Time efficient detection of protein–ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment

The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5-to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G 2 -M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-a-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-a-dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 mmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor-bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K.

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“…T 2 experiments were measured with a CPMG pulse train of 200 ms. WaterLOGSY experiments were measured in sensitive mode as described before (12).…”

Section: Fmentioning

confidence: 99%

Characterization of the Mechanism of Action of the Pan Class I PI3K Inhibitor NVP-BKM120 across a Broad Range of Concentrations

Brachmann

Kleylein-Sohn

Gaulis

et al. 2012

Molecular Cancer Therapeutics

111

100

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“…NMR sequence for screening ligand-protein interactions [40,41,47,48]. The pulse sequence is shown in Fig.…”

Section: Numerical Simulation Of Noe Based On Clay-bound Interactionsmentioning

confidence: 99%

“…This sequence was conceived to exploit the differences in the dynamics between bound water on protein surfaces and free water in solution. When water molecules are bound on a protein, their correlation times will increase temporarily to match that of the protein, thus leading to a change in the sign of their NOE [40,47,48]. Similarly, for the case of clay-bound water molecules, the sign of their NOE will be opposite of that in free water due to the differences in their correlation times.…”

Section: Numerical Simulation Of Noe Based On Clay-bound Interactionsmentioning

confidence: 99%

“…In this case, the relationship between the rate of NOE transfer between xenobiotics and clay-bound water can be described via Eq. (2) [40,47,48] (2) Fig. 3 A schematic illustrating the radio frequency (r.f.)…”

Section: Numerical Simulation Of Noe Based On Clay-bound Interactionsmentioning

confidence: 99%

“…In the present study, we demonstrate the avenue in which water-mediated NOE can be transferred to a clay-bound molecules via an NMR radio frequency (r.f.) sequence called Water Ligand Observed via Gradient Spectroscopy (waterLOGSY) [40,47,48]. In order to illustrate the usefulness of this sequence, a series of xenobiotic compounds will be screened for their affinity for clay particles, allowing us to evaluate its effectiveness in organo-clay research.…”

Section: Introductionmentioning

confidence: 99%

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Water-mediated NOE: a promising tool for interrogating interfacial clay–xenobiotic interactions

Soong

Botana

Wang

et al. 2017

Chem. Biol. Technol. Agric.

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Background:The sorption of anthropogenic compounds on clay minerals is a complex molecular process with important implications for the fate of agrochemicals and organic pollutants in the environment. Results:The present study illustrates the use of a water-mediated NOE approach to study clay binding interactions. This method exploits the interfacial water layer on clay surfaces as a hydrogen reservoir for magnetization transfer. The interactions of four different xenobiotics with clay suspension were investigated through this method to demonstrate its capability to screen for the clay-xenobiotic molecular affinity. Further, based onthe NOE build-up rates, epitope map of clay-xenobiotic interactions can be generated, explaining theorientation and mechanism of the interactions. Conclusions:The water-mediated NOE approach has the potential to reveal key insights into the role that interfacial water plays in the binding process, providing a better understanding of the partitioning of anthropogenic compounds from bulk water into aqueous clay suspensions.

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Saturation Transfer Difference Spectroscopy

Gairì

Feliz

García

2016

Encyclopedia of Analytical Chemistry

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Saturation transfer difference (STD) NMR is a powerful technique for detection and characterization of reversible binding interactions between small molecule ligands and macromolecular receptors. The STD methodology is broadly used for detection of protein–ligand interactions and for fragment screening and has also been successfully applied to complex systems such as reconstituted membranes, intact cells, platelets, or whole viruses. The basic STD experiment relies on1H NMR (Nuclear Magnetic Resonance) difference spectroscopy. Saturation transferred from the receptor to bound ligands in the complex is measured by observing the NMR signal of the ligand. The corresponding STD NMR spectrum reveals only signals of binding compounds. Quantitative analysis of the STD effects allows the identification of the binding epitope of a ligand when bound to its receptor and can also provide affinity information. Water–ligand observed gradient spectroscopy (WaterLOGSY) is a related method for the characterization of transient ligand–receptor interactions, but unlike STD, in WaterLOGSY experiments, the bulk water is the origin of the perturbation transfer.

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Time efficient detection of protein–ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment

Cited by 46 publications

References 15 publications

Characterization of the Mechanism of Action of the Pan Class I PI3K Inhibitor NVP-BKM120 across a Broad Range of Concentrations

Characterization of the Mechanism of Action of the Pan Class I PI3K Inhibitor NVP-BKM120 across a Broad Range of Concentrations

Water-mediated NOE: a promising tool for interrogating interfacial clay–xenobiotic interactions

Saturation Transfer Difference Spectroscopy

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