MOLECULAR SENSOR FOR NMR/MRI BASED ON ANALYTE-DEPENDENT SPECTRAL CHANGES OF TEMPORARILY ENCAPSULATED HYPERPOLARIZED 129XE

Abstract

The present invention relates to a precursor of a molecular sensor for determining analyte concentrations and/or measuring analyte concentration changes comprising a host for an active nucleus, an NMR-modulating moiety and an interacting moiety, wherein said NMR-modulating moiety changes the resonance frequency or the chemical exchange saturation transfer (CEST) signal of the active nucleus-host complex, and wherein said interacting moiety specifically responds to an environmental parameter, to an analyte or to a target molecule that binds the analyte or said interacting moiety specifically binds to a target molecule in an analyte-dependent manner. The present invention further relates to a molecular sensor comprising an active nucleus and said precursor. The present invention further relates to a molecular sensor for determining analyte concentrations and/or measuring analyte concentration changes inside a cell, wherein moiety/ies of the sensor are expressed in said cells and then assembled inside said cell. The present invention further relates to uses of the molecular sensors as well as to an in vitro method for determining metal concentration and/or measuring metal concentration changes and a method for diagnosing and/or monitoring treatment of diseases causing changes in metal concentrations.

Claims

1. A precursor or a molecular sensor selected from: A) a precursor for a molecular sensor for determining analyte concentrations and/or measuring analyte concentration changes comprising (a) a host for an active nucleus, wherein said host enables at least a transient binding of said active nucleus that produces a detectable NMR signal when the sensor binds to the analyte, (b) an NMR-modulating moiety, wherein said NMR-modulating moiety changes the resonance frequency or the chemical exchange saturation transfer (CEST) signal of the active nucleus-host complex, and (c) an interaction moiety, wherein said interaction moiety specifically responds to an environment parameter, an analyte or a target molecule that binds the analyte or said interaction moiety specifically binds to a target molecule in an analyte-dependent manner, wherein the analyte is a metal a peptide or a protein, wherein the NMR-modulating moiety (b) and the interaction moiety (c) reversibly suppress, or are capable of reversibly suppressing, a CEST signal from the otherwise accessible host by a specific conformation of the NMR-modulating moiety (b) in the host vicinity, and an interaction of the interaction moiety (c) with a target molecule in an analyte-dependent manner unsuppresses the CEST-signal; and B) a molecular sensor for determining analyte concentrations and/or measuring analyte concentration changes in a cell, comprising an active nucleus, and said precursor of part A), wherein the analyte is a metal or a peptide or a protein.

2. (canceled)

3. The molecular sensor of claim 1, which is assembled inside a cell or tissue, wherein the active nucleus is delivered across the cellular membrane and the host is delivered across the cellular membrane or is expressed by said cell, wherein said NMR-modulating moiety is expressed by said cell, and wherein said interaction moiety is expressed by said cell.

4. The precursor or the molecular sensor according to claim 1, wherein said host is selected from a cryptophane, cucurbit[n]uriles, pillar[n]arenes, and self-assembling metal-organic cages.

5. The precursor or the molecular sensor according to claim 1, wherein the NMR-modulating moiety (b) and the interaction moiety (c) are attached to each other or form a joint moiety.

6. The precursor or the molecular sensor of according to claim 1, wherein the NMR-modulating moiety (b) and the interaction moiety (c) comprises or is the peptide RS20 or the peptide M13 that specifically binds to an EF hand protein in a calcium-dependent manner.

7. The precursor or the molecular sensor according to claim 1, wherein the NMR-modulating moiety (b) and the interaction moiety (c), are attached to each other, and decrease the CEST signal of the active nucleus, wherein the CEST signal from the otherwise accessible host is (reversibly) suppressed by a specific conformation of the NMR-modulating moiety (b) in the host vicinity, and an interaction of the interaction moiety (c) with a target molecule in an analyte-dependent manner unsuppresses the CEST signal.

8. The precursor or the molecular sensor according to claim 1, wherein the host (a), the NMR-modulating moiety (b) and the interaction moiety (c) are attached to each other.

9. The precursor or the molecular sensor according to claim 1, further comprising: (d) a further sensor moiety, a further active nucleus-host complex, contrast agent(s), chromophore(s) and/or fluorophore(s), MRI or PET agent(s) or chelated transition metal(s), actuator(s), nanostructures, absorber or combinations thereof; and/or (e) a solubilizing and/or biodistribution moiety; and/or (f) further interacting moiety or moieties.

10. A method comprising the use of a molecular sensor of claim 1 for determining analyte concentrations and/or measuring analyte concentration changes, in vitro, ex vivo and in vivo measurements of spatiotemporal analyte distributions, and/or in vivo imaging of analyte distributions in animal models and humans, wherein the analyte is a metal, wherein the method further comprises nuclear magnetic resonance (NMR) spectroscopy and imaging, and optionally, comprising multimodal detection of further sensor moiety/moieties via absorbance/transmission, reflection, fluorescence or optoacoustic or ultrasound measurements and imaging.

11. The method according to claim 10, comprising: ex vivo imaging of tissues and bodily fluids, and/or in vitro measurement and imaging in biomedical or environmental samples, and, optionally, comprises determining further analyte(s).

12. (canceled)

13. A method of diagnosing, treating, and/or monitoring treatment of a disease, wherein said method comprises the use of the molecular sensor of claim 1, and wherein the disease is selected from: diseases in which calcium signaling is affected, and diseases in which calcium uptake, storage, utilization or excretion is affected.

14. An in vitro method for determining metal concentration and/or measuring metal concentration changes in a sample, comprising the steps of: (i) providing the precursor of a molecular sensor according to claim 1, (ii) providing an active nucleus of said molecular sensor to the sample, (iii) performing nuclear magnetic resonance (NMR) spectroscopy and imaging in a sample, optionally performing further measurement(s) to detect the further sensor moiety(moieties) (e), (iv) determining metal concentrations or metal concentration changes of or in the sample.

15. A method for diagnosing and/or monitoring treatment of a disease causing changes in metal concentrations, comprising the steps of: (i) administering to the body of a patient or nun-human animal the precursor of a molecular sensor according to claim 1, (ii) providing an active nucleus of said molecular sensor to the region or tissue of interest, (iii) performing in vivo nuclear magnetic resonance (NMR) spectroscopy and imaging, optionally performing further measurement(s) to detect the further sensor moiety(moieties) (e), (iv) determining metal concentrations or metal concentration changes of or in the body of said patient or nun-human animal, wherein said change in metal concentrations is associated with a disease selected from: diseases in which calcium signaling is affected, and diseases in which calcium uptake, storage, utilization or excretion is affected.

16. The precursor or the molecular sensor according to claim 5, wherein the NMR-modulating moiety (b) and the interaction moiety (c) comprise, or consist of, a calmodulin (CaM)-binding peptide or a linker, wherein said calmodulin (CaM)-binding peptide is selected from RS20, calcineurin A, M13 and other CaM-binding peptides, and wherein said linker is a peptide or polyethylene glycol (PEG).

17. The precursor or the molecular sensor according to claim 1, wherein the NMR-modulating moiety (b) and the interaction moiety (c) comprises, or is, an amino acid sequence selected from SEQ ID NOs: 1 to 3, 14 and 15.

18. The precursor or the molecular sensor according to claim 1, wherein the host (a), the NMR-modulating moiety (b) and the interaction moiety (c) are attached to each other via an amino acid side chain of the peptide, or via a linker or tether, wherein said linker is a peptide chain.

19. The method according to claim 11, comprising: ex vivo imaging of blood, urine, lymph or lymphatic drainage, cerebrospinal fluid, stool/feces, semen, saliva or mucous fluids, or a cell culture sample, or in vitro measurement and imaging in cell or tissue culture, or metal screening in water, air, soil, plants, or food.

20. The method of claim 13, wherein the disease is selected from: neuropsychiatric diseases stroke, brain damage, neurodegenerative diseases, states of altered neuronal processing, cardiovascular diseases, neuromuscular or muscular diseases, endocrinological conditions, malnutrition and gastrointestinal diseases, bone-related diseases, kidney diseases and treatment with diuretics, osteoclastic processes of tumors or infections, cancer, and atherosclerotic alterations or inflammatory processes, due to other medical treatments.

21. A method for diagnosing and/or monitoring treatment of a disease causing changes in metal concentrations, comprising the steps of: (i) administering to the body of a patient or nun-human animal the precursor of a molecular sensor according to claim 1, (ii) providing an active nucleus of said molecular sensor to the region or tissue of interest, (iii) performing in vivo nuclear magnetic resonance (NMR) spectroscopy and imaging, optionally performing further measurement(s) to detect the further sensor moiety(moieties) (e), (iv) determining metal concentrations or metal concentration changes of, or in, the body of said patient or nun-human animal, wherein the active nucleus is hyperpolarized xenon gas, which can be administered through the breathing air, Xe mixture or injection of a Xe-saturated carrier solution, wherein said change in metal concentration is associated with a disease selected from: neuropsychiatric diseases, stroke, brain damage, neurodegenerative diseases, states of altered neuronal processing, intoxication, cardiovascular diseases, neuromuscular or muscular diseases, endocrinological conditions, malnutrition and gastrointestinal diseases, bone-related diseases, kidney diseases and treatment with diuretics, osteoclastic processes of tumors or infections, cancers, and atherosclerotic alterations or inflammatory processes, due to other medical treatments.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0364] FIG. 1: Schematic representation of on Turn-on Mechanism.

[0365] Visualization of the different moieties of the sensor systems that enable the analyte-dependent activation of the NMR signal that is suppressed in the absence of the target condition e.g. presence of the analyte and unsuppressed if this condition is fulfilled.

[0366] FIG. 2: Calcium-dependent spectral changes of temporally encapsulated hyperpolarized 129Xenon.

[0367] (A) The presence of a Xe host-bearing peptidic sequence (labelled with an optical reporter for fluorescence co-registration) is revealed by a certain NMR saturation transfer response along the chemical shift dimension when observing free xenon after RF saturation of bound Xe. The intensity of the spectral response can be weak or strong and does not change significantly upon addition of calmodulin. Only further presence of calcium as analyte induces a conformational change of the peptidic scaffold around the captured Xe host that induces spectral changes for saturation of temporarily bound xenon. The peptidic scaffold therefore serves as the NMR modulating moiety, CaM interacts with the interaction moiety in a calcium-dependent manner. These are identified as altered intensity, frequency, or width of the saturation response or any combinations thereof (B).

[0368] FIG. 3: Scheme depicting the in vitro and in vivo applications of the calcium sensor.

[0369] Dependent on the calcium concentration, a specific peptidic scaffold will be tightly bound by an EF-hand protein such as calmodulin. Hyperpolarized Xenon can temporarily bind to a molecular (cage-shaped) host conjugated to the peptidic scaffold that suppresses the NRM signal in the absence of the analyte calcium. In the presence of calcium, the peptide will bind to calmodulin and Xenon will thus sample a different molecular environment in a calcium-dependent manner resulting in a different NMR signature expressed by the chemical shift and/or saturation transfer (Hyper-CEST).

[0370] (A) This analyte-dependent Hyper-CEST turn-on effect can thus be used for in vitro or ex vivo detection of calcium by NMR/MRI. (B) For cellular and In vivo calcium detection, an engineered variant of the protein can be genetically expressed that binds to the cage-modified peptide delivered to the cell again resulting in the NMR signature.

[0371] Alternatively, the binding partner for the primary analyte can be a surface-expressed protein or lipid modification (not drawn). (C) The entire sensor including the Xenon host is genetically expressed.

[0372] FIG. 4: Calcium-dependent docking of peptides to the calcium-binding protein calmodulin.

[0373] As an example for calcium-dependent protein-peptide binding, the 3D structure of the EF hand protein calmodulin is shown bound to the peptide RS20 that has high similarity to M13 (the orientation of the peptide is indicated by the arrow). The lysine residues used for attachment of the Xenon-binding cage-like molecule cryptophane are underlined (color corresponding to the highlighted residues in the scheme). The position for attaching a fluorophore, a chelated transition metal or PET agent is indicated by the letters Fluo (A). The alternative peptide calcineurin A is depicted that has opposite directionality. The cage-conjugated calcineurin does not exhibit a change in the CEST effect upon binding to calmodulin. Same color coding and highlighting of residues as above (B).

[0374] FIG. 5: CEST signal for different cryptophane-conjugated peptides in the absence and presence of calcium.

[0375] Hyperpolarized Xenon was bubbled into buffered (MOPS) solutions containing different cryptophane bearing peptides indicated in the respective subfigures (1 M) and calmodulin (3 M) in the absence and presence of 100 M calcium and CEST spectra were obtained on a 9.4 T Bruker AV 400 wide bore NMR spectrometer. (E) Effects of C-terminal portion of RS20 on the NMR-signal modulation. Replacement of the maleimide-Fluorescein by a maleimide capping group (NEM) partially unsuppresses the CEST effect. (These data identify in particular the C-terminus of RS20 and M13 to be functioning as the NMR-modulating moiety, whereas the N-terminus is mainly engaged in interacting (interaction moiety) with CaM).

[0376] FIG. 6: Conformational Changes of NMR-modulating moiety of the Sensor System

[0377] (A) Circular Dichroism data showing how with 20% TFE, an -helical structure is promoted, but not fully induced. At 50% TFE, the induction an -helical structure is enforced. (B) Corresponding normalized CEST spectra showing that the CEST effect is strongly switched on when the peptide is forced to assume -helical structure.

[0378] FIG. 7: Quantitative Detection of Calcium through turn-on effect.

[0379] CEST spectra (A) and binding curves (B) of the sensor peptide RS20-cage@K1 (2 M) in the presence of 10 M calmodulin are shown for calcium concentrations ranging from 0 to 39 M (MOPS buffer).

[0380] FIG. 8: Cellular uptake of cage-conjugated peptides.

[0381] (A) Cage-conjugated peptides that were also labeled with fluorescein (25 M) were incubated on HEK293 cells for 20 minutes followed by confocal microscopy imaging (nuclear counterstain with DAPI). (B) Analysis of FACS experiments with cells incubated with different peptides modified with cryptophane cages and fluorescein indicating labeling of the cells with the fluorescent peptides (sequences as indicated in FIG. 2; @K# indicates the lysine residue number used for conjugation of the cryptophane cage). The inset shows a cell after uptake of the fluorescent RS20@K1 peptide (nuclear counterstain with Hoechst dye). The cellular uptake of similar EF-protein-binding peptides is likely due to their high scores on prediction tools for cell-penetration such as CPPpred or CellPPD.

[0382] FIG. 9: Turn-on mechanism from biotinylated-PEG-cryptophane cage.

[0383] The figure displays a switch in the CEST signal from hyperpolarized Xenon interacting with a biotinylated cryptophane cage (CrA-PEG3-biotin) depending on the absence (A) or presence (B) of the high affinity binding partner avidin. The lower row shows the corresponding Dynamic Light Scatter (DLS, averaged data from 850 nm) results showing a reduction of size of the CrA-PEG3-biotin upon binding to avidin most likely as a result of a change in the agglomeration state mediated by a solubility switch.

[0384] FIG. 10: Rendering of different peptides for different sensor versions within calmodulin.

[0385] The different orientations of the peptides RS20, M13 and Calcineurin A are shown upon binding to Calmodulin in a Ca.sup.z+-dependent fashion together with the position of the Cryptophane A cages.

[0386] FIG. 11: NMR data for several peptides demonstrate reversible and quantitative detection of Calcium through turn-on effect. (A) CEST spectra from M13 with a cryptophane Cage and also harboring a fluorescein (G-K(Cr-A)-RRWKKNFIAVSAANRFKKISSSGAL-C(5-Fluo-M)-amid) where Cr-A denotes the cryptophane A cage attached on the lysine residue and 5-Fluo-M a fluorescein attached to a cysteine) in the presence of 2 M calmodulin and in response to free calcium concentrations ranging from 0 to 39 M (MOPS buffer) (B) CEST spectra from RS20 (GRR-K(Cr-A)-WQKTGHAVRAIGRLSSS-C(5-Fluo-M)-amid; same annotation of modifications as in (A)) plotted analogously as in (A). (C) Corresponding binding curves yielding an apparent dissociation constant of 0.91 M and 1 M for M13 and RS20 respectively.

[0387] FIG. 12: Spectroscopic changes upon conformational change. (A and B) Circular Dichroism (CD) spectra of M13 (blue) and RS20 (red) at different concentrations of trifluororethanol (TFE) indicate an increase of the helicity of the peptide moiety, which is known to also occur during the binding to calmoduliln (CaM). The induction of helicity causes a CEST signal without Ca.sup.2+ or CaM, indicating that the conformational change during CaM binding plays a key role. In water, there is no CEST signal (C-D), while at 20% (E-F) and 50% TFE (G-H) the CEST signal is turned on for both M13 and RS20.

[0388] FIG. 13: Functionality of the sensor in cell lysates. The sensor M13-based sensor (blue) and RS20-based sensor (red) were added to lysates obtained from HEK293 cells. Addition of just CaM in the presence of the calcium already present in the lysate turned on the sensor. Addition of saturating amounts of calcium yielded a stronger response.

[0389] FIG. 14: Single-piece variant of the biosensor with the peptide-xenon-host directly conjugated to calmodulin (CaM). (A) Rendering of a version of a single-molecule sensor in which the xenon host (cryptophane-A, Cr-A) is bioconjugated to the genetically encodable calcium-indicator protein GCaMP comprised of a circularly permuted version of green fluorescent protein (GFP) flanked by calmodulin (CaM) and the peptide RS20 either extended by an intein (strategy 1) or terminated by an artificial amino acid such as p-acetyl-L-phenylalanine (Apa) (strategy 2). In strategy 1, Cr-A itself is synthetically coupled to a corresponding intein (N-intein) that undergoes a protein ligation reaction resulting in a scarless ligation of the Cr-A labeled RS20 to the C-terminal part of GCaMP resulting a full Cr-labeled GCaMP protein. In strategy 2, Cr-A is aminooxy-functionalized such that it can react with the bioorthogonal Apa keto group (Sletten E M, Bertozzi C R. Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality. Angew Chem Int Ed. 2009 September 7; 48(38):6974-98.). Here, an Amber STOP codon is placed on the position instead of the lysines determined before as suitable for Cr-A labeling (K1 and K3). (B) SDS-PAGE gel showing the purified GCaMP-intein construct before (lane A) and after (lane B) the intein-mediated protein ligation. Product # denotes the Cr-A labelled full GCaMP protein from the protein ligation. The bands *, $ and & denote C-GCaMP, full-length GCaMP and C-Intein, respectively. (C) Fluorescence measurement of the GCaMP6s-Peptide-CrA (green colors) construct without and with addition of Ca.sup.2. Purified GCamP6s (red colors) before intein ligation is shown in red colors.

EXAMPLES

Example 1

[0390] Materials and Methods

[0391] We have generated peptides of the specific sequences as listed above and have attached a cryptophane cage at exactly the positions indicated to obtain a reversible CEST suppression effect under baseline conditions (in the absence of the analyte) as shown in the figures.

[0392] As can be seen in FIG. 5, a specific amino acid composition is essential for reversible CEST suppression and unsuppression of the Ca.sup.2+-sensor. Attachment of the cryptophane cage at the first lysine of the peptide RS20 exhibits a complete CEST suppression and a strong, Ca.sup.2+-dependent CEST unsuppression (FIG. 5 A). Similar effects can be observed for the peptide M13 when increasing amounts of Ca.sup.2+ (0-100 M) are added (FIG. 5 D). However, the CEST suppression and Ca.sup.2+-mediated unsuppression is weaker if the cryptophane cage is attached at the third lysine (@K3, FIG. 5B). Moreover, a specific calcineurin A peptide with an attachment of the cryptophane cage at a central position ARKEVIRNKIRAIGK-CrytophaneCage-MARVFSVLR (SEQ ID NO. 3) does neither show a suppression nor an unsuppression. (FIG. 5C).

[0393] In addition, slight modifications of the C-terminus (e.g. replacement of the fluorescein by a capping group (N-ethylmaleimide) diminishes the CEST suppression effect (FIG. 5E, blue curve as compared to the black curve in 5D).

[0394] To generate the data shown in the Figures, the following general procedures were applied.

[0395] The production, delivery and application of hyperpolarized Xe for NMR and MRI studies has been described previously including a full list of materials (Witte et al., 2012; Witte et al., 2014). Typically, hyperpolarized .sup.129Xe (20% polarization and more) is generated by spin exchange optical pumping using a 150 W continuous wave laser (795 nm, 0.5 nm bandwidth) in a custom-designed continuous flow setup at 4.5 bar absolute pressure using a gas mixture of 5% Xe (26.4% natural abundance of .sup.129Xe), 10% N.sub.2, and 85% He.

[0396] Experiments benefit from high field MRI scanners because of the increased spectral resolution for resolving the CEST response. As an example, a 9.4 T NMR spectrometer can be used for the NMR experiments. It requires gradient coils for MR imaging and a variable temperature unit for adjusting the sample temperature. A 10 mm inner-diameter double resonant probe (.sup.129Xe (110 MHz) and .sup.iH (400 MHz)), can be used for excitation and detection.

[0397] The freshly hyperpolarized xenon gas mix is directly bubbled into solution for ca. 15 seconds at a total flow rate of 0.1 SLM followed by a 1 s delay (to allow possible remaining bubbles to collapse) before signal acquisition. To reduce foaming, 0.1% of Pluronic L81 may be added to the sample. After bubbling, a saturation RF pulse is irradiated at the desired frequency and the signal of free Xe in solution is observed. .sup.129Xe Hyper-CEST MR images can be acquired using averages of a RARE sequence with a slice-selective 90 gaussian shaped excitation pulse and high RARE factor (up to 32 for a 3232 matrix size) due to sufficiently slow T2 relaxation. Two images are acquired, one with an off-resonant saturation pulse for control and one with an on-resonant saturation pulse to induce signal loss in the vicinity of the sensor. Hyper-CEST MR images are obtained by a pixel-wise subtraction of the noise corrected off- and on-resonant images. All data analysis was performed with MATLAB.

Example 2

[0398] As an additional preferred instantiation of the CEST unsuppression effect, FIG. 9 shows results from a Crytpophane cage modified with a short polyethylene glycol (PEG) chain that contains a biotin moiety at the end. Binding of this compound to avidin (FIG. 9A) leads to a CEST unsuppression that is most likely due to a solubility switch as indicated by the large apparent size of the compound (as measured by Dynamic Light Scattering) in the absence of avidin (most likely indicating agglomerates) that gets reduced upon binding to avidin.

[0399] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

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