WO2021033015A1 - Sonde génétiquement codée pour la détection et la quantification d'un rapport lactate/pyruvate avec une résolution spatiale et temporelle élevée - Google Patents

Sonde génétiquement codée pour la détection et la quantification d'un rapport lactate/pyruvate avec une résolution spatiale et temporelle élevée Download PDF

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WO2021033015A1
WO2021033015A1 PCT/IB2019/057089 IB2019057089W WO2021033015A1 WO 2021033015 A1 WO2021033015 A1 WO 2021033015A1 IB 2019057089 W IB2019057089 W IB 2019057089W WO 2021033015 A1 WO2021033015 A1 WO 2021033015A1
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lactate
seq
pyruvate
nanosensor
pyruvate ratio
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PCT/IB2019/057089
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English (en)
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Alejandro Antonio SAN MARTIN SAN MARTIN
Alex Danilo GALAZ SALAZAR
Luis Felipe Barros Olmedo
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Centro De Estudios Científicos De Valdivia
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Priority to PCT/IB2019/057089 priority Critical patent/WO2021033015A1/fr
Publication of WO2021033015A1 publication Critical patent/WO2021033015A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • 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.
  • 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
  • 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 .
  • 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.
  • 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.
  • 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.
  • the current art does not describe any bacterial protein with the required lactate/pyruvate ligand domain, an essential part of fluorescent nanosensors.
  • 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.
  • 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.
  • FRET Forster resonance energy transfer
  • 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.
  • a non- metabolized pyruvate analogue such as oxamate
  • oxamate analogues such as /V-Ethyl oxamate, N- Propyl oxamate, N- Butyl oxamate, N- isobutyl oxamate, N- Sec-butyl oxamate, etc.
  • 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
  • the use of the nanosensor of the invention in a method allows the detection and quantification of intracellular lactate/pyruvate ratio in intact systems.
  • the use of the nanosensor of the invention in a method allows the evaluation of fermentative and oxidative metabolism in simultaneous fashion.
  • the use of the nanosensor of the invention in a method allows the detection of lactate/pyruvate ratio in biological samples.
  • the use of the nanosensor of the invention in a method allows to evaluate monocarboxylate permeability, using a non-metabolized pyruvate analogue.
  • 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
  • SEQ ID 10 corresponding to variant based on Venus 221.
  • 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.
  • 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.
  • 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;
  • 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 nanosensor can be used in a method to measure of relative lactate/pyruvate ratio.
  • the use of the nanosensor of the invention in a method allows the detection and quantification of intracellular lactate/pyruvate ration in intact systems.
  • the use of the nanosensor of the invention in a method allows the evaluation of fermentative and oxidative metabolism in simultaneous fashion.
  • the use of the nanosensor of the invention in a method allows the detection of lactate/pyruvate ratio in biological samples.
  • the use of the nanosensor of the invention in a method allows to evaluate monocarboxylate permeability, using a metabolized pyruvate analogue.
  • 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.
  • 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.
  • Nickel resin His Bin® from Novagen
  • 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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Abstract

La présente invention comprend un nanocapteur fluorescent génétiquement codé pour détecter et quantifier un rapport lactate/pyruvate dans différents types d'échantillons, tels que de fluides biologiques, de tissus, intracellulaires, et même dans des compartiments subcellulaires, avec une résolution spatiale et temporelle élevée, et des protocoles pour exprimer ce nanocapteur dans des échantillons biologiques, et son utilisation pour la détection et la quantification d'un rapport lactate/pyruvate intracellulaire dans des systèmes intacts, l'évaluation d'un métabolisme fermentatif et oxydatif de manière simultanée, la détection d'un rapport lactate/pyruvate dans des échantillons biologiques et l'évaluation de la perméabilité au monocarboxylate, à l'aide d'un analogue non métabolisé.
PCT/IB2019/057089 2019-08-22 2019-08-22 Sonde génétiquement codée pour la détection et la quantification d'un rapport lactate/pyruvate avec une résolution spatiale et temporelle élevée WO2021033015A1 (fr)

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