NMR magnetization-transfer analysis of rapid membrane transport in human erythrocytes

We have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carriermediated transmembrane transport of fluorinated substrates. The method is based on adapted kinetic theory that describes the concentration dependence of the transmembrane exchange rates of two competing, simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogs in human erythrocytes (red blood cells) using 19 F NMR exchange spectroscopy. An inosine-based glucose-free medium was shown to promote survival and stable metabolism of red blood cells over the duration of the experiments (several hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we could categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 toward them. Differences in the membrane permeability of the three glucose analogs were qualitatively interpreted in terms of local perturbations in the bonding of substrates to key amino acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results can be applied to the design of probes of glucose transport or inhibitors of glucose metabolism in cells, including those exhibiting the Warburg effect.

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Section: Determination Of Exchange Rates Constantsmentioning

confidence: 99%

Transmembrane Exchange of Fluorosugars: Characterization of Red Cell GLUT1 Kinetics Using 19F NMR

Shishmarev

Fontenelle

Kuprov

et al. 2018

Biophysical Journal

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“…[78][79][80] From an experimental standpoint, NMR-based fragment screening allows fragments that bind to the protein target of interest to be discovered mainly by monitoring variations on the fundamental NMR signal: the chemical shift, either of the ligand or of the target. [81] Alternatively, other experimental measurements can be followed, such as NOE effects, [82][83][84][85] relaxation times, [86][87][88] magnetization transfer, [89,90] and the translational diffusion coefficient. [91][92][93] On that respect, NMR fragment screening techniques provide the versatility to allow medicinal chemists to embark on either ligand-or target-based drug design, or on a combination of both.…”

Section: Nmr-based Fragment Screening: a Summary Of The Central Technmentioning

confidence: 99%

Using NMR Spectroscopy in the Fragment‐Based Drug Discovery of Small‐Molecule Anticancer Targeted Therapies

Diethelm‐Varela

2020

ChemMedChem

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Against the challenge of providing personalized cancer care, the development of targeted therapies stands as a promising approach. The discovery of these agents can benefit from fragment‐based drug discovery (FBDD) methods that help guide ligand design and provide key structural information on the targets of interest. In particular, nuclear magnetic resonance spectroscopy is a promising biophysical tool in fragment discovery due to its detection capabilities and versatility. This review provides an overview of FBDD, describes the basis of NMR‐based fragment screening, summarizes some exciting technical advances reported over the past decades, and closes with a discussion of selected case studies where this technique has been used as part of drug discovery campaigns to produce lead compounds towards the design of anti‐cancer targeted therapies.

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“…Solution of the Bloch-McConnell differential equations gives the dependence of the intensities of the NMR signals in a magnetization-transfer experiment on the mixing time (34). In the 1D-EXSY experiment, employed for quantification in the present work, the radio-frequency transmitter was set 'on resonance' with the intracellular spin-population in order to achieve the selective inversion of its magnetization at the beginning of the experiment (pulse sequence of Fig.…”

Section: Determination Of Anomer Exchange Rates Constantsmentioning

confidence: 99%

Transmembrane exchange of fluorosugars: characterization of red cell GLUT1 kinetics using ¹⁹F NMR

Shishmarev¹,

Fontenelle²,

Kuprov³

et al. 2018

Preprint

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We developed a novel approach for quantifying the equilibrium-exchange kinetics of carrier-mediated transmembrane transport of fluorinated substrates. Our method is based on an adapted kinetic theory describing the concentration dependence of the transmembrane-exchange rates of two simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogues in human erythrocytes (red blood cells: RBCs) using 19 F nuclear magnetic resonance (NMR) exchange spectroscopy (EXSY).An inosine-based glucose-free medium was shown to promote survival and stable metabolism of RBCs over the duration of the experiments (a few hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we were able to categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 towards them. Differences in the membrane permeability of the three glucose analogues were qualitatively interpreted in terms of local perturbations in the bonding of substrates to amino-acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results will apply to the design of probes of glucose transport, or inhibitors of glucose metabolism in cells including those exhibiting the Warburg effect.

show abstract

NMR magnetization-transfer analysis of rapid membrane transport in human erythrocytes

Cited by 9 publications

References 87 publications

Transmembrane Exchange of Fluorosugars: Characterization of Red Cell GLUT1 Kinetics Using 19F NMR

Transmembrane Exchange of Fluorosugars: Characterization of Red Cell GLUT1 Kinetics Using 19F NMR

Using NMR Spectroscopy in the Fragment‐Based Drug Discovery of Small‐Molecule Anticancer Targeted Therapies

Transmembrane exchange of fluorosugars: characterization of red cell GLUT1 kinetics using ¹⁹F NMR

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NMR magnetization-transfer analysis of rapid membrane transport in human erythrocytes

Cited by 9 publications

References 87 publications

Transmembrane Exchange of Fluorosugars: Characterization of Red Cell GLUT1 Kinetics Using 19F NMR

Transmembrane Exchange of Fluorosugars: Characterization of Red Cell GLUT1 Kinetics Using 19F NMR

Using NMR Spectroscopy in the Fragment‐Based Drug Discovery of Small‐Molecule Anticancer Targeted Therapies

Transmembrane exchange of fluorosugars: characterization of red cell GLUT1 kinetics using 19F NMR

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Transmembrane exchange of fluorosugars: characterization of red cell GLUT1 kinetics using ¹⁹F NMR