complex were more effective at targeting cancer cells (of cell lines MDA-MB-231 (breast cancer) and A549 (lung cancer) than the micelles with a higher load (HL) of the platinum complex. This is evidenced by the lower IC50 (half maximal inhibitory concentration) values of the LL micelles as compared to the HL micelles. Both of those results could be related to the micellar structure and their potential for interaction between the sugar moieties and the cell wall [362].
Another example of practical application of ssNMR is the work of Lee and colleagues [363] in which they investigated the structure of a designed zinc-binding amyloid fibril that catalyzed ester hydrolysis. Metals ions such as zinc where found to affect the process of protein aggregation which resulted in arise of amyloid like structures. Therefore, understanding the processes of aggregation and the factors related to them is crucial for creation of new drugs for amyloid related diseases [364]. In the experiment Lee et al. used Ac-IHVHLQI-CONH2 peptide (referred as HHQ) to form fibrils with varying Zn2+:HHQ molar ratios. The results showed that Zn2+-bound HHQ fibrils form parallel-in-register form of packing β-strand in each sheet and His residues are coordinated to Zn2+ via Nδ1, while half of the His residues are also coordinated to Zn2+ via Nε2. Additionally, Zn2+ binds in a 1:1 metal ion/peptide ratio. After further analysis using structural bioinformatics, it was concluded that each zinc ion was coordinated by three histidine nitrogens from two adjacent strands. Half of all histidines bridged to Zn2+ ions forming a metal–imidazolate chain [363].