3.5. NMR Validation in Drug Design
A “hit” is a molecule identified from a screening technique (HTS, FBDD, etc) as having a desirable effect (i.e., decreased cellular growth, high affinity score) on a target [365,366]. However, the question of whether the activity is related to actual binding to the target, or to interference with one of the components of the assay readout mechanism, is uncertain. Thus, a validation step is required. Hit-validation is therefore the process of confirming, or validating, that the molecule(s) identified previously have on target activity and selectivity [367,368]. One of the highest-impacts of NMR on drug discovery is the use as a hit-validation tool. Though the hit-validation or confirmation of drugs is mostly limited to the solution state [369], this aspect of NMR truly is a “gold standard” technique in drug discovery.
NMR by itself is a powerful tool for drug validation as in the case of Sharma et al. (2012) [370] who sought to identify potential drug-like inhibitors against L-Aspartate α-Decarboxylase (ADC) an enzyme responsible for the decarboxylation of L-aspartate in order to generate β-alanine and carbon dioxide [371], in Mycobacterium tuberculosis. They began with known inhibitors of ADC, and developed a protocol to measure the enzymatic activity of ADC. Upon addition of ADC to a solution of L-aspartate, L-aspartate gradually disappeared because ADC was converted to L-aspartate to β-alanine; therefore the peak intensity of L-aspartate decreased, and the peak intensity of β-alanine increased in the presence of ADC (no inhibitor drug present). Using this newly developed NMR-based protocol allowed direct measurement of ADC enzymatic activity, and Sharma et al. were able to confirm the enzymatic inhibiting activity of seven previously discovered inhibitors of ADC [370]. This study demonstrated that NMR can be an effective validation tool for known drugs and for new drugs generated by a screening approach.
NMR is also able to remove false positives that emerge from biochemical screens [372]. For example, an aptly named technique called A La Assay to detect Reactive Molecules by Nuclear Magnetic Resonance (ALARM NMR) is able to eliminate false positives from HTS methods [373], and in the presence of a test compound or mixture, measures dithiothreitol (DTT)-dependent 13C chemical shift changes of the human La antigen [373]. Dahlin et al. provided an updated protocol of ALARM NMR to aid researchers in the production of the 13C-labeled La antigen reporter protein, in testing compounds with the La protein, and in the analysis of obtained NMR spectra. Using ALARM NMR prioritized hits identified from HTS screening [374]. An example of ALARM NMR is found in the work of Dahlin et al., where they used this technique to test molecules that were assumed to be inhibitors of histone acetyltransferase (HAT) inhibitors, and from their studies, actually discovered that 65% (15 out of 23) of the most commonly reported HAT inhibitors were actually faulty. They were actually nonselective interference compounds, not necessarily specific to the inhibition of HAT [375]. Thus, ALARM NMR (and NMR in general) served as a useful validation method, especially for unvalidated hits identified from biochemical screens [372] or other screening techniques.
The last example highlights the need for cross validation, or the combination of two or more techniques to verify identified chemical hits. Of course, NMR is not the sole technique used for drug validation. Most often, NMR drug validation is coupled with additional methods [367] such as surface plasmon resonance (SPR) [376,377] X-ray crystallography [377,378,379], isothermal calorimetry (ITC) [379], UV-Vis and/or fluorescence spectroscopy [380].
The work of Goudreau et al. is an excellent example of combining NMR with another biophysical technique, in this case X-ray crystallography, for drug validation [378]. A series of benzodiazepine inhibitors of Human immunodeficiency virus 1 (HIV-1) was identified using an in vitro capsid assembly assay, and further characterized by 19F-NMR. Analysis of the chemical shift perturbation and line broadening effect on the 19F-NMR spectra of the benzodiazepine inhibitors revealed the specificity and reversibility of the binding inhibitors. The same set of 19F-NMR spectra were used to identify the N-terminal domain of the capsid as the binding site of the benzodiazepine inhibitors. The specific amino acids involved in the binding of the benzodiazepine inhibitors were identified from the chemical shift perturbation of 1H,15N-TROSY NMR spectra. Later, use of X-ray co-crystallography confirmed binding locations of the benzodiazepine inhibitors and their binding modes, which was useful for further development and optimization of the benzodiazepine inhibitors [378]. The work of Goudreau et al. therefore showed how NMR could be used as a co-validation technique with another biophysical method [378].
NMR can be also coupled with multiple biophysical techniques to validate a molecule’s ability to inhibit protein-protein interactions (PPIs) [367]. An example of the combination of NMR with SPR and X-ray crystallography can be found in the work of Fry et al., where the authors sought to understand how the nutlin molecule inhibits MDM2-p53, a protein-protein interaction that has been an important cancer therapy target for several years [381,382,383]. Fry et al. [377] gradually deconstructed RG7112, the first nutlin molecule to enter clinical trials [384], into 11 fragments so they could study the inhibitory effect of RG7112 on the MDM2-p53 interaction by SPR, NMR, and X-ray crystallography. SPR was used to determine the Kd values of the RG7112 fragments and confirmed that RG7112 and some of its fragments do bind to MDM2, inhibiting the MDM2-p53 interaction. 1H,15N-HSQC NMR chemical shift perturbation was also used to assess and verify binding identified by SPR. Of the six fragments of RG7112 confirmed by 1H,15N-HSQC NMR as binding to MDM2, SPR showed binding for five of them; thus, the two separate techniques were in good agreement with each other. The fragments of RG7112 that were confirmed to bind by both SPR and 1H,15N-HSQC NMR were further studied with X-ray crystallography, which can tell precisely where and how the molecules bind to the protein. Using co-crystallization, Fry et al. were able to obtain structures for several of the verified binding fragments in complex with MDM2 and were able to visualize the binding of the fragments to the MDM2 protein [377]. NMR is obviously a powerful drug binding validation tool, but it becomes much more powerful when coupled with additional biophysical techniques, as seen in the work of Fry et al. [377].
Dias et al. [379] took a similar approach as Fry et al. [377] in that they took known inhibitors of a protein-protein interaction, and dissected them into individual fragments to assess a protein’s drug-ability. The interaction studied was that between the proteins von Hippel–Lindau (VHL), and the alpha subunit of hypoxia-inducible factor 1 (HIF-1α). Twelve compounds (known inhibitors and derived fragments) were developed using a crystal structure of HIF-1α peptide bound to the stable multiprotein complex pVHL-elongin C:elongin B (VCB). Each of these compounds was screened using three separate NMR techniques, Saturation Transfer Difference (STD), Carr–Purcell–Meiboom–Gill (CPMG) relaxation experiments, and WaterLOGSY (to assess drug binding and to predict drug binding mode. Each compound that was unambiguously detected (i.e., the molecule was identified as successfully binding by at least two of the three NMR methods of STD, CPMG, and WaterLOGSY) was subjected to further analysis by ITC and X-ray crystallography. ITC was used to determine the dissociation constants of binding molecules, and X-ray crystallography was used to confirm the binding mode predicted by the NMR studies. Generally speaking, the designed fragments had similar ligands efficacies compared to the parent molecules but had much higher dissociation constants (Kd values), meaning that the fragments bound less tightly than the original parent molecule [379]. With this example, it is possible to see the strength of using NMR as its own hit-validation tool (i.e., three different NMR techniques were used for screening compounds [379]), and yet, the follow-up of NMR studies with ITC and X-ray crystallography was useful in providing a basis for assessing the drug-ability of a protein-protein interaction [385,386,387,388]. Thus, it is clear to see that NMR is a prominent method of hit-validation in drug discovery research, especially in combination with other biophysical techniques.