NMR Insights into the Inner Workings of Living Cells - American

Review pubs.acs.org/ac

NMR Insights into the Inner Workings of Living Cells Mathilde H. Lerche,† Pernille R. Jensen,† Magnus Karlsson,† and Sebastian Meier*,‡,§ †

Albeda Research, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark § Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 201, 2800 Kongens Lyngby, Denmark ‡

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CONTENTS

Hyperpolarized Small-Molecule NMR Probes for Cell Biology Analytical Aspects of Hyperpolarized NMR Sensitivity Time Scale and Generalizability Substrate Preparations and Biocompatibility Background and Interferences Biochemical Specificity and Mechanistic Understanding Reproducibility Throughput Data Analysis Developments and Directions Biological Problems That Are Addressed with Hyperpolarized NMR Probes Metabolic Pathways Observing Biological Functionality in Different Cell Types and Mutants Cellular Response to Small Molecule Effectors Import and Export Across the Cell Membrane Discovery and Evaluation of Candidate Molecular Markers Concluding Remarks Author Information Corresponding Author Notes Biographies Acknowledgments References

chemical resolution, but current instrumentation is not highly sensitive.3 The scope of NMR spectroscopy for cell biology greatly improves by increasing the signal-to-noise ratio. These improvements are sought both through methodological and technological improvements, including the enhancement of NMR detectable nuclear magnetism in the molecules of interest.4 Such improvements have permitted the noninvasive, selective observation of macromolecules and small molecular probes with enhanced time resolution and sensitivity against cellular backgrounds (Table 1). In this manner, NMR spectroscopy keeps developing into an improved method yielding previously inaccessible information for cell biology with molecular detail and resolution of individual atomic sites. NMR platforms with different scopes for visualizing cellular structures and functions have materialized, including (1) highresolution in-cell NMR of macromolecules using cells as test tubes for determining macromolecular structure, tumbling, dynamics, and interactions in cellular environments; (2) solidstate NMR spectra of whole cell preparations or structures and interactions at the cell surface or in native membranes; (3) the measurement of cell physiological parameters (e.g., pH, [Ca2+], redox state) and dynamic (time-resolved) tracking of metabolite fluxes through cellular reactions with endogenous small molecule probes and high-resolution NMR detection. Using these platforms, a variety of applications have been developed for the probing of macromolecular structure, dynamics, and interactions with liquid state NMR spectroscopy of living cells and with solid-state NMR spectroscopy of cell preparations. An overview of these applications is given in Table 2. Comprehensive reviews covering in-cell and on-cell NMR of macromolecules16−18 have recently been assembled. Here, we therefore focus more specifically on the current promises and challenges for fundamental insights into the inner workings of living cells, using NMR detectable probes generated by “hyperpolarization methods” that maximize the signal of the detected molecules.14,15 Hyperpolarization is a collective term for a physical spin ordering approach that temporarily enhances NMR-detectable nuclear magnetism by several orders of magnitude, either directly or through the coupling of nuclear magnetic moments to other spins with high polarization (Figure 1). Various hyperpolarization methodologies have been described.37−40 While the theoretical basis for hyperpolarization methods is not

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ome of the most important chemical problems involve complex molecular systems such as living cells. Increasingly advanced methodologies facilitate insights into complex systems or enable entirely new types of experiments by visualizing chemical processes that are invisible to the naked eye.1 Technologies for the cataloguing of cellular components by disruption, amplification, and physical separation are well established. In contrast, nondisruptive insights into the functions and interactions of cellular components remain a considerable challenge.2 Within the past few years, however, spectroscopy has evolved to levels of sophistication that form the basis for gaining insights into the inner workings of the living cell. The scrutiny of living systems usually hinges upon nondestructive optical or nuclear spectroscopy using lowenergy radiation. Nuclear magnetic resonance (NMR) detection is the lowest-energy method and provides excellent © XXXX American Chemical Society

Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2015

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Table 1. Approaches Supporting Magnetic Resonance Detection of Biomolecules in Live Cells approach

gain in signal to background

gain in signal-to-noise

detected molecules

∼4× ∼2−10×

macromolecules macromolecules, small molecule probes small molecule probes and macromolecules small molecule probes

cooled detection electronics2 rapid pulsing methods5−7 C, 15N isotope enrichment8−11 or tagging12,13 hyperpolarization14,15

13

supraphysiological concentrations

100−250×

100−250×

up to ∼105×

up to ∼105× relative to single scan 13C/15N NMR experiments at 14.1 T >1×

>1×

small molecule probes and macromolecules

especially at the cell surface and to resolve cellular components by solid-state NMR spectroscopy.19,34−36,41 These solid state methods in frozen cellular preparations overcome sensitivity and size limitation of in-cell liquid state NMR but are unsuitable for tracking dynamic processes in real time. Nuclear spins have been hyperpolarized by the transfer of electron polarization, parahydrogen spin order, or photon angular moment to NMR detectable nuclei (Figure 1A). At the time of writing, dynamic nuclear polarization (DNP) using electron polarization is by far the most widely used means of hyperpolarization for biological applications. DNP proceeds in the solid state by transfer of electron spin polarization to atomic nuclei in amorphous samples at low temperature under microwave irradiation near the electron Larmor frequency. Unpaired electrons are only needed in catalytic amounts in the DNP process and are usually provided by the addition of stable radicals. For the production of hyperpolarized small molecule probes that are useful in cell biological assays, hyperpolarized samples are harvested to ambient temperatures by rapidly washing the probe out of the polarizer with hot buffer.15 Thus, current instrumentation generates enhanced nuclear magnetism in a probe molecule ex situ in a dedicated instrument and analyzes the liquefied probe in conventional NMR or MRI instruments (Figure 1B). This approach, called rapid dissolution DNP, yields molecular probes in liquid samples with enhancements of NMR detectable signal on the order of 105 and has founded a burgeoning field: hundreds of studies have harnessed the power of rapid dissolution DNP during the past 5−10 years using almost 100 different hyperpolarized molecules.42−44 Descriptions of probe formulations,42,43 hyperpolarization theory and techniques, assay types with hyperpolarized probes,44 and their use as contrast agents45−48 have recently been compiled and are outside the scope of this review. Rather, the current review attempts to emphasize the challenges and emerging strategies toward gaining fundamental biological insights. Such fundamental insights into cellular (dys-)function are particularly relevant in disciplines benefiting from systems approaches, including strain improvement for biotechnological applications49 and the study of complex pathologies.50

Table 2. Examples of Macromolecular In-Cell NMR Observations and Solid-State NMR Observations of Cell Preparations • macromolecular structures and folding state19−23 • macromolecular tumbling and dynamics16,22 • protein−protein, protein−nucleic acid, protein−small molecule interaction24−28 • posttranlational modifications29−31 • protein−ligand interactions at the cell surface32 • cell surface glycan structures, on-cell NMR33,34 • recognition at the cell surface35 • protein, nucleic acids and lipids in whole cells36

Figure 1. (A) Nuclear spins can be hyperpolarized by the transfer of electron polarization, photon angular moment, or spin order of parahydrogen (p-H2) to yield an increase in NMR detectable polarization by several orders of magnitude. (B) Schematic representation of a rapid dissolution DNP-NMR setup. Prior to cellular NMR spectroscopy, hyperpolarized probe molecules are generated ex situ (B0, B0′ are the static magnetic fields in the polarizer and spectrometer, respectively, and MW designates microwave irradiation).

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HYPERPOLARIZED SMALL-MOLECULE NMR PROBES FOR CELL BIOLOGY Despite their sensitivity constraints, NMR methods provide considerable opportunities for collecting diverse and unique information within a reasonable time and cost. The noninvasive nature of NMR spectroscopy opens up the possibility for longitudinal studies in single samples without compromising statistical significance.51 The real-time tracking of endogenous molecules in working cells is generally fraught by the challenges of (1) selectivity for molecules of interest against cellular

a recent discovery, these approaches have found biological application and subsequent commercialization and have become wider spread only during the past decade. Hyperpolarization of cellular preparations in the solid-state has been employed to study macromolecular structures and interaction B

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Analytical considerations for the use of hyperpolarized probes are therefore detailed in the following. Sensitivity. NMR detection has been a principal means for tracking highly concentrated biomolecules in living cells for more than 4 decades, due to the aptitude for selective (highresolution) analyte detection in aqueous solutions.8,99−102 The limited sensitivity of conventional NMR, however, implies the need for signal accumulation. In consequence, the method fails in the detection of rapid cellular processes and low-populated metabolic intermediates owing to lacking sensitivity. The use of hyperpolarized metabolites addresses this limitation and allows the observation of transient cellular reactions, including the detection of reaction intermediates. Polarization levels approach the theoretical maximum in some instances and generally leave only limited room for further improvement.4 NMR detectors are however not highly sensitive. Currently, hyperpolarized molecules are rapidly detectable at concentrations in the sample volume of slightly below micromolar, corresponding to proportionally higher concentrations in the intracellular volume of dilute cell suspensions.103 Usually, only a minor fraction of the substrate is taken up and metabolized during the assay time scale of a few minutes (see below). At the time of writing, hyperpolarized substrates are therefore used at least in millimolar concentrations and hence do not qualify as “massless tracers” with no effect on the biological system under study. Hyperpolarized NMR only remedies the sensitivity shortcomings of conventional NMR in part. While relaxation replenishes thermal equilibrium polarization and permits signal accumulation through cycles of excitation and relaxation in conventional NMR, both excitation and T1 relaxation toward thermal equilibrium reduce the hyperpolarized signal. The polarization approaches its thermal equilibrium inside the NMR spectrometer with a characteristic time constant T1 and hyperpolarization is not replenished in the spectrometer upon excitation. Hence, time-resolved measurements using hyperpolarized probes are commonly conducted with a small-flip angle excitation that only uses part of the hyperpolarization for each time point (5° or 10° flip angles preserve 99.6%/98.4% of the longitudinal magnetization and result in a transverse magnetization of 8.7%/17.4%), thereby allowing for the acquisition of a series of spectra before hyperpolarization is lost. Hyperpolarization of the substrate can be protected from losses due to excitation through the use of selective excitation of the products.104 This approach improves the collection of metabolic signals without enforcing detrimental polarization losses onto the substrate. Time Scale and Generalizability. Hyperpolarization methods for optimizing polarization in small molecule probes are ex situ approaches that do not currently lend themselves to replenishing probe signal or turn-on sensing. Biological assays therefore need to be conducted on the time scale of the hyperpolarization lifetime. In contrast, hyperpolarization can be generated and replenished in situ in solid-state DNP-NMR systems. The time scale of assays using hyperpolarized NMR probes is limited to ∼3−5 × T1, as more than 99% of the hyperpolarized magnetization is faded after a time of 5 × T1. Biological probes with long T1, and rapid transfer of the probe from the ex situ polarization to the detecting NMR spectrometer are therefore advantageous. While endogenous metabolites can reach T1 times of up to 1−3 min at available NMR spectrometer magnetic fields (∼7−20 T), designed probe molecules have

backgrounds, (2) sufficient time resolution and sensitivity near physiological concentrations, (3) noninvasiveness, and (4) the preferred use of the natural, endogenous molecules, in order to abolish any doubts about the physiological relevance of the probe replacing the natural substrate (tracee). Hyperpolarization has been used as a tracer technique, devising NMR detectable small molecule probes for the visualization of cellular functions that are not observable by other means, for instance, by detection of fluorescence markers. Using hyperpolarization, probe signals can be selectively enhanced by more than 6 orders of magnitude through the combined effect of hyperpolarization and 13C or 15N isotope enrichment. Such enhancements can be sufficient to track endogenous molecules at close-to-natural concentrations. Until recently, nuclear spin hyperpolarization has largely developed outside analytical biochemistry and analytical chemistry.43,45 The potential of hyperpolarized endogenous molecules has been especially recognized in noninvasive functional studies of tissues.52−94 Rapidly after real-time metabolic imaging of tissues with hyperpolarized metabolites was conceived,95,96 the application of hyperpolarization approaches to visualizing transient phenomena in simpler systems was suggested.3 Simple biological model systems arguably have a role to play in making hyperpolarized probes useful as screening methods and in predicting cellular behavior. The application of hyperpolarized probes to cell suspensions (and perfused tissues ex vivo) benefits from several advantages in experiment design and data interpretation. Cells form the functional and structural unit of life. Not only are cell cultures vital for the synthesis of biotechnological products but also for studies of cellular responses to well-defined environmental effectors, stresses, and genetic alterations. The experiments are easy to control and are not compromised by interindividual differences, effects of anesthesia or adrenergic response, or other physiological changes that occur over time in vivo. In addition, cell cultures require less ethical consideration than in vivo studies while permitting integration with established proteomic and genomic data.97 Bioassays in cell culture specifically allow higher throughput, improved probe delivery to cells, improved sensitivity, lower cost, multiplexed parallel detections, and a simpler quantitative interpretation of real-time data for probe transport and enzymatic probe conversion.98 Because of their noninvasiveness, the experiments can be conducted in single samples using various hyperpolarized probes simultaneously or by maintaining cells in bioreactors for sequential experiments.43 Nevertheless, dedicated applications to suspensions of model organisms and cell line models have received increasing attention only rather recently.

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ANALYTICAL ASPECTS OF HYPERPOLARIZED NMR Hyperpolarized probe molecules have been used in various assay types, such as (1) the real-time tracking of metabolism by reaction progress curves, (2) tagging of amino acids, (3) ratiometric assays, mostly of state (pH, redox state, reactive oxygen species), (4) binding or diffusion assays, (5) fusion of hyperpolarized probes to variable sensing units (enzyme activities, Ca2+ concentrations, reactive oxygen species), and (6) fusion of reporter enzymes to genes of interest and subsequent assay of the reporter enzyme with a bio-orthogonal hyperpolarized probe. In all of these assay types, hyperpolarized probes have distinguishing advantages and shortcomings relative to other members of the cellular biology toolbox. C

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abundances of NMR active 13C and 15N are 1.1% and 0.4%, respectively), and the low sensitivity of NMR detectors result in very small background signals from most cellular components. Isotope enrichment of molecular probes with 13C and 15N (100−250-fold) in conjunction with hyperpolarization selectively increases the 13C and 15N signals of interest by 6−7 orders of magnitude. Background signals are virtually entirely absent in published DNP NMR assays and are usually smaller than the noise. Interferences in form of signal overlap are less of a problem than for conventional 1H NMR spectroscopy due to (1) the higher signal dispersion (∼20-fold larger chemical shift range for 13C than for 1H NMR), (2) the narrow line widths from commonly employed small molecule metabolites, and (3) the absence of solvent signals. The principal sources of overlap are substrate signals or impurities in substrate preparations. Arguably, the generation of tracers and resolution advantages in 13 C NMR spectra provides the principal advantage of hyperpolarized 13C NMR spectroscopy, while sensitivity advantages relative to conventional 1H NMR spectroscopy are more moderate or even absent. NMR spectroscopy has a very rich information content compared to other spectroscopic methods. Spectral frequencies are accurately measured with parts-per-billion accuracy (relative to a ∼200 ppm spread in signals) and report on the composition, constitution, and configuration of detected molecules. Signals are quantifiable and scale linearly with the molar concentration over a wide dynamic range. Chemical structure and biosynthetic pathways can be deduced from line splittings,124 while line widths and the loss of hyperpolarization report on molecular tumbling.103,125 The resolution of individual atomic sites permits the simultaneous detection of multiple probes. Signal assignments are commonly conducted based upon chemical shift measurements and known metabolic network structures. The singlescan nature of hyperpolarized NMR complicates the acquisition of multidimensional spectra for signal assignment, albeit such methods have proven principally feasible.126−128 The identification of metabolites contributing to the detected signals can therefore sometimes remain ambiguous.67,129 Assignments depend on standard reference spectra recorded near physiological pH or on the establishment of consistently referenced chemical shift databases of intracellular signals. Kinetic profiles of metabolites and signal multiplicity can aid the identification of a metabolite’s chemical structure. A persisting challenge lies in the distinction of intracellular and extracellular probe signals but also in the distinction of intracellular enzyme activities and cell necrosis or leakage. At the relatively low cell densities that have been used in hyperpolarized NMR, the detection of reactions that require cofactors strongly indicates the detection of intracellular enzyme activities rather than leakage of catalysts from cells. Biochemical Specificity and Mechanistic Understanding. Endogenous molecules as hyperpolarized probes permit tracking cellular functions without doubts about the physiological relevance of the substrate. In contrast, fluorophores or other chemically introduced reporter groups affect the biological function of small molecules. Assays employing endogenous molecules as hyperpolarized probes are intrinsically specific due to the specificity of enzyme−substrate recognition. Despite the use of endogenous molecules, the mechanistic details of the observations often remain difficult to determine. For instance, it is not a priori clear if the cellular uptake of a probe molecule or its reaction in the cell is rate

been devised that contain a nuclear position with T1 times of up to 15 min.105−107 Unsurprisingly, efforts are being undertaken toward prolonging the assay lifetime by storing hyperpolarization in nuclear singlet order, which is protected against many relaxation mechanisms108−113 and toward establishing hyperpolarization in situ in the liquid state in order to replenish hyperpolarization.114,115 So far, these approaches have, however, not been established for metabolic studies. The T1 time is increased for symmetric sites without nuclear dipoles in their vicinity and for small molecules with shorter rotational correlation times. Thus, T1 relaxation does not only set limits for the assay time scale, but also means that cellular assays are best conducted with substrates of molecular weight below ∼200−300 Da. An analysis of molecular weight distributions of metabolites in Saccharomyces cerevisiae and Escherichia coli indicates that up to two-third of all metabolites are under MW 300.116 This restriction to small molecules renders hyperpolarized probes complementary to optical probes: unlabeled metabolites are usually difficult to detect with optical methods, while markers such as fluorophores are larger than most metabolites and prevent the metabolite from performing its biological function in living cells.62,117 Thus, marker-free approaches are especially favorable for small molecules. The temporal resolution (and lower limit for the assay time scale) is given by the acquisition of the NMR signal during milliseconds to hundreds of milliseconds for real-time biological assays. As hyperpolarized NMR is a powerful method for probes that are rapidly taken up and modified by the cell, approaches for improving cellular uptake and retention, for instance by probe derivatization and facilitation of passive influx, would greatly benefit biological applications of hyperpolarized NMR. Substrate Preparations and Biocompatibility. The polarization process itself is generalizable and thus permits the generation of a variety of hyperpolarized substrates for metabolic studies in living cells: specifically the success of the DNP methods for hyperpolarization hinges on their general applicability, and no small molecule probe, to our knowledge, poses insurmountable challenges for DNP enhancement. The probe should, however, be soluble and biochemically stable under the potentially harsh dissolution conditions. Biocompatibility is a minor issue for endogenous substances used at near physiological concentrations. DNP preparations contain, however, slight amounts of stable radicals and additives, which are ideally removed prior to biological assays. Recently, it was shown that addition of persistent radical sources can be avoided altogether118 by photoinduced radicals of α-ketoacid substrates. This approach generates radical pairs from the probe molecule itself with UV-irradiation of frozen probe preparations. The radicals then recombine upon dissolution of the probe and result in solutions of purely endogenous products. Alternative approaches have scavenged free radicals and paramagnetic ions by reduction with vitamin C, ion exchange, entrapment in gels, or rapid extraction into a different solvent phase.119−123 In addition to removing potentially toxic radicals, these approaches avoid a shortening in the lifetime of liquefied hyperpolarized probes by paramagnetic impurities. Background and Interferences. Spectroscopy of living cells is often hampered by the signal-to-background ratios rather than signal-to-noise ratios. That is usually not the case for hyperpolarized NMR using small molecule probes. The low natural abundance of well-suited NMR nuclei (the natural D

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methods enable assay throughputs approaching the hyperpolarization lifetime (few minutes per assay). Improvements in throughput are significant, as rapid metabolic methods are highly desirable in times where genomics and proteomics outpace metabolic characterizations.2 In parallel to exploring fundamental cell biology, small molecule probes could thus also permit high-throughput screening of cellular responses. Such transformations toward screening methods still lie ahead.139 It is noteworthy that parahydrogen induced hyperpolarization (PHIP) currently can be generated more rapidly than DNPinduced hyperpolarization, but this advantage in throughput is offset by the limited set of substrates that is amenable to PHIP. Notwithstanding, an optimized throughput in biological assays could therefore, in principle, also arise from a broader applicability of PHIP hyperpolarization.45,140 Data Analysis. Mathematical models for the interpretation of kinetic data obtained with hyperpolarized probes have to incorporate relaxation rates, flip angles, and repetition rates.68,75,85,141−143 The loss of hyperpolarization by T1 relaxation contributes information to hyperpolarized NMR assays but at the same time complicates the extraction of quantitative information from the observations, especially as intracellular relaxation rate constants and metabolite concentrations are a priori unknown. In addition, hyperpolarization loss is accelerated during transport, catalysis, especially in the vicinity of paramagnetic prosthetic groups, and other binding events involving macromolecular hosts. While model-independent quantitative data interpretation thus remains a challenge, reaction usage and relative changes in fluxes upon extra- or intracellular interventions are more straightforward to derive. Magnetic resonance techniques provide versatile detection schemes and permit tailoring experimental information content. Adapted NMR detection schemes allow the amplified detection of signals by saturation transfer methods,67,144 enhanced temporal and spatial resolution, as well as improved information content even in single-scan schemes.78,126−128,145−151 Innovative spectroscopic schemes include multidimensional spectra for identifying analytes or independent measurements of parameters affecting the detected signal amplitudes (flip angles and relaxation times) in order to facilitate determinations of intracellular reaction rates. Nevertheless, reaction progress curves, in some instances of entire pathways (see Figure 3 below), contain a deluge of information and extracting all conceivable information from hyperpolarized NMR data probably remains a long-term challenge. Several applications of hyperpolarized NMR are therefore based on ratiometric assays.44,64,143,152 Developments and Directions. Instrumentation and technology for hyperpolarization have been transformed into commercially available products during the last years, thus supporting the rapid development of the method in interdisciplinary research groups. While hyperpolarization equipment is commercially available, it requires expert personnel for optimal output and ease of use is not a principal stronghold. The standardization of sample preparation and experimental protocols could facilitate the comparison of results from different laboratories. Efforts toward the improvement of detectors include miniaturized NMR detectors, detector arrays, and novel detection schemes including magnetic force microscopy or superconducting quantum interference devices.3 In parallel to biological studies, the realtime probing with hyperpolarized NMR has recently expanded

limiting. Increased metabolite formation from a hyperpolarized substrate can result from altered transporter activity, enzyme activity, cofactor pools, or cellular metabolite pools.85 The contributions from these influences are therefore often delineated by invasive biological validation. Integration of NMR probing with other functional information deserves greater emphasis in the future, especially the integration with transcriptome, proteome, metabolome, and fluxome data. Another question arising for reversible reactions is whether a net chemical conversion of probes to metabolites is observed or whether isotope exchange into cellular metabolite pools occurs. In the conversion of the hyperpolarized 13C labeled pyruvate to lactate, the mechanism of label exchange has been experimentally studied in cell suspensions. Co-injection of unlabeled lactate leads to increased exchange of 13C pyruvate to lactate and is incompatible with the significant net synthesis of lactate.54,85 Introduced, noncellular enzyme activities have been used as specific reporter systems in conjunction with suitable hyperpolarized NMR probes. These systems have been assayed against backgrounds that show no activity.72,130 A reporter enzyme system based on transgenic lactate dehydrogenase was recently developed and assayed by substrates, which were specifically designed to react with the transgenic but not hostcell variant of the enzyme.131 Other magnetic resonance reporter genes exploiting manageable cellular NMR backgrounds signals have previously been developed using conventional 31P or 19F NMR, some of them based on enzymes.132 Reproducibility. The simplicity and rapidity of cellular approaches using hyperpolarized NMR has resulted in published assays that have generally been conducted in replicate. In a study specifically performed to assess the quantitative value of the method, hyperpolarized signal amplitudes were directly translated into analyte concentrations using an internal molecular standard. This approach indicated an excellent suitability of hyperpolarized NMR for reproducible quantitative measurements in the quantification of analytes in duplicate samples. Comparison of quantitations with hyperpolarized NMR and as a standard method (liquid chromatography−mass spectrometry) yielded a Pearson correlation coefficient of 0.99, thus underlying the possibility for robust measurements of hyperpolarized NMR signal areas.133 The reproducibility of hyperpolarized NMR measurements was also quantitatively assessed with hyperpolarized pyruvate applied to cells maintained in a bioreactor. In this study, the reproducibility was shown to be good when normalizing metabolite signal relative to the substrate signal.68 31P NMR can independently assess the maintenance and reproducibility of cellular states in the same samples, specifically of cellular energetics.43,68 Throughput. Until recently, throughput arguably has remained a major shortcoming in hyperpolarized NMR assays for cellular biology. Ex situ hyperpolarization by the DNP step in the solid state is usually performed for approximately an hour and thus constitutes a bottleneck. Recent efforts address this bottleneck for instance by allowing multiple samples to be polarized simultaneously70,134,135 or alternatively by the transfer of polarization via rapidly polarized 1H nuclei to 13C or 15N nuclei.136−138 Increasingly rapid methods for probe generation could be paralleled by faster liquid-state NMR assays using these probes. Multiplexed detection in multichamber bioreactors has recently been suggested as a means toward improved assay throughput and reproducibility.98 These E

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to the study of other complex systems in materials and chemistry fields, including chemical reaction mechanisms153 and conformational transitions in protein folding.154 Hyperpolarization technologies benefit from interdisciplinary improvements on various levels. Critical developments have occurred in NMR theory and methodology (e.g., storage of hyperpolarization in long-lived states and spin-locking radiofrequency pulses to maintain the long-lived states109), engineering (high-field polarizers,47,48 simplified hardware engineering through 3D-printing,155 miniaturized and inexpensive equipment), cell biology (genome editing, strain collections, cell culture conditions), and probe design and synthesis (hyperpolarization lifetime, cellular uptake, and retention). The inventiveness of researchers in designing optimized probes offers enticing opportunities for unravelling cellular biochemistry.

Table 3. Examples of Hyperpolarized Probes Used in Biological Assays

BIOLOGICAL PROBLEMS THAT ARE ADDRESSED WITH HYPERPOLARIZED NMR PROBES In order to address the function and dysfunction of organisms, a systems approach using living cells is required. The investigation of cellular (dys-)function with hyperpolarized NMR probes is in its infancy and methodological developments as those outlined above continue to extend the scope of hyperpolarized probes. Hyperpolarized pyruvate has been the prototype probe due to its ease of hyperpolarization, rapid cellular uptake, and central biochemical position as a hub between different pathways. Meanwhile, a host of other probes has emerged that can be used to characterize the phenotype of cells under a particular set of conditions (Table 3, Figure 2). Metabolic Pathways. Real-time tracking is particularly suitable for cellular phenomena that are in a nonequilibrium state and require time-resolved analyses. Among these phenomena are metabolic pathways in open, dissipative systems such as the living cell. The molecular resolution of substrates, intermediates, and products allows NMR to probe functional modules of cellular reaction networks, given that reaction fluxes through these modules are rapid and that signal is sufficient. Glycolysis is recognized as a rapid pathway and therefore is suitable for the study with hyperpolarized NMR. Pathway bottlenecks, off-pathway reactions, and enzymatic reaction mechanisms have been probed with hyperpolarized carbohydrates, [2-13C] fructose and [U-13C, U-2H] glucose, as substrates.67,158,159,161,162,179 The use of endogenous macronutrients as DNP substrates that are effectively metabolized in rapid pathways clearly supports the detection of pathways on a fast time scale. Figure 3 displays the real-time tracking of glycolysis and fermentation by hyperpolarized NMR using hyperpolarized glucose probes in Escherichia coli, S. cerevisiae, and human cells. Differences in kinetic profiles of mixed acid fermentation (E. coli),158,161 alcoholic fermentation and carbon dioxide formation (S. cerevisiae),159 and lactate fermentation (human cancer cells)67,129,163 are evident and reflect metabolic survival strategies of different genomes. Chemical detail in the observation of pathway reactions extends to the distinction of isomers and their susceptibility to enzymatic turnover. The use of site-specifically labeled [2-13C] fructose has permitted the real-time observation of probe flux into gluconeogenic reactions and nonproductive off-pathway intermediates such as the hydrate form of dihydroxyacetonephosphate.159 In addition, [2-13C] fructose rules out possible kinetic isotope effects that could affect the reaction progression when using perdeuterated glucose.

short chain fatty acids89,90,160

probe molecule class Endogenous Molecules amino acids42,63,73,156,157

monosaccharides57,67,129,158−163

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TCA cycle substrates65,164,165 pH indicators64,84,160,166

ascorbate/dehydroascorbate74,167 α-keto acids61,68,76,79,83,86,88,131,168−171

ketone bodies42,172 choline/betaine92,173−176 urea177 Designed Molecules esters94,164,131 carboxypeptidase prodrug72 N-acetyl methionine130 benzoyl formic acid152 89 Y-complexes107 p-anisidine (methoxy compound)178 trimethylphenylammonium platform106

biological assay/information content transaminases tumor metabolism treatment response glycolytic pathways metabolic regulation genetic and genomic differences intracellular association of metabolites with metabolic effectors pathway fluxes, metabolic bottlenecks, reaction reversibility redox state indication of intracellular cosubstrate pools uptake accumulation intracellular pH change intracellular cosubstrate pools multistep reactions in fatty acid metabolism citric acid cycle flux cell necrosis sample pH, tumor pH intracellular pH treatment intracellular cosubstrate pools and redox state transaminases dehydrogenases decarboxylases oncogene signaling treatment response pyruvate carboxylase and phosphoenolpyruvate carboxykinase isocitrate dehydrogenase mutational status limited metabolism observed so far limited metabolism observed so far transport in erythrocytes enzyme activity reporter for target protein expression enzyme activity protein expression H2O2 pH HOCl Ca2+ enzyme activity H2O2

Hyperpolarized probes have been used to probe other pathways. The enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate was tracked with ∼1 s temporal resolution using [2-13C] pyruvate in isolated perfused hearts to study healthy and pathological states.165 Recently, hyperpolarized probes have been used to track intracellular pathways of short chain fatty acid and ketone body metabolism in real time. A butyrate probe has visualized the flux of fatty acids to acetoacetate and several tricarboxylic acid cycle intermediates in cardiac muscle cells.89 F

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responsible for covalent modifications observed for recombinant proteins expressed in E. coli BL21.181,182 Whole genome alignment validates that the lactonase gene catalyzing the hydrolysis of 6-phosphogluconolactone is lacking in the BL21 strain due to a deletion. Such molecular phenotypes can be observed in the absence of phenotypic variations, for instance altered growth rates.161 Metabolic differences in different cell types were recently compared in human cells by tracking the glycolytic pathway (Figure 4).163 Ratiometric measurements of lactate and pyruvate signals in two different proliferating cell types were used to noninvasively detect differences in the cytosolic redox state. In the cytosol, lactate and pyruvate form a redox couple that rapidly equilibrates in a reaction dependent on the cytosolic NAD+/NADH ratio. A prostate cancer cell type (PC3) showed a 4-fold increased intracellular ratio of free cytosolic NAD+/NADH compared to a breast cancer cell line in a functional assay using a hyperpolarized glucose probe. This increased NAD+/NADH ratio reflects a distinct metabolic phenotype consistent with previously reported alterations in the energy metabolism of prostate cells. Vitamin C and its oxidized form have been established as an alternative means for probing redox biology with the [1-13C] ascorbate and [1-13C] dehydroascorbate isotopomers.74,167 Here, the kinetic phenotype of ascorbate and dehydroascrobate interconversion correlates strongly with glutathione concentration.183 Recently, the value of hyperpolarized NMR probes as functional genomics tools for disease biology was underlined in human cell types differing only in isocitrate dehydrogenase 1 (IDH1) mutational status using hyperpolarized [1-13C] αketoglutarate. IDH1 catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate in the cytosol. Gain-of-function mutations in IDH1 (most commonly at residue 132) result in the new ability of the enzyme to catalyze the NADPHdependent reduction of α-ketoglutarate to the (R)-enantiomer of 2-hydroxyglutarate.184 Accordingly, isogenic glioblastoma cells differing only in IDH1 status show differences in the conversion of the hyperpolarized α-ketoglutarate signal to the onco-metabolite (R)-2hydroxyglutarate (Figure 5).171 Correlations between genes and functional phenotypes should become more standard by the advent of genome editing technologies. Gene deletion libraries and extended culture collections already exist for microbial organisms including yeasts and bacteria. These collections could facilitate the identification or improvement of cell factories and production strains, especially when molecular probes have become applicable in high-throughput screening methods. Cellular Response to Small Molecule Effectors. Among the most interesting applications of NMR probes is the

Figure 2. (A) Hyperpolarization from the perspective of the probe, encompassing ex situ preparation, harvest, and subsequent application to cellular NMR observations. (B) A host of information, much of it previously inaccessible, emerges from hyperpolarized NMR probing in living cells.

A recent approach combines hyperpolarized dynamic measurements with steady state labeling, metabolite extraction, isotopomer evaluation, and flux analysis.104 This approach uses dynamic measurements of pyruvate metabolism in live cells and relative flux measurements for more extended reaction pathways to obtain quantitative flux data of several pyruvatedependent pathways in two cell types with divergent pyruvate metabolism. Observing Biological Functionality in Different Cell Types and Mutants. The presence of multiple interlocked pathways with common metabolites and higher order genetic and metabolic regulation in the cell makes it difficult to predict how cell physiology and intracellular metabolism will respond to a genetic modification.180 Hyperpolarized NMR spectroscopy of genetically well-defined and homogeneous cell suspensions is a prospective approach to response analysis under genetic modifications. The most commonly used E. coli laboratory strains BL21 and K-12 show strong differences in the reaction progression of the pentose phosphate pathway. In strain BL21, a reactive intermediate accumulates that is

Figure 3. Real-time observations of glycolysis and fermentation using hyperpolarized glucose and 13C NMR. Accumulating glycolytic intermediates and fermentation products are resolved due to the sensitivity of 13C NMR signal to the chemical environment of the nuclei. The sum of signals fades due to the loss of hyperpolarization. Composition and kinetics of the intermediates and products formed in different cell types differ strongly due to different flux bottlenecks and pathways. G

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Figure 4. Measurement of free cytosolic NAD+/NADH ratios in different human cancer cell types using a hyperpolarized real-time assay of cancer cell glycolysis (left). The free cytosolic NAD+/NADH ratio can be derived from measured signal ratios of pyruvate and lactate as shown on the right. This research was originally published in The Journal of Biological Chemistry. Christensen, C. E.; Karlsson, M.; Winther, J. R.; Jensen, P. R.; Lerche, M. H. Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes. The Journal of Biological Chemistry. 2013; 289, 2344−2352 (ref 163). Copyright 2013 The American Society for Biochemistry and Molecular Biology.

pH changes, and effects on reaction balances in glycolysis and alcoholic fermentation (e.g., acetaldehyde accumulation) were probed with hyperpolarized acetate and glucose.160 These findings rationalize the presence of acetaldehyde in some alcoholic beverages due to suboptimal production technology.185 Altered metabolic balances were also observed in Escherichia coli in the presence of a protonophore 2,4dinitrophenol causing a dramatic metabolic switch in the metabolism downstream of pyruvate, most likely as the result of an altered intracellular redox state.158 Metabolic switches function as a microbial survival strategy in changing environments.180,186 The adaptation to altered nutrient availability in different growth phases of microbial batch cultures has been probed with hyperpolarized carbohydrates. Direct observation of glucose metabolism in Escherichia coli at different growth phases directly shows a ∼4-fold down regulation of glucose-6 phosphate dehydrogenase activity in intact cells at stationary compared to midexponential phase.158 Glucose-6 phosphate dehydrogenase regulates the influx of carbohydrates into the biosynthetic pentose phosphate pathway, which is down regulated as cell reach the stationary phase

Figure 5. Mutated isocitrate dehydrogenase 1 (IDH1) acquires a novel activity producing 2-hydroxyglutarate (2-HG). The reaction can be probed in living cells with the hyperpolarized version of the IDH1mut substrate α-ketoglutarate (α-KG).

observation of a cell’s chemical strategy for coping with environmental stress and chemical effectors.117 Hyperpolarized probe molecules have been extensively used in this context for probing the effect of xenobiotics on cells. Studies of disease metabolism in the presence of effectors with known, intended targets are paralleled by mechanistic studies of stressors with unknown targets in microbial cells. Intracellular complexes between effectors and metabolites at glycolytic bottlenecks were detected in the presence of sulphite.162 Use of various molecular probes allows a more extended series of cellular events to be deduced. For instance, the real-time influx of glycolytic inhibitor, resulting intracellular

Figure 6. Comparison of healthy and proliferative cell state. Glutaminolysis and anaerobic glycolysis are metabolic hallmarks of proliferative cells. The interdependency of metabolic changes and changes to the microenvironment are indicated. Inhibitors of signaling pathways are used to detect correlations between metabolism, signaling, and proliferation. H

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pyruvate to lactate metabolism.82,83 Inhibition of plateletderived growth factor receptor with imatinib proved to reduce the conversion of hyperpolarized pyruvate to lactate by lowering the expression of lactate dehydrogenase, presumably mediated by reduced expression of their transcription factors HIF-1 and Myc.194 More varied effects were observed upon treatment of different cancer cell types (PC3 and MCF-7) with a mitogenactivated protein kinase inhibitor (U0126). Treatment had opposite effects on hyperpolarized 13C label flux from pyruvate to lactate in the two cell types due to decreased expression of the monocarboxylate transporter 1 in MCF-7 cells. Surprisingly, lactate dehydrogenase expression and activity were increased in both of these cellular models in the presence of U0126.79 The latter example signifies the ability for unexpected observations on intracellular metabolic fluxes and the need for biological validations of the underlying molecular causes.195 Import and Export Across the Cell Membrane. Metabolite exchange by transport processes can be monitored with hyperpolarized probes, as a variety of NMR detectable parameters can change upon transport. Hyperpolarized probes have for instance been used for the detection of passive and active transport in living cells by using translational diffusion properties, 196 rotational diffusion and T 1 relaxation changes,163,197 pH dependent extra- and intracellular signals,160 competition experiments,85 or differences in magnetic susceptibilities inside and outside human erythrocytes (red blood cells).177 Increasingly, quantitative measures of molecular transport (both influx and efflux) have been obtained, thus enhancing the understanding of metabolite exchange kinetics for a growing number of molecules.163,198 As an example, the kinetic modeling of hyperpolarized NMR data indicated that pyruvate conversion to lactate in cancer cells is determined by pyruvate uptake.68,85,141 Active lactate export from cancer cells was recently probed using various methods. Data obtained using hyperpolarized glucose probes in different cell types163 was subjected to relaxation-kinetic modeling to show different exchange kinetics in the different cells. Transport of extracellular lactate in perfused cells was used to determine the intracellular conversion of lactate and subsequent export at modulated flow rates in a bioreactor. Compared to localized cells, metastatic cells showed higher lactate efflux, which is important for maintaining a high glycolytic rate in these cells.198 Kinetic glucose uptake parameters were determined with hyperpolarized NMR, indicating that glucose uptake remained rate limiting for proliferative metabolism to lactate in a human model.67 Despite these successes, generalizable and robust distinction of extra- and intracellular metabolites remains a challenge. Discovery and Evaluation of Candidate Molecular Markers. Biomarkers provide a molecular readout of cellular physiology and are often derived with genetic, genomic, or proteomic methods.199 Neither mRNA nor protein levels are necessarily quantitatively correlated with intracellular enzyme activities, not least as a variety of posttranscriptional and posttranslational processes regulate these activities.200,201 Metabolites and other small molecules are key players in many life processes. They, directly affect cell phenotypes202 and hence are suitable diagnostic, prognostic, and mechanistic probes of cellular function. Informative component measurements of metabolites do not contain functional information per se, however.203

under nutrient limitation. Cell culture assays studying the metabolic effect of controlled perturbations to the cell environment (pH, hypoxia, nutrients) in proliferating cells have received surprisingly little attention, so far, while the effects of vasculature have been studied in vivo with hyperpolarized NMR.187,188 An emerging focus area of hyperpolarized NMR probing in disease biology is the visualization of treatment effects. The direct interference of xenobiotics with fermentative glycolysis was visualized with hyperpolarized pyruvate for two scenarios that favor “healthy” pyruvate usage in quiescent cells (Figure 6). The inhibition of lactate dehydrogenase in a lung cancer cell line resulted in apoptosis and increased sensitivity to the chemotherapeutic paclitaxel. Inhibition of fermentative glycolysis was also achieved by activation of the pyruvate dehydrogenase complex with dichloroacetate.189 Induced cell death was observed with hyperpolarized NMR and treatment with the DNA-damaging agent etoposide.190 In murine lymphoma cells and tumors, the decrease in pyruvate to lactate flux was comparable to the decrease in radiolabeled fluorodeoxyglucose uptake upon treatment with etoposide.191 Etoposide treatment was subsequently assessed by the use of hyperpolarized fumarate to supposedly detect tumor cell death65,86 and by the use of hyperpolarized glutamine to probe glutaminolysis (catabolism of glutamine) upon treatment.59 While glutaminolysis takes place in all proliferating cells, some cancer cells exhibit a particular glutaminolytic phenotype and depend on glutamine for energy production. The degree of glutaminolysis, measured as the dependence of cancer cells on glutamine in the growth medium, could be correlated to the metabolic readout from hyperpolarized [5-13C]glutamine in two prostate cancer cell types. Under proliferating growth conditions, glutamine metabolism was found to be 4 times higher in the more glutaminolytic DU145 cells than in PC3 cells. Upon drug treatment with two natural drugs, a significant decrease in the glutamine metabolism was measured.192 In addition to the metabolic hallmarks of malignant transformation, the correlations between metabolism, signaling, and proliferation have been probed by hyperpolarized NMR. This correlation has been studied in mammalian systems using a switchable model of Myc expression, hyperpolarized NMR, and gene expression data. Myc regulates lactate dehydrogenase A. Metabolic changes were thus probed with hyperpolarized pyruvate and were found to precede morphological changes in the formation and regression under the influence of a single oncogene.69 In addition, a downstream target of Myc, branched chain amino acid transferase, was used in the functional profiling of proliferating cells. Different fluxes through the branched chain amino acid transferase reaction were detected for different cell types and validated by enzyme activity measurements in cell lysates.42 For inhibitors targeting cellular signaling pathways, the dependencies between the signaling pathways and metabolism get amenable to noninvasive probing with hyperpolarized NMR. Several drugs are in clinical trials that target the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway.82,193 Inhibition of the PI3K/Akt/mTOR pathway with the PI3K inhibitor LY294002 leads to a significant drop in hyperpolarized 13C label flux from pyruvate to lactate in various glioblastoma and breast adenocarcinoma cells. Using a DNA damaging agent instead of mechanism-based inhibition did not have an effect on I

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combination of NMR-derived molecular detail with rapid sampling and quenching methods,206 expressed fluorescent biosensors for key metabolites,139 and click-chemistry based labeling with activity-based probes and optical reporter tags201,207 provides remarkable prospects for unravelling the biochemistry of living cells.

Hyperpolarized NMR is among the noninvasive methods deriving from functional imaging that can monitor candidate biomarkers (mostly enzyme activities) of physiological and pathological cellular function. Noninvasive probes have wellknown advantages in avoiding pitfalls in quenching, sample workup, extractions, or metabolite stability. The probing of novel molecular markers of pathological alterations and treatment successes by hyperpolarized NMR imaging are principal driving forces for preclinical and clinical applications.68,79,163,169 The majority of hyperpolarized NMR studies evaluates the chemical environment (pH, ion concentrations, reactive oxygen species, redox state) or intracellular reaction progress (transporter activity, enzyme activity, cosubstrate pools, competing reactions, or a combination of these) in physiological and pathological states. Taken together, functional changes in response to stress, therapeutics, mutation, evolutionary (genomic) adaptations, and disease can be probed and often understood at a deterministic level with hyperpolarized NMR in the disease biology and microbiology fields. As a consequence, hyperpolarized NMR probes are among the transformative molecular tools that could vastly improve our ability to design means for manipulating cellular functions and that could influence workstreams in disease biology and biotechnology. So far, the predicting power of whole-cell models is limited by insufficient information on intracellular kinetics.49 Using the untapped potential of intracellular kinetic measurements for modeling and controlling bioprocesses thus could accelerate product and process development cycles.204

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Mathilde H. Lerche earned her M.S. (1995) in Chemistry from the University of Roskilde and her Ph.D. (2000) in Protein Chemistry from University of Copenhagen. Presently, she is co-owner of Albeda Research, which specializes in hyperpolarization research. In 2003, she coauthored the pioneering work on liquid state hyperpolarization using dynamic nuclear polarization, demonstrating a >10 000-fold enhancement of probe molecules for NMR. Since then, her research has focussed on the development of hyperpolarized metabolic contrast agents for in cell and in vivo magnetic resonance investigations. Her research is particularly focussed on the fields of biotechnology and studies of complex pathologies aiming at early diagnosis and monitoring of therapeutic effects. Pernille Rose Jensen earned her B.A. (1996), M.S. (1999) in Chemistry and her Ph.D. (2003) in Protein Chemistry from the University of Copenhagen, Denmark. From 2003 to 2006 she was a postdoctoral researcher at the University of Basel, Switzerland. In 2006 she began working with hyperpolarization methods at Imagnia AB, Sweden, and in 2009 she cofounded Albeda Research. During the last 8 years, her main research interest has been the development of hyperpolarized molecular probes for cellular studies of cancer metabolism.

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CONCLUDING REMARKS Magnetic resonance spectroscopy is a routine method for chemical structure determination, structural biology, medical imaging, and compositional analysis of complex samples. Rapid developments have recently addressed the inherent insensitivity of NMR and have transformed real-time magnetic resonance spectroscopy into appropriate tools for gaining insight into physiological and pathological processes of living cells. These methods are still in their infancy and profound biological knowledge gained with them is arguably sparse. Nevertheless, a variety of previously impossible assays for detecting cellular physiology and pathology are likely to improve the systems level understanding of cells and open up new avenues of biological research. Hyperpolarized probes, as an example, have excellent potential for enabling surprise observations, once indisputable experimental setups are established. Arguably, breakthroughs will require interdisciplinary work to derive general principles of how cellular systems function. Not surprisingly, challenges of macromolecular in-cell NMR and hyperpolarized NMR probing of cellular functions are similar, when in-cell assays are compared to test tube NMR studies in solution. Specifically the limited signal lifetime in the intracellular environment (especially in the case of binding to macromolecular targets) and the limited sensitivity at physiological concentrations have deleterious effects on NMR signals. Protein leakage from cells is a problem for the interpretation of in-cell macromolecular NMR18 and for the interpretation of enzyme activities that do not require cofactors. As of today, we still lack an understanding of most cellular and subcellular processes at high temporal and spatial resolution. Significant methodological foundations have been laid for bridging the gap toward understanding the interactions and function of cellular components.205 Comparison and

Magnus Karlsson earned his M.S. (1995) in Chemistry and his Ph.D. (2000) in Inorganic Chemistry from Lund University, Sweden. From 2000 to 2005 he was a researcher at Amersham Health/GE Healthcare in Malmø, Sweden. In 2006 he cofounded Imagnia AB, Sweden, and in 2009 Albeda Research, Denmark. He has been working with hyperpolarized NMR for more than 10 years. His main interest lies in developing DNP applications for studying metabolic reactions in in vitro systems. Sebastian Meier received his M.S. in Biochemistry from the University of Regensburg, Germany (2000) and his Ph.D. in Biophysics from the University of Basel, Switzerland (2004). After postdoctoral work in Basel, Grenoble, and Copenhagen, he joined the Carlsberg laboratory in 2007 to establish in situ and in vivo NMR methods for the study of complex biological and biotechnological systems. In 2014, he accepted a position at the Technical University of Denmark in Lyngby. His principal research interests are the direct observation of polysaccharide metabolism, the characterization of complex carbohydrate mixtures, and the observation of carbohydrate degradation pathways in living cells and their response to internal and external stimuli by NMR spectroscopy.

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ACKNOWLEDGMENTS We gratefully acknowledge Sophie R. Beeren for critically commenting on the manuscript. REFERENCES

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dx.doi.org/10.1021/ac501467x | Anal. Chem. XXXX, XXX, XXX−XXX