Patent Application
20190307907
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.
Metal ions are of fundamental importance for biological processes since they function as essential constituents of structural proteins and enzymes, or as signaling molecules and second messengers. The most prominent example of the last category is calcium, which is important in many signal-transduction cascades and also essential for the signaling in electrically excitable cells, in particular the electrochemical conversion at the presynaptic synapses of neurons mediated through voltage gated influx and intracellular release of calcium as a function of membrane potential. Organ(ism)s thus have to tightly control the concentration of metals and their spatiotemporal distribution as many factors including e.g. increased metabolic demand, malnutrition or contact with toxic concentrations of metals present for instance in environmental pollutants can cause deflections from the physiological range.
Synthetic metal sensors are available for optical readouts based on absorbance (Durham et al., 1983) and fluorescence (Carter et al., 2014). There also exist genetically encoded sensors for readout via fluorescence (Looger et al., 2013) and bioluminescence (in conjunction with appropriate synthetic substrates). These photon-detected metal sensors however all suffer from the poor penetration of photons in biological tissue or in opaque and/or turbid samples. The penetration depth depends on the specific method and desired resolution; even optimal use of near-infrared illumination and independence from scattering as achieved by optacoustics however still limits the maximum penetration depth in biological tissues to a few centimeters (Ntziachristos et al., 2010). For these photon-based imaging methods (the wavelengths being in the visible range) to measure deeper structures in vivo, invasive methods are necessary that e.g. surgically insert optical guides such as fiber bundles into tissue. For diagnostic measurements of in vitro or ex-vivo samples (such as biological fluids and tissue as well also non-medical samples such as waste water) photon-dependent methods are also severely limited in their performance by the samples' opacity and turbidity.
It would therefore be of great value for biomedical diagnostics as well as for applications in food and environmental testing if robust photon-independent detection of metals in opaque and turbid media and non-invasive in vivo imaging of their distribution in organ(ism)s could be achieved at sub-millimeter resolution.
One non-invasive method that fulfills these criteria is nuclear Magnetic Resonance Imaging (MRI) that routinely achieves whole-body coverage in humans. Metal-sensitive contrast agents for NMR and MRI have been developed previously. They fall into four categories according to their physical mechanism: T2 relaxation agents, TI relaxation agents, chemical shift and CEST agents. However, these methods suffer from a lack of sensitivity, obviously one of the major obstacles for in vivo MRI in general, necessitating the use of higher micromolar if not millimolar concentrations of the contrast agent.
To improve MRI's sensitivity, several so-called hyperpolarization methods have been developed that can increase the polarization of nuclear spins and thus the available signal (Viale et al. 2009, Viale et al. 2010). In the case of the noble gases helium (3He) and xenon (129Xe), hyperpolarization can be achieved via optical pumping.
Caged xenon has been described as MRI reporter or biosensor. See e.g. Klippel et al., 2014; SchrBder 2007; Rose et al., 2014.
Klippel et al. (2014) describe MRI localization of cells labeled with caged xenon in a packed-bed bioreactor working under perfusion with hyperpolarized-xenon-saturated medium. Xenon hosts enable NMR/MRI experiments with switchable contrast and selectivity for cell-associated versus unbound cages.
Rose et al. (2014) describe a functional xenon NMR biosensor that can identify the cell surface protein CD14 by targeted 129Xe MRI. Cells expressing CD14 can be spatially distinguished from control cells with incorporation of the xenon MRI readout unit, cryptophane-A. The recognition of CD14 is accomplished via a CD14-specific antibody.
Witte et al. (2015) describe Xenon-MRI on living cells with hyper-CEST biosensors for metabolically labelled glycans on the cell surface.
U.S. Pat. No. 6,652,833 B2 describes a functionalized active-nucleus complex that selectively associates with a biological target species, wherein the functionalized active-nucleus complex comprises: a) an active nucleus and b) a cryptophane family member targeting carrier comprising: i) a first binding region having at least a minimal transient binding of said active-nucleus to form the functionalized active-nucleus complex that produces a detectable signal when the functionalized active-nucleus complex associates with the target species and ii) a second binding region, non-coextensive with said first binding region, that selectively associates with the target species.
Here as well as in other earlier presented sensors, the cryptophane family member or Xe host is always accessible for reversibly bound Xe atoms and thus provides a measurable CEST effect even under conditions where the sensor is dissociated from the target or not in an environment that has certain biochemical “target conditions” (such as temperature, viscosity, pH, a certain ion concentration, O2 level, etc.). The sensing capability of such earlier presented sensors therefore does not rely on changing the Xe access to the host. Thus, there is a need for improved means and methods for analyte sensing for in vitro and in vivo applications.
According to the present invention this object is solved by a precursor for a molecular sensor for determining analyte concentrations and/or measuring analyte concentration changes comprising
According to the present invention this object is solved by a precursor for a molecular sensor for determining analyte concentrations and/or measuring analyte concentration changes
According to the present invention this object is solved by the use of a molecular sensor of the present invention for
According to the present invention this object is solved by the molecular sensor of the present invention for use as a medicament.
According to the present invention this object is solved by the molecular sensor of the present invention for use in a method of diagnosing and/or monitoring treatment of diseases causing changes in metal concentrations.
According to the present invention this object is solved by an in vitro method for determining metal concentration and/or measuring metal concentration changes in a sample, comprising the steps of
According to the present invention this object is solved by a method for diagnosing and/or monitoring treatment of diseases causing changes in metal concentrations, comprising the steps of
Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.
Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “5 to 40 amino acids” should be interpreted to include not only the explicitly recited values of 5 to 40, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 5, 6, 7, 8, 9, 10 . . . 38, 39, 40 and sub-ranges such as from 10 to 30, 15 to 25, from 20 to 35, etc. This same principle applies to ranges reciting only one numerical value, such as “at least 5 amino acids”. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
Molecular analyte sensors and their precursors The present invention provides a precursor to a molecular sensor for determining analyte concentrations and/or measuring analyte concentration changes.
The present invention provides a molecular sensor for determining analyte concentrations and/or measuring analyte concentration changes.
The analyte is preferably a metal (or metal ion), more preferably calcium (or calcium ions). The analyte can be any ion/atomic/molecular structure that induces a conformational change that has impact on the NMR-modulating moiety (b) and interaction moiety (c).
The analyte can be a protein.
Said precursor to a molecular sensor comprises
(a) a host for an active nucleus,
(b) an NMR-modulating moiety, and
(c) an interaction moiety.
Said molecular sensor comprises
(a) a host for the active nucleus,
(b) an NMR-modulating moiety, and
(c) an interaction moiety.
Said precursor can comprise in addition to (a) to (c) or said molecular sensor can comprise in addition to (a) to (c) and the active nucleus:
(d) optionally, further sensor moiety/ies,
(e) optionally, solubilizing and/or biodistribution moiety/ies,
(f) optionally, further interacting moiety/ies.
In a preferred embodiment, the molecular sensor is assembled inside a (target) cell or tissue.
Active Nucleus
The active nucleus is preferably xenon 129Xe, more preferably hyperpolarized xenon.
The active nucleus can be any other nucleus participating in reversible exchange and CEST detection, such as 13C, 15N, 19F, 29Si, 31P, 89Y.
(a) Host for the Active Nucleus
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.
The host is preferably a cage-like molecule.
The host depends on the active nucleus to be hosted.
The active nucleus and the host form an active nucleus-host complex.
In said active nucleus-host complex the nucleus changes its NMR resonance condition upon binding to the host.
In a preferred embodiment, the active nucleus is xenon and the host is a xenon host.
A xenon host is preferably selected from a cryptophane, cucurbit[n]uriles, pillar[n]arenes, or self-assembling metal-organic cages,
including genetically encoded compartments consisting of proteins, peptides, DNA and RNA.
See e.g. Lowery et al. (2004) that describe a conformation-sensitive xenon-binding cavity in the ribose-binding protein. See e.g. Rubin et al. (2002) that describe Xenon-binding sites in Proteins which can be detected by 129Xe NMR spectroscopy.
Cryptophanes are a class of organic supramolecular compounds for molecular encapsulation and recognition. Cryptophane cages are formed by two cup-shaped [1.1.1]-orthocyclophane units connected by three bridges (denoted Y). There are also choices of the peripheral substitutes R1 and R2 attached to the aromatic rings of the units. Most cryptophanes exhibit two diastereomeric forms (syn and anti), distinguished by their symmetry type. This general scheme offers a variety of choices (Y, R1, R2, and symmetry type) by which the shape, the volume, and the chemical properties of the generally hydrophobic pocket inside the cage can be modified, making cryptophanes suitable for encapsulating many types of small molecules and even chemical reactions.
Cryptophanes form with xenon host-guest complexes that are of reversible nature, i.e. the noble gas is in continuous exchange between its bound state in the host and the unbound state in solution.
Cucurbiturils are macrocyclic molecules made of glycoluril (═C4H2N4O2═) monomers linked by methylene bridges (—CH2—). The oxygen atoms are located along the edges of the band and are tilted inwards, forming a partly enclosed cavity. Cucurbiturils are commonly written as cucurbit[n]uril, where n is the number of glycoluril units (and n can be e.g. 5, 6, 7, 8, and 10). Two common abbreviations are CB[n], or simply CBn. Cucurbiturils are efficient host molecules in molecular recognition and have a particularly high affinity for positively charged or cationic compounds. They have been identified as efficient Hyper-CEST agents (Kunth et al. 2015).
Pillararenes or pillar[n]arenes are macrocycles composed of hydroquinone units (n=5 to 10) linked by methylene bridges in the para-position. They are structurally similar to the cucurbiturils and have been studied as Xe hosts (Aldiri et al, 2013).
(b) NMR-Modulating Moiety
The NMR-modulating moiety changes the resonance frequency or the chemical exchange saturation transfer (CEST) signal of the active nucleus-host complex.
This modulation can be influenced by an interaction of the interacting moiety.
In one embodiment, 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 to unsuppresses the CEST-signal.
(c) Interaction Moiety
The interaction moiety mediates interactions with environmental parameters, target molecules, or analytes.
The interaction moiety specifically responds to an environmental 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.
Environmental parameters can be temperature or viscosity.
In a preferred embodiment, the NMR-modulating moiety (b) decreases the CEST signal of the active nucleus-host complex.
More preferably, the NMR-modulating moiety (b) and the interaction moiety (c) (reversibly) suppress the CEST signal from the otherwise accessible host by a specific conformation of the NMR-modulating moiety (b) in the host vicinity, and an interaction with a target molecule via the interaction moiety (c) in an analyte-dependent manner unsuppresses the CEST signal/effect.
“Chemical exchange saturation transfer (CEST)” imaging is a relatively new MRI contrast approach in which exogenous or endogenous compounds containing either exchangeable protons or exchangeable molecules are selectively saturated and, after transfer of this saturation, detected indirectly through the water signal with enhanced sensitivity. In CEST MRI, transfer of magnetization is studied from a dilute to an abundant pool. CEST imaging requires sufficiently slow exchange on the MR time scale to allow selective irradiation of the protons of interest. As a consequence, magnetic labeling is not limited to radio-frequency saturation but can be expanded with slower frequency-selective approaches such as inversion and frequency labeling.
The “hyper-CEST effect” as used herein refers to the use of spin-hyperpolarized nuclei as the exchanging species.
The present invention uses an “analyte-triggered CEST effect” which refers to a change in the (hyper-)CEST performance, i.e., a modulation of the exchange properties in the reversible binding of the nuclei, induced by the interaction of moiety (c) with the analyte and mediated by moiety (b).
The molecular sensors of the present invention, preferably the Xenon-based sensors, operate by a reversible (un)suppression of a HyperCEST effect.
The interaction moiety (c) specifically binds to the analyte or to a target molecule that binds the analyte.
The interaction moiety (c) can specifically bind to a target molecule in an analyte-dependent manner.
The interaction moiety (c) can be or comprise a protein, polypeptide or peptide, such as
which are each specific to the analyte or to a target molecule that binds the analyte or which each specifically bind to a target molecule in an analyte-dependent manner.
The analyte is preferably a metal (or metal ion), more preferably calcium (or calcium ions).
In one embodiment, the NMR-modulating moiety (b) and the interaction moiety (c) are attached to each other or form a “joint moiety”.
Thereby, the NMR-modulating moiety (b) and the interaction moiety (c) are preferably comprised in or consist of a protein, a polypeptide or a peptide or a linker, such as
which are each specific to the analyte or to a target molecule that binds the analyte or which each specifically bind to a target molecule in an analyte-dependent manner.
In one embodiment, said linker is a peptide chain or is selected from polyethylene glycol (PEG) In an embodiment, where the analyte is calcium, and where the NMR-modulating moiety (b) and the interaction moiety (c) are attached to each other (in a “joint moiety”), the NMR-modulating moiety (b) and the interaction moiety (c) comprise or consist of a calmodulin (CaM)-binding peptide, preferably a calmodulin (CaM)-binding peptide selected from
A “calmodulin-binding peptide”, as used herein, is a peptide that is capable of binding, or a peptide that binds, to calmodulin.
RS20 is a synthetic peptide derived from the calmodulin-binding region of smooth muscle myosin light chain kinase (smMLCK).
M13 is a synthetic peptide. Its sequence is the same as the calmodulin-binding domain of skeletal muscle myosin light chain kinase (skMLCK) (residues 577-602). Calmodulin can bind the M13 peptide without the presence of the entire protein.
Calcineurin A is the calmodulin-binding catalytic subunit of calcineurin, which is a calcium and calmodulin dependent serine/threonine protein phosphatase (also known as protein phosphatase 3, and calcium-dependent serine-threonine phosphatase).
Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca2+, and the binding of Ca+ is required for the activation of Calmodulin. Once bound to Ca2+, Calmodulin acts as part of a calcium signal transduction pathway by modifying its interactions with various target proteins, such as myosin light chain kinase (MLCK), calmodulin-dependent kinases, protein phosphatase calcineurin, phosphodiesterase, nitric oxide synthase, Ca2+-ATPase pumps, as well as cytoskeletal structural proteins.
Examples for said other CaM-binding peptides or proteins are e.g. described in Barnes and Gomes (1995), Ashfar et al. (1994), Vetter and Leclerc (2003), Erickson-Viitanen and DeGrado (1987).
CaM-kinase II (CAMK2) is a prominent kinase in the central nervous system that may function in long-term potentiation and neurotransmitter release. Member of the NMDAR signaling complex in excitatory synapses it may regulate NMDAR-dependent potentiation of the AMPAR and synaptic plasticity.
Troponin (Tn) is the central regulatory protein of striated muscle contraction. Tn consists of three components: Tn-I which is the inhibitor of actomyosin ATPase, Tn-T which contains the binding site for tropomyosin and Tn-C. The binding of calcium to Tn-C abolishes the inhibitory action of Tn on actin filaments.
The sensitivity of the molecular sensor of the present invention, such as a calcium sensor, can be changed/tuned by using variants of the target molecule or protein, such as calmodulin, with higher or lower affinities for the analyte (such as calcium).
The sensitivity of the molecular sensor can be tuned by using different target molecules or proteins that exhibit lower affinity for the analyte, such as proteins interacting with calcium at higher concentrations present in e.g. synaptic vesicles (e.g. C2A domain containing, lipid-binding proteins). This allows for tuning the sensor to extracellular calcium concentrations, thus avoiding the need for cellular delivery of the agent.
In one embodiment, the host (a), the NMR-modulating moiety (b) and the interaction moiety (c) are attached or connected to each other,
such as
In a preferred embodiment, the NMR-modulating moiety (b) and the interaction moiety (c) comprises or consists of/is the peptide RS20 or the peptide M13 that specifically binds to an EF hand protein, such as calmodulin, in a calcium-dependent manner.
Preferably, the NMR-modulating moiety (b) and the interaction moiety (c), when attached/connected to each other/“joint moiety”) comprises or consists of the amino acid sequence selected from SEQ ID NOs. 1 to 3, 14 and 15
In a preferred embodiment the NMR-modulating and binding moiety (c) comprises or consists of the amino acid sequence of SEQ ID NO. 1 [RS20] or SEQ ID NO. 2 [M13] and is attached/linked via lysine side chains to the host.
The peptides can be linked to further sensor moiety/ies (as described below); such as fluorescein (“5-Fluo-M”), (“Btn-M”), or a second Xenon-host molecule such as Cucnrbit[6]uril. The preferred host whose signal is modulated is cryptophane A (“Cr-A”.) The maleimide-Fluorescein can be replaced by a maleimide capping group (“NEM”).
In one embodiment, wherein the NMR-modulating moiety (b) and the interaction moiety (c) are attached to each other or form a joint moiety, preferably the NMR-modulating moiety (b) and the interaction moiety (c) are comprised in or consist of a linker, such as polyethylene glycol (PEG), or a peptide chain.
In a preferred embodiment, the NMR-modulating moiety (b) and the interaction moiety (c) are attached to each other and comprise or consist of a peptide as defined herein, decrease the CEST signal of the active nucleus/nucleus-host complex in aqueous solution and in absence of other compounds.
More preferably the CEST signal from the otherwise accessible host is suppressed by a specific conformation of the peptide in the host vicinity.
In the absence of the analyte (such as calcium ions) the saturation transfer signal from the cryptophane-encapsulated hyperpolarized 129Xenon is suppressed by the peptide, with some contribution of the C-terminal modification (e.g. the further sensor moiety, such as fluorescein). If the analyte is added, the peptide binds tightly with the protein calmodulin and undergoes a conformational change, thereby turning on (unsuppressing) the saturation transfer signal in a calcium-dependent manner. The analyte-dependent NMR signal activation of this preferred embodiment thus occurs as schematized in
Intracellular Assembly of the Molecular Sensor Inside the Cell
As discussed above, in a preferred embodiment the molecular sensor for determining analyte concentrations and/or measuring analyte concentration changes in a cell/in a target cell, is assembled inside said cell or target cell or said tissue.
This can be realized by delivering the host for the active nucleus functionalized with respective moieties across the cellular membrane where it specifically bioconjugates with the other moieties of the sensor that are genetically expressed.
In another embodiment all moieties of the sensor including the host for the active molecule are genetically expressed.
The components (a) to (c) and the active nucleus can be delivered to the (target) cells. Moieties (a), (b) and/or (c) can be expressed by said (target) cells.
In one embodiment, the complex of the active nucleus and its host (a) or the host (a) are e.g. used and delivered to the target cells. The NMR-modulating moiety (b) and/or the interaction moiety (c), optionally fused to the target molecule of the analyte, will be provided by the target cells, such as via intracellular expression.
For example, the xenon-binding host molecule (a) can be delivered across the cellular membrane to “find” a specific genetically expressed calcium-binding protein such as a calmodulin-peptide fusion.
This can be achieved via bioconjugation via inteins, specific amino acid sequences including artificial amino acids and known chemistry such as click chemistry or known ligand to protein interaction such as biotin to avidin, all of which are known to people informed with the basic knowledge available in the field.
In a preferred embodiment, the modified peptides can be directly conjugated to calmodulin (CaM) as the interacting molecule via an intein reaction of an artificial aminoacid, where CaM may be integrated in a fluorescent calcium indicator such as GCaMP A. Miyawaki, J. Llopis, R. Heim, J. M. McCafery, J. A. Adams, M. Ikura, R. Y. Tsien, Nature 1997, 388, 882-887. G. S. Baird, D. A. Zacharias, R. Y. Tsien, Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11241-11246. J. Nakai, M. Ohkura, K. Imoto, Nat. Biotechnol. 2001, 19, 137-141.) using intein-based protein ligation (GCaMP-DnaB-Intein and the corresponding intein-modified peptide Cr-A@K1_N-GCaMP:N-Intein) or biorthogonal conjugation based on unnatural lamino-acids integrated via the expanded genetic code (
In such SEQ ID NO:14, amino acids 1-108 denotes the auxiliary protein part to allow high expression with high solubility, its purification using Ni-NTA columns and its cleavage after successful purification; amino acids 109-251 denotes the DnaB Intein from Synechocystis sp. PCC6803 (SspDnaB) with all amino acid N-terminal trunctation; amino acids 252-644 denotes the C-terminal GCaMP6 to which the synthetized N-GCaMP6:N-Intein peptide is post-translationally ligated
In such SEQ ID NO: 15, Cr-A@K1_N-GCaMP:N-Intein; amino acids 1-19 denotes the N-terminal amino acids of GCaMP and (same sequences as RS20); amino acids 20-30 denote the amino acids 1-11 from SspDnaB Intein
Further Components
In one embodiment, the precursor or the molecular sensor of the present invention further comprises
Said further sensor moiety/moieties (d) and/or said solubilizing and/or biodistribution moiety/ies (e) can be covalently attached to the NMR-modulating moiety (b) and/or the interaction moiety (c).
Said further sensor moiety or moieties (d) can be
or combinations thereof.
Said further sensor moiety can serve as reference signal, e.g. for quantifying the precursor or the molecular sensor of the invention.
For example, a ratiometric readout can be achieved by addition of a second (Xe) host (e.g. cucurbit or cryptophane) whose environment does not change as a function of analyte (calcium) binding (or other of the above-mentioned stimuli) such that it can be used to normalize the concentration of the sensor.
Said further sensor moiety can be used for localization of the precursor or the molecular sensor of the invention, e.g. in in vivo applications.
For example, localization of the precursor or the sensor by an independent read-out can be built in via a fluorophore, an MRI agent such as a TI agent represented by gadolinium DPTA (or similar chelates of paramagnetic ions), or a radioactive probe for PET detection.
This multimodal readout is possible by the precise knowledge of the three-dimensional structure of a primary analyte or of a target molecule, such as calmodulin, that affords the extension of the peptidic scaffold out of the binding pocket; external modifications by the modifiers mentioned above can thus be realized without interfering with the calcium-mediated peptide binding mechanisms.
Said further sensor moiety or moieties (d) can also enhance the NMR signal suppression effect.
Said solubilizing and/or biodistribution moiety or moieties (e) preferably is/are for modifying the solubility and biodistribution of the active-nucleus host complex, e.g. cell permeability. Preferably, it improves or enables cellular uptake and/or enables delivery over barriers, such as the blood-brain-barrier.
Said solubilizing and/or biodistribution moiety (e) aids in the delivery of the sensor of the present invention to a desired target organ (including the brain), target tissue and target cells.
The solubilizing and/or biodistribution moiety (e) can comprise respective receptor components, such as transferrin receptor or insulin receptor, or further specific binding moieties, such as antibodies or antibody fragments, which are specific for the desired target organ, tissue, cells.
In one embodiment, the precursor or the molecular sensor comprises a further interacting moiety or moieties (f).
Preferably, said further interacting moieties (f) aid in the binding of the precursor or the molecular sensor to the target molecule in in vivo application or in cellular applications or in applications where the target molecules are genetically modified and provided by intracellular expression.
Examples for said further interacting moieties (f) are
For example, the precursor or molecular sensor of the invention which is to be delivered into the target cells comprises a biotin. The target molecule comprises an (strep)avidin.
For example, specificity for genetically defined cellular populations can be achieved by genetically expressing a variant of the target molecule, such as a calcium-binding protein, e.g. calmodulin, that exhibits a specific affinity for the peptide-cage construct (i.e. the molecular sensor) being delivered to the cell. This can be achieved by e.g. using high-affinity interacting moieties, such as SpyTag/SpyCatcher, Biotin/Avidin or antiparallel homodimeric protein interfaces.
Preferred Example for a Calcium Sensor:
and the joined NMR-modulating moiety (b) and the interaction moiety (c) binds to calmodulin in a calcium-dependent manner,
The respective precursor of the calcium sensor comprises the above components without the active nucleus 129Xenon, preferably spin-hyperpolarized 129Xenon.
A particularly advantageous feature of a preferred embodiment of the sensor is a complete suppression of the CEST signal in the absence of calcium. As demonstrated in
Imaging and Medical Uses of the Molecular Sensor
The present invention provides the use of the molecular sensor of the present invention for
The analyte is preferably a metal (or metal ion), more preferably calcium (or calcium ions).
The use according to the invention preferably comprises nuclear magnetic resonance (NMR) spectroscopy and imaging (MRS, MRI, Hyper-CEST).
The use according to the invention can comprise determining further analyte(s), such as further metal(s). In such an embodiment, the sensor of the present invention comprises further sensor moiety/moieties (d) that may e.g. operate via a different contrast agent property such as a change in the fluorescence or photoacoustic signal.
For example, the molecular sensor of the present invention can be used for sensing other external stimuli, such as the presence of other metals, and changes in environmental parameters, such as, temperature, viscosity or effects from electromagnetic waves, mechanical waves, or particles irradiation by using respective protein-peptide pairs (as the NMR-modulating and interaction moiety (b), (c) and optionally solubilizing and/or biodistribution moiety (e)).
The use according to the invention optionally comprises multimodal detection of further sensor moiety/moieties, preferably as defined herein above, via absorbance/transmission, reflection, fluorescence or optoacoustic or ultrasound measurements and imaging.
As used herein “multimodal detection” refers to the combined detection of the sensor of the present invention via a detection method that is not be based on the NMR, such as PET, ultrasound or photon-dependent methods.
In one embodiment, the magnetic resonance readout can be combined with photon-dependent imaging via transmission, fluorescence, or optoacoustic imaging techniques detecting a chromophore inserted.
As used herein “optoacoustic or photoacoustic” measurements or imaging refer to the detection of the mechanical waves generated by the optoacoustic, or photoacoustic effect (Ntziachristos et al., 2010)
As used herein “fluorescent or absorption” measurements or imaging refer to the detection of fluorescent and/or absorbed photons for point measurements or spatiotemporally resolved measurements/imaging.
The use according to the invention preferably comprises
The administration of the hyperpolarized 129Xenon can be achieved for in vitro applications via bubbling/dispersing it into solution or via administration through the breathing air/Xe mixture or injection of a Xe-saturated carrier solution such as saline solution, blood plasma etc. for preclinical in vivo studies.
For applications in cells, cellular delivery of the precursor of the molecular sensor (or of the molecular sensor) of the present invention is aided/enabled in preferred embodiments by the cell penetrating properties of the calmodulin-binding peptides mediated by the presence of several positively charged amino acids.
For applications in neuroscience, trans blood-brain-barrier (BBB) uptake can be achieved via active transport (e.g. coupling to transferrin receptor or insulin receptor binding moieties such as antibodies) or after application of BBB opening techniques, such as ultrasound-mediated BBB opening, or pharmacological intervention, such as hyperosmolar shock.
The present invention provides the molecular sensor of the present invention for use as a medicament.
The present invention provides the molecular sensor of the present invention for use in a method of diagnosing and/or monitoring treatment of diseases causing changes in metal concentrations.
Thereby, the progress of a disease and/or the treatment of a disease can be monitored.
Preferably, a “disease causing changes in metal concentrations” is selected from:
Preferably, the imaging is real-time.
Preferably, the uses comprise imaging of tissues and bodily fluids,
Methods for Determining Metal Concentration and/or Measuring Metal Concentration Changes
The present invention provides an in vitro method for determining metal concentration and/or measuring metal concentration changes in a sample.
Said method comprises the steps of
The active nucleus is preferably a spin-hyperpolarized nucleus, such as 129Xe or other reversibly binding nuclei.
The provision of the active nucleus, such as hyperpolarized 129Xenon, to the sample can be achieved via e.g. bubbling/dispersing it into solution.
Step (iii) can optionally comprise performing further measurement(s) to detect the further sensor moiety(moieties) (d).
Preferably, the sample is a biological fluid, such as blood, urine, lymph or lymphatic drainage, cerebrospinal fluid, stool/feces, semen, saliva or mucous fluids, or a cell culture sample, such as derived from the human or non-human animal body, ex vivo tissue, cell culture.
Methods for Diagnosing and/or Monitoring Treatment of Diseases Causing Changes in Metal Concentrations
The present invention provides a method for diagnosing and/or monitoring treatment of diseases causing changes in metal concentrations.
Said method comprises the steps of
Preferably, said method is in real-time.
In step (ii), the active nucleus is preferably a spin-hyperpolarized nucleus, such as 129Xe or other reversibly binding nuclei.
Preferably, the active nucleus is hyperpolarized xenon gas, which can be e.g. inhaled.
The administration of the active nucleus, preferably the hyperpolarized 129Xenon, can be achieved via administration through the breathing air (e.g. by inhalation), Xe mixture or injection of a Xe-saturated carrier solution such as saline solution, blood plasma etc.
Step (iii) can optionally comprise performing further measurement(s) to detect the further sensor moiety(moieties) (d).
As discussed above, a “disease causing changes in metal concentrations” is preferably selected from:
The molecular sensor of the present invention differs from sensors described before, such as in Klippel et al., (2014), Rose et al. (2014) or Witte et al. (2015), since they rely on increased uptake into the cells or specific binding to a cellular (surface) target in as much as the CEST effect for activating the image contrast is always available, even if the sensor is not yet bound to the target. In the molecular sensor of the invention the CEST effect is suppressed in the absence of the interaction with the target and gets unsuppressed in an analyte-dependent manner.
The present invention describes a sensor for Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI) based on spectral changes of the NMR signature (such as the NMR saturation transfer) of temporarily encapsulated hyperpolarized 129Xenon caused by analyte-dependent changes of the local environment of a Xenon-binding molecule coupled interaction moieties, such as proteins or peptides. Such changes in the NMR signature can be a change in the resonance frequency (so-called chemical shift) or the intensity or width of a saturation response through chemical exchange saturation transfer onto free hyperpolarized Xe (Hyper-CEST). The Hyper-CEST effect is typically probed by radio frequency (RF) saturation schemes, either continuous wave (cw) or pulsed irradiation.
The present invention relates to a novel analyte-triggered NMR signal turn on mechanism and specifically designed sensor compound that supports it.
The sensor of the present invention needs to provide the following functionalities:
(a) an active nucleus (e.g. xenon);
(b) a host for the active nucleus (e.g. xenon host (such as cryptophane, cucurbit[n]uril etc.),
(c) an NMR signal-modulating moiety (e.g. a peptide)
(d) an interaction moiety
(e) optionally, a contrast agent moiety or further sensor moiety
(f) an optional solubilizing and/or biodistribution moiety
In a preferred embodiment of the invention, a xenon-binding cage-like compound called the “host” (such as cryptophane or similar entities, e.g. macrocyclic units like cucurbit[n]uriles, pillar[n]arenes, or self-assembling metal-organic cages etc.) is conjugated to a peptide (such as RS20, M13) that binds to an EF hand protein (such as calmodulin) in a calcium-dependent manner.
In the absence of the analyte (in this case calcium ions) the saturation transfer signal from the cryptophane-encapsulated hyperpolarized 129Xenon is suppressed by the peptide acting as “the NMR-signal modulating moiety”. If the analyte is added, the peptide binds tightly with the protein calmodulin and undergoes a conformational change, thereby turning on (unsuppressing) the saturation transfer signal in a calcium-dependent manner. The analyte-dependent NMR signal turn on of this preferred embodiment thus occurs as schematized in
In the preferred embodiment, the peptide fulfills the function of NMR modulation and binding. It also enables cellular uptake (see
A particularly advantageous feature of a preferred embodiment of the sensor is a complete suppression of the CEST signal in the absence of calcium. As demonstrated in
Aided by the well-characterized crystal structures of calmodulin with peptides (see
A previously published Calcium sensor had a turn-on signal in the presence of Ca that was used to generate a 19F CEST response with specificity for Ca ions over Mg and Zn ions (Bar-Shir et al., 2013). However, since this sensor did not employ spin-hyperpolarized Xe and therefore requires concentrations in the mM range. Moreover, it does not rely on a modulation of the the exchange kinetics of reversibly binding nuclei (Xe in our case) but rather on the kinetics of conformational changes of a chelate that comprises permanently bound 19F nuclei for NMR/MRI detection.
Whereas the cellular delivery of the hyper-CEST sensor described in this application can occur over hours to be later ‘interrogated’ by rapidly administered hyperpolarized Xenon, sensors that work via a chemical shift of a hyperpolarized 13C or 15N moiety only provide increased signal for very short time interval limited by the relaxation rates of the heteronuclei (Nonaka et al., 2015; Hata et al., 2013). Synthetic compounds have been made to sense toxic metals but via a conventional chemical shift mechanism (Tassali et al., 2014) not the CEST un(suppression) described herein here.
Using this robust turn-on CEST effect, we can demonstrate quantitative calcium detection in the lower micromolar range with an excellent dynamic range for physiological concentrations during signal transduction processes (
We furthermore conducted confocal microscopy studies confirming that the fluorophore-labeled peptides are readily taking up into cells (
The means and methods of the present invention offer the following unique combination of advantages:
Instead, the delivery of the xenon-binding host to the target organ (including the brain) and into the cell can occur at slow kinetics. The interrogation of the distribution and analyte-dependent state of the host can then take place in a second step within the optimal time windows defined by the decay rates of the hyperpolarized signal. The administration of the hyperpolarized 129Xenon can be achieved for in vitro applications via bubbling/dispersing it into solution or via administration through the breathing air/Xe mixture or injection of a Xe-saturated carrier solution such as saline solution, blood plasma etc. for preclinical in vivo studies.
The means and methods described herein could be used for in vitro detection of calcium in opaque specimens.
For applications in cells, cellular delivery of the scaffold-host conjugate (peptide-cage) is enabled by the cell penetrating properties of the calmodulin-binding peptides mediated by the presence of several positively charged amino acids. For applications in neuroscience, trans blood-brain-barrier (BBB) uptake can be achieved via active transport (e.g. coupling to transferrin receptor or insulin receptor binding moieties such as antibodies) or after application of BBB opening techniques, such as ultrasound-mediated BBB opening, or pharmacological intervention, such as hyperosmolar shock.
Specificity for genetically defined cellular populations can be achieved by genetically expressing a variant of the calcium-binding protein, such as calmodulin, that exhibits a specific affinity for the peptide-cage construct being delivered to the cell (
The sensitivity of the calcium sensor can be tuned by using variants of the calcium protein such as calmodulin with higher or lower affinities for calcium. Also orthogonal calmodulin/peptide pairs could be used alternatively to ensure orthogonality, i.e. that do only interact with each other but do not have high affinities for the respective endogenous binding partners.
Alternatively, the sensitivity of the sensor can be tuned by using different proteins that exhibit lower affinity, such as proteins interacting with calcium at higher concentrations present in e.g. synaptic vesicles (e.g. C2A domain containing, lipid-binding proteins). This allows for tuning the sensor to extracellular calcium concentrations, thus avoiding the need for cellular delivery of the agent.
Similarly, the means and methods described herein can be extended to sensing other external stimuli, such as the presence of other metals, and changes in environmental parameters, such as pH, by using the respective alternative protein-peptide pairs.
A ratiometric readout can be achieved by addition of a second Xe host (e.g. cucurbit or cryptophane) whose environment does not change as a function of calcium binding (or other of the above-mentioned stimuli) such that it can be used to normalize for the concentration of the sensor.
Furthermore, multimodal calcium sensing can be achieved by attaching an environmentally sensitive fluorophore to the calmodulin-binding peptide. Similarly, localization of the sensor by an independent read-out can be built in via a fluorophore, an MRI agent such as a Ti agent represented by gadolinium DPTA (or similar chelates of paramagnetic ions), or a radioactive probe for PET detection. This multimodal readout is made possible by the precise knowledge of the three-dimensional structure of a primary analyte such as calmodulin that affords the extension of the peptidic scaffold out of the binding pocket; external modifications by the modifiers mentioned above can thus be realized without interfering with the calcium-mediated peptide binding mechanisms.
As stated above, the moiety attached in addition to the xenon-binding cage (such as a chelated transition metal or a molecular such as a planar fluorophore) can influence the chemical shift of xenon thereby supporting its calcium-dependent change via peptide-protein binding (that e.g. increases the average distance between the cryptophane cage and the additional moiety)
The semi-genetic method featuring a genetically expressed protein and synthetically modified peptide to be delivered to the sample (either extracellularly or intracellularly) could also be made a fully-genetic solution by genetically expressing a Xenon-host (e.g. gas binding proteins or self-assembling macromolecular structures) with function similar to that of the synthetic cryptophane cage.
The calcium sensor of the present invention has a few orders of magnitude higher sensitivity in terms of the signal to noise ratio over any existing calcium sensor for NMR/MRI (see e.g. Schröder et al., 2006; Schröder et al., 2008-1; Schröder et al., 2008-2; Kunth et al., 2012). This is caused by the ‘analyte-triggered CEST effect’, as described herein. The sensor has furthermore an optimal dynamic range to detect relevant intracellular concentrations of calcium and we show that it easily enters into cells in stark contrast to many of the other concepts for NMR sensors (see e.g. Caravan 2006; Caravan et al., 1999).
The calcium sensor of the present invention utilizes Xenon hyperpolarization and the hyper-CEST mechanism that has been described before by Schröder et al. (2006).
The very specific advantageous properties of the means and methods of the invention are:
that will enable in vitro, ex vivo and in vivo use of the analyte sensor (calcium sensor) in biomedical applications.
The molecular calcium sensor specified in the present invention disclosure has a broad range of possible applications in in vitro, in vivo and ex vivo detection of calcium concentration changes as well as measurements of spatiotemporal distribution of calcium via Nuclear Magnetic Resonance (NMR) spectroscopy and imaging (MRS, MRI, Hyper-CEST).
These photon-independent detection methods have great advantages over methods that rely on fluorescence, absorbance or bioluminescence for measuring metal concentration changes in opaque or turbid samples and live organ(ism)s. As compared with existing NMR-based metal-sensitive contrast agents, the present invention disclosure describes the generation of a molecular cage-based sensor that can be interrogated via addition of hyperpolarized xenon generating orders of magnitude higher NMR signal. This increased SNR is a necessary precondition to perform in vivo measurements of spatiotemporal metal distributions, preferably with direct quantification via ratiometric capabilities and preferably with the ability identify specific metals via their characteristic chemical shift.
The preferred field of application for the metal-biosensors described herein is in vivo imaging of calcium distributions in preclinical animal models and humans, in particular for diagnosing and/or monitoring treatment of diseases associated with changes in metal concentrations, such as diseases:
The molecular sensor can furthermore be applied in, ex vivo (pre)clinical imaging of tissues and bodily fluids (such as blood, urine, feces, CSF, lymphatic drainage) and explanted tissues or cells, and in in vitro measurement and imaging in biomedical (cell and tissue cultures) or environmental samples (such as metal screening in e.g. water, air, soil, plants, food).
The following examples and drawings illustrate the present invention without, however, limiting the same thereto.
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.
(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).
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).
(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.
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.
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).
Hyperpolarized Xenon was bubbled into buffered (MOPS) solutions containing different cryptophane bearing peptides indicated in the respective subfigures (1 μM) and calmodulin (3 μM) in the absence and presence of 100 μM 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).
(A) Circular Dichroism data showing how with 20% TFE, an α-helical structure is promoted, but not fully induced. At 50% TFE, the induction an α-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 α-helical structure.
CEST spectra (A) and binding curves (B) of the sensor peptide RS20-cage@K1 (2 μM) in the presence of 10 μM calmodulin are shown for calcium concentrations ranging from 0 to 39 μM (MOPS buffer).
(A) Cage-conjugated peptides that were also labeled with fluorescein (25 μM) 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
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.
The different orientations of the peptides RS20, M13 and Calcineurin A are shown upon binding to Calmodulin in a Caz+-dependent fashion together with the position of the Cryptophane A cages.
Materials and Methods
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.
As can be seen in
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 (
To generate the data shown in the Figures, the following general procedures were applied.
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 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 129Xe), 10% N2, and 85% He.
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 (129Xe (110 MHz) and iH (400 MHz)), can be used for excitation and detection.
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. 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 32×32 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®.
As an additional preferred instantiation of the CEST unsuppression effect,
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16181994.1 | Jul 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/069213 | 7/28/2017 | WO | 00 |
