FIELD OF THE INVENTION

The present invention comprises a genetically-encoded fluorescent nanosensor for detecting and quantifying lactate/pyruvate ratio in different types of samples, such as biological fluids, tissues, intra-cellular, and even in subcellular compartments, with high spatial and temporal resolution, and protocols to express this nanosensor in biological samples, and its use for the detection and quantification of intracellular lactate/pyruvate ratio in intact systems, evaluation of fermentative and oxidative metabolism in simultaneous fashion, detection of lactate/pyruvate ratio in biological samples and evaluation of monocarboxylate permeability, using a non- metabolized analogue.

BACKGROUND OF THE INVENTION

Lactate and pyruvate are key monocarboxylates produced by fermentative and oxidative metabolism in unicellular microorganism, insects, fish and mammalian systems. Pyruvate is produced from catabolized glucose and oxidized by mitochondrial metabolism to produce chemical energy in form of ATP. Lactate is produced from pyruvate and it is a direct readout of fermentative metabolism. Lactate/pyruvate ratio is a critical parameter for clinical research and prognosis of secondary cerebral and systemics insults in acute brain injury, subarachnoid hemorrhage and intracerebral hemorrhage ¹ · ² . Also lactate/pyruvate ratio is valuable readout to assess cellular redox, since the conversion of pyruvate-lactate-pyruvate, an LDH (Lactate Dehydrogenase) catalyzed reaction, is driven by NADH and NAD+ cofactors ³ . Furthermore, real time monitoring of lactate/pyruvate ratio should be critical to evaluate Warburg phenotype highly activated in tumoral cells ⁴ . Tools that allow to detect and quantify lactate/pyruvate ratio with single resolution in culture cells, tissue and in vivo will be useful to monitor and evaluate fermentative/oxidative metabolism in real-time fashion in living systems.

Standard methods do not allow to measured lactate and pyruvate simultaneously, precluding the possibility to have lactate/pyruvate ratio in a single-step measurement. Current methodologies to detect pyruvate and lactate separately are based on enzymatic reactions, which must be followed by photometric, amperometric or other devices. Enzyme-based electrodes have been developed that can detect both molecules with high-temporal resolution. Another approach to measure lactate and pyruvate is high performance liquid chromatography (HPLC), where the monocarboxylates are separated from other compounds by passing the sample through a stationary phase stored in a column. There is a problem in the prior art, however, that the existing methods are invasive as they require the extraction of samples or consume pyruvate and lactate, and therefore, they change the concentration of the analytes in the sample. A second problem is their sensitivity, since they cannot detect the minute amount of pyruvate or lactate present in a single cell or in subcellular organelles.

In the current state-of-the-art there is no evidence of an optical tools based on proteins or organic molecules for detecting and quantifying lactate/pyruvate ratio in a single-step measurement in biological fluids, tissues and cellular and subcellular compartments, with high spatial and temporal resolution. Also, there are no available techniques to quantitate with single cell resolution lactate/pyruvate ratio. Nevertheless, there are related documents in the art, which will be described below.

Sensors for different metabolites are described in W02006096213A1 , W02006096214A1 , W02006044612A2 and W02007046786A2 that involve a FRET donor, a FRET acceptor and a member of the class of periplasmic binding proteins (PBPs), proteins located in outside bacterial plasma membranes involved in chemotaxis. The periplasmic binding protein serves as the specific recognition element. As there is no known rule to predict whether a given protein may serve as an effective recognition element, these proteins have been the result of informed trial and error, semi-rational design. The current invention does not used any of the recognition elements described W02006096213A1 , W02006096214A1 , W02006044612A2 or

W02007046786A2. Moreover, the current invention is not based on any members of the periplasmic binding protein family but rather on a member of the GntR family, a subclass of transcription factors involved in adaptation of bacteria to changing environmental conditions. Surprisingly, the bacterial binding protein described in the present invention was found to detect the lactate/pyruvate ratio, which makes it unique. The current art does not include a genetically encoded fluorescent nanosensor to detect and quantify the lactate/pyruvate ratio. Additionally, the current art does not describe any bacterial protein with the required lactate/pyruvate ligand domain, an essential part of fluorescent nanosensors. DISCLOSURE OF THE INVENTION

The subject of the present invention is to provide a nanosensor, which allows minimally-invasive measurement of lactate/pyruvate ratio with high sensitivity regardless of the concentration of the probe, which does not consume pyruvate or lactate during measurement, and that can be used to measure lactate/pyruvate ratio in samples, in cells and in subcellular compartments. Further, the subject of the present invention is to provide a measuring method of lactate/pyruvate ratio using the nanosensor. Said method can be used to detect and quantify the intracellular lactate/pyruvate ratio in intact systems, evaluate fermentative and oxidative metabolism, detect lactate/pyruvate ratio in biological samples and evaluate monocarboxylate permeability, using a no-metabolized pyruvate analogue.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is related to a genetically encoded Forster resonance energy transfer (FRET)-based indicator composed of the bacterial transcriptional factor LutR from Bacillus licheniformis sandwiched between any suitable donor and acceptor fluorescent proteins moieties that are capable in combination of serving as donor and acceptor moieties in FRET. Preferred donor and acceptor moieties are selected from the group consisting of mTFP (monomeric teal fluorescent protein), CFP (cyan fluorescent protein), BFP (blue fluorescent protein), GFP (green fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent proteins), enhanced variations thereof such as enhanced YFP (EYFP), Citrine or Venus, or infrared fluorescent proteins from bacterial phytochromes, with a particularly preferred embodiment provided by the donor/acceptor mTFP/YFP Venus, a variant of YFP with improved pH tolerance and maturation time or circularly permuted version of Venus. An alternative is the use of a single fluorescent moiety such as circularly-permuted variations, e.g. GFP or mRuby or other suitable circularly- permuted fluorescent protein, inserted into the backbone of LutR or other suitable pyruvate/lactate-binding protein, which undergoes a change in fluorescence intensity in response to binding of pyruvate and lactate to the LutR moiety or to other suitable pyruvate/lactate-binding protein.

The present invention is based on the finding of reciprocal changes in mTFP/Venus ratio induced by pyruvate and lactate. This behavior shows that pyruvate and lactate bind to LutR. This finding was completely unexpected since previous reports has been suggested lactate as the sole ligand for this bacterial transcriptional factor ^{5 8} . To the best of our understanding, no information is available about pyruvate binding to LutR from any species. The present invention shows a dose response curve with apparent dissociation constant (K _D ) for lactate of 2 mM and 12 mM for pyruvate. Lactate/pyruvate ratio sensing is possible due to the reciprocal effects of lactate and pyruvate on the fluorescence ratio and a decrease affinity for lactate in the presence of pyruvate. Pyruvate concentrations of 0, 1 , 10, 100 and 1000 mM produced a decrease in the affinity of the probe for lactate, with respective K _D values of 2.03 ± 0.1 , 5.4 ± 0.5, 25.9 ± 4.3 and 268 ± 71. The dependency of mTFP/Venus fluorescence to lactate/pyruvate ratio has a K _D of 0.4 being capable to detect in the range of 0.04 - 4.

The measurements can be performed in biological fluids, single cells or cell populations, adherent cells or in suspension, in a cell culture, a tissue culture, a mixed cell culture, a tissue explant, or it can also be applied to animal tissues in vivo. The method comprises the expression of the lactate/pyruvate ratio sensor of the present invention in individual cells or its use as a purified protein for metabolite detection in biological fluids. Once the sensor is expressed in single cells or cell populations, adherent cells or in suspension, in a cell culture, a tissue culture, a mixed cell culture, a tissue explant, or in animal tissues in vivo, the sensor is calibrated according to pre-established conditions in order to assess the lactate/pyruvate ratio. A two-point calibration protocol is applied at the beginning of each experiment. Briefly, intracellular lactate/pyruvate ratio is lowered by depriving the cells of any carbon sources and using a non- metabolized pyruvate analogue such as oxamate, which is detected by the nanosensor in the same manner of pyruvate, to mimic a low lactate/pyruvate. Alternatively, can be use oxamate analogues such as /V-Ethyl oxamate, N- Propyl oxamate, N- Butyl oxamate, N- isobutyl oxamate, N- Sec-butyl oxamate, etc. Then a pulse of high lactate is applied in the absence of non- metabolized pyruvate analogue such as oxamate in order to displace it from the sensor and reach the maximal lactate/pyruvate ratio.

Depending on the configuration for lactate/pyruvate ratio measurements:

In a first embodiment, the use of the nanosensor of the invention in a method allows the detection and quantification of intracellular lactate/pyruvate ratio in intact systems. In a second embodiment, the use of the nanosensor of the invention in a method allows the evaluation of fermentative and oxidative metabolism in simultaneous fashion.

In a third embodiment, the use of the nanosensor of the invention in a method allows the detection of lactate/pyruvate ratio in biological samples.

In a fourth embodiment, the use of the nanosensor of the invention in a method allows to evaluate monocarboxylate permeability, using a non-metabolized pyruvate analogue.

Both the foregoing summary and the following detailed description provide examples and are explanatory only. Accordingly, the foregoing summary and the following detailed description should not be considered to be restrictive. Further, features or variations may be provided in addition to those set forth herein. For example, certain embodiments may be directed to various feature combinations and sub-combinations described in the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated by the accompanying drawings wherein:

Figure 1

It shows the predicted tridimensional structure of the transcriptional regulator LutR from Bacillus licheniformis.

Figure 2

It shows the small library screening to evaluate the effector molecule of LutR using foster resonance energy transfer.

Figure 3

It shows the alignment of the amino acid sequences of four variants of the prototype of lactate/pyruvate ratio nanosensor. SEQ ID 01 corresponding to variant 1 , SEQ ID 02 corresponding to variant 2, SEQ ID 03 corresponding to variant 3 and SEQ ID 04 corresponding to variant 4. Figure 4

It shows the constructed variants to optimize the lactate and pyruvate response using circularly permuted versions of Venus. SEQ ID 05 corresponding to variant based on Venus 49, SEQ ID 06 corresponding to variant based on Venus 145, SEQ ID 07 corresponding to variant based on Venus 157, SEQ ID 08 corresponding to variant based on Venus 173, SEQ ID 09 corresponding to variant based on Venus 195 and SEQ ID 10 corresponding to variant based on Venus 221.

Figure 5

It shows the alignment of the aminoacid sequence of six variants of lactate/pyruvate ratio responsive sensor.

Figure 6

Its shows the effect of lactate and pyruvate on the fluorescence emission spectrum of the most responsive variant of the sensor which is encoded by SEQ ID N08.

Figure 7

It shows the change of fluorescence of variant SEQ ID 08 in response to increase of lactate and pyruvate concentrations.

Figure 8

It shows the effect of increasing pyruvate concentration over the fluorescence change induced by lactate on variant SEQ ID 08, which is a lactate/pyruvate ratio sensor.

Figure 9

It shows the effect of increasing lactate/pyruvate ratios over the fluorescence change of variant SEQ ID 08.

Figure 10

It shows the specificity of variant SEQ ID 08 to lactate and pyruvate and structurally related molecules. Figure 11

It shows the effect of monocarboxylate transporter substrates and non-metabolized pyruvate analogue on lactate-induced fluorescence change.

Figure 12

Its shows the effect of pH on the fluorescence change induced by increasing lactate/pyruvate ratios of variant SEQ ID 08.

Figure 13

Its shows the effect of 25 and 37 °C on the fluorescence change induced by increasing lactate/pyruvate ratios of variant SEQ ID 08.

Figure 14

It shows the mTFP and Venus signal from the sensor variant SEQ ID 08 expressed in a cellular system with single-cell resolution.

Figure 15

It shows a real-time monitoring of uncalibrated lactate/pyruvate ratio fluorescent signal in intact system, e.g. HEK293 epithelial mammalian cells. Fluorescent signal change as is expected to a giving perturbation of intracellular redox state.

Figure 16

It shows a real-time monitoring of calibrated lactate/pyruvate ratio fluorescent signal in an intact system.

Figure 17

It shows the mTFP and Venus signal from the sensor variant SEQ ID 08 expression in a unicellular system, e.g. Saccharomyces cerevisiae with single-cell resolution.

Figure 18

It shows a real-time monitoring of uncalibrated lactate/pyruvate ratio fluorescent signal in a unicellular microorganism, e.g. Saccharomyces cerevisiae. Fluorescent signal acutely increase as is expected to OXPHOS inhibitor such as azide. Figure 19

It shows the use of the nanosensor to evaluate monocarboxylates transport through MCT, measuring the response of the nanosensor a non-metabolize pyruvate analogue oxamate.

Figure 20

It shows the use of the nanosensor to evaluate fermentative and oxidative metabolism in epithelial and tumoral cells lines.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description refers to the accompanying drawings. While embodiments of the nanosensor of the invention may be described, modifications, adaptations, and other implementations are possible. For example, substitutions, additions, or modifications may be made to the elements illustrated in the drawings, and the methods described herein may be modified by substituting, reordering, or adding stages to the disclosed methods. Accordingly, the following detailed description does not limit the scope of the invention. While the nanosensor and the methods are described in terms of “comprising” various elements or steps, the nanosensor and the methods can also “consist essentially of” or “consist of’ the various elements or steps, unless stated otherwise. Additionally, the terms “a,” “an,” and “the” are intended to include plural alternatives, e.g., at least one, unless stated otherwise.

The nanosensor quantifies lactate/pyruvate ratio, allowing single-cell minimally invasive measurement of lactate/pyruvate ratio.

The nanosensor of the present invention is a Forster Resonance Energy Transfer (FRET)-based lactate/pyruvate ratio nanosensor based on LutR, a bacterial transcriptional regulator that has two modules, a lactate/pyruvate-binding domain and a DNA-binding domain. The LutR gen was selected from Bacillus licheniformis.

In a further embodiment, the present invention includes lactate/pyruvate ratio nanosensors described according to the nucleic acid sequences and have at least 60%, 70%, 80% 85%, 90%, 95%, or 99% sequence identity with SEQ ID 01 , SEQ ID 02, SEQ ID 03, SEQ ID 04, SEQ ID 05, SEQ ID 06, SEQ ID 07, SEQ ID 08, SEQ ID 09 or SEQ ID 10. The present invention also considers the amino acid sequences having at least 60%, 70%, 80% 85%, 90%, 95%, or 99% sequence identity with SEQ ID 11 , SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID 19 or SEQ ID 20.

The sequences described in SEQ ID 8 to SEQ ID 18 are only particular embodiments of the present invention, provided as way of exemplification of the present invention and should not be considered to limit the scope of the invention.

The invention further comprises methods using the aforementioned nanosensor for determination of lactate/pyruvate ratio in single cells or cell populations, adherent cells or in suspension, in a cell culture, a tissue culture, a mixed cell culture, a tissue explant, or in animal tissues in vivo.

The method comprises the general steps of: a) Expressing the nanosensor of the invention, in a desired host, such as single cells or cell populations, adherent cells or in suspension, in a cell culture, a tissue culture, a mixed cell culture, a tissue explant, or in animal tissues in vivo ; b) Calibrating the host with predetermined values of intracellular, extracellular, subcellular lactate/pyruvate concentrations, recording lactate/pyruvate ratio concentrations in time; c) Recording the output from the nanosensor calculating the lactate/pyruvate ratio at different time points;

In the step b), corresponding to calibrating the host, the nanosensor of the invention is calibrated in cells using two calibrations points and the kinetic constants of the sensor from in vitro assay. Lowest lactate/pyruvate ratio level is reached using 6 mM oxamate and the maximum level of lactate/pyruvate ratio is determined by a pulse of 10 mM lactate.

The general method can be applied in different configurations, for example, in a first embodiment, the nanosensor can be used in a method to measure of relative lactate/pyruvate ratio. Embodiments

The following examples are provided to help in the understanding of the present invention and should not be considered a limitation to the scope of the invention.

In a first embodiment, the use of the nanosensor of the invention in a method allows the detection and quantification of intracellular lactate/pyruvate ration in intact systems.

In a second embodiment, the use of the nanosensor of the invention in a method allows the evaluation of fermentative and oxidative metabolism in simultaneous fashion.

In a third embodiment, the use of the nanosensor of the invention in a method allows the detection of lactate/pyruvate ratio in biological samples.

In a fourth embodiment, the use of the nanosensor of the invention in a method allows to evaluate monocarboxylate permeability, using a metabolized pyruvate analogue.

EXAMPLES

In order to help understanding the invention, the present invention will be explained with reference to specific examples

Example 1 : A two-point calibration protocol is applied at the beginning of each experiment. Briefly, intracellular lactate/pyruvate ratio is lowered by depriving the cells of carbon sources and used of non-metabolized pyruvate analogue oxamate which is detected by the nanosensor in the same manner of pyruvate to mimic a low lactate/pyruvate ratio. Alternatively, can be used oxamate analogues such as N- Ethyl oxamate, N- Propyl oxamate, N- Butyl oxamate, N- isobutyl oxamate, N- Sec-butyl oxamate, etc. A pulse of high lactate is applied in order to pump out oxamate or its analogues, through monocarboxylates transporter and displace it from the sensor to reach a maximal lactate/pyruvate ratio. The in vitro calibration curve is interpolated to calculate the lactate/pyruvate ratio of the sample. Example 2: The capability to accumulate lactate over pyruvate as a result of mitochondrial poisoning is a useful readout to evaluate glycolytic and oxidative metabolism. To be in detection range, assays should be performed in zero carbon sources. Highly glycolytic cells such as cancer cells accumulate more lactate than epithelial or primary cells, when they are exposed to mitochondrial poison azide, and the difference can be used to evaluate cell metabolism.

Example 3: To obtain high quality purify nanosensor plasmid constructs were transformed into E. coli BL21 (DE3). A single colony was inoculated in 100 ml of LB medium with 100 mg/ml ampicillin (without IPTG) and shaken in the dark for 2-3 days. Cells were collected by centrifugation at 5000 rpm (4 °C) for 10 min and disrupted by sonication (Hielscher Ultrasound Technology) in 5 mL of Tris-HCI buffer pH 8.0. A cell-free extract was obtained by centrifugation at 10,000 rpm (4 °C) for 1 hour and filtering of the supernatant (0.45 pm). Proteins were purified using a Nickel resin (His Bin® from Novagen) as recommended by the manufacturer. Eluted proteins were quantified using the Biuret method and stored at -20 °C in 20% glycerol. The variants were cloned into pRSETB for expression in Escherichia coli using the restriction sites BamHI and Hindlll.

Purify protein is diluted in Tris-HCI buffer pH 7.0 and mixed with test samples in a multi-well plate. In parallel calibration curve is simultaneously measured. Results are interpolated in the calibration curve to compute the lactate/pyruvate ratio.

Example 4: To assess monocarboxylates transport activity using a no-metabolized pyruvate analogue. Culture cell are exposed to a constant superfusion of KRH HEPES buffer with 5 mM glucose and 1 mM lactate, condition in which the sensor is fully saturated. Exposed the cells to rapid pulse of 6 mM oxamate to decrease the lactate/pyruvate ratio and induced an acute decrease of mTFP/Venus173 ratio. To evaluate transport inhibition in reference buffer exposed the cells to a putative inhibitor and a pulse of 6 mM oxamate. The monocarboxylate transport inhibition induced a decrease of rate of fluorescence change in comparison with the oxamate pulse without inhibitor. REFERENCES

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