Development of an Optimized Protocol for NMR Metabolomics Studies of Human Colon Cancer Cell Lines and First Insight from Testing of the Protocol Using DNA G-Quadruplex Ligands as Novel Anti-Cancer Drugs.

The study of cell lines by nuclear magnetic resonance (NMR) spectroscopy metabolomics represents a powerful tool to understand how the local metabolism and biochemical pathways are influenced by external or internal stimuli. In particular, the use of adherent mammalian cells is emerging in the metabolomics field in order to understand the molecular mechanism of disease progression or, for example, the cellular response to drug treatments. Hereto metabolomics investigations for this kind of cells have generally been limited to mass spectrometry studies. This study proposes an optimized protocol for the analysis of the endo-metabolome of human colon cancer cells (HCT116) by NMR. The protocol includes experimental conditions such as washing, quenching and extraction. In order to test the proposed protocol, it was applied to an exploratory study of cancer cells with and without treatment by anti-cancer drugs, such as DNA G-quadruplex binders and Adriamycin (a traditional anti-cancer drug). The exploratory NMR metabolomics analysis resulted in NMR assignment of all endo-metabolites that could be detected and provided preliminary insights about the biological behavior of the drugs tested.

Use of NMR in profiling of cocaine seizures.

Cocaine is the most widely used illicit drug, and its origin is always the focus of intense investigation aimed at identifying the trafficking routes. Since NMR represents a unique methodology for performing chemical identification and quantification, here it is proposed a strategy based on (1)H NMR spectral analysis in conjunction with multivariate analysis to identify the chemical "fingerprint" of cocaine samples, and to link cocaine samples based on this information. The most relevant spectral regions containing the fingerprint have been identified: δH 0.86-0.96, 1.50-1.56, 5.90-5.93, 6.48-6.52, 7.31-7.34, 7.61-7.63, 7.68-7.72 ppm. The strategy has been applied on samples seized in different times and places in Naples (Italy). The chemical "fingerprint" depend on what plant they were extracted from, where it was cultivated, and which procedures were used for extraction and purification, thus adding significant information in the process toward identification of the trafficking routes for this drug.

Application of "magnetic tongue" to the sensory evaluation of extra virgin olive oil.

The perception of odour and flavour of foods is a complicated physiological and psychological process that cannot be explained by simple models. Unfortunately, taste is not objective, but partially subjective and it depends also on the mood of the taster. Generally, sensory analysis is used to describe sensory features. The availability of a number of instrumental techniques has opened up the possibility to calibrate the sensory perception. Here we have tested the potentiality of nuclear magnetic resonance spectroscopy as "magnetic tongue" to measure sensory descriptors in extra-virgin olive oil. We were able to correlate the NMR metabolomic fingerprints of extra-virgin olive oil to the sensory descriptors: tomato, bitter, pungent, rosemary, artichoke, sweet, grassy and leaf.

Shooting for selective druglike G-quadruplex binders: evidence for telomeric DNA damage and tumor cell death.

Targeting of DNA secondary structures, such as G-quadruplexes, is now considered an appealing opportunity for drug intervention in anticancer therapy. So far, efforts made in the discovery of chemotypes able to target G-quadruplexes mainly succeeded in the identification of a number of polyaromatic compounds featuring end-stacking binding properties. Against this general trend, we were persuaded that the G-quadruplex grooves can recognize molecular entities with better drug-like and selectivity properties. From this idea, a set of small molecules was identified and the structural features responsible for G-quadruplex recognition were delineated. These compounds were demonstrated to have enhanced affinity and selectivity for the G-quadruplex over the duplex structure. Their ability to induce selective DNA damage at telomeric level and to induction of apoptosis and senescence on tumor cells is herein experimentally proven.

State-of-the-art methodologies for the discovery and characterization of DNA G-quadruplex binders.

Nowadays, the molecular basis of interaction between low molecular weight compounds and biological macromolecules is the subject of numerous investigations aimed at the rational design of molecules with specific therapeutic applications. In the last decades, it has been demonstrated that DNA quadruplexes play a critical role in several biological processes both at telomeric and gene promoting levels thus providing a great stride in the discovery of ligands able to interact with such a biologically relevant DNA conformation. So far, a number of experimental and computational approaches have been successfully employed in order to identify new ligands and to characterize their binding to the DNA. The main focus of this review is the description of these methodologies, placing a particular emphasis on computational methods, isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopies.

NMR spectrometers as "magnetic tongues": prediction of sensory descriptors in canned tomatoes.

The perception of odor and flavor of food is a complicated physiological and psychological process that cannot be explained by simple models. Quantitative descriptive analysis is a technique used to describe sensory features. Nevertheless, the availability of a number of instrumental techniques has opened up the possibility to calibrate the sensory perception. In this frame, we have tested the potentiality of nuclear magnetic resonance spectroscopy as a predictive tool to measure sensory descriptors. In particular, we have used an NMR metabolomic approach that allowed us to differentiate the analyzed samples based on their chemical composition. We were able to correlate the NMR metabolomic fingerprints recorded for canned tomato samples to the sensory descriptors bitterness, sweetness, sourness, saltiness, tomato and metal taste, redness, and density, suggesting that NMR might be a very useful tool for the characterization of sensory features of tomatoes.

A more detailed picture of the interactions between virtual screening-derived hits and the DNA G-quadruplex: NMR, molecular modelling and ITC studies.

The growing amount of literature about G-quadruplex DNA clearly demonstrates that such a structure is no longer viewed as just a biophysical strangeness but it is instead being considered as an important target for the treatment of various human disorders such as cancers or venous thrombosis. In this scenario, with the aim of finding brand new molecular scaffolds able to interact with the groove of the DNA quadruplex [d(TGGGGT)](4), we recently performed a successful structure-based virtual screening (VS) campaign. As a result, six molecules were found to be somehow groove binders. Herein, we report the results of novel NMR titration experiments of these VS-derived ligands with modified quadruplexes, namely [d(TGG(Br)GGT)](4) and [d(TGGGG(Br)T)](4). The novel NMR spectroscopy experiments combined with molecular modelling studies, allow for a more detailed picture of the interaction between each binder and the quadruplex DNA. Noteworthy, isothermal titration calorimetry (ITC) measurements on the above-mentioned compounds revealed that 2, 4, and 6 besides their relatively small dimensions bind the DNA quadruplex [d(TGGGGT)](4) with higher affinity than distamycin A, to the best of our knowledge, the most potent groove binder identified thus far.