CN114958901B - In-vitro preparation, activity detection and application of antifungal drug action target point - Google Patents
In-vitro preparation, activity detection and application of antifungal drug action target point Download PDFInfo
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- C12Y204/01034—1,3-Beta-glucan synthase (2.4.1.34)
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Abstract
The invention relates to in-vitro preparation, activity detection and application of an antifungal drug action target point, and belongs to the field of drug target point materials and preparation and application thereof. The drug target point in the invention is a key enzyme FKS (beta-1, 3-glucan synthase) for inhibiting the synthesis of fungal cell walls. The active drug action target protein is prepared in vitro through a eukaryotic expression system, an effective enzyme activity analysis system is established by utilizing the characteristic that the active drug action target protein can convert a substrate into UDP (uridine diphosphate), and the system can be applied to detection of drugs or compounds with enzyme activity inhibition and has the feasibility of drug screening; in addition, the application of the FKS enzyme activity detection method in the aspect of identifying fungal drug-resistant mutant strains is also developed.
Description
Technical Field
The invention belongs to the field of medicine target point materials and preparation and application thereof, and in particular relates to in-vitro preparation, activity detection and application of an antifungal medicine action target point.
Background
The wide use of broad spectrum antibiotics, immunosuppressants, steroids, etc. has led to an upward trend in fungal infections. Invasive fungal infections cause more than 150 tens of thousands of deaths each year, and such infections are more detrimental to immunocompromised populations and to new crown (COVID-19) patients. The emergence of drug-resistant fungus strains presents a more serious challenge for treating infected patients. Therefore, there is an urgent need to develop various novel antifungal drugs to cope with the current demand.
The fungus cell wall is an important composition structure of fungus cells and is composed of special essential components of fungi such as beta-1, 3-glucan, so that glucan synthase FKS for catalyzing the synthesis of beta-1, 3-glucan is an ideal target for the development of antifungal drugs. FKS is a membrane-bound beta-1, 3-glucan synthase containing 17 transmembrane helices that catalyzes the biochemical reaction: glucose is transferred from donor UDP-glucose to growing glucan chains as beta-1, 3-glycosidic linkages, which allows the glucan chains to be extended. The catalytic function of fungal FKS is regulated by cofactors GTP-gamma-S and Rho 1.
FKS and its homologs play an important role in the survival of a variety of fungal pathogens. For example, in clostridium slimichium and saccharomyces cerevisiae, simultaneous deletion of FKS1 and FKS2 results in bacterial death; FKS1 in candida albicans and cryptococcus neoformans is critical to the viability of the bacteria; aspergillus fumigatus with FKS1 deletion has serious growth defects and cell lysis. The FKS in fungi is highly conserved, and the conservation of the structure enables the medicine developed aiming at the target to have broad-spectrum antifungal effect. Currently, there are two types of antifungal agents on the market for FKS, namely the first-line antifungal agents echinocandins (including caspofungin and micafungin, etc.) widely used in clinical practice, and newly approved oral fungicidal agents Ibrexafungerp (approval by the FDA in the united states of 2021).
The continued development of novel antifungal drugs for inhibiting the synthesis of fungal cell walls aiming at the targets is a long-term research hotspot in the fields of drug basic research and clinical application research.
Disclosure of Invention
The invention relates to in-vitro preparation, activity detection and application of an antifungal drug action target, and the target drug action target is purified fungus FKS1 protein.
The purified fungus FKS1 protein has the activity of catalyzing the substrate UDP-glucose to be converted into the product UDP in vitro under the action of cofactors GTP-gamma-S and Rho1, and an in vitro activity detection system for the FKS1 protein is established.
The invention also aims at screening antifungal drugs by using an in vitro established FKS1 protease activity detection method.
The invention also aims at applying the in-vitro established FKS1 protease activity detection method to the identification of fungal drug-resistant mutant strains.
According to a first aspect of the present invention, there is provided an in vitro preparation method of FKS1 as an antifungal drug target, comprising the steps of firstly expressing FKS1 protein containing a recombinant tag at genome level by using a fungal cell expression system, then dissolving fungal cell membrane by using a membrane dissolving buffer, and finally purifying FKS1 protein by using an affinity chromatography; the method specifically comprises the following steps:
(1) Using a PCR-based gene recombination approach, a FLAG tag was ligated to the C-terminus of the saccharomyces cerevisiae strain chromosome FKS1 sequence;
(2) Culturing the strain obtained in the step (1), collecting cells by centrifugation, adding a lysis buffer, and then performing lysis by a high-pressure crushing method;
(3) Centrifuging to remove cell debris, centrifuging the supernatant to collect cell membranes;
(4) Dissolving the membrane collected in the step (3) in a membrane dissolving buffer solution; FKS1 and mutant proteins thereof are purified by using anti-FLAG M2 affinity gel, and elution is carried out by using an elution buffer solution containing FLAG peptide, thus obtaining the FKS1 as an antifungal drug target.
According to another aspect of the present invention, an in vitro enzyme activity determination method for an antifungal drug target FKS1 is provided, wherein a purified FKS1 protein is used as an antifungal drug detection target, and the yield of uridine diphosphate, which is a product of FKS1 catalytic reaction, is used as a detection index; the method specifically comprises the following steps:
(1) Adding FKS1 protein into a reaction system, wherein the reaction system contains cofactors GTP-gamma-S and Rho1; then adding uridine diphosphate-glucose substrate to perform enzyme activity reaction;
(2) Adding a fluorescent chromogenic reagent, reading a fluorescent value by using an enzyme-labeled instrument, calculating the level of uridine diphosphate generated by the reaction according to the fluorescent chromogenic level, and converting to obtain the in vitro enzyme activity of the antifungal drug target FKS 1.
Preferably, the step (2) specifically comprises: adding a glycosyltransferase detection kit fluorescent chromogenic reagent, reading a fluorescence value by using a PHERASTAR FS enzyme-labeled instrument system, calculating to obtain the level of uridine diphosphate generated by the reaction, and converting to obtain the in vitro enzyme activity of the antifungal drug target FKS 1.
According to another aspect of the present invention, there is provided the use of an in vitro established method for detecting FKS1 protease activity for testing the inhibition of a drug and/or compound against a target FKS1, after incubation of an antifungal drug with FKS1, detecting the in vitro enzyme activity level of FKS1 after dosing relative to the in vitro enzyme activity level of FKS1 before dosing; the method specifically comprises the following steps:
(1) Firstly, incubating a medicine with FKS1, and then adding Rho1, GTP-gamma-S, potassium fluoride and uridine diphosphate-glucose to perform enzyme activity reaction; reading the fluorescence value by using an enzyme-labeled instrument;
(2) The in vitro enzyme activity measured fluorescence value of FKS1 as an antifungal drug target is taken as a standard value 100, the ratio of the fluorescence value measured by FKS1 protein after drug addition to the fluorescence value measured by FKS1 before drug addition is defined as the relative activity of FKS1 after drug inhibition, and the relative activity value of FKS1 after inhibition is lower than 50, which is considered to obviously inhibit the activity of FKS1 by the corresponding drug, so that the inhibition effect of the drug on FKS1 is determined.
According to another aspect of the present invention, there is provided the use of an in vitro established method of detecting FKS1 protease activity for the identification of fungal resistant mutants, comprising the steps of:
(1) Expressing and purifying wild type fungus FKS1 protein and mutant fungus FKS1 corresponding protein by a gene recombination expression and affinity chromatography method, and respectively adding the protein and the protein into a reaction system, wherein the reaction system contains auxiliary factors GTP-gamma-S and Rho1; then adding uridine diphosphate-glucose substrate to perform enzyme activity reaction; adding a fluorescent chromogenic reagent, and reading a fluorescent value by using an enzyme-labeled instrument;
(2) Incubating the antifungal drug with wild fungi and mutant fungi respectively by using gradient dilution, then adding Rho1, GTP-gamma-S, potassium fluoride and uridine diphosphate-glucose to perform enzyme activity reaction, and reading fluorescence values by using an enzyme-labeling instrument;
(3) Taking a fluorescence value measured by an enzyme activity system before dosing as a standard value 100, and defining the ratio of the fluorescence value measured by the protein after dosing to the fluorescence value measured by the protein before dosing as the relative activity of FKS1 or a mutant after dosing; determining the median inhibitory concentration IC 50 value of the drug according to the concentration of the antifungal drug corresponding to 50% of enzyme activity; wild-type ScFKS protein corresponds to an IC50 value of 0.55. Mu.M; when the measured IC 50 value is greater than 0.55. Mu.M, the corresponding mutant protein is resistant to the mutation.
In general, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) The existing method for detecting the FKS activity of the beta-1, 3-glucan synthase comprises the steps of 1) extracting fungal cell membranes and detecting byproducts with [ 3 H ] radioactivity by an isotope labeling method; 2) And detecting the survival rate of the fungi after knocking out related genes. The two methods have the defects of weak target specificity, radiation pollution, complicated operation, time consumption and the like. The detection method is used for detecting the UDP product of the FKS1 catalytic reaction, and has the advantages of no pollution, simplicity and convenience in operation and time saving.
(2) The detection method is based on the detection of the unfolding activity of the purified FKS1 protein, the detection process is not interfered by other proteins in fungal cell membranes, and the specificity is strong.
(3) The detection method can establish a drug-resistant mutant library by purifying mutant proteins, can establish a high-throughput detection method based on the library, can quickly determine corresponding sensitive drugs by combining with the PCR results of strains in clinical patients, and has potential value of being widely applied to clinic.
Drawings
Fig. 1: relative enzyme activity of in vitro purified FKS1 wild type and mutant.
Fig. 2: the inhibition of the target FKS1 by the drug/compound was tested using an in vitro enzyme activity assay for FKS1, demonstrating that this method can be effectively used to screen for antifungal compounds targeting FKS 1.
Fig. 3: the effect of the fungal drug caspofungin on the enzyme activity of FKS1 mutants was tested using an FKS1 in vitro enzyme activity assay.
Fig. 4: analysis of the tolerance of fungal strains bearing FKS1 mutations (corresponding to fig. 3) to caspofungin.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The invention relates to in-vitro preparation, activity detection and application of an antifungal drug action target, wherein the drug target is key enzyme FKS (beta-1, 3-glucan synthase) for inhibiting fungal cell wall synthesis. The active drug action target protein is prepared in vitro through a eukaryotic expression system, an effective enzyme activity analysis system is established by utilizing the characteristic that the active drug action target protein can convert a substrate into uridine diphosphate, the active enzyme activity analysis system can be applied to detection of drugs or compounds with enzyme activity inhibition, and the system has feasibility of drug screening.
Example 1: in vitro preparation of FKS1 as antifungal drug target
1) Using a PCR-based gene recombination approach, the 3×FLAG tag was ligated to the C-terminus of the Saccharomyces cerevisiae strain chromosomal FKS1 (ScFKS 1) sequence.
2) The strain was cultured in YPD medium at 30℃for 20 hours. Cells were then collected by centrifugation and homogenized with lysis buffer (50 mM Tris-HCl (pH 7.4), 150mM NaCl, 2mM MgCl 2 (supplemented with protease inhibitor)) followed by lysis by high pressure disruption.
3) Cell debris was removed by centrifugation at 15000 Xg for half an hour. The supernatant was further ultracentrifuged at 100000 Xg for 1 hour to collect cell membranes.
4) The collected membranes were dissolved in a membrane-dissolving buffer (50 mM Tris-HCl pH 7.4, 1mM EDTA and 33% (v/v) glycerol, detergent, 4uM GTP-. Gamma. -S, protease inhibitor cocktail).
5) FKS1 and its mutant proteins were purified using anti-FLAG M2 affinity gel (Sigma) and eluted with an elution buffer (50 mM Tris-HCl pH 7.4, 1mM EDTA, 33% (v/v) glycerol, detergent) containing 150. Mu.g/ml 3 XFLAG peptide.
6) FKS1 was concentrated to 0.1mg/ml and frozen in liquid nitrogen.
7) The purification method of FKS1 corresponding mutant protein expression is described in example 1 1) -6).
Example 2: establishment of in vitro enzyme activity measuring system of antifungal drug target FKS1
1) 2.5. Mu.L of FKS1 or a mutant thereof was added to the reaction system (30. Mu.L of elution buffer added Rho1, 4. Mu.M GTP-. Gamma. -S, 20mM KF). The reaction was started by the subsequent addition of UDP-glucose substrate to a final concentration of 2.5mM at 37 ℃.
2) After 60min of reaction, 30. Mu.L of a glycosyltransferase detection kit (Promega) was added and the reaction was carried out at room temperature for 30min.
3) The fluorescence values were read using PHERASTAR FS microplate reader system (BMG Labtech) and the UDP level generated by the reaction was measured.
4) The fluorescence value measured by the mutant protein of FKS1 is taken as a standard value 100: the ratio of fluorescence values measured for wild-type FKS1 is defined as the relative activity of mutant FKS1, with different mutants exhibiting different activities. (FIG. 1)
Example 3: testing the inhibition of FKS1 on target by drugs/compounds using in vitro established FKS1 enzyme activity detection method
1) The echinocandin antifungal drug caspofungin (caspofungin) or micafungin (micafungin) is a clinical antifungal drug, and the effectiveness of the method applied to antifungal drug screening is determined by using the two drugs. The drug (concentration 100. Mu.M) was first incubated with FKS1 for 10 minutes at room temperature, followed by addition of Rho1, 4. Mu.M GTP-. Gamma. -S, 20mM KF, 2.5mM UDP-glucose, and starting the enzyme reaction at 37 ℃.
2) The fluorescence values were read using PHERASTAR FS enzyme-labeled instrument system (BMG Labtech) to determine the reduced level of FKS1 activity following addition of the antifungal agent. Taking the fluorescence value measured by the FKS1 enzyme activity system without any antibacterial agent as a standard value 100, and measuring the fluorescence value of FKS1 protein after adding the antibacterial agent: the ratio of fluorescence values measured for FKS1 prior to dosing is defined as the relative activity of FKS1 after inhibition of the drug, and a relative activity value of FKS1 below 50 after inhibition is considered to be significantly inhibited for FKS1, thereby determining the inhibition effect of the drug on FKS 1. (FIG. 2)
Example 4: application of in vitro established FKS protease activity detection method to identification of fungal drug-resistant mutant strain
1) Candida albicans FKS1 mutations (CaFKS 1) F641S and S645P are reported mutant strains insensitive to caspofungin drug effects, and these two mutation sites correspond to ScFKS F639S and S643P, respectively, of the FKS1 protein of Saccharomyces cerevisiae. And expressing and purifying ScFKS F639S and S643P by a gene recombination expression method. Reference example 1, steps 1) -6).
2) The enzyme activity was started at 37℃using gradient diluted caspofungin (0.001-100. Mu.M) incubated with Saccharomyces cerevisiae ScFKS, scFKS F639S, scFKS S643P at room temperature for 10 minutes, respectively, followed by addition of Rho1, 4. Mu.M GTP-. Gamma. -S, 20mM KF, 2.5mM UDP-glucose.
3) The fluorescence value was read using PHERASTAR FS enzyme labelling system (BMG Labtech) and was measured with the wild type FKS1 enzyme activity system before dosing as standard value 100, and after dosing the FKS1 or mutant proteins: the ratio of fluorescence values measured for FKS1 or mutant proteins prior to dosing is the relative activity of FKS1 or mutant after dosing. And determining the median inhibitory concentration IC 50 of the drug according to the caspofungin concentration corresponding to 50% of enzyme activity. The wild-type ScFKS protein corresponds to IC 50 at a value of 0.55. Mu.M. When the IC 50 value was greater than 0.55. Mu.M, the corresponding mutant protein was resistant to mutation (FIG. 3).
4) The consistency and effectiveness of the assay results of this method were determined by the viability level of the mutant strain. The ScFKS F639S and ScFKS S643P mutant gene sequences are introduced into corresponding sites of saccharomyces cerevisiae by a homologous recombination method to establish the chromosome mutation of FKS1, and the sequence is verified by sequencing. After addition of a gradient of caspofungin (0-16. Mu.M) to yeast medium and incubation at 30℃for 48 hours, the viability of Saccharomyces cerevisiae was observed to determine the effectiveness of the method for identifying fungal drug resistant mutations (FIG. 4).
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (2)
1. The application of the in-vitro established FKS1 protease activity detection method for testing the inhibition effect of drugs and/or compounds on target FKS1 is characterized in that after the antifungal drugs are incubated with FKS1, the in-vitro enzyme activity of FKS1 after drug addition is detected relative to the in-vitro enzyme activity level of FKS1 before drug addition; the method specifically comprises the following steps:
(1) Firstly, incubating a medicine with FKS1, and then adding Rho1, GTP-gamma-S, potassium fluoride and uridine diphosphate-glucose to perform enzyme activity reaction; reading the fluorescence value by using an enzyme-labeled instrument;
(2) Taking a fluorescence value measured by in-vitro enzyme activity of an antifungal drug target FKS1 as a standard value 100, defining a ratio of the fluorescence value measured by FKS1 protein after drug addition to the fluorescence value measured by FKS1 before drug addition as relative activity of FKS1 after drug inhibition, wherein the relative activity value of FKS1 after inhibition is lower than 50, and the relative activity value of FKS1 after inhibition is considered to be obviously inhibited by the corresponding drug, so that the inhibition effect of the drug on FKS1 is determined;
the in vitro established FKS1 protease activity detection method specifically comprises the following steps:
The purified FKS1 protein is used as an antifungal drug detection target, and the purification method of the FKS1 protein comprises the following steps: FKS1 and its mutant proteins were purified using anti-FLAG M2 affinity gel and eluted with an elution buffer containing FLAG peptide; taking the yield of uridine diphosphate which is a product of FKS1 catalytic reaction as a detection index; the method specifically comprises the following steps:
s1: adding FKS1 protein into a reaction system, wherein the reaction system contains cofactors GTP-gamma-S and Rho1; then adding uridine diphosphate-glucose substrate to perform enzyme activity reaction;
S2: adding a fluorescent chromogenic reagent, reading a fluorescent value by using an enzyme-labeled instrument, calculating the level of uridine diphosphate generated by the reaction according to the fluorescent chromogenic level, and converting to obtain the in vitro enzyme activity of the antifungal drug target FKS 1;
the step S2 specifically comprises the following steps: adding a glycosyltransferase detection kit fluorescent chromogenic reagent, reading a fluorescence value by using a PHERASTAR FS enzyme-labeled instrument system, calculating to obtain the level of uridine diphosphate generated by the reaction, and converting to obtain the in vitro enzyme activity of the antifungal drug target FKS 1.
2. The application of the in vitro established FKS1 protease activity detection method for identifying fungal drug-resistant mutant strains is characterized by comprising the following steps:
(1) Expressing and purifying wild type fungus FKS1 protein and mutant fungus FKS1 corresponding protein by a gene recombination expression and affinity chromatography method, and respectively adding the protein and the protein into a reaction system, wherein the reaction system contains auxiliary factors GTP-gamma-S and Rho1; then adding uridine diphosphate-glucose substrate to perform enzyme activity reaction; adding a fluorescent chromogenic reagent, and reading a fluorescent value by using an enzyme-labeled instrument;
(2) Incubating the antifungal drug with wild fungi and mutant fungi respectively by using gradient dilution, then adding Rho1, GTP-gamma-S, potassium fluoride and uridine diphosphate-glucose to perform enzyme activity reaction, and reading fluorescence values by using an enzyme-labeling instrument;
(3) Taking a fluorescence value measured by an enzyme activity system before dosing as a standard value 100, and defining the ratio of the fluorescence value measured by the protein after dosing to the fluorescence value measured by the protein before dosing as the relative activity of FKS1 or a mutant after dosing; determining the median inhibitory concentration IC 50 value of the drug according to the concentration of the antifungal drug corresponding to 50% of enzyme activity; wild-type ScFKS protein corresponds to an IC50 value of 0.55. Mu.M; when the measured IC 50 value is greater than 0.55 mu M, the corresponding mutant protein is drug-resistant mutation;
the in vitro established FKS1 protease activity detection method specifically comprises the following steps:
The purified FKS1 protein is used as an antifungal drug detection target, and the purification method of the FKS1 protein comprises the following steps: FKS1 and its mutant proteins were purified using anti-FLAG M2 affinity gel and eluted with an elution buffer containing FLAG peptide; taking the yield of uridine diphosphate which is a product of FKS1 catalytic reaction as a detection index; the method specifically comprises the following steps:
s1: adding FKS1 protein into a reaction system, wherein the reaction system contains cofactors GTP-gamma-S and Rho1; then adding uridine diphosphate-glucose substrate to perform enzyme activity reaction;
S2: adding a fluorescent chromogenic reagent, reading a fluorescent value by using an enzyme-labeled instrument, calculating the level of uridine diphosphate generated by the reaction according to the fluorescent chromogenic level, and converting to obtain the in vitro enzyme activity of the antifungal drug target FKS 1;
the step S2 specifically comprises the following steps: adding a glycosyltransferase detection kit fluorescent chromogenic reagent, reading a fluorescence value by using a PHERASTAR FS enzyme-labeled instrument system, calculating to obtain the level of uridine diphosphate generated by the reaction, and converting to obtain the in vitro enzyme activity of the antifungal drug target FKS 1.
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