Probes for detecting chemotactic factors and uses thereof

Abstract

The present application relates to a probe protein capable of binding to a chemokine, in particular a probe protein capable of binding to CCL2, a method and a kit for detecting a chemokine. The probe protein binds to a chemokine to cause a change in fluorescence intensity, which detection has high sensitivity and specificity, and therefore the present application also relates to the use of the probe protein for high-throughput screening of drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to fusion protein constructs encoding biological probes and their applications. Background Technology

[0002] Chemokines are a class of small cytokines with molecular weights of 8-12 kDa that play a crucial role in regulating and promoting the immune response in mammals by chemotactically attracting different subsets of leukocytes. They exert their biological functions by binding to specific receptors (represented by seven-transmembrane G protein-coupled receptors (GPCRs)) and promoting the migration of leukocytes to sites of inflammation. Beyond their involvement in inflammatory responses, chemokines are increasingly recognized to participate in a wider range of biological functions, including homeostasis regulation, tissue repair, cell adhesion, and the development and maintenance of the immune system. Furthermore, the interactions between secreted chemokines and their receptors are involved in various physiological and pathological processes, including tumor metastasis and brain function regulation.

[0003] Numerous studies have shown that chemokines in inflamed tissues are produced by multiple cell types involved in innate and adaptive immunity in response to exogenous factors such as bacteria, viruses, fungi, and environmental stimuli (e.g., thermal injury, chemical stimuli) as well as endogenous factors such as cytokines, growth factors, and other stress signals. However, accurately depicting the spatiotemporal patterns of chemokine release and distribution in the extracellular space remains a challenging task. For example, traditional methods such as qPCR quantify chemokine expression by monitoring mRNA transcription levels, but do not directly measure extracellular release levels. Immunoassays, including ELISA and Western blotting, have been widely used for the quantitative detection of specific chemokines. However, the specificity of existing antibodies is often insufficient to distinguish closely related chemokine family members. Furthermore, Western blotting is time-consuming and less quantifiable and sensitive than ELISA. On the other hand, techniques such as mass spectrometry can simultaneously identify multiple chemokines directly and (semi-)quantitatively, but are limited by low throughput and slow processing times. In summary, all of the above methods are performed on homogenized tissue as endpoints, lacking the temporal and spatial resolution necessary for long-term, real-time analysis of chemokine secretion in vivo.

[0004] Therefore, there is a need to develop biological probes with higher sensitivity and selectivity so that they can be better applied to the development of chemokine-related drugs and detection reagents. Summary of the Invention

[0005] This disclosure provides a detection protein with high selectivity and sensitivity to specific chemokines, which can be used for real-time monitoring of chemokines in vitro and in vivo, and has high spatiotemporal resolution.

[0006] Biological probes based on heritable genetically encoded fluorescent GPCRs are well-suited for real-time visualization of extracellular signaling molecule release in model organisms, offering high sensitivity, selectivity, and spatiotemporal resolution. Therefore, this invention utilizes the human CCL2 receptor CCR2 as a backbone to develop the first heritably encoded CCL2 probe, named hCRAFi-CCR2, which exhibits approximately 300% fluorescence signal change (ΔF / F) and approximately 2.5 nM affinity for mCCL2 in primary mouse astrocytes, along with high selectivity and sensitivity. The probe of this invention is suitable for real-time monitoring of extracellular mCCL2 fluctuations during inflammatory stimulation lasting up to 20 hours in vitro and in vivo. Furthermore, this invention relates to a FLIPR-based mid-throughput screening platform for screening compounds targeting CCR2.

[0007] In some aspects, the present invention provides a detection protein comprising a chemokine receptor and a fluorescent reporter protein inserted into the chemokine receptor. In some embodiments, the chemokine receptor is selected from CCR2, CCR3, CCR6, and CXCR4. The fluorescent reporter protein may be inserted into a third intracellular loop between the fifth and sixth transmembrane regions of the chemokine receptor. Optionally, the fluorescent reporter protein is a cyclically rearranged fluorescent protein.

[0008] In some embodiments, the cyclic rearranged fluorescent protein is selected from cyclic rearranged green fluorescent protein, cyclic rearranged red fluorescent protein, cyclic rearranged yellow fluorescent protein, and cyclic rearranged blue fluorescent protein. In some embodiments, the reporter protein is cyclic rearranged green fluorescent protein; preferably, the cyclic rearranged green fluorescent protein has the amino acid sequence shown in SEQ ID NO: 3.

[0009] In some embodiments, the chemokine receptor is human CCR2B or a variant thereof, and the fluorescent reporter protein is inserted between amino acid residues 235 and 236 from the N-terminus to the C-terminus of the chemokine receptor. In some embodiments, the detector protein comprises, from the N-terminus to the C-terminus, amino acid sequences 1-235 of the chemokine receptor operably linked to the fluorescent reporter protein, and the fluorescent reporter protein operably linked to the amino acid sequence following residue 236 of the chemokine receptor. In some embodiments, the human CCR2B or a variant thereof has the amino acid sequence shown in SEQ ID NO: 20 or 21.

[0010] In some embodiments, the chemokine receptor is mouse CCR2, and the fluorescent reporter protein is inserted between amino acid residues 248 and 249 from the N-terminus to the C-terminus of the chemokine receptor. In some embodiments, the detector protein comprises, from the N-terminus to the C-terminus, amino acid sequences 1-248 of the chemokine receptor operably linked to the fluorescent reporter protein, and the fluorescent reporter protein operably linked to the amino acid sequence after residue 249 of the chemokine receptor.

[0011] In some embodiments, the fluorescent reporter protein is inserted into the chemokine receptor via an N-terminal and a C-terminal adapter. Optionally, the N-terminal adapter is LSSLI, LSSSV, or LEEGG. Optionally, the C-terminal adapter is NHDQL, NH, or ID. In some embodiments, the amino acid sequence of the N-terminal adapter is LSSLI, and the amino acid sequence of the C-terminal adapter is NHDQL or NH; or, the amino acid sequence of the N-terminal adapter is LSSSV or LEEGG, and the amino acid sequence of the C-terminal adapter is ID.

[0012] In some embodiments, the C-terminus of the probe protein further comprises a signal peptide sequence, such as a Golgi output signal (KGC) sequence and an endoplasmic reticulum output signal (ER2) sequence. Optionally, the probe protein comprises one or more (e.g., 1, 2, 3 or more) copies of the KGC sequence. In some embodiments, the KGC sequence has the amino acid sequence shown in SEQ ID NO: 4, and the ER2 sequence has the amino acid sequence shown in SEQ ID NO: 5.

[0013] In some embodiments, the C-terminus of the detector protein further includes an amino acid sequence of an indicator fluorescent protein that does not overlap with the fluorescence spectrum of the fluorescent reporter protein; optionally, the other fluorescent protein is a red fluorescent protein, such as FusionRed. Preferably, the indicator fluorescent protein has the amino acid sequence shown in SEQ ID NO:10. In some embodiments, the KGC sequence is operatively linked to the indicator fluorescent protein, and the indicator fluorescent protein is operatively linked to the ER2 sequence.

[0014] In some embodiments, the fluorescence intensity changes after the probe protein binds to a chemokine targeted by the chemokine receptor, wherein the ΔF / F is about 50% or higher, about 100% or higher, about 150% or higher, about 200% or higher, or about 250% or higher.

[0015] In some embodiments, the probe protein comprises one of the following: (a) an amino acid sequence as shown in SEQ ID NO: 1 or 2; (b) an amino acid sequence as shown in SEQ ID NO: 15, 16 or 17; (c) an amino acid sequence as shown in SEQ ID NO: 8 or 9; (d) an amino acid sequence as shown in SEQ ID NO: 19; (e) an amino acid sequence encoded by a nucleotide sequence as shown in SEQ ID NO: 6 or 7; or (f) an amino acid sequence having at least 95% sequence identity with any one of (a)-(e).

[0016] In an alternative embodiment, the detector protein comprises a chemokine receptor and a fluorescent reporter protein, wherein the chemokine receptor is mouse-derived mCCR3, mCXCR4, or mCCR6, and wherein the amino acid sequence of the chemokine receptor from the N-terminus to the C-terminus, positions 5.59 to 6.34 (i.e., positions 224-242 of mCCR3, positions 227-245 of mCXCR4, and positions 227-245 of mCCR6), is partially replaced by a transplanted sequence comprising the fluorescent reporter protein. Optionally, the detector protein comprises, from the N-terminus to the C-terminus, amino acid sequences 1-223 of the chemokine receptor operably linked to the portion containing the fluorescent reporter protein, and the portion containing the fluorescent reporter protein operably linked to the amino acid sequence after position 243 of the chemokine receptor; or, the detector protein comprises, from the N-terminus to the C-terminus, amino acid sequences 1-226 of the chemokine receptor operably linked to the portion containing the fluorescent reporter protein, and the portion containing the fluorescent reporter protein operably linked to the amino acid sequence after position 246 of the chemokine receptor. Optionally, the transplanted sequence portion containing the fluorescent reporter protein has the sequence shown in SEQ ID NO:11. In some embodiments, the detector protein comprises amino acid sequences as shown in SEQ ID NO:12, 13, or 14.

[0017] In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a detector protein disclosed herein.

[0018] In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a detector protein comprising an indicator fluorescent protein as disclosed herein, wherein the nucleotide sequence encoding the indicator fluorescent protein, which does not overlap with the fluorescence spectrum of the fluorescent reporter protein, is linked to the nucleotide sequence encoding the detector protein by a nucleotide sequence encoding a self-cleaving peptide; optionally, the self-cleaving peptide is selected from P2A, F2A, T2A, and E2A.

[0019] In some aspects, the present invention provides a vector comprising the nucleic acid molecules disclosed herein. In some embodiments, the vector is an adenovirus vector or an adeno-associated virus vector.

[0020] In one aspect, the present invention provides a host cell comprising a nucleic acid molecule or a vector disclosed herein.

[0021] In one aspect, the present invention provides a method for detecting changes in the concentration of chemokines in a sample, comprising: culturing host cells disclosed herein under suitable conditions; adding a test sample to the cell culture, and recording the fluorescence intensity F0 before the addition of the sample, the fluorescence intensity at a first time point after the addition of the sample, and the fluorescence intensity at a second time point; and analyzing the fluorescence intensity change ΔF / F at the first time point and the second time point relative to F0, thereby determining the change in the concentration of chemokines in the test sample at the first time point relative to the second time point. In some embodiments, the test sample is selected from cell cultures or extracts thereof, biological tissues or extracts thereof, and biological fluids or extracts thereof.

[0022] In one aspect, the present invention provides a method for detecting the level of a chemokine in a sample, comprising: culturing a host cell disclosed herein under suitable conditions; adding a test sample to the cell culture, recording the fluorescence intensity F0 before and after the addition of the sample; and analyzing the change in fluorescence intensity ΔF / F0 relative to F0 after the addition of the sample, thereby determining the level of the chemokine in the test sample. In some embodiments, the test sample is selected from cell cultures or extracts thereof, biological tissues or extracts thereof, and biological fluids or extracts thereof.

[0023] The host cell referred to in this invention can be a mammalian cell, such as a human cell, including but not limited to HEK cells, neuronal cells, and astrocytes.

[0024] In one aspect, the present invention provides a kit for detecting changes in the level or concentration of chemokines in biological samples, comprising the vectors or host cells disclosed herein.

[0025] In one aspect, the present invention provides a method for monitoring the spatiotemporal pattern of chemokine release in cells, comprising: expressing a detector protein disclosed herein in cells; recording the fluorescence intensity of the detector protein over time within one or more fields of view over a time interval; and analyzing the change in fluorescence intensity over time ΔF / F in each of the one or more fields of view to obtain the spatiotemporal pattern of chemokine release. In some embodiments, the time interval is about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 20 hours, or longer.

[0026] In some embodiments, the cells express the probe protein via transient transfection, adenovirus transfection, or adeno-associated virus transfection. In some embodiments, the cells are induced to secrete chemokines by a stimulant, wherein the stimulant is a pro-inflammatory stimulant.

[0027] In some aspects, the present invention provides a method for monitoring the secretion of chemokines by organs, tissues, or cells expressing the detector proteins disclosed herein, including recording the fluorescence intensity of the detector proteins over time. In some embodiments, the organs, tissues, or cells are derived from the brain or a portion thereof, such as the cortex. The cells may be, for example, neurons and astrocytes.

[0028] In one aspect, the present invention provides a method for screening probe proteins for detecting chemokines, comprising: inducing cells to express the probe protein to be screened; applying a chemokine to the cells and recording the change in fluorescence intensity ΔF / F of the probe protein; and determining that the probe protein can be used to detect chemokines by ΔF / F ≥ 50%.

[0029] In one aspect, the present invention provides a method for screening antagonists and / or agonists of chemokine receptors using the detector proteins disclosed herein, comprising: constructing a cell line expressing the detector protein; adding a library of compounds to the cell line; and detecting a change in fluorescence intensity ΔF / F of the detector protein; wherein ΔF / F>0 indicates that the compound is an agonist, and ΔF / F<0 indicates that the compound is an antagonist.

[0030] In one aspect, the present invention provides a fluorescence-based drug screening system comprising a drug delivery module, a response module, and an imaging module, wherein the drug delivery module is a multi-channel drug delivery module, the response module comprises a cell culture device and cells expressing the detector protein disclosed herein, and the imaging module records the fluorescence intensity of the cells before and after drug delivery in real time. Attached Figure Description

[0031] Figure 1-7The development of a CCR2-based probe protein is shown. Figure 1 The development process of the CCR2-based probe protein is shown: insertion site selection, adapter optimization, and ICL3 optimization. The 3xKGC and ER2 motifs were fused to the C-terminus of hCRAFi-CCR2* to improve membrane localization. The hCRAFi-CCR2 probe protein motif (-G-LEEGG-cpGFP-ID-) was transferred into mCCR2 to generate mCRAFi-CCR2 (SEQ ID NO:19). A binding pocket mutation Y49A / Y120A was introduced to generate a nonfunctional mutant hCRAFi-CCR2null as a control.

[0032] Figure 2 The development and optimization of the hCRAFi-CCR2 probe protein prototype were demonstrated. Figure 2 a shows the insertion of the cpGFP reporter portion (green) and the original adapter sequence (red) (top) from dLight1.1 into five sites of ICL3 (bottom, TM5 and TM6 amino acids are black, ICL3 amino acids are blue) of hCCR2B. Figure 2 b showed Figure 2 Monolayer confocal fluorescence images of five variants with different insertion sites in image a. The chimera with insertion site 3 shows better membrane localization and higher brightness compared to the other four chimeras. Scale bar, 50 μm. Figure 2 c shows the ΔF / F ratio for 16 variants with different connector lengths. The variant with the L1:LSSLI / L2:NH connector exhibits the largest ΔF / F ratio and is marked with a circle. This variant is named hCRAFi-CCR2_0.1. Figure 2 The image shows (d) a wide-field microscopy image and (e) the response after application of 0.5 μM hCCL2 to hCRAFi-CCR2_0.1 expressed in HEK cells (n = 15 cells from one independent transfection). Data are presented as mean (solid line), with shaded areas indicating SEM. The peak ΔF / F was 32.7 ± 1.7%.

[0033] Figure 3 The schematic diagram illustrates the iDrug system, which uses microscopy to automate the screening of CCR2-based probe protein variants. The inset in the lower right corner shows a representative trajectory from an HEK cell exhibiting a fluorescent response to hCCL2 (0.5 μM).

[0034] Figure 4 The comparison of baseline brightness and fluorescence response of the hCRAFi-CCR2 detector protein mutant is shown. Figure 4Image a shows a representative image illustrating the basal expression levels of hCCR2B-GFP, hCRAFi-CCR2_0.2, and hCRAFi-CCR2_0.3 in HEK cells. Figure 4 b shows the statistical analysis of the baseline luminance of hCCR2B-GFP, hCRAFi-CCR2_0.2, and hCRAFi-CCR2_0.3. Figure 4 c shows the fluorescence trajectory of the response of the probe proteins hCRAFi-CCR2_0.2 and hCRAFi-CCR2_0.3. Figure 4 Figure d shows the fluorescence response (ΔF / F) of hCRAFi-CCR2_0.2 and hCRAFi-CCR2_0.3.

[0035] Figure 5 Screening fluorescence images of hCRAFi-CCR2 and mCRAFi-CCR2 are shown. Scale bar, 50 μm.

[0036] Figure 6 The figures show the responses of hCRAFi-CCR2 and mCRAFi-CCR2 to the ligands hCCL2 or mCCL2. The average fluorescence trajectory in the top figure shows the responses of hCRAFi-CCR2, mCRAFi-CCR2, and hCRAFi-CCR2null to hCCL2 (0.1 μM) or mCCL2 (0.1 μM) (the trajectory represents the average response of n = 13–15 cells in a single transfection; solid line, mean; shaded area, sem). The figure below shows the dose-response curves of hCCL2 and the EC50 of hCRAFi-CCR2 and mCRAFi-CCR2 in HEK cells (hCRAFi-CCR2 and mCRAFi-CCR2 were derived from 2 and 1 independent cultures, respectively, n = 35 and 13 cells). Square, mean; error bars, sem; solid line, fitted curve using Hill equation (hCRAFi-CCR2 and mCRAFi-CCR2 R-squared). 2 =0.999).

[0037] Figure 7 Showing Figure 6 Quantitative analysis of the maximum fluorescence response of hCRAFi-CCR2, mCRAFi-CCR2, and hCRAFi-CCR2null to hCCL2 or mCCL2 in the experiments shown (n = 14–60 cells, each from 2–4 independent experiments). Box plots with incisions: narrow portion of the incision, median; top and bottom of the incision, 95% confidence interval of the median; upper and lower horizontal lines, 25th and 75th percentiles of the data; whiskers for values ​​extended from the 25th and 75th percentiles to 1.5 × interquartile range; horizontal line, mean; diamond, outliers.

[0038] Figure 8 The results showed that, compared to hCRAFi-CCR2, the Y49A / Y120A mutant exhibited significantly decreased responses to all hCCR2 ligand chemokines (n = 19–60 cells, from 2–5 independent cultures). The p-values ​​for the responses of hCRAFi-CCR2 to hCCL2, hCCL7, hCCL8, hCCL13, mCCL2, mCCL7, and mCCL8 compared to Y49AY120A were 7.4 × 10⁻⁶. -78 2.6×10 -10 2.9×10 -12 2.0×10 -33 2.4×10 -25 2.2×10 -29 5.0×10 -5 (Two-tailed Student's t-test, ***, p<0.001).

[0039] Figure 9 The sequence arrangement information of the two developed ratiometric probe protein constructs, pN1-hCRAFi-CCR2-P2A-FusionRed and pN1-hCRAFi-CCR2-Linker-FusionRed, is shown. Specifically, pN1-hCRAFi-CCR2-P2A-FusionRed is an operatively linked hCRAFi-CCR2-KGC-ER2 sequence-P2A-FusionRed from the N-terminus to the C-terminus, while pN1-hCRAFi-CCR2-Linker-FusionRed is an operatively linked hCRAFi-CCR2-KGC-FusionRed-ER2 sequence from the N-terminus to the C-terminus.

[0040] Figure 10 The dose-response curves of pN1-hCRAFi-CCR2-KGC-FusionRed-ER2 (KGC connector), pN1-hCRAFi-CCR2-3KGC-FusionRed-ER2 (3KGC connector), and hCRAFi-CCR2-KGC-ER2 with mCCL2 are shown. The three have similar EC50 values ​​(1KGC connector: 10.74±1.43 nM; 3KGC connector: 6.29±3.00 nM; hCRAFi-CCR2: 7.56±0.85 nM).

[0041] Figure 11The expression of hCRAFi-CCR2 in HEK cells and its response to hCCL2 are shown. Fluorescence intensity increased after application of hCCL2 (590 nM). Scale bar, 50 μm.

[0042] Figure 12 The excitation and emission spectra of hCRAFi-CCR2 are shown in the presence and absence of hCCL2 (590 nM).

[0043] Figure 13 The time constant of the hCRAFi-CCR2 response to hCCL2 (100 nM) is shown in the soaking perfusion experiment with hCCL2. The left panel shows the mean response of hCRAFi-CCR2 to hCCL2 (100 nM) and its antagonist MK-0812 (4 μM) (n = 15 cells, from a single transfection); the time constant was determined by single exponential fitting (top panel τ = 2.4 s, R0). 2 =0.9999; in the figure below, τ = 39.3s, R 2 =0.998). The right figure is a summary of the on and off time constants (n=30 cells, from two independent transfections).

[0044] Figure 14 The time constants of the hCCL2 response on the cell membrane to hCCL2 during microdose administration to single cells are shown. The left panel shows a representative response of hCCL2 (1000 nM) on the HEK cell membrane, recorded using confocal line scanning mode; the time constants were determined by single exponential fitting. The right panel summarizes the on and off time constants. Data are expressed as mean ± sem from three independent experiments.

[0045] Figure 15 The relative ΔF / F of hCRAFi-CCR2 for different human and mouse chemokines are shown (n = 15–60 cells, data for each chemokine from 2–4 independent experiments).

[0046] Figure 16 The dose-response curves and EC50 of hCRAFi-CCR2 for different CCR2 receptor ligands are shown. 50 hCCL2, hCCL8, hCCL7, hCCL13, mCCL2, mCCL7 (n = 29-60 cells, from 27 independent transfections of one culture; data are presented as mean ± sem; solid line, fitted curve using Hill's equation, regression coefficient R0). 2 The values ​​are 0.996, 0.988, 0.99, 0.99, 0.998, and 0.991, respectively.

[0047] Figure 17 Intracellular calcium response was shown in cells co-transfected with R-GECO1.2 and wild-type hCCR2B or co-transfected with R-GECO1.2 and hCRAFi-CCR2 probe protein after application of hCCL2 (500 nM) (n = 35–39 cells, each from 2–3 independent transfections). Two-tailed Student t-test, p = 3.4 × 10⁻⁶. -11 .

[0048] Figure 18 The fluorescence response of HEK cells expressing hCRAFi-CCR2 after 2 hours of co-incubation with hCCL2 (0.5 μM) is shown (n = 45 cells, from three independent transfections of one cell culture). Box plots with notches: the notch represents the median, and the top and bottom of the notch represent the 95% confidence intervals of the median; the upper and lower horizontal lines represent the 25th and 75th percentiles of the data. Data were analyzed using one-way ANOVA followed by Bonferroni's multiple comparison test; ns, not significant. ***, p < 0.001.

[0049] Figure 19 a shows the mean response of hCRAFi-CCR2 expressed on neurons to mCCL2 (0.5 μM) and its antagonist MK-0812 (4 μM) (n = 4 and 25 cells, each from an independent transfection). ΔF / F0 is expressed as mean ± sem. Figure 19 b shows the maximum ΔF / F0 of hCRAFi-CCR2 or hCRAFi-CCR2null against mCCL2 (0.3–0.5 μM) in cultured neurons (n ​​= 9–10 neurons from two independent cell cultures, two-tailed Student's t-test, p = 2.17 x 10⁻⁶). -5 ). Figure 19 c shows the dose-response curves of hCRAFi-CCR2 in cultured neurons. n = 36 neurons, from three independent transfections from one culture, mCCL2EC 50 6.07±1.06, R 2 =0.995. The solid line is the fitted curve using Hill's equation.

[0050] Figure 20 a shows the mean response of hCRAFi-CCR2 expressed on astrocytes to mCCL2 (0.5 μM) and its antagonist MK-0812 (4 μM) (n = 4 and 25 cells, each from an independent transfection). ΔF / F0 is expressed as mean ± sem. Figure 20b shows the maximum ΔF / F0 of hCRAFi-CCR2 or hCRAFi-CCR2null against mCCL2 (0.3–0.5 μM) in cultured astrocytes (n = 50–59 astrocytes from two independent cultures, two-tailed Student's t-test, p = 5.61 x 10⁻⁶). -29 ). Figure 20 c shows the dose-response curve of hCRAFi-CCR2 on cultured astrocytes. n = 15–60 astrocytes, 1–4 independent transfections from one astrocyte culture, mCCL2 EC50: 1.91 ± 0.10 nM, R 2 =0.998, mCCL7, EC50: 47.26±10.35nM, R 2 =0.990. The solid line is the fitted curve using Hill's equation.

[0051] Figure 21 The ΔF / F0 of hCRAFi-CCR2 in cultured (a) hippocampal neurons and (b) astrocytes is shown. The ΔF / F0 values ​​were calculated for different co-incubation times in the presence of hCCL2 (500 nM, neurons) or mCCL2 (500 nM, astrocytes). The antagonist MK-0812 (4 μM) was added to the culture medium at the end of imaging. n = 5–11 neurons and 30 astrocytes, each from two independent cultures. One-way ANOVA was performed, followed by a Bonferroni post-hoc test. *p<0.05, **, p<0.01, ***, p<0.001.

[0052] Figure 22 The release of endogenous mCCL2 was shown in cultured astrocytes and mice subjected to inflammatory stimulation. Figure 22 Image a shows representative images of cultured astrocytes expressing GfaABC1D-hCRAFi-CCR2 at 0 and 8 hours after treatment with LPS, Poly(I:C), and ODN 1668. The concentration of the pro-inflammatory agents was 10 μg / ml. Fifteen hours after stimulation, the CCR2 antagonist MK-0812 (4 μM) was added to the cells. Scale bar: 100 μm. Figure 22 Image b shows dynamic fluorescence elevation in cultured astrocytes 15 hours after various inflammatory stimuli. N = 8 FOVs from 2 independent cultures. Data are presented as mean values, and shaded areas indicate SEM. Figure 22 c is Figure 22The time constant of the increase in hCRAFi-CCR2 fluorescence intensity in b was fitted by an exponential equation. A one-way ANOVA was then performed followed by a Bonferroni multiple comparison post-hoc test. The p-value for LPS compared to Poly(I:C) was 1.2 × 10⁻⁶. -4 The p-value of Poly(I:C) compared to ODN 1668 is 0.049. Figure 22 d represents the time progression of hCRAFi-CCR2 fluorescence intensity in astrocytes after the addition of the CCR2 antagonist MK-0812 (4 μM). Figure 22 As in a, astrocytes were co-incubated with LPS, Poly(I:C), and ODN 1668 for 15 hours (n = 4 FOVs from one culture). Data are presented as mean (solid line), with shaded areas indicating SEM. Figure 22 e shows the maximum fluorescence changes of the hCRAFi-CCR2 probe protein and the hCRAFi-CCR2 null mutant after 15 hours of co-incubation with inflammatory stimuli. Figure 22 f shows the dynamic fluorescence changes in cultured astrocytes during imaging 17 hours after treatment with different concentrations of poly(I:C). n = 5–6 FOVs from a single culture. Data are presented as mean values, and shaded areas indicate SEM. Figure 22 g shows the maximum fluorescence change ΔF / F in cultured astrocytes expressing the hCRAFi-CCR2 probe protein after 17 hours of co-incubation with different concentrations of poly(I:C). n = 5–6 FOVs from a single culture. One-way ANOVA was subsequently performed using a Bonferroni post-hoc test for multiple comparisons. Figure 22 h represents a representative single-plane image of HEK cells transfected with hCRAFi-CCR2 cultured in mouse plasma before (top) or after (bottom) treatment with the CCR2 antagonist MK-0812 (4 μM MK). Figure 22 i shows the hCRAFi-CCR2 fluorescence intensity normalized relative to FusionRed intensity. Plasma was collected from 10–12 mice in each group after treatment with LPS (2 mg / kg) for 0, 6, and 24 hours. Bonferroni multiple comparison post-hoc test was then performed using one-way ANOVA. Figure 22 j shows the standard mCCL2 dose-response curve of hCRAFi-CCR2 imaged on the same day as the measurement of plasma-induced fluorescence changes (EC50: 5.8 ± 0.8 nM, regression coefficient R). 2=0.99 (each concentration was repeated 3 times). HEK cells expressing hCRAFi-CCR2 were incubated with different concentrations of mCCL2 or mouse plasma. The fluorescence intensity of hCRAFi-CCR2 was normalized relative to FusionRed. ΔF / F was calculated by subtracting the normalized fluorescence intensity of control cells. The ΔF / F of LPS-6h plasma and LPS-24h plasma are included in the figure. The calculated ΔF / F of 6h plasma and 24h plasma were 0.79 and 0.42, respectively, corresponding to 6.5 nM and 3.81 nM mCCL2 when registered with the curve.

[0053] Figure 23 This study demonstrates the detection of mCCL2 in conditioned medium of primary mouse astrocytes using a mutant of the hCRAFi-CCR2 probe protein expressed in HEK cells. Figure 23 ab shows (a) a representative wide-field fluorescence image and (b) a single-test fluorescence trajectory of the hCRAFi-CCR2_0.2 probe protein in HEK cells in response to hCCL2 (0.5 mM). Figure 23 c shows the maximum ΔF / F of hCRAFi-CCR2_0.2 in HEK cells (n = 30 cells, 2 independent transfections from one culture). Figure 23 Figure d shows the normalized fluorescence intensity of hCRAFi-CCR2_0.2 incubated in astrocyte conditioned medium. Astrocytes were treated with TNFα or LPS at the indicated concentrations for 18 hours. The medium was collected and applied to HEK cells expressing the hCRAFi-CCR2_0.2 probe protein, and imaged under a microscope (n = 40 cells, from two independent transfections from one culture). Control medium compared to other conditioned media showed p = 1 and 3 × 10⁻⁶. -6 7.4×10 -5 6.2×10 -5 0.004 (TNFa 5 ng / ml, TNFa 10 ng / ml, TNFa 50 ng / ml, LPS 10 mg / ml, LPS 50 mg / ml, respectively; one-way ANOVA followed by Bonferroni multiple comparison post-hoc test, **, p < 0.01, ***, p < 0.001). Figure 23 e shows the time progression of fluorescence intensity in hCRAFi-CCR2_0.2 cells incubated in LPS (50 mg / ml) astrocyte conditioned medium or hCCL2 (0.5 mM) after the addition of the CCR2 antagonist MK-0812 (2 mM) (n = 5 cells, each from 1 transfection). Data are shown as mean (solid line), and shaded areas indicate SEM.

[0054] Figure 24 The data show time-dependent fluorescence changes in mice expressing the probe protein or expressing the mutant after LPS stimulation induced systemic inflammation (left panel), and group analysis of LPS-induced fluorescence elevation between 19 and 20 hours (right panel, hCRAFi-CCR2 probe protein mice n=3, mutant mice n=2; p=0.041).

[0055] Figure 25 Spatial heterogeneity of fluorescence response after LPS injection is shown. (a) Patch-like regions indicate a higher increase in fluorescence. Typical patch-like regions and their sizes are shown on the left side of the figure; (b) Comparison of peak fluorescence elevation (ΔF / F0, left panel) and basal fluorescence (F0, right panel) of ROIs within (In) or outside (Out) of the patch (n=3 mice; p=0.020 for ΔF / F0, p=0.94 for F0); and (c) Distribution of size (left panel), amplitude (middle panel), or signal onset time (time to reach 10% maximum amplitude, right panel) among different patches (n=50 patches).

[0056] Figure 26 The fluorescence intensity change ΔF / F during a 4.5-hour recording period following photoablation-induced injury is shown (n = 6 ROIs from 1 mouse; dots represent the mean; error bars represent SEM). Photoablation was performed using a 920 nm laser at 0.8 W for 92 ms.

[0057] Figure 27 The hCRAFi-CCR2 probe protein was used as a drug screening tool to screen compounds targeting chemokine receptors. (a) Ten antagonistic compounds and one agonist (J-11383) were screened from 59 compounds using the FLIPR high-throughput real-time fluorescence detection system based on hCRAFi-CCR2; (b) CCR2 antagonistic compounds identified by FLIPR (blue) and CCR2 antagonists reported in the literature (red); (c) and (d) the screening results in (a) were verified by wide-field microscopy; (e) the dose-response curves of the newly identified CCR2 antagonists using the hCRAFi-CCR2 probe protein were compared with the known antagonist MK-0812; (f) the dose-response curve of hCRAFi-CCR2 to J-11383; (g) the increase in hCRAFi-CCR2 fluorescence intensity induced by J-11383 could be blocked by MK-0812.

[0058] Figure 28 Figures a and b show the amino acid sequence of the TM5-linker-cpEGFP-linker-TM6 motif of the hCRAFi-CCR2 probe protein (a) and its position in the three-dimensional structure of the probe protein (b). Figure 28c shows the response of other probe proteins to its major ligand dF / F of the transplanted TM5-adaptor-cpEGFP-adaptor-TM6 motif. Figure 28 The data in df shows the dose-response curves of the probe proteins CRAFi-mCCR3 (17.72±3.30 nM), CRAFi-mCXCR4 (5.60319±1.10287 nM), and CRAFi-mCCR6 (23.05593±3.00151 nM), respectively.

[0059] Figure 29 a shows the ΔF / F of library compound screening based on various chemokine probe proteins (hCRAFi-CCR2, mCRAFi-CCR3, mCRAFi-CXCR4, and mCRAFi-CCR6) using wide-field microscopy; data were obtained from two replicates (n = 2 wells, each from an independent culture). Figure 29 b shows the representation in heatmap form. Figure 29 The result of a. Figure 29 c shows a Venn diagram of the compounds identified by various chemokine probe proteins. Detailed Implementation

[0060] In this invention, unless otherwise stated, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, patent applications, and other publications cited herein are incorporated herein by reference in their entirety. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are widely used terms and routine procedures in their respective fields. If any definition presented herein conflicts with a definition presented in a patent, patent application, or other publication incorporated herein by reference, the definition presented herein shall prevail.

[0061] Unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context explicitly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a mixture of cells, etc. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms (such as “including” and “containing”) is not restrictive. Moreover, the scope provided in the specification and the appended claims includes all values ​​between endpoints.

[0062] Numerical ranges include numbers within defined ranges. Taking into account significant figures and measurement-related errors, measured and measurable values ​​should be understood as approximate values. As used in this application, the terms "about" and "approximately" have their meanings as understood in the art. Unless otherwise specified, figures used in this application, with or without modifiers such as "about" or "approximately," should be understood to cover normal divergence and / or fluctuations, as will be understood by one of ordinary skill in the art. In some embodiments, the terms "about" or "approximately" refer to a range of values ​​(greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction of the reference value, unless otherwise stated or otherwise apparent from the context (except where such values ​​would exceed 100% of possible values).

[0063] As used herein, the term "detector protein" is used interchangeably with "biosensor" and refers to a fusion protein comprising a chemokine receptor and a fluorescent reporter protein for detecting chemokines. The fluorescent reporter protein may be inserted into the chemokine receptor or attached to the terminal end of the chemokine receptor. Preferably, the fluorescent reporter protein is a cyclically rearranged fluorescent protein. When inserted into the chemokine receptor, the fluorescent reporter protein inserts in a manner that does not affect the interaction between the chemokine receptor and the chemokine, for example, by inserting into an intracellular loop within the receptor.

[0064] As used herein, the term "G protein-coupled receptor (GPCR)" refers to a membrane receptor protein present on the cell surface, belonging to the largest protein superfamily in eukaryotes. The three-dimensional structure of a GPCR contains seven transmembrane α-helices, and the C-terminus of its peptide chain and the intracellular loop (the third intracellular loop) connecting the 5th and 6th transmembrane helices (also called transmembrane regions) contain G protein (guanylate-binding protein) binding sites. Known ligands binding to GPCRs include odorants, pheromones, hormones, neurotransmitters, and chemokines. The activation and conformational change of ligand-bound GPCRs makes them potentially valuable for development as biomolecular probe proteins. Previous studies have resolved information related to the crystal structure of GPCRs. The sites of greatest conformational change during activation are the 5th and 6th transmembrane regions, while the third intracellular loop, connecting the 5th (TM5) and 6th (TM6) transmembrane regions, extends as the transmembrane regions undergo conformational changes. In some implementations, inserting a fluorescent protein into the third intracellular loop of the GPCR can still maintain the correct folding and cell membrane positioning of the receptor itself, and result in good fluorescence signal changes when the receptor conformation changes.

[0065] Those skilled in the art are familiar with methods for determining the N-terminus, transmembrane region, intracellular loop, and C-terminus of G protein-coupled receptors (GPCRs), for example, based on their amino acid sequence and similarity to the transmembrane regions of known GPCRs. Various bioinformatics methods can be used to determine the location and structure of transmembrane regions in proteins; for example, alignment and amino acid sequence comparison can be performed using conventional methods in the art, such as the BLAST or CLUSTAL W programs. Based on alignment with known GPCRs containing transmembrane regions, those skilled in the art can predict the location and structure of transmembrane regions in other GPCRs. Many other programs are also available for predicting the location and structure of transmembrane regions in proteins. For example, one or a combination of the following programs can be used: TMpred, which predicts transmembrane protein fragments; TopPred, which predicts the topology of membrane proteins; PREDATOR, which predicts secondary structures from single and multiple sequences; TMAP, which predicts transmembrane regions of proteins from multiple aligned sequences; and AL0M2, which predicts transmembrane regions from a single sequence. According to standard nomenclature, the transmembrane region and intracellular loop are numbered relative to the N-terminus of the GPCR. To determine the numbering of amino acid residues in the transmembrane region of GPCR proteins, a general numbering scheme for GPCRs can be used, such as the ballesteros-harelweinstein numbering scheme (see Ballesteros, Juan A. and H. Weinstein. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods in Neuroences 25.05(1995):366-428, which is incorporated herein by reference in its entirety). In this numbering scheme, the most conserved amino acid in the transmembrane region is determined by multiple sequence alignment using GPCR. Based on this, the position of an amino acid residue in the transmembrane region is represented relative to the position of the most conserved amino acid in that region, which is labeled 50. Each amino acid is then assigned a number consisting of two digits separated by a decimal point, where the first digit indicates the transmembrane region and the second digit indicates the position corresponding to the most conserved amino acid. For example, residue "4.50" indicates the most conserved amino acid in the fourth transmembrane region, "4.55" indicates the position of the fifth amino acid at the C-terminus following 4.50, and similarly, "4.45" indicates the position of the fifth amino acid at the N-terminus preceding 4.50. For example, "5.59" indicates the position of the ninth amino acid at the C-terminus of the most conserved amino acid in the fifth transmembrane region, and "6.34" indicates the position of the sixteenth amino acid at the N-terminus of the most conserved amino acid in the sixth transmembrane region.

[0066] As used herein, the term "circularly permutated fluorescent protein (cpFP)," as is well known to those skilled in the art, refers to a fluorescent protein formed by linking the N-terminus and C-terminus of the original fluorescent protein molecule (e.g., directly or via a linker sequence) and then cleaving the protein at any other arbitrary site to form new C-termini and N-termini. Fluorescent proteins possess their own three-amino acid chromophore center, the chemical reactions of which determine the spectral properties and fluorescence intensity of the fluorescent protein. The chromophore of a fluorescent protein is mostly located inside the protein and protected by a barrel-like structure of surrounding β-sheets. When a wild-type fluorescent protein is fused to a target protein, there is essentially no change to the internal chromophore and its surrounding environment; therefore, the fluorescence intensity of the fluorescent protein remains unaffected upon fusion with the target protein. In cyclically rearranged fluorescent proteins (cpFPs), the chromophore is relatively close to the newly formed end. When cpFPs bind to a target protein, conformational changes in the target protein pull on the end of the cpFP, causing changes in the environment around its chromophore and thus altering the fluorescence intensity of the cpFP (increasing or decreasing). This translates the conformational change of the target protein into a change in fluorescence intensity, allowing for real-time detection using optical imaging methods. cpFPs were originally derived from green fluorescent protein (GFP), and the amino acid sequence of cyclically rearranged GFP (cpGFP) is highly homologous to GFP. Currently, various cpFPs have been constructed for protein detection; they exhibit high sensitivity to protein conformational changes and characterize these changes through fluorescence alterations.

[0067] As used herein, the term "identity" or "homology" refers to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or nucleotide monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences are 60% identical. Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using methods well known to those skilled in the art, for example, conveniently performed using computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48:443-453.

[0068] As used herein, "ΔF / F0" and "ΔF / F" are synonymous in relation to fluorescence intensity, referring to the relative change in fluorescence intensity of the biodetector protein after binding to the ligand relative to before binding. F0 refers to the average fluorescence value of the detector protein before binding to the ligand, and ΔF is the difference between the average fluorescence value F of the detector protein after binding to the ligand and the average fluorescence value F0 of the detector protein before binding to the ligand (ΔF = F - F0). In this invention, ΔF is also referred to as dF.

[0069] As used herein, the term "vector" refers to a nucleic acid medium in which polynucleotides can be inserted. When a vector allows the expression of a protein by the polynucleotide sequence inserted therein, the vector is called an expression vector. A vector can express a carried genetic material element in a host cell by transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (such as SV40). A vector may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may contain an origin of replication.

[0070] As used herein, when referring to amino acid sequences (e.g., peptides, polypeptides, or proteins), the term "fusion" or "fused" refers to a combination of two or more amino acid sequences, formed, for example, through chemical bonding or recombination, to form a single amino acid sequence that does not naturally exist. Fusion amino acid sequences can be generated by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell. In this context, "fusion" includes the connection of two parts directly or via a linker, such as a peptide linker, i.e., one sequence is attached to the N-terminus or C-terminus of another sequence, or the insertion of one sequence into a non-functional region of another sequence, i.e., the insertion does not affect the normal functioning of both sequences, or, in the case of fusion of more than one amino acid sequence, a combination of both.

[0071] The terms "connector" or "peptide connector" used herein are used interchangeably and refer to a short peptide that connects different polypeptide moieties in a fusion protein. For fusion proteins composed of two or more polypeptide moieties, the "connector" described herein includes an N-terminal connector located at the N-terminus of the polypeptide and a C-terminal connector located at the C-terminus of the polypeptide. In addition to connecting the moieties, the connector also helps the fusion protein fold correctly. In this invention, the connector allows for the association of receptor conformational changes and fluorescent protein brightness changes. Therefore, the connector used herein should be a connector capable of performing the aforementioned function. In some embodiments, when a cyclic rearranged fluorescent protein is inserted into the third intracellular loop of a chemokine receptor, the N-terminus of the cyclic rearranged fluorescent protein is connected to the third intracellular loop via an N-terminal connector, and the C-terminus of the cyclic rearranged fluorescent protein is connected to the third intracellular loop via a C-terminal connector.

[0072] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; bacteriophages; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0073] As used herein, the term "host cell" refers to a cell that can be used to introduce the vector, including but not limited to prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells. Host cells can include single cells or cell populations. There are no particular limitations on the host cells used in this invention, as long as they can express the probe protein of this invention. In some embodiments, the host cell is HEK293 cell. In some embodiments, the host cell is a neuronal cell or an astrocyte.

[0074] Probe proteins / biosensors for detecting chemokines

[0075] Chemokines are a class of small molecules (mostly 8–10 kDa) that can attract and direct cells to move in a directional manner. Based on the number and position of the N-terminal cysteine ​​residues in their primary structure, chemokines can be classified into four classes: C, CC, CXC, and CX3C. Chemokine receptors are seven-transmembrane G protein-coupled receptors (GPCRs) typically expressed on the surface of specific cells such as immune cells, neutrophils, and endothelial cells. Depending on the subfamily of chemokines they bind to, these receptors are named CXCR, CCR, CX3CR, and XCR, respectively. Once activated, chemokine receptors primarily bind to intracellular G proteins or repressor proteins (such as β-arrestin), initiating downstream signal transduction and thereby regulating cellular chemotaxis, proliferation, and changes in the affinity of cell adhesion molecules.

[0076] This disclosure provides a probe protein comprising a chemokine receptor and a fluorescent reporter protein for detecting or monitoring the release and fluctuations of chemokines in vitro and in vivo in real time. This detection and monitoring can be short-term or long-term, for example, monitoring for more than 20 hours. The probe protein can be readily used for in vivo imaging to observe level changes in the spatiotemporal patterns of chemokines. Preferably, the fluorescent reporter protein is inserted into the receptor of the chemokine, for example, into an intracellular loop (ICL) between transmembrane regions; this insertion does not affect the normal interaction between the receptor and the chemokine.

[0077] In some embodiments, the chemokine receptor is selected from CCR2, CCR3, CCR6, and CXCR4.

[0078] Preferably, the reporter protein is a circularly permuted fluorescent protein. Examples of circularly permuted fluorescent proteins that can be used in the detection proteins of the present invention include, but are not limited to, cpGFP and circularly permuted red fluorescent protein (cpRFP) and circularly permuted yellow fluorescent protein (cpYFP). cpGFP can be cpGFP derived from GCaMP6s or GCaMP6m (Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300 (2013)), or cpGFP derived from GECO1.2 (Zhao, Y. et al. An Expanded Palette of Genetically Encoded Ca2+ Indicators. Science 333, 1888-1891 (2011)). cpRFPs can be cpmApples, such as those from R-GECO1 (Yongxin Zhao et al., An Expanded Palette of Genetically Encoded Ca2+ Indicators, Science, 2011). Their sequences can be obtained from the NCBI database or the addgene database.Those skilled in the art will understand that any other cpFP can also be used in this invention, including but not limited to, for example, cyclically rearranged superfolder GFP, cyclically rearranged mApple (cpmApple), cyclically rearranged mCherry (cpmCherry), cyclically rearranged mKate (cpmKate), cyclically rearranged Venus (cpVenus), cyclically rearranged Citrin (cpCitrine), cyclically rearranged yellow fluorescent protein (cpYFP), and cyclically rearranged infrared fluorescent protein (cpiRFP, see Daria M Shcherbakova et al., Near-infrared fluorescent proteins for multicolor in vivoimaging, Nature methods, 2013; Pandey N et al., Tolerance of a Knotted Near-Infrared fluorescent protein torandomcircular permutation, Biochemistry, 2016). In some embodiments, the detector protein includes a cyclically rearranged green fluorescent protein having the amino acid sequence shown in SEQ ID NO: 3.

[0079] This disclosure also provides methods, systems, or platforms for screening probe protein candidates for detecting chemokines (e.g., CCL2), which detect the response of the probe protein candidates to the chemokines, preferably in batches using automated microscopy. The probe protein candidates may have different fluorescent reporter proteins, different adapters at both ends, and different insertion sites. The methods of this disclosure can detect multiple parameters, including baseline brightness, ΔF / F, and signal-to-noise ratio, thereby screening for probe protein candidates with optimal performance.

[0080] Detection protein / biosensor for CCL2

[0081] In some aspects, this disclosure provides a probe protein for detecting the chemokine CCL2, which is obtained by inserting a cyclically rearranged green fluorescent protein into the CCR2 receptor, referred herein as CRAFi-CCR2. C hemokine- R eceptor A motivation-based FThe luorescent indicator (a fluorescent indicator based on human chemokine receptor activation) is a detection protein or CRAFi-CCR2 sensor. When constructed based on the human CCR2 receptor, it is called hCRAFi-CCR2, and when constructed based on the mouse CCR2 receptor, it is called mCRAFi-CCR2.

[0082] CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), is an important member of the CC subfamily of chemokines. It mediates the migration and invasion of monocytes / macrophages and participates in brain inflammation and many other neurodegenerative diseases. Numerous studies have shown elevated CCL2 levels in the tissues or serum of patients with cancers such as breast cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, gastric cancer, and lung cancer, and these levels are closely related to tumor progression, stage, invasion, metastasis, and patient prognosis. CCL2 primarily exerts its biological effects by binding to its receptor CCR2. The human CCR2 gene is located on chromosome 3 and includes two subtypes: CCR2A and CCR2B. CCR2 is also the receptor for chemokines MCP-2, MCP-3, and MCP-4.

[0083] In some embodiments, the detector protein comprises a chemokine receptor CCR2 and a reporter protein, wherein the reporter protein is inserted into a third intracellular loop between the fifth and sixth transmembrane regions of the CCR2. Upon binding to a corresponding chemokine ligand, the fluorescence intensity of the reporter protein in the detector protein changes, for example, increases or decreases, expressed as ΔF / F. In some embodiments, the binding of the ligand to the detector protein causes an increase in fluorescence intensity, wherein ΔF / F is about 100% or higher, about 150% or higher, about 200% or higher, about 250% or higher, or about 300% or higher. For example, △F / F is approximately 100%, approximately 110%, approximately 120%, approximately 130%, approximately 140%, approximately 150%, approximately 200%, approximately 250%, approximately 260%, approximately 270%, approximately 280%, approximately 290%, approximately 300%, approximately 310%, approximately 320%, approximately 330%, approximately 340%, or approximately 350%.

[0084] In some embodiments, the chemokine receptor is a human or mouse CCR2. Preferably, the human CCR2 is human CCR2B, the exemplary sequence of which can be found in NCBI reference sequence number: NP_001116868.1. The CCR2 can be wild-type or a variant thereof. In some embodiments, the chemokine receptor is a human CCR2B variant containing the E235G mutation. In some embodiments, the CCR2 comprises an amino acid sequence as shown in any one of SEQ ID NO:20-22. In some embodiments, the CCR2 comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:20-22.

[0085] Preferably, the reporter protein is inserted after the “RNE” or “RNG” sequence in the third intracellular loop of the chemokine receptor. In some specific embodiments, the chemokine receptor is the human chemokine receptor CCR2, and the reporter protein is inserted between amino acid residues 235 and 236 of the chemokine receptor. In some embodiments, the reporter protein is inserted between amino acid residues 9 and 10 of the third intracellular loop of the chemokine receptor. In some embodiments, when inserted into CCR2, the reporter protein has an N-terminal adapter and a C-terminal adapter, respectively. In some embodiments, the N-terminal adapter is LSSLI, LSSSV, or LEEGG, and the C-terminal adapter is NHDQL, NH, or ID. Other flexible peptide adapters can also be used to construct the probe protein of the present invention, such as (G4S)n or (Gly)n, as long as they do not affect the correct folding and performance of the probe protein of the present invention. The selection of the adapter sequence can be determined by methods well known in the art, such as detecting whether the probe protein is correctly folded and detecting whether the binding of the probe protein to its ligand causes a change in fluorescence intensity.

[0086] The probe protein of the present invention can be expressed on the cell membrane. In some embodiments, the C-terminus of the probe protein of the present invention further includes a Golgi export transport signal (KGC) sequence and / or an endoplasmic reticulum export (ER2) sequence for better membrane localization. Optionally, the C-terminus of the probe protein includes one or more (e.g., 1, 2, 3 or more) copies of the KGC sequence. More specifically, the KGC sequence is shown in SEQ ID NO: 4, and the ER2 sequence is shown in SEQ ID NO: 5.

[0087] In some embodiments, the C-terminus of the detector protein of the present invention further comprises an indicator fluorescent protein, wherein the fluorescence spectrum of the indicator fluorescent protein does not overlap with that of the reporter protein. In some embodiments, the indicator fluorescent protein is linked to the C-terminus of the ER2 sequence via a self-cleaving peptide, and the detector protein has the following structure: an hCRAFi-CCR2-KGC sequence-ER2 sequence-self-cleaving peptide-indicator protein operably linked from the N-terminus to the C-terminus. In some embodiments, the indicator fluorescent protein is operably linked to the C-terminus of the KGC sequence, and the detector protein has the following structure: an hCRAFi-CCR2-KGC sequence-indicator protein-ER2 sequence operably linked from the N-terminus to the C-terminus. The operable linking can be direct linking or linking via a peptide linker. The KGC sequence can be one or three copies of the KGC sequence (e.g., three copies of KGC are three KGC sequences tandemly, referred to herein as 3KGC or 3xKGC). Optionally, the self-cleaving peptide is P2A, F2A, T2A, or E2A; optionally, the indicator fluorescent protein is a red fluorescent protein, such as FusionRed. In a specific embodiment, the detector protein comprises an operatively linked hCRAFi-CCR2-1KGC / 3KGC-FusionRed-ER2. In some embodiments, the indicator fluorescent protein comprises the amino acid sequence shown in SEQ ID NO: 10.

[0088] In some embodiments, the probe protein comprises an amino acid sequence as shown in SEQ ID NO: 15, 16 or 17 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 15, 16 or 17.

[0089] In some embodiments, the probe protein comprises: (a) an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1; (b) an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2; or (c) an amino acid sequence as shown in SEQ ID NO: 19 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 19.

[0090] In some embodiments, the probe protein comprises an amino acid sequence as shown in SEQ ID NO: 8 or 9, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 8 or 9.

[0091] In some implementations, the probe protein has an EC50 of approximately 9 nM for CCL2.

[0092] As demonstrated in the examples, the hCRAFi-CCR2 sensor, derived from the human CCR2B receptor, exhibited approximately 300% fluorescence change for mCCL2 in primary mouse astrocytes, demonstrating high selectivity and high affinity for the ligand in the nanomolar range (~2.5 nM). Activation of the hCRAFi-CCR2 sensor did not affect downstream signaling pathways such as calcium mobilization and receptor internalization. Using this probe protein, long-term imaging (e.g., 17–20 hours) of endogenous mCCL2 release under inflammatory conditions was possible in cell culture and in vivo in mice. In the mouse brain, spatial heterogeneity of CCL2 signaling response was observed at a scale of approximately 20–50 μm, highlighting the complexity of spatiotemporal signaling in the immune system.

[0093] Although the probe protein exhibits similar sensitivity to human MCP1-4, it demonstrates higher selectivity for mouse MCP1 in mouse MCP. In mouse astrocytes, the probe protein is approximately 25 times more sensitive to mCCL2 than to mCCL7.

[0094] Detection proteins / biosensors for detecting other chemokines

[0095] In some aspects, this disclosure provides a detector protein for detecting other chemokines, comprising a reporter protein located between the fifth and sixth transmembrane regions of a receptor for said other chemokine. Preferably, the reporter protein is a cyclic rearrangement fluorescent protein. The other chemokines include, for example, CCL3, CCL5, CCL11, CCL17, CCL20, CCL25, CXCL12α, or CXCL12β. Accordingly, the chemokine receptor may be selected from CCR1, CCR3, CCR4, CCR5, CCR6, CCR9, and CXCR4. In some embodiments, the other chemokine is CCL11, and the chemokine receptor is CCR3. In some embodiments, the other chemokine is CCL20, and the chemokine receptor is CCR6. In some embodiments, the other chemokine is CXCL12α or CXCL12β, and the chemokine receptor is CXCR4. The chemokine receptor for the detector protein of the present invention may be wild-type or a variant thereof. Preferably, the other chemokines are mouse-derived mCCR3, mCXCR4, and mCCR6. In some embodiments, the amino acid sequence of the chemokine receptor variant has at least 90%, at least 95%, and at least 99% sequence identity compared to its wild-type amino acid sequence.

[0096] In some embodiments, a reporter protein is inserted into the third intracellular loop (ICL3) of the chemokine receptor to obtain the detector protein for detecting other chemokines; preferably, the reporter protein is inserted into the ICL3 of the chemokine receptor via N-terminal and C-terminal adapters. Further, a portion of the amino acid sequence between TM5 and TM6 of the chemokine receptor is replaced by the corresponding amino acid sequence of a CCR2-based detector protein. In some embodiments, the detector protein comprises a chemokine receptor and a reporter protein, wherein the sequence of amino acid residues 5.59 to 6.34 of the chemokine receptor is replaced with the portion containing the reporter protein. In some embodiments, the chemokine receptor is mCCR3, and the sequence of amino acid residues 224 to 242 is replaced with the portion containing the reporter protein. In some embodiments, the chemokine receptor is mCXCR4 or mCCR6, and the sequence of amino acid residues 227 to 245 is replaced with the portion containing the reporter protein. In some embodiments, the portion containing the reporter protein is the TM5-adaptor-cpEGFP-adaptor-TM6 motif (SEQ ID NO:11) from the hCRAFi-CCR2 probe protein.

[0097] Upon binding to a corresponding chemokine ligand, the fluorescence intensity of the reporter protein in the detector protein changes, for example, increases or decreases, denoted as ΔF / F. In some embodiments, the binding of the ligand to the detector protein causes an increase in fluorescence intensity, wherein ΔF / F is about 100% or higher, about 150% or higher, about 200% or higher, about 250% or higher, or about 300% or higher. For example, ΔF / F is about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 200%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, or about 350%.

[0098] In some embodiments, when inserted into a chemokine receptor, the reporter protein has N-terminal and C-terminal adapters, respectively. In some embodiments, the N-terminal adapter is LSSLI, LSSSV, or LEEGG, and the C-terminal adapter is NHDQL, NH, or ID. Other flexible peptide adapters can also be used to construct the probe protein of the present invention, such as (G4S)n or (Gly)n, as long as they do not affect the correct folding and performance of the probe protein. The selection of the adapter sequence can be determined by methods well known in the art, such as detecting whether the probe protein is correctly folded and detecting whether the binding of the probe protein to its ligand causes a change in fluorescence intensity.

[0099] In some embodiments, the probe protein comprises: (a) an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 12; (b) an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 13; and (c) an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 14.

[0100] The probe protein of this invention can be expressed on the cell membrane. In some embodiments, the C-terminus of the probe protein of this invention further includes one or more copies of a Golgi export transport signal (KGC) sequence and / or an endoplasmic reticulum export (ER2) sequence to achieve better membrane localization. More specifically, the KGC sequence is shown in SEQ ID NO: 4, and the ER2 sequence is shown in SEQ ID NO: 5.

[0101] In some embodiments, the C-terminus of the detector protein of the present invention further comprises an indicator fluorescent protein, wherein the fluorescence spectrum of the indicator fluorescent protein does not overlap with that of the reporter protein. In some embodiments, the indicator fluorescent protein is linked to the C-terminus of the ER2 sequence via a self-cleaving peptide, and the detector protein has the following structure: detector protein-KGC sequence-ER2 sequence-self-cleaving peptide-indicator protein operably linked from the N-terminus to the C-terminus. In some embodiments, the indicator fluorescent protein is operably linked to the C-terminus of the KGC sequence, and the detector protein has the following structure: detector protein-KGC sequence-indicator protein-ER2 sequence operably linked from the N-terminus to the C-terminus. The operably linked structure can be direct or via a peptide linker. The KGC sequence can be one or three copies of the KGC sequence (e.g., three KGC sequences tandemly, denoted herein as 3KGC or 3xKGC). Optionally, the self-cleaving peptide is P2A, F2A, T2A, or E2A; optionally, the indicator fluorescent protein is a red fluorescent protein, such as FusionRed. In some embodiments, the indicator fluorescent protein comprises an amino acid sequence as shown in SEQ ID NO: 10.

[0102] In some embodiments, the probe protein has an EC50 of about 30 nM or less for the chemokine ligand, such as less than 29 nM, less than 28 nM, less than 27 nM, less than 26 nM, less than 25 nM, less than 20 nM, less than 19 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, or less than 5 nM.

[0103] Characterization of probe protein function

[0104] One method for evaluating the function of a probe protein is by detecting the change in fluorescence intensity ΔF / F0 after the probe protein binds to a ligand. At the same ligand concentration, a greater change in fluorescence intensity indicates higher sensitivity of the probe protein, making it more likely for intracellular or in vivo detection. In some embodiments, the absolute value of ΔF / F0 is about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 30% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater, about 100% or greater, about 200% or greater, about 300% or greater, about 350% or greater, or about 500% or greater. The change can be an increase or decrease in fluorescence intensity.

[0105] Polynucleotides, vectors and host cells

[0106] In some aspects, the present invention also provides polynucleotides encoding the detector proteins disclosed herein. These polynucleotides can be used to express the detector proteins in cells.

[0107] The polynucleotide encoding the probe protein can be isolated and sequenced using conventional procedures known in the art. The polynucleotide can also be obtained by synthetic methods. Preferably, the polynucleotide is codon-optimized for expression in eukaryotic host cells, particularly mammalian cells. In some embodiments, the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO: 6 or 7.

[0108] Using known recombination techniques, polynucleotides encoding probe proteins can be inserted into vectors for further cloning (DNA amplification) or expression. Vector components typically include, but are not limited to, one or more of the following: signal sequences, origin of replication, one or more marker genes, enhancer elements, promoters (e.g., SV40, CMV, EF-1α, hSyn, GfABC1D), and transcription termination sequences.

[0109] In some embodiments, this disclosure provides a vector (e.g., an expression vector) comprising a polynucleotide encoding a probe protein provided herein, at least one promoter operatively linked to the polynucleotide (e.g., SV40, CMV, EF-1α, hSyn, GfABC1D), and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (e.g., SV40), λ phages, and M13 phages, liposomes, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, and pGEM. EX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0110] Vectors containing multinucleotide sequences encoding probe proteins can be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors described herein are prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive bacteria like *Escherichia coli*. Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the provided vectors. *Saccharomyces cerevisiae* or common baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and useful herein.

[0111] Suitable host cells for expressing the probe proteins provided herein can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. In some preferred embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some other preferred embodiments, the host cell is another mammalian cell line, such as a human cell line. In some embodiments, the host cell is a nerve cell, such as a neuron or astrocyte.

[0112] Host cells used to express the probe proteins provided herein can be cultured in a variety of culture media. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., the drug Gentamycin), trace elements (defined as inorganic compounds typically present in micromolar concentrations), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression and are readily apparent to those skilled in the art.

[0113] Application of the detection protein of this invention

[0114] In one aspect, the present invention also relates to a method for qualitative or quantitative detection of chemokine concentrations in a test sample or tissue using the detection protein disclosed herein. In this detection method, detection or screening results are obtained by measuring changes in fluorescence signal intensity, wherein the change in fluorescence intensity can be an increase or a decrease. Those skilled in the art can readily determine the detection or screening results based on changes in fluorescence intensity. The term "test sample" as used herein can include samples outside a living organism, including but not limited to cell cultures or extracts thereof; biopsy materials obtained from mammals or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof. Detection of the test sample can be performed in vitro. The term "test tissue" as used herein can include any tissue within a living organism, including but not limited to heart tissue, brain tissue, etc. Detection of the test tissue can be performed in vivo.

[0115] In one aspect, the present invention also relates to a method for detecting changes in chemokine concentration in a sample or tissue using the detector protein disclosed herein, the method comprising: culturing host cells expressing the detector protein disclosed herein under suitable conditions; adding a sample or tissue to be tested to the cell culture, and recording the fluorescence intensity F0 before sample addition, the fluorescence intensity at a first time point after sample addition, and the fluorescence intensity at a second time point; and analyzing the fluorescence intensity change ΔF / F at the first time point and the second time point relative to F0, thereby determining the concentration change of the chemokine in the sample or tissue to be tested at the first time point relative to the second time point. The first time point and the second time point can be selected according to the detection purpose. Optionally, the first time point and the second time point are each independently any time after sample addition, for example, the first time point is 10 minutes after sample addition and the second time point is 11 minutes after sample addition; or, the first time point is 30 minutes after sample addition and the second time point is 1 hour after sample addition. Optionally, the time interval between the first time point and the second time point can be approximately 1 minute, approximately 5 minutes, approximately 10 minutes, approximately 20 minutes, approximately 30 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 20 hours, or longer.

[0116] In some aspects, the present invention also relates to a method for detecting the level of chemokines in a sample or tissue using the detector protein disclosed herein, comprising: culturing host cells expressing the detector protein disclosed herein under suitable conditions; adding the sample or tissue to be tested to the cell culture, recording the fluorescence intensity F0 before adding the sample or tissue and the fluorescence intensity after adding the sample or tissue; analyzing the change in fluorescence intensity ΔF / F0 relative to F0 after adding the sample, thereby determining the level of chemokines in the sample or tissue to be tested. More than one sample, such as samples collected at different time points, can be added to the cell culture in parallel to detect changes in chemokines in the sample over time or after specific stimulation.

[0117] In one aspect, the invention also relates to spatiotemporal patterns of monitoring chemokine release using the probe proteins disclosed herein.

[0118] The detector protein disclosed herein exhibits high sensitivity, specificity, and stability, making it particularly suitable for high-resolution, long-term continuous imaging recording. Therefore, this invention also relates to a method for monitoring the spatiotemporal pattern of chemokine release from tissues, organs, or cells, comprising: expressing the detector protein disclosed herein in an organ, tissue, or cell; recording the fluorescence intensity of the detector protein over time within one or more fields of view; and analyzing the change in fluorescence intensity over time, ΔF / F, for each of the one or more fields of view, thereby obtaining the spatiotemporal pattern of chemokine release.

[0119] Methods for expressing the detector protein disclosed herein in organs, tissues, or cells include, but are not limited to, transient transfection, adenovirus transfection, or adeno-associated virus transfection. Those skilled in the art will understand that, for an organ or tissue, expression of the detector protein means that 50% or more of the cells in the organ or tissue express the detector protein, preferably 60% or more, 70% or more, 80% or more, or 90% or more of the cells express the detector protein. In some embodiments, the organ, tissue, or cells are derived from the brain or a portion thereof, such as the cortex.

[0120] The organ, tissue, or cell is induced by a stimulus, or the organ, tissue, or cell originates from an organism induced by a stimulus, thereby enabling it to secrete chemokines. In some embodiments, the stimulus is a chemical stimulus with pro-inflammatory effects, including but not limited to lipopolysaccharide (LPS), ovalbumin (OVA), titanium dioxide (TiO2) particles, infectious bacteria, etc. In some embodiments, the stimulus is a physical stimulus such as electricity, light, sound, heat, cold, or machinery.

[0121] The spatiotemporal patterns of chemokine release are monitored by continuously acquiring fluorescence signals of the detection protein within one or more fields of view over a long period of time. In some implementations, the time interval for imaging recording can be approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 20 hours, or longer.

[0122] In some implementations, the frequency at which fluorescence signals (fluorescence intensity) are acquired during imaging can be once per hour, once every 30 minutes, once every 20 minutes, once every 10 minutes, once every 5 minutes, or once every 1 minute or higher. In some implementations, the frequency at which fluorescence signals are acquired can be 1 Hz, 5 Hz, 10 Hz, 15 Hz, 20 Hz, 25 Hz, 30 Hz, 31 Hz, 32 Hz, 33 Hz, 34 Hz, 35 Hz or higher.

[0123] Spatial patterns of chemokine release are obtained by analyzing the distribution of fluorescence signals within one or more fields of view. Typically, acquired images are segmented based on fluorescence signal levels, with positive regions exhibiting fluorescence intensity above the baseline. The spatiotemporal patterns of chemokine release are derived by analyzing the changes in fluorescence signal characteristics (including, for example, maximum response, fluorescence intensity magnitude, and response onset time) over time in regions of interest within one or more fields of view. As used herein, "maximum response" refers to the maximum difference between fluorescence intensity and baseline within a given time period. As used herein, "response onset time" refers to the time required for fluorescence intensity to rise above baseline after stimulation.

[0124] Reagent test kit

[0125] In one aspect, the present invention provides a kit for detecting the content, level, or concentration of chemokines or changes thereof in biological fluids, comprising a vector or host cell expressing the probe protein disclosed herein.

[0126] For detecting chemokine levels in biological fluids using the kit of this invention, host cells can be cultured under suitable conditions to express the probe proteins disclosed herein, or a cell line expressing the probe proteins disclosed herein can be constructed by introducing the vector into cells. The test biological fluid is then applied to the host cells or the cell line, and the fluorescence intensity of the probe proteins on the cell membrane before and after the addition of the test biological fluid is recorded using a fluorescence imaging device (e.g., wide-field microscopy, confocal microscopy, or two-photon microscopy), thereby detecting the chemokine levels in the biological fluid. Optionally, the chemokine levels in the biological fluid are determined by analyzing the change in fluorescence intensity ΔF / F of the probe proteins on the cell membrane before and after the addition of the test biological fluid, and matching the obtained ΔF / F value to a standard concentration-response curve.

[0127] For detecting changes in chemokine concentration in biological fluids using the kit of this invention, host cells can be cultured under suitable conditions to express the probe protein disclosed herein, or the vector can be introduced into cells to construct a cell line expressing the probe protein disclosed herein, and then the biological fluid to be tested can be applied to the host cells or the cell line. The fluorescence intensity of the probe protein on the cell membrane before and after the addition of the biological fluid to be tested can be recorded using a fluorescence imaging device (e.g., wide-field microscope, confocal microscope, or two-photon microscope) to detect the level of chemokines in the biological fluid.

[0128] In some implementations, the biofluid may be one or more samples from one or more samples. For example, the biofluid may be multiple samples from one or more samples, wherein the multiple samples may be samples collected at different time points in the same experiment or samples collected under different experimental conditions.

[0129] The above-mentioned host cells can be cultured, cell lines constructed using vectors, fluorescence imaging performed, and fluorescence intensity analyzed using the exemplary methods provided in the embodiments herein or conventional techniques known to those skilled in the art.

[0130] Drug screening methods and screening platforms

[0131] In one aspect, the invention also relates to a method for screening drugs using the detector proteins disclosed herein, wherein the drugs target chemokine receptors. In some embodiments, the drug is a chemokine receptor agonist. In some embodiments, the drug is a chemokine receptor antagonist.

[0132] The method includes: constructing a cell line expressing the detector protein; adding a library of compounds to be screened to the cell line; and detecting the change in fluorescence intensity ΔF / F of the detector protein. Wherein, a ΔF / F > 0 for the detector protein caused by a compound indicates that the compound is an agonist, and a ΔF / F < 0 for the detector protein indicates that the compound is an antagonist.

[0133] Cells used to construct cell lines expressing the probe protein include, but are not limited to, prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells. Cells may include single cells or cell populations. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are neuronal cells or astrocytes.

[0134] In some aspects, the present invention provides a method for screening compounds targeting CCR2, comprising: constructing a cell line expressing an hCRAFi-CCR2 probe protein; adding a library of compounds to be screened to the cell line; and detecting the change in fluorescence intensity ΔF / F of the probe protein. Wherein, a ΔF / F > 0 for the probe protein indicates that the compound is a CCR2 agonist, and a ΔF / F < 0 indicates that the compound is a CCR2 antagonist. In some embodiments, the cell line used to construct the probe protein expression is HEK293 cells. In some embodiments, the cell line used to construct the probe protein expression is a neuron or astrocyte.

[0135] In some aspects, the present invention provides a system for screening compounds targeting CCR2, comprising a drug delivery module, a response module, and an imaging module, wherein the drug delivery module is a multi-channel drug delivery module, the response module includes a cell culture device and cells expressing the hCRAFi-CCR2 detector protein, and the imaging module records the fluorescence intensity of the cells before and after drug delivery in real time. Existing multi-channel drug perfusion delivery devices or automated sample loading devices can be used as the drug delivery module of the system of the present invention, including but not limited to, the automated sample loading module of FLIPR, the ALAOCtaFlow™ multi-channel multifunctional perfusion drug delivery device, etc. The imaging module that can be used in the system of the present invention is a module or device with fluorescence imaging capabilities, including but not limited to, a wide-field microscope, a fluorescence imaging plate reader, a confocal microscope, or a two-photon microscope. Those skilled in the art know how to adjust the parameters of the imaging module to acquire the fluorescence signal of the detector protein.

[0136] The method and system described above can perform screening at medium or high throughput using automated microscopy.

[0137] As verified in the examples, a library of 59 compounds targeting chemokine receptors was screened using the described method in agonist and antagonist modes. Ten compounds were identified as antagonists of the hCRAFI-CCR2 response, and one compound (J-11383) was identified as a CCR2 agonist. Of these antagonists, nine out of ten have been reported to antagonize the CCR2 receptor. During the screening process, the compound AZD-5069 was newly discovered to antagonize CCR2, reducing the fluorescence intensity of hCRAFI-CCR2 at a micromolar concentration (IC50 = 2.01 ± 0.33 μM). Furthermore, J-113863, considered a CCR1 and CCR3 antagonist, increased the fluorescence intensity of hCRAFI-CCR2, with an EC50 of 4.1 ± 2.0 μM. Therefore, the described method can be used reliably and efficiently to screen compounds targeting CCR2.

[0138] Beneficial effects of the present invention

[0139] 1. The inventors have developed a novel, ultrasensitive probe protein, hCRAFi-CCR2, for real-time monitoring of endogenous CCL2 release, for example, in mammalian cell cultures and in mice. Using the human chemokine receptor CCR2B as a backbone, the CCL2 probe protein was developed and characterized in cultured HEK cells, neurons, and astrocytes. hCRAFi-CCR2 exhibits high affinity for hCCL2 / mCCL2 (EC50 of 2-10 nM), a relatively large dynamic range (300-400%), and minimal impact on downstream signaling pathways. This probe protein enables real-time monitoring of extracellular mCCL2 fluctuations during inflammatory stimulation lasting up to 20 hours, both in vitro and in vivo.

[0140] 2. The present invention also provides a FLIPR-based medium-throughput screening platform for screening compounds targeting CCR2.

[0141] 3. This invention also develops a probe protein targeting other chemokines based on hCRAFi-CCR2, obtained by transplanting the reporter protein portion of hCRAFi-CCR2 onto other chemokine receptors such as CCR3, CCR6, or CXCR4. This probe protein achieves similar effects to hCRAFi-CCR2, exhibiting high affinity (EC50 of 5-24 nM) and high sensitivity to its chemokine ligands, and can be applied to a FLIPR-based drug screening platform for screening compounds targeting CCR3, CCR6, or CXCR4.

[0142] Example

[0143] The sources of chemical reagents in the following examples are summarized as follows:

[0144]

[0145]

[0146] Example 1. General Experimental Procedure

[0147] Molecular cloning and mutagenesis

[0148] Human CCR2A (GenBank NP_001116513.2), human CCR2B (GenBank NP_001116868.1), and mouse CCR2 (GenBank NP_034045.1) were synthesized de novo by Tsingke Biotechnology Co., Ltd. Cyclic rearranged GFP was inserted into the ICL3 region of the chemokine receptor and subcloned into the pN1 vector (Clontech) using AgeI and NotI sites. Site-directed mutagenesis using custom-designed primers generated the hCRAFi-CCR2Y49A / Y120A mutant. To improve the cell membrane localization of the chemokine probe protein in vivo, the KGC and ER2 motifs were added to the C-terminus of the probe protein. The hCRAFi-CCR2 gene and its nonfunctional mutant gene were subcloned into the pAAV-hSyn vector (using WeKwikGene plasmid #0000133 pAAV-hSyn-GRAB_ATP1.0 as a template) and the pAAV-GfABC1D vector (using WeKwikGene plasmid #0000135 pAAV-GfaABC1D-GRAB_ATP1.0 as a template) for expression in neurons and astrocytes, respectively. All constructs contained the same Kozak sequence. High-fidelity PCR amplification was performed using PrimeStar Max premix (PrimeStar Maxmaster mix, Clontech). Restriction endonucleases were purchased from New England BioLabs (USA) and used according to the manufacturer's instructions. Top 10 E. coli cells (Biomed) were used as host cells for plasmid amplification. Small-scale plasmid DNA isolation was performed using the commercially available Mini-Prep kit (Sangon Biotech); medium-scale DNA plasmid purification was performed using the midi-Prep kit (Qiagen). The purified plasmids and bacterial colonies were sequenced using the Sanger method (Zhejiang Youkang Biotechnology Co., Ltd.).

[0149] A site-directed mutagenesis linear PCR library spanning the CMV promoter and SV40 polyA sequence was generated using an overlap extension PCR method. Synthesized DNA primers were purchased from Qingke Biotechnology Co., Ltd. One-quarter of the PCR products were randomly selected for verification by gel electrophoresis or DNA sequencing. The resulting PCR mixture required no further purification and was used for cell culture transfection and imaging.

[0150] mammalian cell culture

[0151] All cells were cultured in a 37°C / 5% CO2 incubator. HEK293FT cells (Invitrogen) were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) on 24-well glass-bottom plates (Cellvis, Cat: P24-0-N) or Matrigel (BD Biosciences, 356235) coated 12-mm glass coverslips. When confluence reached 80-90%, cells were transiently transfected with plasmids using Hieff Trans Liposomal transfection reagent (YeasenBiotechnology, 40802ES02) according to the manufacturer's instructions, and imaging was performed 36-48 hours later. For PCR library screening, cells were grown in Matrigel-coated 96-well glass plates and transfected with Lipofectamine 3000 (Life Technologies) (~100 ng DNA, 0.4 μL p3000 reagent, 0.4 μL Lipofectamine). The medium was changed after 4-12 hours, and imaging was performed 2 days after transfection.

[0152] Hippocampal neurons were cultured as follows. In short, the unilateral hippocampus of P0 rat pups was isolated under a stereomicroscope in cold HBSS. The isolated hippocampal tissue was digested with 0.25% trypsin (GIBCO, 25200056) at 37°C for 12 min, gently agitated every 4 minutes. Digestion was terminated with complete medium preheated at 37°C in DMEM supplemented with 10% FBS. The digested tissue was then gently dissociated and filtered through a 40-μm nylon mesh. The dissociated neurons were seeded at a density of 150,000 cells per well on pre-coated Matrigel coverslips (Thermo Fisher Scientific). TM Cells were cultured at 37°C / 5% CO2 for 12 hours to allow cell adhesion. Half of the culture medium was then replaced with Neurobasal medium (GIBCO, 21103049) supplemented with 1% GlutaMax (GIBCO, 35050061) and 2% B27 (GIBCO, 17504044). After 5-6 days of in vitro culture (DIV), when the glial cell density reached 70-80%, AraC (4μM, Sigma, C6645) was added. Subsequently, half of the culture medium was replaced with fresh Neurobasal medium every 4-5 days. At DIV 6-7, neurons were transfected for 50-60 minutes using a calcium phosphate transfection kit (Invitrogen, Cat#K278001) or infected with rAAV. Neurons were imaged 5-7 days after transfection or transduction.

[0153] Primary mouse astrocytes were prepared from the cortex similarly to neuronal cultures. Astrocytes were cultured in T25 flasks in DMEM supplemented with 10% FBS. At DIV 5-8, when the astrocytes reached 90-100% confluence, the flasks were shaken at 180 rpm for 1 hour to remove microglia. The remaining astrocytes were dissociated with 0.25% trypsin and seeded onto 12-mm Matrigel-coated glass coverslips. One day later, the astrocytes were transduced with rAAV-2 / 5-GfABC1D-hCRAFi-CCR2 or rAAV-2 / 5-GfABC1D-hCRAFi-CCR2. The astrocytes were used for imaging 5-7 days post-transduction.

[0154] PCR mutant library screening

[0155] PCR library screening was performed as described previously (see Villallette, V. et al., Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice. Cell 179, 1590-1608.e23 (2019)), with slight modifications. In short, cells were grown in Matrigel-coated 96-well glass-bottomed culture plates and transfected with Lipofectamine 3000 (Life Technologies; ~100 ng DNA, 0.4 μL p300 reagent, 0.4 μL Lipofectamine), with the medium replaced after 4–12 h. For imaging 36–48 h post-transfection, the medium was replaced with an extracellular solution (150 g NaCl, 5 g KCl, 1 g MgCl2, 2 g CaCl2, 10 g HEPES, 10 g D-glucose, mM, pH adjusted to 7.4 with Tris base). The 96-well glass-bottom plate was then mounted on a custom-modified CellSorter system (CellSorter Company for Innovations) for imaging. In addition to cell analysis, detection, and cell picking, the system supports millisecond-level precise synchronization between the camera and drug delivery controlled by Valve 1, and imaging of each of the 96 wells. The trigger input line of the Andor Zyla 4.2 camera was connected to the TTL output of the high-speed electromagnetic coil Valve 1. Turning on Valve 1 triggered the acquisition of a series of image frames at a preset delay in the camera. The response time of Valve 1 (~10 ms) was considered a constant delay. The volume of injected drug could be set to 5 μl, which saved on costly drugs. During screening, cells were first imaged for 10–20 s to record baseline fluorescence, then a manipulator guided the glass electrode down into the well, positioned 300–400 μm above the cells. 10 μl of 500 nM chemokine solution was released into the cells through a 70 μm diameter glass capillary connected to the injection pump, and a 60 s time-lapse video was recorded. Screening can be performed in automatic or semi-automatic mode. In automatic mode, automatic imaging and drug delivery can be performed simultaneously on 8 wells during a single screening cycle. For analysis of time-lapse datasets, fluorescence trajectories of 10–15 cells are calculated within a single field of view (FOV).

[0156] Fluorescence imaging of cultured cells

[0157] HEK cells cultured as described above, as well as cultured primary neurons and astrocytes, were imaged under a Nikon Ti2-E wide-field microscope equipped with a SpectraIII Light Engine (LumenCore), an ORCA-Flash 4.0V3s CMOS camera (Hamamatsu), 20x / 0.75NA objectives, a 40x / 1.15NA water immersion objective, and controlled by NIS Elements (AR 5.21.00). cpGFP-based probe proteins and FusionRed were imaged using the green (excitation 475 / 28nm, emission 535 / 46nm) and red channels (excitation 561nm laser, emission 595 / 50nm), respectively. Cells were washed and incubated in extracellular solution (ECS), and compounds were applied manually (96-well and 24-well plates) or via a gravity-driven system (RSC200 BioLogic). Chemokines were manually applied to 96-well plates for specificity measurements using a single concentration. Figure 2 The dose-response curves and kinetic measurements in C were performed using the RSC200 perfusion system with local application of chemokines and antagonists. For rapid probing of protein kinetic measurements (… Figure 12 HEK293T cells were transfected with hCRAFi-CCR2 using NEOFECT (Neofect, #TF20121201), plated on coverslips, and imaged using a laser scanning confocal microscope (LSM 880, Zeiss) 48 h post-transfection. CCL2 (1 μM, Cat#Z03292, GenScript) was mixed with Cy3.5 (Cat#Q-0027523, Qiyuebio) and introduced into a 0.05 mm diameter glass electrode for local delivery, programmed and controlled by a microoperating system. Culture medium was rapidly exchanged via perfusion to ensure rapid ligand removal. Fluorescence responses on the cell membrane surface were recorded using confocal line scanning mode. All experiments were performed at 23–25 °C.

[0158] For high-quality long-term time-lapse imaging of cultured cells, mouse brain slices expressing hCRAFi-CCR2, and chemokine release from cultured astrocytes, a Nikon CSU-W1SoRa confocal microscope equipped with a 20x / 0.75NA objective, a 40x / 1.15NA water immersion objective, a 488-nm laser, and a 561-nm laser was used. For long-term time-lapse imaging of astrocytes, appropriate concentrations of LPS (Sigma, Cat#L4130), poly(I:C) (Tocris, Cat#4287 / 10), and ODN 1668 (MCE, Cat#HY-150726) were added to the wells, and imaging was performed continuously for 15-18 hours in a live-cell chamber (OkaLab) at 37°C / 5% CO2. For the development and optimization of the hCRAFi-CCR2 probe protein prototype, HEK cells were imaged using a Zeiss LSM 800 with Airyscan and 40X / 1.20NA objectives. The GFP channel used an Ex / Em of 493 / 517nm and the DAPI channel used an Ex / Em of 353nm / 465nm.

[0159] Spectral measurement

[0160] HEK293 cells were transfected with hCRAFi-CCR2 probe protein in 6 mm dishes. After 36–48 h, the cells were digested with 0.25% trypsin and washed three times in PBS. Excitation (λem = 560 nm) and emission (λex = 470 nm) spectra of the cell suspension before and after the addition of 500 nM hCCL2 were then measured using an FLS1000 spectrometer (Edinburgh Instruments) in 1 nm increments.

[0161] Animal husbandry and AAV virus injection surgery

[0162] All mouse husbandry and experimental procedures were conducted in accordance with the Westlake University Laboratory Animal Management Guidelines. All animal studies were approved by the Westlake University Laboratory Animal Management and Use Committee (AP no. 19-044-KP-2) or according to the protocol approved by the CIBR Laboratory Animal Management and Use Committee (#CIBR-IACUC-007). C57BL / 6 mice provided by the Westlake University Laboratory Animal Center were used, without sex differentiation. Mice aged 5-14 weeks were used for in vivo surgery and two-photon imaging. All mice were home-housed or housed in pairs in a temperature-controlled room with a 12h-12h light-dark cycle. Animals were randomly assigned to groups, and there was no distinction between experimental and control groups during experiments and analyses.

[0163] for Figure 23In vivo expression of the hCRAFi-CCR2 probe protein in the mouse brain was determined by injecting rAAV2 / 5-GfaABC1D-hCRAFi-CCR2, rAAV2 / DJ-hSyn-hCRAFi-CCR2-P2A-FusionRed, and the corresponding nonfunctional mutant rAAV encoding each probe protein (>10) into the pancortex (AP: 0.8-1.2 mm, ML: 0.5-1 mm) of P0-P2 pups using a Hamilton microsyringe. 12 Viral genome / mL; Shanghai Sunbio Medical Biotechnology). Young mice were placed on ice for 3-5 minutes to induce hypothermic anesthesia. 0.4-0.6 μl of a viral solution containing 10% FastGreen dye (Sigma-Aldrich, USA) was manually injected into the cortex. After injection, the young mice were moved to a heating pad for recovery and then returned to their rearing cages.

[0164] Craniotomy implantation and two-photon microscopy

[0165] The craniotomy implantation procedure was similar to that previously reported (Wu, Z. et al., A sensitive GRAB sensor for detecting extracellular ATP in vitro and in Vivo, Neuron, 2022). Mice aged 5–14 weeks (sex-neutral) were anesthetized by intraperitoneal injection of avertin (250 mg / kg body weight, Sigma-Aldrich, #T48402), and a metal head fixation recording cavity was attached to the skull. After 3–4 days of recovery, the mice were re-anesthetized and placed on a stereotaxic instrument (RWDInstruments) for craniotomy implantation. During implantation, AAV (400–500 nL) was injected into the V1 visual cortex. A 4 mm × 4 mm coverslip was used to replace the skull. In vivo imaging of awake mice was performed using a two-photon laser scanning microscope (FVMPE-RS; Olympus) equipped with a pulsed Ti sapphire laser (Insight Deep See; Newport Spectra-Physics). For recording the protein response after LPS (Sigma, #L4130) injection, images were recorded every 10 minutes in a 500 μm × 500 μm field, with Z-stack scans at depths of 50–150 μm. hCRAFi-CCR2 was imaged using a 920 nm laser at a frequency of 32 Hz and a resolution of 512 x 512 pixels. To induce acute brain injury, a region approximately 10 μm in diameter was ablated intracortically through a cranial window using a two-photon laser. Photoablation was performed using a 0.8 W 920 nm laser for 92 ms. Images were acquired every half hour.

[0166] Immunohistochemistry

[0167] For immunohistochemistry, four months after AAV injection, mice were sacrificed by intraperitoneal injection of sodium pentobarbital (100-150 mg / kg), regardless of sex, and then perfused with PBS and 4% PFA (Beyotime) sequentially. The brain was fixed overnight in 4% PFA. Coronal brain sections with a thickness of 50 μm were prepared using a vibratory microtome (Leica VT1000S). The sections were first washed three times with PBS, then soaked in blocking buffer (PBS solution containing 5% BSA / 0.1% Triton X-100) for 2 hours, and then incubated overnight at 4°C with chicken anti-GFP antibody (diluted 1:500 in PBS solution containing 1% BSA / 0.1% Triton X-100; Abcam, Cat#ab13970, lot#1018753-8). Then, the sections were washed three times with PBS and incubated for 2 hours at 23-25°C with Alexa-488-labeled goat anti-chicken secondary antibody (1:1000 dilution, Abcam, Cat#ab150173). Afterward, the sections were washed three times with PBS and incubated with DAPI, then... TM Gold Antifade (Thermo Fisher, Cat#P36930) was used for sealing.

[0168] LPS-induced systemic inflammation model

[0169] Systemic inflammation was induced in mice by intraperitoneal injection of a single dose of LPS dissolved in physiological saline (0.5 mg / ml; Sigma, #L4130). The median lethal dose (LD50) of LPS in mice ranged from 10–25 mg / kg body weight (BW). We selected a dose of 2 mg / kg to induce systemic inflammation in mice and collected blood samples at 6 h and 24 h post-LPS stimulation. All blood samples were collected via cardiac sampling into EDTA-K2 tubes (Konsfi). Plasma was separated from the blood samples by centrifugation (2000 rpm, 15 min, 4 °C). The separated plasma was transferred to clean tubes and stored at -80 °C for subsequent analysis.

[0170] Fluorescence changes induced by plasma were measured. 50 μl of plasma from each sample was directly added to HEK cells expressing hCRAFi-CCR2 in 96-well plates. Fluorescence images were acquired before and after the addition of the CCR2 antagonist MK-0812 (4 μM). The fluorescence intensity of hCRAFi-CCR2 was normalized to FusionRed intensity. ΔF / F was calculated by subtracting the normalized fluorescence intensity from that of control cells.

[0171] Compound library screening

[0172] Pharmacological screening experiments were performed using HEK cells stably expressing hCRAFi-CCR2. HEK cells were transiently transfected with the pN1-hCRAFi-CCR2-P2A-FusinRed construct. Green-positive cells were selected by two rounds of FACS (BD FACSAriaFusion) at 10-13 day intervals. Positive cells were then cultured for 4 days for proliferation and frozen in 10% DMSO for subsequent experiments. Before screening, 96-well plates with glass bottoms (P96-1.5HN, Cellvis, for microscopy imaging) or ordinary 96-well plates (Corning, #3340, for FLIPR assays) were coated with Matrigel (BD Biosciences, 356235), and hCRAFi-CCR2-expressing cells were seeded at a density of 20,000 cells / well in DMEM containing 10% FBS. Cells were used 24 hours after seeding.

[0173] To validate the effectiveness of hCRAFi-CCR2 for drug screening, a commercially available chemokine receptor inhibitor library (TargetMol, #L7600, USA) containing 59 compounds was screened. For FLIPR-based screening, cells in 96-well plates were washed twice with ECS and then incubated in 50 μl of ECS (agonist mode) or 100 nM hCCL2 (antagonist mode). The drug stock solution from the library was dissolved in DMSO to obtain a 10 mM solution and stored at -70 °C. On the day of the screening experiment, the stored solution was diluted to 100 μM using ECS. During screening, 20 μl of working solution was added to each well (28.6 μM as the final concentration). After the FLIPR assay was completed, the data were exported and analyzed using Excel.

[0174] Data analysis, image processing, statistics and data visualization

[0175] Data were analyzed offline using NIS Elements Advance Research software (versions 5.21.00 and 5.30.00), Excel (Microsoft), OriginPro (2019b, OriginLab), and Fiji ImageJ 2.9.01 / 1.53t. The 3D model of the hCRAFi-CCR2 probe protein predicted by AlphaFold2 was processed using UCSF Chimera software (v1.17.3). For fluorescence imaging data of HEK cells, cultured mouse hippocampal neurons, and cortical astrocytes, cells containing ROIs, or (for neurons) cell bodies, or (for overnight long-term imaging of cultured astrocytes) the entire FOV and adjacent cell-free regions (as background) were manually selected using Elements Advance Research software or Fiji ImageJ 2.9.01 / 1.53t. The mean fluorescence intensity (F) of each ROI was extracted in the FITC and FusionRed channels (if applicable), and the corresponding background was subtracted.

[0176] ΔF / F0 was calculated using the formula [(F-F0) / F0], where F0 is the baseline fluorescence intensity. The signal-to-noise ratio (SNR) was calculated as the peak response divided by the standard deviation of the baseline fluorescence fluctuation. For two-photon imaging data analysis, ΔF / F was calculated after motion correction using the ImageJ plugin Timeseries Analyzer V3 (https: / / imagej.nih.gov / ij / plugins / time-series.html). All ΔF / F and SNR values ​​were calculated in Excel and presented in OriginPro software.

[0177] The dose-response curves of different chemokines were fitted using Hill's equation (F = Fmax * [ligand]^n / (EC50^n + [ligand]^n)), where F is the fluorescence intensity at the ligand-limited concentration, Fmax is the maximum fluorescence intensity, and EC50^n is the maximum fluorescence intensity. 50 is the half-maximal effect concentration, and n is the Hill coefficient.

[0178] Proteins were identified using LC-MS / MS analysis data files processed with Proteome Discoverer (version 2.5) and protein intensities were calculated using the Sequest HT search engine, with default parameters for label-free quantification and an FDR of <0.01 for both protein and peptide levels. Mouse (Mus musculus) proteome obtained from Uniprot was used for the search.

[0179] For in vivo imaging data processing, Bigstream registration (https: / / github.com / GFleishman / bigstream) was applied to register the field of view (FOV) deformation and 3D offset between frames during long-term in vivo imaging. Using an image volume at a fixed time point as a reference, the image volume at each time point was registered based on the GFP channel. Patch-like ROI identification was performed using Imaris 10.1.0 (Bitplane) with a three-step analysis scheme. First, the ΔF / F0 of the time-series images was calculated, and the maximum projection of ΔF / F0 was performed over the entire 20-hour recording. This projected image served as input to the surface model in the Imaris software, and intensity-based ROIs were identified and segmented. Finally, patch ROI regions were excluded, and regions outside the patches were segmented and annotated as ROIs outside the patches for further analysis.

[0180] Data are presented as mean ± SEM or as box plots with notches in the legend. Two-tailed Student's t-tests or one-tailed ANOVA tests were performed, along with Bonferroni's multiple comparison test. P-values ​​are expressed as: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

[0181] Example 2. Development of CCR2-based chemokine biodetector proteins

[0182] 2.1 Design and Screening

[0183] The human CCR2 receptor isoform CCR2B was used as the detection protein backbone for the CCL2 chemokine. cpGFP (LSSLI-cpGFP-NHDQL) flanked by adapter sequences was inserted into five different positions (sites 1-5) of the intracellular loop 3 (ICL3) of human CCR2B. Figure 1 , Figure 2 a) The obtained construct was expressed in HEK cells. In this embodiment and below, the screening of chemokine detector proteins was performed in HEK cells using hCCL2. The chimera with insertion site 3 exhibited better membrane localization and higher fluorescence intensity compared to other mutants produced. Figure 2 b), therefore it was chosen for further optimization, including changing the length of the adapter sequence, performing site-directed mutagenesis on the adapter and ICL3, etc. Figure 1 , Figure 2 c) This resulted in several variants. First, by altering the linker length, the first functional mutant probe protein, named hCRAFi-hCCR2_0.1 (LSSLI-cpGFP-NH, full-length SEQ ID NO: 15), was screened and showed a positive response of approximately 33% to hCCL2. Figure 2 de).

[0184] Using an improved automated drug delivery system, ligand solutions as low as 5 μL can be delivered to mammalian cells cultured in 96-well plates, while simultaneously recording changes in fluorescence intensity. Figure 3 Using this system, we further screened approximately 331 variants with mutated adapters and / or site-directed ICL3 mutations. A subset of the selected functional variants were used to further evaluate baseline brightness, ΔF / F, and signal-to-noise ratio (SNR) to select mutants with the optimal combination of several parameters. No significant differences were observed in cell membrane localization among the screened mutants. The substrate fluorescence and fluorescence response characteristics of the hCRAFi-hCCR2 mutants hCRAFi-hCCR2_0.2 (sequence shown in SEQ ID NO: 16) and hCRAFi-hCCR2_0.3 (sequence shown in SEQ ID NO: 17) were compared. Figure 4 ).

[0185]

[0186] To further improve cell membrane localization and promote in vivo expression, the aforementioned probe protein can be further fused at its C-terminus with the Golgi export transport signal (KGC, SEQ ID NO: 4) and the endoplasmic reticulum export (ER2, SEQ ID NO: 5) sequences. The amino acid sequence of hCRAFi-CCR2 after fusion with KGC and ER2 (i.e., hCRAFi-CCR2-KGC-ER2) is shown in SEQ ID NO: 2. Furthermore, we engineered the mCRAFi-CCR2 variant (SEQ ID NO: 19; m indicates mouse, and since all screening was performed with hCCL2, this variant may improve the response to mCCL2) by replacing hCCR2B with the mouse CCR2 receptor, and obtained a CCL2-insensitive variant, named hCRAFi-CCR2null, by mutating residues Y49 and Y120 in the CCL2 binding pocket (studies have reported this to produce a nonfunctional CCR2 receptor). Figure 1 ).

[0187] After expression in HEK cells, hCRAFi-CCR2 and mCRAFi-CCR2 exhibited cell membrane localization and both showed stable positive fluorescent responses to hCCL2 and mCCL2. Figure 5-6 The response of hCRAFi-CCR2 to hCCL2 was 1.25 times greater than that to mCCL2, and the fluorescence dynamic range of mCRAFi-CCR2 to mCCL2 was 1.7 times lower than that of hCRAFi-CCR2 (105±11% and 181±12%, respectively). Figure 6 The half-maximal effect concentrations (EC50) of hCCL2 and mCCL2 in response to hCCL2. 50 It is comparable, within the range of 7-10 nM. Figure 7 hCRAFi-CCR2null exhibits good cell membrane localization and does not respond to mCCL2 and hCCL2, or other CCR2 ligands (including h / mCCL7, h / mCCL8, and hCCL13). Figure 8 Therefore, hCRAFi-CCR2null can be used as a negative control for hCRAFi-CCR2 in subsequent in vitro and in vivo experiments.

[0188] 2.2 Development of Ratiometric Probe Proteins

[0189] Considering that increased basal expression levels during long-term in vivo imaging may lead to increased GFP intensity, which would complicate the interpretation of the hCRAFi-CCR2 signal, we also explored potential strategies to transform the intensity-metric probe protein hCRAFi-CCR2 into a ratiometric probe protein. Therefore, two constructs were created: pN1-hCRAFi-CCR2-P2A-FusionRed (amino acid sequence SEQ ID NO: 8) and pN1-hCRAFi-CCR2-Linker-FusionRed (amino acid sequence SEQ ID NO: 9), as follows... Figure 9 As shown. The advantage of the P2A-construct is that, since FusionRed itself does not bind to the probe protein after translation, it has a lower possibility of interfering with the probe protein's membrane localization. When expressed in 293T cells, hCRAFi-CCR2-KGC-FusionRed-ER2 localizes to the cell membrane, and pN1-hCRAFi-CCR2-KGC-FusionRed-ER2 has similar EC50 values ​​to hCRAFi-CCR2-KGC-ER2 (1KGC adapter: 10.74±1.43 nM; 3KGC adapter: 6.29±3.00 nM; hCRAFi-CCR2: 7.56±0.85 nM); Figure 10 These results indicate that these ratiometric probe proteins can be used for long-term imaging without concern about the expression levels of the probe proteins.

[0190] Example 3. In vitro characterization of hCRAFi-CCR2 in mammalian cells

[0191] Since hCRAFi-CCR2 exhibits higher fluorescence changes in response to hCCL2 and mCCL2 than mCRAFi-CCR2, in this embodiment, the functional properties of hCRAFi-CCR2 in mammalian cells are characterized and verified, with hCRAFi-CCR2null and wild-type hCCRB2 as controls.

[0192] Accordingly, after transient expression of hCRAFi-CCR2 in HEK cells, we characterized the following aspects: localization, spectral properties, response kinetics, selectivity, affinity, and downstream signaling activation, and evaluated the availability of hCRAFi-CCR2 in nerve cells.

[0193] 3.1 Membrane localization, spectral characteristics and response kinetics

[0194] The hCRAFi-CCR2 probe protein exhibits cell membrane localization and no intracellular accumulation was observed, demonstrating a functional response along the membrane of a single cell. Figure 11 The fluorescence spectrum of hCRAFi-CCR2 was not affected by the binding of hCCL2, and was characterized by an excitation peak at 500 nm, an emission peak at 514 nm, and an isoabsorption point at approximately 426 nm. Figure 12 To evaluate the kinetics of the fluorescence response, we performed a bath perfusion experiment using hCCL2 to simulate the volumetric diffusion of chemokines in tissues. In this scenario, the fluorescence response of hCCL2 exhibited relatively slower kinetics compared to neurotransmitter indicators due to the combined effects of ligand diffusion and probe protein kinetics. The rise time constant (τ) was significantly slower when hCCL2 and the CCR2 antagonist MK-0812 were applied, respectively. on The decay time constant is 2.4s, and the decay time constant is τ. off ) is 40s ( Figure 13 However, single-cell microdose administration of the ligand produced a faster fluorescence response, τ on It is 19ms, τ off The amplitude of the fluorescence change was 190 ms, although it was more than ten times smaller than that of immersion perfusion. Figure 14 ).

[0195] 3.2 Selectivity and Affinity

[0196] Redundancy exists in the binding patterns of chemokines and their receptors, meaning that multiple chemokines can bind to the same receptor, and the same chemokine can bind to multiple receptors. While CCL2 is the primary ligand for CCR2, several other chemokines, such as CCL7 (MCP-3), CCL8 (MCP-2), and CCL13 (MCP-4), can bind CCR2, albeit with approximately 10-fold lower affinity. In fact, when testing the fluorescence response of hCRAFi-CCR2 to other chemokines, none of the non-CCR2 ligands besides CCL7, CCL8, and CCL13 induced any fluorescence change. Figure 15 Similarly, mouse MCPs (it should be noted that there are no analogues for hCCL13 (MCP-4) in mice) showed a trend toward fluorescence response amplitudes similar to those of human CCLs, but with smaller amplitudes. Figure 15 At the same concentration of 100 nM, hCCL2 induced the largest response among all tested chemokines. Figure 15 We further measured the EC of these CCR2 ligands in the range up to approximately 600 nM. 50 ( Figure 16 hCCL2 increases the fluorescence intensity of hCRAFi-CCR2 in a concentration-dependent manner, EC 0.5. 50 The value was 11.2 ± 0.1 nM, which is consistent with the EC50 values ​​obtained by measuring wild-type hCCR2A and hCCR2B in oocytes (10–15 nM) and HEK cells (7–30 nM) using a calcium ion mobilization assay. 50Similar (for EC50 of oocytes, see Charo, IF et al., Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Proc. Natl. Acad. Sci. 91, 2752-2756 (1994); for EC50 of HEK cells, see Wong, LM et al., Organization and Differential Expression of the Human Monocyte Chemoattractant Protein 1 Receptor Gene: EVIDENCE FOR THE ROLE OF THE CARBOXYL-TERMINAL TAIL IN RECEPTORTRAFFICKING. J. Biol. Chem. 272, 1038-1045 (1997)). Correspondingly, hCRAFi-CCR2 has approximately 2-2.5 times lower affinity for hCCL7, hCCL8, and hCCL13. Figure 16 For mouse CCR2 ligands, we found that hCRAFi-CCR2 was 7.6 times more sensitive to mCCL2 than to mCCL7. Figure 16 ).

[0197] The results above show that the hCRAFi-CCR2 probe protein reproduces the pharmacological characteristics of the CCR2 receptor, indicating that the dynamic range of this probe protein is within the biologically relevant range of CCL2 concentration.

[0198] 3.3 Downstream signal transduction activation

[0199] To test whether the hCRAFi-CCR2 probe protein is coupled to downstream pathways, we examined whether hCRAFi-CCR2 activation affects cytoplasmic calcium signaling. HEK cells were co-transfected with the wild-type CCR2 receptor or hCRAFi-CCR2 and the red calcium indicator R-GECO1.2. In cells expressing CCR2, application of CCL2 elicited a robust fluorescent response from the calcium probe protein, while no increase in calcium was observed in cells transfected with hCRAFi-CCR2. Figure 17This result indicates that activation of the hCRAFi-CCR2 probe does not induce downstream calcium signaling. Furthermore, we found that the hCRAFi-CCR2 probe exhibited stable membrane fluorescence during 2 hours of co-incubation with CCL2, suggesting that desensitization or internalization of the hCRAFi-CCR2 probe after activation is negligible. Figure 18 ).

[0200] 3.4 Characterization in nerve cells

[0201] Next, we characterized the hCRAFi-CCR2 probe protein in cultured hippocampal neurons and astrocytes to assess its in vivo imaging availability in the brain. Additionally, we used a null mutant as a negative control; mCCL2 was used in the characterization experiments to match in vivo conditions in mice. When expressed in cultured neurons using recombinant adeno-associated virus (rAAV) under the regulation of the neuron-specific hSyn promoter, hCRAFi-CCR2 and the hCRAFinull mutant were uniformly distributed across the cell membrane, exhibiting a higher response than in the cell body, particularly at neural axons. hCRAFi-CCR2 was also expressed in cultured astrocytes using the astrocyte-specific GfaABC1D promoter. The hCRAFi-CCR2 probe protein in both neurons and astrocytes stably responded to mCCL2, with maximum ΔF / F ratios of 181.1 ± 20.7% and 340.7 ± 13.2%, respectively. However, no change in fluorescence was measured in the hCRAFi-CCR2 null mutant probe protein. Figures 19-20 In cultured neurons, the EC50 value of hCRAFi-CCR2 was 6.07 ± 1.06 nM. Figure 19 c) Similar to those measured in HEK cells in Example 3.2. In astrocytes, hCRAFi-CCR2 was 25 times more sensitive to mCCL2 than to mCCL7 (EC50, mCCL2 1.91 ± 0.10 nM, mCCL7 47 ± 10 nM); Figure 20 c). Furthermore, mCCL8 did not elicit a significant fluorescence response in astrocytes. These results indicate that hCRAFi-CCR2 is more selective for mCCL2 than for mCCL7 and mCCL8 in mouse astrocyte cultures, and therefore can be used as an mCCL2 detector protein in mice. In cultured neurons and astrocytes, prolonged co-incubation with hCCL2 was also observed to not affect the fluorescence intensity or membrane localization of the detector protein, consistent with HEK cells, and also indicating no significant photobleaching during imaging at 10-minute intervals for 2 hours. Figure 21 ).

[0202] Example 4. Detection of endogenous mCCL2 release

[0203] 4.1 Detection of endogenous mCCL2 release in primary cultured astrocytes

[0204] Since previous examples demonstrated the high selectivity and sensitivity of hCRAFi-CCR2 in detecting mCCL2 in astrocytes, this example evaluated whether hCRAFi-CCR2 could record the real-time release of endogenous mCCL2 from primary cultured mouse astrocytes. Studies have shown that infection can induce the release of CCL2 from multiple cell types in vitro, including astrocytes, pericytes, microglia, and neurons. To mimic infection, cultured mouse astrocytes expressing hCRAFi-CCR2 or the hCRAFi-CCR2null mutant were treated with lipopolysaccharide (LPS), double-stranded RNA (Poly(I:C)), and CpG DNA (ODN 1668) to activate the endogenous Toll-like receptor TLR4, TLR3, and TLR9 pathways, respectively.

[0205] Approximately 2 hours after treatment with these inflammatory stimuli, the fluorescence intensity in astrocytes expressing hCRAFi-CCR2 gradually increased compared to control cells that did not receive stimulation. Figure 22 ab). Under the experimental conditions tested, the increase in fluorescence induced by LPS was 2.6-fold and 1.7-fold faster than that induced by poly(I:C) and ODN1668, respectively, both of which were statistically significant. Figure 22 c). This increase in fluorescence could be quenched by the CCR2 antagonist MK-0812, indicating the presence of MCP in the astrocyte culture medium, most likely mCCL2 ( Figure 22 d). The differences in fluorescence changes between different stimulus conditions were not biologically or statistically significant. Figure 22 e). Similar results were obtained using the mutant probe protein hCRAFi-CCR2_0.2 in the same experiment, demonstrating the reproducibility of the above results. Figure 23 Conversely, these inflammatory stimuli did not induce fluorescence changes in hCRAFi-CCR2, further demonstrating that the observed increase in hCRAFi-CCR2 fluorescence was not due to elevated expression levels or other factors (e.g., pH, temperature). Figure 22 e).

[0206] To evaluate the availability of hCRAFi-CCR2 in quantifying inflammatory responses, astrocytes were treated with gradient concentrations of LPS (10–10000 ng / mL) or Poly(I:C) (1–100 μg / mL) and serial live-cell imaging was performed for 17 h. Within the tested concentration range, LPS treatment showed similar kinetics and fluorescence amplitudes, indicating a saturation response of cells to inflammatory stimuli. For Poly(I:C), we observed that the fluorescence response was 1.2–1.5 times slower and the amplitude was approximately 1.5 times lower at the lowest concentration compared to 10 and 100 μg / mL (7.5 h / 142% and 6 h / 134%, respectively) (i.e., a rising half-life of 9.2 h and a ΔF / F of 93% at 1 μg / mL).

[0207] Because hCRAFi-CCR2 is sensitive to all MCPs, it was necessary to verify whether the increased fluorescence observed in astrocytes after inflammatory stimulation was caused by the release of MCP-1 (mCCL2). To this end, proteomic analysis was performed on the collected culture medium at the end of the experiment (approximately 24 h post-stimulation). Quantitative untargeted mass spectrometry revealed a 30-fold increase in mCCL2, and CXCL1, CXCL3, and CXCL10 were also present in the LPS-treated samples, while the CCL5 concentration remained unchanged. mCCL7 was not detected under our experimental setup. These results indicate that hCRAFi-CCR2 is suitable for real-time detection of the relative levels of endogenous mCCL2 release in cultured astrocytes during 17 h of continuous imaging (10 min imaging frequency).

[0208] 4.2 Detection of endogenous mCCL2 release in mouse plasma

[0209] During systemic inflammation, CCL2 levels in the blood of mice increased after LPS injection. To test whether hCRAFi-CCR2 could detect biologically relevant concentrations of mCCL2 in vivo, we intraperitoneally injected mice with an appropriate amount of LPS (2 mg / kg, see Example 1 for details) and collected blood samples from mice at 6 and 24 hours post-injection. The plasma was then added to HEK cells expressing hCRAFi-CCR2. Figure 22 Hi). We found that plasma collected from mice treated with LPS for 6 h and 24 h both increased the fluorescence intensity of cells expressing hCRAFi-CCR2. Figure 22 i). Applying MK-0812 can eliminate this fluorescence change (i). Figure 22 (hi), indicating its specificity. Then, the plasma-induced ΔF / F was calculated, and the calculated values ​​were mapped to a standard mCCL2 dose-response curve obtained using parallel experiments with the same cell culture. Figure 22(j) The estimated concentrations of mCCL2 in plasma at 6 hours and 24 hours after LPS treatment were 12 nM and 5 nM, respectively. These concentrations are similar to the range of CCL2 levels reported in other studies using LPS stimulation (25–40 ng / ml corresponding to 3.5 nM–5.5 nM). 1,2 Furthermore, the concentration measured at 24 hours was lower than that measured at 6 hours, which is consistent with literature reports that CCL2 levels in mouse blood peak 3-6 hours after LPS stimulation and then gradually decrease. 2,3 The above results indicate that the hCRAFi-CCR2 probe protein can be used to quantify mCCL2 in biological fluids, similar to ELISA.

[0210] Example 5. In vivo monitoring of dynamic changes in endogenous MCP in the mouse brain

[0211] In this embodiment, we verified whether hCRAFi-CCR2 could measure the dynamic changes in mCCL2 release in awake mice. Under normal physiological conditions, CCL2 levels in the brain are low, but increase after inflammatory stimulation. A systemic inflammation model using LPS was used to study the spatiotemporal patterns of CCL2 release in the mouse cerebral cortex. The hCRAFi-CCR2 probe protein was expressed in astrocytes of the mouse visual cortex. After 3 weeks, a strong green fluorescence signal was observed under a two-photon microscope, and the specific localization of the probe protein expression was further confirmed by immunohistochemistry of brain tissue. Within 5 hours after LPS injection, the fluorescence intensity of hCRAFi-CCR2 began to gradually increase, reaching approximately 40 ± 10% of baseline in the following 15 hours (n = 3 mice). Figure 24 Conversely, under the same experimental conditions, the fluorescence of the hCRAFi-CCR2null detector protein initially decreased linearly. Figure 24 We further characterized the spatial distribution of the signal changes in the hCRAFi-CCR2 probe protein. After LPS treatment, the increase in the probe protein response exhibited spatial heterogeneity, displaying a patchy distribution that could be segmented based on the response level and surrounding region. Figure 25 a). This spatial heterogeneity in response is not due to differences in the expression of the probe protein, because the basal fluorescence inside and outside the plaque is similar ( Figure 25 b). Analysis of the characteristics of these signal patches, including maximum response, size, and onset time (above baseline), supports the conclusion that the dynamic changes of chemokines exhibit rich spatial and temporal complexity. Figure 25 c). The above results indicate that the probe protein of the present invention can detect spatiotemporally specific dynamic changes of mCCL2 in awake mice.

[0212] Further investigation was conducted to determine whether the hCRAFi-CCR2 probe protein could also reflect mCCL2 release in an acute brain injury model. To precisely control the site of injury, two-photon laser ablation was employed (see Davalos, D. et al., ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752-758 (2005)). We observed that the fluorescence intensity of hCRAFi-CCR2 began to increase 2.5 h after photoablation, reaching a plateau around 4 h, with a ΔF / F ratio of approximately 50% (…). Figure 26 The timing pattern of mCCL2 release is quite different from that of ATP, which can be released rapidly within 20 minutes after injury.

[0213] Example 6. Medium- to high-throughput drug screening assay based on hCRAFi-CCR2 probe protein

[0214] Since hCRAFi-CCR2 has pharmacological characteristics similar to its parent wild-type CCR2 receptor, we explored the possibility of using hCRAFi-CCR2 to screen drugs targeting CCR2 in this embodiment.

[0215] First, a cell line expressing hCRAFi-CCR2 was constructed. HEK cells were transiently transfected with the pN1-hCRAFi-CCR2-P2A-FusinRed construct. Green-positive cells were selected by two rounds of FACS (BD FACSAria Fusion) at 10-13 day intervals. The positive cells were then cultured for 4 days for proliferation and cryopreserved for subsequent experiments.

[0216] To improve screening throughput, a FLIPR (fluorescence imaging plate reader) instrument was used to detect changes in hCRAFI-CCR2 fluorescence during automated compound library addition. The screening platform was validated using hCCL2 and MK-0812 as positive and negative controls (z-factor = 0.8, n = 3), demonstrating sufficient sensitivity for high-throughput screening. We then screened a library of 59 compounds targeting chemokine receptors in agonist and antagonist modes. Ten compounds were found to antagonize the hCRAFI-CCR2 response, and one compound acted as an agonist (J-11383). Figure 27 a). We then used a wide-field microscope ( Figure 27 These results were validated by cd. Of these antagonists, 9 out of 10 were reported to antagonize the CCR2 receptor (cd). Figure 27b) indicates that the probe protein perfectly retains the pharmacological properties of wild-type CCR2. Only one known CCR2 inhibitor, cenicriviroc, was not screened during the screening process because its endogenous autofluorescence is too high to be suitable for detecting its effect on the fluorescence of the probe protein. During the screening process, a previously identified CXCR2 antagonist compound, AZD-5069, was newly discovered, which can reduce the fluorescence intensity of hCRAFi-CCR2 at a micromolar concentration (IC50 = 2.01 ± 0.33 μM). Figure 27 e). Furthermore, J-113863, considered an antagonist of CCR1 and CCR3, was found to increase the fluorescence intensity of hCRAFI-CCR2, with an EC50 of 4.1 ± 2.0 μM. Figure 27 f). This activation can be inhibited by the CCR2 antagonist MK-0812, indicating that the increase in fluorescence intensity is due to the activation of the probe protein. Figure 27 g). In fact, our findings are consistent with previously reported studies showing that J113863 can activate CCR2 and CCR5. In summary, hCRAFi-CCR2 can serve as a reliable tool for flux pharmacological screening of CCR2 receptors.

[0217] Example 7. Engineered hCRAFi-CCR2-based detector proteins based on other chemokine receptors and their application in drug screening.

[0218] 7.1 Construction of detector proteins based on other chemokine receptors

[0219] Furthermore, detector proteins based on other chemokine receptors (hCCR5, mCCR1, mCCR3, mCCR4, mCCR5, mCCR6, mCCR9, mCXCR4) were constructed. This was achieved by transplanting the TM5-adaptor-cpEGFP-adaptor-TM6 motif (SEQ ID NO:11) from the hCRAFi-CCR2 detector protein onto other chemokine receptors. Figure 28 (ab) The chemokine receptor region at residues 5.59 to 6.34 was replaced with the hCRAFi-CCR2 sequence. The resulting transplanted variant was then expressed in HEK293 cells for functional testing.

[0220] Of the eight transplantation variants, three showed a strong response to their ligands (ΔF / F>50%). Figure 28c). The epigenetic affinity of these probe proteins for their respective endogenous ligands was further evaluated. We found that each transplanted probe protein had a high affinity in the nanomolar range: CRAFi-mCCR3 (17.72 ± 3.30 nM), CRAFi-mCXCR4 (5.60319 ± 1.10287 nM), and CRAFi-mCCR6 (23.05593 ± 3.00151 nM). Figure 28 df).

[0221] 7.2 Chemokine receptor-based detector proteins for drug screening

[0222] Similarly, we also used the probe proteins constructed in Example 7.1 for high-throughput drug screening. Compound libraries were screened using probe proteins based on mCCR3, mCCR6, and mCXCR4, and the results are as follows... Figure 29 As shown in the table, the screening results show that 8 CXCR4-specific compounds, 5 CCR3-specific compounds, and 3 CCR2-specific compounds were identified using the above-mentioned probe protein, indicating that the constructed probe protein is also suitable for high-throughput pharmacological screening of the corresponding chemokine receptors.

[0223] Sequence Summary

[0224] Information on some sequences involved in this invention is provided in the table below.

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237] References

[0238] 1. Cerri, C. et al. The Chemokine CCL2 Mediates the Seizure-enhancing Effects of Systemic Inflammation. J. Neurosci. 36, 3777-3788 (2016).

[0239] 2. Liu, J. et al. Screening cytokine / chemokine profiles in serum and organs from an endotoxic shock mouse model by LiquiChip. Sci. China Life Sci. 60, 1242-1250 (2017).

[0240] 3. Thompson, W.L., Karpus, W.J. & Van Eldik, L.J. MCP-1-deficient mice show reduced neuroinflammatory responses and increased peripheral inflammatory responses to peripheral endotoxin insult. J. Neuroinflammation 5, 1-13 (2008).

Claims

1. A detection protein comprising a chemokine receptor and a fluorescent reporter protein inserted into the chemokine receptor, wherein the fluorescent reporter protein is inserted into a third intracellular loop between a fifth transmembrane region and a sixth transmembrane region of the chemokine receptor; The chemokine receptors are selected from CCR2, CCR3, CCR6, and CXCR4; Optionally, the fluorescent reporter protein is a cyclically rearranged fluorescent protein.

2. The detection protein according to claim 1, wherein the cyclic rearrangement fluorescent protein is selected from cyclic rearrangement green fluorescent protein, cyclic rearrangement red fluorescent protein, cyclic rearrangement yellow fluorescent protein, and cyclic rearrangement blue fluorescent protein.

3. The detector protein according to claim 1 or 2, wherein the reporter protein is a cyclic rearranged green fluorescent protein; Preferably, the cyclically rearranged green fluorescent protein has the amino acid sequence shown in SEQ ID NO:

3.

4. The detector protein according to any one of claims 1-3, wherein the chemokine receptor is human CCR2B or a variant thereof, and the fluorescent reporter protein is inserted between amino acid residues 235 and 236 from the N-terminus to the C-terminus of the chemokine receptor. Optionally, the detector protein includes, from the N-terminus to the C-terminus, an amino acid sequence from position 1 to 235 of the chemokine receptor operably linked to the fluorescent reporter protein, and the fluorescent reporter protein operably linked to the amino acid sequence from position 236 onwards of the chemokine receptor.

5. The probe protein of claim 4, wherein the human CCR2B or a variant thereof has an amino acid sequence as shown in SEQ ID NO: 20 or 21.

6. The detector protein according to any one of claims 1-3, wherein the chemokine receptor is mouse CCR2, and the fluorescent reporter protein is inserted between amino acid residues 248 and 249 from the N-terminus to the C-terminus of the chemokine receptor. Optionally, the detector protein includes, from the N-terminus to the C-terminus, amino acid sequences 1-248 of the chemokine receptor operably linked to the fluorescent reporter protein, and the fluorescent reporter protein operably linked to amino acid sequences from position 249 onwards of the chemokine receptor.

7. The detector protein according to any one of claims 1-6, wherein the fluorescent reporter protein is inserted into the chemokine receptor via an N-terminal adapter and a C-terminal adapter; Optionally, the amino acid sequence of the N-terminal linker is LSSLI, LSSSV, or LEEGG; Optionally, the amino acid sequence of the C-terminal linker is NHDQL, NH, or ID.

8. The detection protein according to claim 7, wherein: (a) The amino acid sequence of the N-terminal linker is LSSLI, and the amino acid sequence of the C-terminal linker is NHDQL or NH; or (b) The amino acid sequence of the N-terminal linker is LSSSV or LEEGG, and the amino acid sequence of the C-terminal linker is ID.

9. The detector protein according to any one of claims 1-8, wherein its C-terminus further comprises a signal peptide sequence, such as a Golgi output signal (KGC) sequence and an endoplasmic reticulum output signal (ER2) sequence. Optionally, the probe protein comprises 1 to 3 copies of the KGC sequence.

10. The probe protein according to claim 9, wherein the KGC sequence has the amino acid sequence shown in SEQ ID NO: 4, and the ER2 sequence has the amino acid sequence shown in SEQ ID NO:

5.

11. The detector protein according to claim 9 or 10, wherein its C-terminus further comprises an amino acid sequence of an indicator fluorescent protein that does not overlap with the fluorescence spectrum of the fluorescent reporter protein; Optionally, the indicator fluorescent protein is a red fluorescent protein, such as FusionRed; Preferably, the indicator fluorescent protein has an amino acid sequence as shown in SEQ ID NO:

10.

12. The detector protein of claim 11, wherein the KGC sequence is operatively linked to the indicator fluorescent protein, and the indicator fluorescent protein is operatively linked to the ER2 sequence.

13. The detector protein according to any one of claims 1-12, wherein the fluorescence intensity changes upon binding to a chemokine targeted by the chemokine receptor, wherein ΔF / F is about 50% or higher, about 100% or higher, about 150% or higher, about 200% or higher, or about 250% or higher.

14. The detector protein according to any one of claims 4-13, comprising one of the following: (a) An amino acid sequence as shown in SEQ ID NO: 1 or 2; (b) An amino acid sequence as shown in SEQ ID NO: 15, 16 or 17; (c) An amino acid sequence as shown in SEQ ID NO: 8 or 9; (d) The amino acid sequence as shown in SEQ ID NO: 19; (e) An amino acid sequence encoded by a nucleotide sequence as shown in SEQ ID NO: 6 or 7; or (f) has an amino acid sequence that is at least 95% sequence identical to any one of (a)-(e).

15. The detector protein according to any one of claims 1-3, wherein the chemokine receptor is mouse-derived CCR3 or a variant thereof, and the chemokine receptor having amino acid residues from the N-terminus to the C-terminus 224th to 242nd positions partially replaced by a fluorescent reporter protein. Optionally, the detector protein includes, from the N-terminus to the C-terminus, amino acid sequences 1-223 of the chemokine receptor operably linked to the portion containing the fluorescent reporter protein, and the portion containing the fluorescent reporter protein operably linked to the amino acid sequence of the chemokine receptor from position 243 onwards. Optionally, the portion containing the fluorescent reporter protein has the sequence shown in SEQ ID NO:

11.

16. The detector protein according to any one of claims 1-3, wherein the chemokine receptor is mouse-derived CXCR4 or CCR6 or a variant thereof, and amino acid residues 227-245 from the N-terminus to the C-terminus of the chemokine receptor are partially replaced by a fluorescent reporter protein. Optionally, the detector protein includes, from the N-terminus to the C-terminus, amino acid sequences 1-226 of the chemokine receptor operably linked to the portion containing the fluorescent reporter protein, and the portion containing the fluorescent reporter protein operably linked to the amino acid sequence of the chemokine receptor from position 246 onwards. Optionally, the portion containing the fluorescent reporter protein has the sequence shown in SEQ ID NO:

11.

17. The probe protein according to claim 15 or 16, comprising an amino acid sequence as shown in SEQ ID NO: 12, 13 or 14.

18. A nucleic acid molecule comprising a nucleotide sequence encoding a detector protein according to claims 1-17.

19. A nucleic acid molecule comprising a nucleotide sequence encoding a detector protein according to claims 1-10 and a nucleotide sequence encoding an indicator fluorescent protein whose fluorescence spectrum does not overlap with that of the fluorescent reporter protein, the two nucleotide sequences being linked by a nucleotide sequence encoding a self-cleaving peptide. Optionally, the self-cleaving peptide is selected from P2A, F2A, T2A, and E2A.

20. A vector comprising a nucleic acid molecule according to claim 18 or 19.

21. The vector according to claim 20, wherein it is an adenovirus vector or an adeno-associated virus vector.

22. A host cell comprising a nucleic acid molecule according to claim 18 or 19 or a vector according to claim 20 or 21; Optionally, the host cell is a mammalian cell, such as a human cell, including neurons and astrocytes.

23. A method for detecting changes in the concentration of chemokines in a sample, comprising: Culturing the host cells according to claim 22 under suitable conditions; Add the sample to be tested into the cell culture and record the fluorescence intensity F0 before the sample is added, the fluorescence intensity at the first time point after the sample is added, and the fluorescence intensity at the second time point. The fluorescence intensity change ΔF / F relative to F0 at the first and second time points were analyzed to determine the concentration change of the chemokine in the sample at the first time point relative to the second time point.

24. A method for detecting the level of a chemokine in a sample, comprising: Culturing the host cells according to claim 22 under suitable conditions; Add the sample to be tested into the cell culture and record the fluorescence intensity F0 before adding the sample and the fluorescence intensity after adding the sample. The fluorescence intensity change ΔF / F0 after sample addition is analyzed to determine the level of chemokines in the sample.

25. The method according to claim 23 or 24, wherein the sample to be tested is selected from cell cultures or extracts thereof, biological tissues or extracts thereof, and biological fluids or extracts thereof.

26. A kit comprising the vector according to claim 20 or 21 or the host cell according to claim 22.

27. A method for monitoring spatiotemporal patterns of chemokine release in cells, comprising: To induce cells to express the probe protein according to any one of claims 1-16; Record the fluorescence intensity of the detector protein over time within one or more time intervals in the field of view; and The change in fluorescence intensity over time, ΔF / F, is analyzed for one or more fields of view to obtain the spatiotemporal pattern of chemokine release. Optionally, the time interval is approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 20 hours, or longer.

28. The method of claim 27, wherein the cells express the probe protein via transient transfection, adenovirus transfection, or adeno-associated virus transfection.

29. The method according to claim 27 or 28, wherein the cells are induced by a stimulant to secrete chemokines, said stimulant being a pro-inflammatory stimulant.

30. A method for monitoring the secretion of chemokines from organs, tissues or cells expressing the detector protein according to any one of claims 1-16, comprising recording the fluorescence intensity of the detector protein over time.

31. The method of claim 30, wherein the organ, tissue, or cell is derived from the brain or a portion thereof, such as the cortex.

32. A method for screening probe proteins for detecting chemokines, comprising: Inducing cells to express the target protein to be screened; A chemokine was applied to the cells and the change in fluorescence intensity ΔF / F of the detector protein was recorded; and The detection protein was determined to be suitable for detecting chemokines by a ΔF / F ≥ 50%.

33. A method for screening antagonists and / or agonists of chemokine receptors using the probe protein of claims 1-16, comprising: Construct a cell line expressing the probe protein; Add the compound library to the cell line; and The change in fluorescence intensity ΔF / F of the probe protein was detected; Where △F / F>0 indicates that the compound is an agonist, and △F / F<0 indicates that the compound is an antagonist.

34. A fluorescence-based drug screening system comprising a drug delivery module, a response module, and an imaging module, wherein the drug delivery module is a multi-channel drug delivery module, the response module comprises a cell culture device and cells expressing the detector protein according to any one of claims 1-16, and the imaging module records the fluorescence intensity of the cells before and after drug delivery in real time.

Images