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PMC:7594251 / 82756-91482 JSONTXT 10 Projects

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Id Subject Object Predicate Lexical cue
T541 0-4 Sentence denotes 3.5.
T542 5-34 Sentence denotes NMR Validation in Drug Design
T543 35-218 Sentence denotes 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].
T544 219-400 Sentence denotes 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.
T545 401-437 Sentence denotes Thus, a validation step is required.
T546 438-601 Sentence denotes Hit-validation is therefore the process of confirming, or validating, that the molecule(s) identified previously have on target activity and selectivity [367,368].
T547 602-690 Sentence denotes One of the highest-impacts of NMR on drug discovery is the use as a hit-validation tool.
T548 691-863 Sentence denotes 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.
T549 864-1206 Sentence denotes 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.
T550 1207-1314 Sentence denotes They began with known inhibitors of ADC, and developed a protocol to measure the enzymatic activity of ADC.
T551 1315-1607 Sentence denotes 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).
T552 1608-1836 Sentence denotes 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].
T553 1837-1974 Sentence denotes This study demonstrated that NMR can be an effective validation tool for known drugs and for new drugs generated by a screening approach.
T554 1975-2061 Sentence denotes NMR is also able to remove false positives that emerge from biochemical screens [372].
T555 2062-2398 Sentence denotes 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].
T556 2399-2626 Sentence denotes 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.
T557 2627-2696 Sentence denotes Using ALARM NMR prioritized hits identified from HTS screening [374].
T558 2697-3025 Sentence denotes 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.
T559 3026-3138 Sentence denotes They were actually nonselective interference compounds, not necessarily specific to the inhibition of HAT [375].
T560 3139-3318 Sentence denotes 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.
T561 3319-3458 Sentence denotes The last example highlights the need for cross validation, or the combination of two or more techniques to verify identified chemical hits.
T562 3459-3525 Sentence denotes Of course, NMR is not the sole technique used for drug validation.
T563 3526-3768 Sentence denotes 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].
T564 3769-3936 Sentence denotes 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].
T565 3937-4110 Sentence denotes 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.
T566 4111-4307 Sentence denotes 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.
T567 4308-4451 Sentence denotes 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.
T568 4452-4615 Sentence denotes 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.
T569 4616-4838 Sentence denotes 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].
T570 4839-4973 Sentence denotes The work of Goudreau et al. therefore showed how NMR could be used as a co-validation technique with another biophysical method [378].
T571 4974-5121 Sentence denotes NMR can be also coupled with multiple biophysical techniques to validate a molecule’s ability to inhibit protein-protein interactions (PPIs) [367].
T572 5122-5427 Sentence denotes 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].
T573 5428-5672 Sentence denotes 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.
T574 5673-5842 Sentence denotes 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.
T575 5843-5948 Sentence denotes 1H,15N-HSQC NMR chemical shift perturbation was also used to assess and verify binding identified by SPR.
T576 5949-6139 Sentence denotes 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.
T577 6140-6346 Sentence denotes 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.
T578 6347-6564 Sentence denotes 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].
T579 6565-6750 Sentence denotes 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].
T580 6751-6962 Sentence denotes 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.
T581 6963-7103 Sentence denotes The interaction studied was that between the proteins von Hippel–Lindau (VHL), and the alpha subunit of hypoxia-inducible factor 1 (HIF-1α).
T582 7104-7293 Sentence denotes 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).
T583 7294-7535 Sentence denotes 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.
T584 7536-7779 Sentence denotes 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.
T585 7780-7947 Sentence denotes 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.
T586 7948-8197 Sentence denotes 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].
T587 8198-8563 Sentence denotes 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].
T588 8564-8726 Sentence denotes 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.