CN112858653A - High throughput screening method for screening compounds that affect collagen stability - Google Patents
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Abstract
The invention discloses a high-throughput screening method for screening compounds influencing collagen stability, which is based on fluorescence resonance energy transfer or reporter gene segment complementation, after plasmid co-transfections of cells, P4HA1 inhibitor s4682 with different concentrations is added into a culture medium, an ELIAS reader detects FRET after 24h of medicine addition, or fluorescence data or chemiluminescence, FRET, Venus segment complementation and Gluc segment complementation test measure the IC50 that s4682 inhibits the interaction between collagen chain molecules, and the practicability of the screening method is verified by the above results. Also discloses an application of the method in the quantitative analysis of the drug effect of the drug for determining the stability of the triple helix of the collagen and an application in the quantitative analysis of the compound for determining the stability of the triple helix of the collagen.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a high-throughput screening method for screening compounds influencing collagen stability.
Background
Tissue cell damage caused by any cause can result in tissue cell degeneration, necrosis and inflammation. If the damage is small, the normal parenchymal cells around the damaged cells will undergo proliferative repair, which can completely restore normal structure and function. However, if the damage is large or repeated beyond the ability of the parenchymal cells surrounding the damage to regenerate, the extracellular matrix will proliferate to repair the defective tissue, i.e., the pathological change of fibrosis occurs. Fibrosis is thus essentially a repair response after tissue has been damaged to preserve the relative integrity of the tissue and organs. The hyperplastic fibrous connective tissue, although repairing the defect, does not possess the structure and function of the parenchymal cells of the original organ. If this repair reaction is excessive, too strong and uncontrolled, it can cause fibrosis and lead to reduced organ function.
In the world, tissue fibrosis is the main cause of disability and death caused by many diseases, and statistics show that nearly 45% of patients who are fatal to various diseases can be attributed to tissue fibrosis diseases, such as liver failure caused by hepatic fibrosis or liver cancer, respiratory failure caused by pulmonary fibrosis, renal failure caused by renal fibrosis, uremia and the like, which are high-fatal complications.
After organ injury, some cells capable of producing collagen are transformed into fibroblast type under the stimulation of factors and synthesize a large amount of collagen, such as stellate cells, liver sinus endothelial cells and the like in the liver, primitive mesenchymal cells, alveolar type II epithelial cells, arterial vascular endothelium and smooth muscle cells and the like in the lung, cells such as mesangial cells and renal interstitial fibroblasts are activated and produce collagen fibers, and fibrosis can be caused. The types of such collagen fibers are mainly I, III and type IV. The synthetic steps of collagen type I and type III molecules can be targeted as anti-organ fibrosis drugs.
It has been found that there are some cytokines involved in the process of generating organ fibrosis, such as transforming growth factor TGF-beta, platelet growth factor PDGF, connective tissue growth factor CTGF, gamma-interferon (IFN-gamma), tumor necrosis factor TNF-alpha, etc., wherein TGF-beta, PDGF and CTGF are the common indicators of organ fibrosis detection in laboratory and clinic, and are the common factors used in laboratory to activate fibroblast-like cells to synthesize collagen. Cytokines increase the transcription of collagen prolyl hydroxylase and lysine hydroxylase, which catalyze proline/lysine hydroxylation in the collagen peptide chain, resulting in type I collagen (. alpha.1)2 Alpha 2 triple helix and type III collagen (alpha 1)3Triple helix stability is increased.
Based on the above mechanisms, there is a need to develop a high throughput screening method for screening compounds that affect collagen stability.
Disclosure of Invention
The invention aims to solve the technical problem of providing a high-throughput screening method for screening compounds influencing collagen stability, wherein the stability of the triple helix is reflected by the detection of the interaction condition between collagen peptide molecules by a Fluorescence Resonance Energy Transfer (FRET) or reporter gene (Venus fluorescent protein or Gaussia luciferase) fragment complementation method.
In order to solve the technical problems, the first technical scheme adopted by the invention is as follows: a high throughput screening method for screening compounds that affect collagen stability is provided, comprising the steps of:
step 1: constructing a fusion expression plasmid mVenus-Col1A1 of human type I collagen alpha 1 and mVenus without signal peptide, a fusion expression plasmid mVenus-Col1A 2 of human type I collagen alpha 2 and mApple without signal peptide, a fusion expression plasmid mVenus-Col3A1 of human type III collagen alpha 1 and mVenus without signal peptide, a fusion expression plasmid mVenus-Col3A1 of human type III collagen alpha 1 and mApple without signal peptide, and obtaining a plasmid for transfection by transforming DH5 alpha bacteria and a plasmid extraction step;
step 2: co-transfecting mVenus-Col1A1 and mApple-Col1A2 or mVenus-Col3A1 and mApple-Col3A1 by using fibroblasts, and adding P4HA1 inhibitor s4682 with the concentration of 0.01-500 mu M12 h after the cells are co-transfected by plasmids;
and step 3: after the medication intervention is carried out for 24-48 h, the FRET is detected by a microplate reader, and the IC50 of the inhibitor s4682 for inhibiting the interaction between the collagen chain molecules is obtained according to the measured energy transfer efficiency.
In a preferred embodiment of the invention, in step 3, the specific steps for detecting FRET are excitation at 500-515 nm, detection of fluorescence intensity FI at 525-545 nm and 580-620 nm530And FI590。
In order to solve the above technical problems, the second technical solution adopted by the present invention is: a high throughput screening method for screening compounds that affect collagen stability is provided, comprising the steps of:
step 1: constructing a fusion expression plasmid VN-Col1A1 of human type I collagen alpha 1 with signal peptide removed and an mVenus-N end (2-173aa) segment, and constructing a fusion expression plasmid VC-Col1A2 of human type I collagen alpha 2 with signal peptide removed and an mVenus-C end (156-; human type III collagen alpha 1 with signal peptide removed and mVenus-N end fragment fusion expression plasmid mVN-Col3A1 and human type III collagen alpha 1 with signal peptide removed and mVenus-C end fragment fusion expression plasmid mVC-Col3A1 are transformed into DH5 alpha bacteria and plasmid extraction steps are carried out to obtain plasmid for transfection;
step 2: co-transfecting VN-Col1A1 and VC-Col1A2 or VN-Col3A1 and VC-Col3A1 by using fibroblasts, and adding a P4HA1 inhibitor s4682 with the concentration of 0.01-500 mu M12 hours after the cells are co-transfected by plasmids;
and step 3: after 24-48 h of drug intervention, detecting the Venus fluorescence by a microplate reader, and obtaining the IC50 of the inhibitor s4682 for inhibiting the interaction between collagen chain molecules according to the measured relative fluorescence intensity of Venus.
In a preferred embodiment of the present invention, in step 3, Venus fluorescence is detected using an excitation wavelength of 510-520 nm and an emission wavelength of 528-560 nm.
In order to solve the above technical problems, the third technical solution adopted by the present invention is: a high throughput screening method for screening compounds that affect collagen stability is provided, comprising the steps of:
step 1: constructing a fusion expression plasmid GlucN-Col1A1 of human type I collagen alpha 1 with signal peptide removed and a Gluc-N end (2-416 aa) segment, and a fusion expression plasmid GlucC-Col1A2 of human type I collagen alpha 2 with signal peptide removed and a Gluc-C end (398-550 aa) segment; human III type collagen alpha 1 with signal peptide removed and a Gluc-N end fragment are fused to express a plasmid glucN-Col3A1, human III type collagen alpha 1 with signal peptide removed and a Gluc-C end fragment are fused to express a plasmid glucC-Col3A1, and plasmids for transfection are obtained through transformation of DH5 alpha bacteria and plasmid extraction steps;
step 2: co-transfecting GluCN-Col1A1 and GluCC-Col1A2 or GluCN-Col3A1 and GluCC-Col3A1 by using fibroblasts, and adding P4HA1 inhibitor s4682 with the concentration of 0.01-500 mu M12 hours after the cells are co-transfected by plasmids;
and step 3: adding luciferase substrate after the drug intervention is carried out for 24-48 h, detecting Gluc chemiluminescence by using a microplate reader, and obtaining the IC50 of the inhibitor s4682 for inhibiting the interaction between collagen chain molecules according to the measured relative chemiluminescence intensity.
In order to solve the technical problems, the fourth technical scheme adopted by the invention is as follows: provides an application of the method in the quantitative analysis of the drug effect of the drug for determining the stability of the triple helix of the collagen.
In a preferred embodiment of the invention, the medicament for reducing the stability of the collagen triple helix is used for treating collagen proliferative diseases, including organ fibrosis and scar.
In order to solve the above technical problems, a fifth technical solution adopted by the present invention is: provides an application of the method in quantitative analysis for determining the compound for increasing the stability of the collagen triple helix.
In a preferred embodiment of the present invention, the compound for increasing the stability of collagen triple helix comprises drugs and cosmetics for treating diseases and symptoms caused by collagen production deficiency, including osteogenesis imperfecta, retinal detachment, and skin laxity.
The invention has the beneficial effects that: the screening of the compound influencing the stability of the collagen is based on Fluorescence Resonance Energy Transfer (FRET) or reporter gene fragment complementation, and the result shows that the IC50 of the prolyl hydroxylase inhibitor s4682 inhibiting the interaction between collagen chain molecules proves the practicability of the screening method.
Drawings
FIG. 1 is a graph of IC50 inhibition of intermolecular interactions of collagen chains by s4682, plotted on the abscissa of the concentration of the compound and on the ordinate of the energy transfer efficiency, measured in example 1;
FIG. 2 is a graph of IC50 showing the inhibition of intermolecular interactions of collagen chains by s4682, measured in example 2, on the abscissa, the concentration of the compound and on the ordinate, the relative fluorescence intensity of Venus;
FIG. 3 is a graph of IC50 inhibition of collagen chain intermolecular interactions by s4682, plotted on the abscissa of compound concentration and on the ordinate of relative chemiluminescence intensity, measured in example 3;
FIG. 4 is a graph showing the effect of decreasing collagen expression using drug BRL-2021101, GAPDH being the internal control;
FIG. 5 is a graph of the effect of compounds on increasing collagen stability, FIG. 5A is an EC50 curve of Rutin and BRL-2021103 versus increasing collagen stability, with the abscissa representing compound concentration and the ordinate representing relative light intensity; FIG. 5B is a photograph of immunoblotting to examine the effect of Rutin and BRL-2021103 on collagen (Col1A1) expression, with β -actin as an internal reference.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
Example 1:
a high throughput screening method for screening compounds that affect collagen stability, comprising the steps of:
step 1: constructing a fusion expression plasmid mVenus-Col1A1 of human type I collagen alpha 1 and mVenus without signal peptide, a fusion expression plasmid mVenus-Col1A 2 of human type I collagen alpha 2 and mApple without signal peptide, a fusion expression plasmid mVenus-Col3A1 of human type III collagen alpha 1 and mVenus without signal peptide, a fusion expression plasmid mVepple-Col 3A1 of human type III collagen alpha 1 and mApple without signal peptide, and obtaining a plasmid for transfection by a large-scale extraction step of transformed DH5 alpha bacteria and plasmid;
step 2: NIH-3T3 cells in a 6-pore plate co-transfect mVenus-Col1A1 and mApple-Col1A2 respectively at 0.1-4 mu g, or mVenus-Col3A1 and mApple-Col3A1 respectively at 0.1-4 mu g, and P4HA1 inhibitor s4682 with the concentration of 0.01 mu M is added 12h after the cells are co-transfected by the plasmids;
and step 3: after the pharmacological intervention is carried out for 24 hours, the FRET (fluorescence resonance energy transfer) is detected by an enzyme-labeling instrument, the excitation is carried out at 515nm, and the fluorescence intensity FI at 530nm (peak value) and 590nm (peak value) is detected530And FI590The energy transfer efficiency E ═ FI590/(FI530+FI590)。
Example 2:
a high throughput screening method for screening compounds that affect collagen stability, comprising the steps of:
step 1: constructing a fusion expression plasmid VN-Col1A1 of human type I collagen alpha 1 with signal peptide removed and an mVenus-N end (2-173aa) segment, and constructing a fusion expression plasmid VC-Col1A2 of human type I collagen alpha 2 with signal peptide removed and an mVenus-C end (156-; human type III collagen alpha 1 with signal peptide removed and mVenus-N end fragment fusion expression plasmid mVN-Col3A1 and human type III collagen alpha 1 with signal peptide removed and mVenus-C end fragment fusion expression plasmid mVC-Col3A1 are transformed into DH5 alpha bacteria and plasmid mass extraction steps to obtain plasmid for transfection;
step 2: 0.1-4 mu g of each of VN-Col1A1 and VC-Col1A2 co-transfected by NIH-3T3 cells in a 6-hole plate or 0.1-4 mu g of each of mVN-Col3A1 and mVC-Col3A1 in a 6-hole plate, and P4HA1 inhibitor s4682 with the concentration of 10 mu M is added 12 hours after the cells are co-transfected by plasmids;
and step 3: after the medication intervention for 30h, the Venus fluorescence is detected by an enzyme-labeling instrument, the excitation wavelength is 510nm, and the emission wavelength is 530 nm.
Example 3:
a high throughput screening method for screening compounds that affect collagen stability, comprising the steps of:
step 1: constructing a fusion expression plasmid GlucN-Col1A1 of human type I collagen alpha 1 with signal peptide removed and a Gluc-N end (2-416 aa) segment, and a fusion expression plasmid GlucC-Col1A2 of human type I collagen alpha 2 with signal peptide removed and a Gluc-C end (398-550 aa) segment; human III type collagen alpha 1 with signal peptide removed and a Gluc-N end fragment are fused to express a plasmid glucN-Col3A1, human III type collagen alpha 1 with signal peptide removed and a Gluc-C end fragment are fused to express a plasmid glucC-Col3A1, and plasmids which can be used for transfection are obtained through a step of transforming DH5 alpha bacteria and extracting the plasmids in a large quantity;
step 2: NIH-3T3 cells in a 6-pore plate co-transfect glucN-Col1A1 and glucC-Col1A2 respectively at 0.1-4 mu g, or glucN-Col3A1 and glucC-Col3A1 respectively at 0.1-4 mu g, and P4HA1 inhibitor s4682 with the concentration of 500 mu M is added 12h after the cells are co-transfected by plasmids;
and step 3: adding luciferase substrate after 48h of drug intervention, and detecting Gluc chemiluminescence by using an enzyme-labeling instrument.
Example 4: s4682 inhibits collagen triple helix molecular stability
12h after the plasmid co-transfects the cells, P4HA1 inhibitor s4682 with different concentrations is added into the culture medium, and after 24h of adding the drug, FRET is detected by an enzyme-labeling instrument, or fluorescence data or chemiluminescence is detected, and the results are shown in figures 1-3. FRET, Venus fragment complementation and Gluc fragment complementation tests found that s4682 inhibited type I collagen triple helix formation with an IC50 of 23.14. mu.M, 52.59. mu.M and 48.89. mu.M, respectively. Both methods, except the FRET method, are closer to the known empirical values for s4682 for inhibition of type I collagen expression. Considering that the signal-to-noise ratio Gluc fragment complementation method is better.
Example 5: determination of collagen stability reduction by drug BRL-2021101
The compound BRL-2021101 was screened from its own library using the above method to inhibit collagen type I intermolecular interaction IC50 ═ 31 μ M, with a maximum efficacy of 134% of s 4682. Immunoblotting verified that 30 μ M BRL-2021101 significantly reduced collagen expression, by affecting the stability of the collagen molecule, because the lysosomal inhibitor Bafilomycin a1 inhibited collagen degradation. As shown in FIG. 4, 30 μ M BRL-2021101 significantly reduced collagen expression. MG, proteasome inhibitor MG 132; BafA1, lysosomal inhibitor Bafilomycin a 1; GAPDH was used as an internal control.
Example 6: determination of Compound increased collagen stability
The compound BRL-2021103, which increased the type I collagen intermolecular interaction EC50 ═ 20.59 μ M, was selected from the own compound library using example 3, while the positive control drug Rutin increased the type I collagen intermolecular interaction EC50 ═ 41.61 μ M, with BRL-2021103 maximal efficacy being 174% of that of Rutin (fig. 5A). Immunoblot analysis demonstrated that 20 μ M BRL-2021103 significantly increased collagen expression by affecting the stability of the collagen molecule (fig. 5B).
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent flow transformations made by using the contents of the specification and drawings, or applied directly or indirectly to other related technical fields, are included in the scope of the present invention.
Claims (9)
1. A high throughput screening method for screening compounds that affect collagen stability, comprising the steps of:
step 1: constructing a fusion expression plasmid mVenus-Col1A1 of human type I collagen alpha 1 and mVenus without signal peptide, a fusion expression plasmid mVenus-Col1A 2 of human type I collagen alpha 2 and mApple without signal peptide, a fusion expression plasmid mVenus-Col3A1 of human type III collagen alpha 1 and mVenus without signal peptide, a fusion expression plasmid mVenus-Col3A1 of human type III collagen alpha 1 and mApple without signal peptide, and obtaining a plasmid for transfection by transforming DH5 alpha bacteria and a plasmid extraction step;
step 2: co-transfecting mVenus-Col1A1 and mApple-Col1A2 or mVenus-Col3A1 and mApple-Col3A1 by using fibroblasts, and adding P4HA1 inhibitor s4682 with the concentration of 0.01-500 mu M12 h after the cells are co-transfected by plasmids;
and step 3: after the medication intervention is carried out for 24-48 h, the FRET is detected by a microplate reader, and the IC50 of the inhibitor s4682 for inhibiting the interaction between the collagen chain molecules is obtained according to the measured energy transfer efficiency.
2. The high throughput screening method of claim 1, wherein in step 3, the specific step of detecting FRET is 500-515 nm excitation, and the fluorescence intensities FI of 525-545 nm and 580-620 nm are detected530And FI590。
3. A high throughput screening method for screening compounds that affect collagen stability, comprising the steps of:
step 1: constructing a fusion expression plasmid VN-Col1A1 of human type I collagen alpha 1 with signal peptide removed and an mVenus-N end (2-173aa) segment, and constructing a fusion expression plasmid VC-Col1A2 of human type I collagen alpha 2 with signal peptide removed and an mVenus-C end (156-; human type III collagen alpha 1 with signal peptide removed and mVenus-N end fragment fusion expression plasmid mVN-Col3A1 and human type III collagen alpha 1 with signal peptide removed and mVenus-C end fragment fusion expression plasmid mVC-Col3A1 are transformed into DH5 alpha bacteria and plasmid extraction steps are carried out to obtain plasmid for transfection;
step 2: co-transfecting VN-Col1A1 and VC-Col1A2 or VN-Col3A1 and VC-Col3A1 by using fibroblasts, and adding a P4HA1 inhibitor s4682 with the concentration of 0.01-500 mu M12 hours after the cells are co-transfected by plasmids;
and step 3: after 24-48 h of drug intervention, detecting the Venus fluorescence by a microplate reader, and obtaining the IC50 of the inhibitor s4682 for inhibiting the interaction between collagen chain molecules according to the measured relative fluorescence intensity of Venus.
4. The high throughput screening method of claim 1, wherein in step 3, Venus fluorescence is detected at an excitation wavelength of 510-520 nm and an emission wavelength of 528-560 nm.
5. A high throughput screening method for screening compounds that affect collagen stability, comprising the steps of:
step 1: constructing a fusion expression plasmid GlucN-Col1A1 of human type I collagen alpha 1 with signal peptide removed and a Gluc-N end (2-416 aa) segment, and a fusion expression plasmid GlucC-Col1A2 of human type I collagen alpha 2 with signal peptide removed and a Gluc-C end (398-550 aa) segment; human III type collagen alpha 1 with signal peptide removed and a Gluc-N end fragment are fused to express a plasmid glucN-Col3A1, human III type collagen alpha 1 with signal peptide removed and a Gluc-C end fragment are fused to express a plasmid glucC-Col3A1, and plasmids for transfection are obtained through transformation of DH5 alpha bacteria and plasmid extraction steps;
step 2: co-transfecting GluCN-Col1A1 and GluCC-Col1A2 or GluCN-Col3A1 and GluCC-Col3A1 by using fibroblasts, and adding P4HA1 inhibitor s4682 with the concentration of 0.01-500 mu M12 hours after the cells are co-transfected by plasmids;
and step 3: adding luciferase substrate after the drug intervention is carried out for 24-48 h, detecting Gluc chemiluminescence by using a microplate reader, and obtaining the IC50 of the inhibitor s4682 for inhibiting the interaction between collagen chain molecules according to the measured relative chemiluminescence intensity.
6. Use of a high throughput screening method for screening compounds affecting collagen stability according to any one of claims 1 to 5 in a quantitative pharmacodynamic assay for the determination of a drug that decreases collagen triple helix stability.
7. The use according to claim 6, wherein the medicament for decreasing the stability of the collagen triple helix is for the treatment of collagen proliferative diseases, including organ fibrosis, scarring.
8. Use of a high throughput screening method for screening compounds affecting collagen stability according to any one of claims 1 to 5 in a quantitative assay for compounds increasing collagen triple helix stability.
9. The use according to claim 8, wherein the compound increasing the stability of the collagen triple helix comprises drugs, cosmetics for the treatment of diseases and conditions resulting from a low collagen production, including osteogenesis imperfecta, retinal detachment, skin laxity.
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| CN103159857A (en) * | 2007-05-21 | 2013-06-19 | 财团法人工业技术研究院 | Compound based on recombinational triple helix support |
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| CN103159857A (en) * | 2007-05-21 | 2013-06-19 | 财团法人工业技术研究院 | Compound based on recombinational triple helix support |
| CN104232588A (en) * | 2014-09-12 | 2014-12-24 | 中国医学科学院医药生物技术研究所 | Construction and application of anti-hepatic fibrosis drug high-throughput screening cell model |
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