Polarization Transfer from Ligands Hyperpolarized by Dissolution Dynamic Nuclear Polarization for Screening in Drug Discovery

Min, Hlaing; Sekar, Giridhar; Hilty, Christian

doi:10.1002/cmdc.201500241

Cited by 31 publications

(52 citation statements)

References 34 publications

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“…Due to the hyperpolarization of the ligand, polarization transfer to the protein leading to visible signals even in a single scan can be expected (Min et al 2015). Enhanced signals in spectral regions characteristic of the protein, in particular side-chain protons, are indeed readily seen upon admixing of DHFR.…”

Section: Resultsmentioning

confidence: 99%

“…Ultrafast 2D NMR spectra of polarization transferred from water to exchangeable amide protons in proteins were reported (Olsen et al 2016). In our previous work, we have demonstrated that polarization transfer from a specifically binding hyperpolarized ligand shows a spectrum similar to the frequency dependence of the transfer of saturation from the protein to the ligand (Min et al 2015). Using D-DNP hyperpolarization, a two-step polarization transfer between competitively binding ligands, mediated by the protein, can be observed similar to the Interligand NOE for Pharmacophore Mapping (INPHARMA) experiment (Orts et al 2009; Lee et al 2012).…”

Section: Introductionmentioning

confidence: 92%

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Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase

2016

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Protein–ligand interaction is often characterized using polarization transfer by the intermolecular nuclear Overhauser effect (NOE). For such NOE experiments, hyperpolarization of nuclear spins presents the opportunity to increase the spin magnetization, which is transferred, by several orders of magnitude. Here, folic acid, a ligand of dihydrofolate reductase (DHFR), was hyperpolarized on 1H spins using dissolution dynamic nuclear polarization (D-DNP). Mixing hyperpolarized ligand with protein resulted in observable increases in protein 1H signal predominantly in the methyl group region of the spectra. Using 13C single quantum selection in a series of one-dimensional spectra, the carbon chemical shift ranges of the corresponding methyl groups can be elucidated. Signals observed in these hyperpolarized spectra could be confirmed using 3D isotope filtered NOESY spectra, although the hyperpolarized spectra were obtained in single scans. By further correlating the signal intensities observed in the D-DNP experiments with the occurrence of short distances in the crystal structure of the protein–ligand complex, the observed methyl proton signals could be matched to the chemical shifts of six amino acids in the active site of DHFR-folic acid binary complex. These data demonstrate that 13C chemical shift selection of protein resonances, combined with the intrinsic selectivity towards magnetization originating from the initially hyperpolarized spins, can be used for site specific characterization of protein–ligand interactions.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%

Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase

2016

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“…8 Transfer of ligand hyperpolarization via nuclear Overhauser effect to the protein or to a competing ligand can further provide a means to prove binding and potentially characterize the binding site. 9,10 While the latter may already extend beyond the most basic screening application, a parameter that is often of interest in drug discovery is the dissociation constant of the ligand for the protein. Typically, the determination of binding affinities using NMR spectroscopy is achieved by a titration of protein or ligand concentration.…”

Section: Introductionmentioning

confidence: 99%

Parallelized Ligand Screening Using Dissolution Dynamic Nuclear Polarization

2016

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Protein−ligand interactions are frequently screened using nuclear magnetic resonance (NMR) spectroscopy. The dissociation constant (KD) of a ligand of interest can be determined via a spin-spin relaxation measurement of a reporter ligand, in a single scan when using hyperpolarization by means of dissolution dynamic nuclear polarization (D-DNP). Despite nearly instantaneous signal acquisition, a limitation of D-DNP for the screening of protein−ligand interactions is the required polarization time on the order of tens of minutes. Here, we introduce a multiplexed NMR experiment, where a single hyperpolarized ligand sample is rapidly mixed with protein injected into two flow cells. NMR detection is achieved simultaneously on both channels, resulting in a chemical shift resolved spin relaxation measurement. Spectral resolution allows the use of reference compounds for accurate quantification of concentrations. Simultaneous use of two concentration ratios between protein and ligand broadens the range of KD that is accurately measurable in a single experiment to at least an order of magnitude. In a comparison of inhibitors for the protein trypsin, the average KD values of benzamidine and benzylamine were found to be 12.6±1.4 μM and 207±22.3 μM from three measurements, based on KD = 142 μM assumed known for the reporter ligand 4-(trifluoromethyl)benzene-1-carboximidamide. Typical confidence ranges at 95% evaluated for single experiments were (8.3 μM, 20 μM) and (151 μM, 328 μM). The multiplexed detection of two or more hyperpolarized samples increases throughput of D-DNP by the same factor, improving the applicability to most multi-point measurements that would traditionally be achieved using titrations.

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“…F spectra of TFA after mixing with hyperpolarized water using the pulse sequence in Figure 1a. b) Stacked plots of the successively acquired 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 11 parameter is σ = 2.6110 -4 s -1 M -1 for the data set shown here.…”

Section: Resultsmentioning

confidence: 99%

“…In this case, the cross-relaxation parameter (3) can be defined, which takes on the same value irrespective of the concentrations in the sample. The 19 F signal intensities were modeled during each time period free of rf pulses with analytical solutions of these equations with arbitrarily selectable initial signal intensities for both spins:…”

Section: Methodsmentioning

confidence: 99%

Determination of Intermolecular Interactions Using Polarization Compensated Heteronuclear Overhauser Effect of Hyperpolarized Spins

et al. 2015

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The nuclear Overhauser effect (NOE) has long been used as a selective indicator for intermolecular interactions. Due to relatively small changes of signal intensity, often on the order of several percent, quantitative NOE measurements can be challenging. Hyperpolarization of nuclear spins can dramatically increase the NOE intensity by increasing population differences, but poses its own challenge in quantifying the original polarization level. Here, we demonstrate a method for the accurate measurement of intermolecular heteronuclear cross-relaxation rates by simultaneous acquisition of signals from both nuclei. Using this method, we measure cross-relaxation rates between water protons and (19)F of trifluoroacetic acid at concentrations ranging from 23 to 72 mM. A concentration-independent value of 2.46 × 10(-4) ± 1.02 × 10(-5) s(-1) M(-1) is obtained at a temperature of 301 K and validated using a nonhyperpolarized measurement. In a broader context, accurate measurement of heteronuclear cross-relaxation rates may enable the study of intermolecular interactions including those involving macromolecules where (19)F atoms can be introduced as site-selective labels.

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Polarization Transfer from Ligands Hyperpolarized by Dissolution Dynamic Nuclear Polarization for Screening in Drug Discovery

Cited by 31 publications

References 34 publications

Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase

Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase

Parallelized Ligand Screening Using Dissolution Dynamic Nuclear Polarization

Determination of Intermolecular Interactions Using Polarization Compensated Heteronuclear Overhauser Effect of Hyperpolarized Spins

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