CN113380343B - Rapid screening method of hepatotoxic compounds in polygonum multiflorum based on OATP1B1 and OATP1B3 - Google Patents
Rapid screening method of hepatotoxic compounds in polygonum multiflorum based on OATP1B1 and OATP1B3 Download PDFInfo
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- CN113380343B CN113380343B CN202110654288.4A CN202110654288A CN113380343B CN 113380343 B CN113380343 B CN 113380343B CN 202110654288 A CN202110654288 A CN 202110654288A CN 113380343 B CN113380343 B CN 113380343B
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- G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
- G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
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Abstract
The application relates to a screening method of hepatotoxic compounds in polygonum multiflorum based on a computer molecule docking technology, which comprises the following steps: (1) Obtaining the three-dimensional chemical structure of at least one compound to be detected in polygonum multiflorum; (2) Constructing protein homologous modeling structures of OATP1B1 and OATP1B3, and respectively determining active sites of OATP1B1 and OATP1B 3; (3) Respectively carrying out computer molecular docking on the compound to be detected and a positive compound serving as a common substrate of OATP1B1 and OATP1B3 with the active site to obtain docking scoring; (4) The conditions under which the test compound is judged to be a hepatotoxic compound are: and the docking score of the compound to be detected reaches a docking judgment value. The screening method of the application is quick, economical and simple to operate, and can realize high-throughput screening of a plurality of compounds.
Description
Technical Field
The application relates to a rapid screening method of hepatotoxic compounds in polygonum multiflorum, in particular to a screening method of hepatotoxic compounds in polygonum multiflorum, which is based on a molecular docking technology and takes organic anion transport polypeptide 1B1 (organic anion transporting polypepide 1B1, OATP1B 1) and organic anion transport polypeptide 1B3 (organic anion transporting polypepide 1B3, OATP1B 3) as acting targets.
Background
The polygonum multiflorum is a dry root tuber of polygonum multiflorum (Polygonum multiflorum thunb.) of Polygonaceae, has the effects of tonifying liver and kidney, replenishing essence and blood and blackening beard and hair, is a tonic and anti-aging medicament widely promoted by the traditional Chinese medicine, has very wide clinical application, but in recent years, the toxicity problem of the polygonum multiflorum is gradually highlighted, the adverse reaction report of polygonum multiflorum preparation (more than 1000 parts is related) is collected in recent years by the national food and drug administration (SFDA) adverse reaction monitoring center, and liver function damage is the main adverse reaction. The drug-induced liver injury caused by polygonum multiflorum accounts for 1.06 percent (rank 15) of all drug liver injury cases and accounts for 5.69 percent (rank 1) of all drug liver injury cases.
The polygonum multiflorum has a plurality of ingredients, mainly comprising anthraquinone, stilbene, phospholipid, tannins, phenols, sterols and other compounds, but the hepatotoxicity of the polygonum multiflorum cannot be effectively characterized by a chemical component analysis method. Therefore, it is necessary to define the hepatotoxic substances, and guide the clinical safety and reasonable medication.
The reported screening method of hepatotoxic components in polygonum multiflorum mainly adopts in vitro cell or micro-tissue experiments or animal experiments. Yang Min (J. Chinese J. 2016,41 (7): 1289-1296.DOI:10.4268/cjcmm 20160721.) reports the use of MTT method to detect toxicity of free anthraquinone, bound anthraquinone and naphthalene 11 monomer components in Polygonum multiflorum on Hep G2 cells, and the method comprises co-culturing clear cytotoxic components with rat liver slices to prepare liver tissue homogenate, and determining protein content in the homogenate by BCA method to verify cytotoxic components to investigate toxic effect of these components on liver tissue. Wei Peifeng et al (research on the correlation between the induction of hepatic apoptosis by different ingredients of prepared fleece flower root and tumor necrosis factor alpha, sichuan Chinese medicine, 2009,27 (10): 47) report that the correlation between TNF-alpha and the induction of hepatic apoptosis by different ingredients of prepared fleece flower root is detected by gastric lavage administration of prepared fleece flower root, rhein, emodin and chrysophanol to rats for 3 months. The screening method adopting in vitro cell and micro-tissue experiments has the problems of undefined target point of hepatotoxic components, long time consumption, high cost and the like. Therefore, it is a urgent need in the art to provide a rapid and accurate method for screening hepatotoxic molecules in polygonum multiflorum.
The organic anion transport polypeptides OATP1B1 and OATP1B3 belong to the solute carrier superfamily, and can mediate transmembrane transport of drugs or endogenous substances and are highly expressed in the liver. Since drugs are taken up into the liver by OATP1B1 and OATP1B3 before metabolic excretion occurs, the process of transport mediation is critical. The substrates of OATP1B1 and OATP1B3 are numerous, and the substrates of these two transporters, when overlapping, can trigger drug interactions and even toxic reactions.
The molecular docking technology is mainly applied to the field of drug molecule design, and is a method for carrying out drug design by the characteristics of a receptor and the interaction mode between the receptor and drug molecules based on computer science. A theoretical simulation of intermolecular interactions (e.g., ligands and receptors) and predicting binding patterns and avidity. Meanwhile, molecular docking can also be used to predict binding between small molecules. By simulating the structure of the polypeptide in the peptide library, the method for predicting the combination of the polypeptide and the corresponding target molecule can save a great deal of manpower and material resource consumption in the screening process of the peptide aptamer, avoid the harm of toxic reagents in the screening process to experimental personnel, and greatly improve the screening efficiency, limit the capacity of the peptide library and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the application provides a screening method of hepatotoxic compounds in polygonum multiflorum based on a computer molecule docking technology, which comprises the following steps: (1) Obtaining the three-dimensional chemical structure of at least one compound to be detected in polygonum multiflorum; (2) Constructing protein homologous modeling structures of OATP1B1 and OATP1B3, and respectively determining active sites of OATP1B1 and OATP1B 3; (3) Respectively carrying out computer molecular docking on the compound to be detected and a positive compound serving as a common substrate of OATP1B1 and OATP1B3 with the active site to obtain docking scoring; (4) The conditions under which the test compound is judged to be a hepatotoxic compound are: and the docking score of the compound to be detected reaches a docking judgment value.
In some embodiments, step (1) further comprises the step of pre-treating the test compound, the pre-treatment comprising three-dimensional structure generation, structure optimization, and/or deletion of a repeating structure.
In some embodiments, the test compound is pretreated with Discovery studio2.5 software.
In some embodiments, the chemical structure of the test compound is obtained by literature retrieval.
In some embodiments, the test compound may be at least 1, at least 10, at least 100, or all ingredient compounds in polygonum multiflorum.
In some embodiments, trRossetta is used to construct protein homologous modular structures for OATP1B1, OATP1B 3.
In some embodiments, the homologous modeling structure is assessed using Lawster's plot and Profile-3D.
In some embodiments, the validation Score (Verify Score) for OATP1B1 is 215.04, the validation desired high Score (Verify Expected High Score) is 316.539, and the validation desired low Score (Verify Expected Low Score) is 142.442.
In some embodiments, the validation Score (Verify Score) for OATP1B3 is 217.21, the validation desired high Score (Verify Expected High Score) is 321.618, and the validation desired low Score (Verify Expected Low Score) is 144.728.
In some embodiments, step (2) further comprises a step of pre-treating the homoplastic molded structure, the pre-treating comprising structure optimization, and/or protonation.
In some embodiments, the method of identifying an active site is selected from the group consisting of: literature or database investigation methods, experimental screening methods and software prediction methods.
In some embodiments, the primary function and family of the protein can be learned from the experimental results of others/predecessors by literature investigation to find itActive site information. For example, OATP1B1 has a radius ofThe three-dimensional coordinate positions of the loci are 8.709612, -19.331732, -10.620168, and the key amino acid residues are R93, H92, K90, L550 and R580; OATP1B3 has a radius of +.>The three-dimensional coordinate positions of the loci are-36.679656, 30.015304 and 21.209486, and the key amino acid residues are R93, H92, K90, T550 and R580. By comparing the homologous proteins of other species which have been studied more, the corresponding pocket information is found. The UniprotKB database (https:// www.uniprot.org /) integrates rich protein structure-function information, and sometimes contains mutation site information.
In some embodiments, the experimental screening method includes a site-directed mutagenesis method and a fluorescent probe labeling method.
In some embodiments, the active sites on OATP1B1 and OATP1B3 (also referred to as active pockets) can be predicted by computational methods, and software for predicting pockets includes, but is not limited to: fpocket, POCASA.
In some embodiments, the OATP1B1 active site is defined by the key amino acid residues R93, H92, K90, L550, R580.
In some embodiments, the OATP1B3 active site is defined by the key amino acid residues R93, H92, K90, T550, R580.
In some embodiments, the OATP1B1 active site is defined as: radius is ofThe three-dimensional coordinate positions of the sites are 8.709612, -19.331732, -10.620168.
In some embodiments, the OATP1B3 active site is defined as: radius is ofThe three-dimensional coordinate position of the site is-36.679656,30.015304，21.209486。
in some embodiments, the computer molecule docking is performed by software selected from the group consisting of: dock, autodock, libDock, flexX, glide, GOLD, MOE Dock, surflex-Dock and LigandFit, libDock being preferred.
In some embodiments, the method of molecular docking is selected from the group consisting of rigid docking, semi-flexible docking, and flexible docking, preferably rigid docking.
In some embodiments, algorithms for molecular docking include, but are not limited to: matching algorithm, monte Carlo simulation and genetic algorithm, and integrated docking.
In some embodiments, the docking score is calculated by a scoring function. Scoring functions include, but are not limited to: a scoring function based on force field, a scoring function based on priori knowledge, a scoring function based on experience.
In some embodiments, the docking determination in step (4) is at least 50%, at least 60%, at least 70%, at least 80%, or at least 85% of the docking score for a positive compound.
In some embodiments, the positive compound that is a co-substrate for OATP1B1 and OATP1B3 is bilirubin.
In some embodiments, when the positive compound is bilirubin, the dock-judging value is at least 80%, preferably at least 85%, more preferably at least 90% of the dock-scoring of the positive compound.
In some embodiments, the method of screening for hepatotoxic compounds in polygonum multiflorum based on the computer molecular docking technology of the application comprises the following steps: (1) Obtaining the three-dimensional chemical structure of at least one compound to be detected in polygonum multiflorum; (2) Constructing protein homologous modeling structures of OATP1B1 and OATP1B3, and respectively determining active sites of OATP1B1 and OATP1B 3; (3) Respectively carrying out computer molecular docking on the compound to be detected and the positive compound bilirubin with the active site through Libdock software to obtain docking scoring; (4) The conditions under which the test compound is judged to be a hepatotoxic compound are: if the test compound binds to OATP1B1, a docking score of at least 86 or at least 91 is reached; if the test compound binds to OATP1B3, a docking score of at least 99 or at least 105 is achieved.
In some embodiments, the conditions under which the test compound is determined to be a hepatotoxic compound are: if the test compound binds to OATP1B1, a docking score of at least 90, at least 95, or at least 100 is achieved;
in some embodiments, the conditions under which the test compound is determined to be a hepatotoxic compound are: if the test compound binds to OATP1B3, a docking score of at least 100, at least 105, at least 110, at least 115, or at least 120 is achieved.
In some embodiments, the method further comprises: (5) And (3) a test step of determining whether or not the hepatotoxicity of the hepatotoxic compound is consistent with the determination in the step (4), for example, by a hepatotoxicity screening model test.
In some embodiments, the hepatotoxicity screening model is a hepavg cell.
In some embodiments, the hepatotoxicity is through IC of hepavg cell survival 50 Characterization.
In some embodiments, cell viability is calculated as follows: cell viability= (a Administration group －A Blank control group )/(A Vehicle control group －A Blank control group )×100％。
The rapid screening method of hepatotoxic compounds in polygonum multiflorum provided by the application at least realizes one of the following beneficial effects:
the application provides the thinking of developing the research on hepatotoxic components in polygonum multiflorum aiming at an OATP1B1/3 target spot for the first time, constructs an OATP1B1/3 protein homologous modeling structure by correlating OATP1B1/3 inhibition with hepatotoxicity, adopts a computer molecular docking technology to screen components with OATP1B1/3 inhibition activity in polygonum multiflorum in a high throughput manner, and directly judges the hepatotoxicity according to docking scoring, thereby realizing targeted, rapid and effective screening of hepatotoxic compounds in polygonum multiflorum, being particularly suitable for early toxicity screening of medicines, and greatly saving time and reducing manpower and material resource investment compared with the traditional in vitro cell/tissue experiment or animal experiment;
OATP1B1 active site: radius of radiusThree-dimensional coordinate position of site (8.709612, -19.331732, -10.620168) and OATP1B3 active site: radius->The three-dimensional coordinate position of the locus is (-36.679656, 30.015304, 21.209486) with good accuracy and repeatability, the butt joint mark can be accurately correlated with the liver toxicity, the potential drug use risk of the drug can be directly and effectively predicted, and a new thought is provided for the safety evaluation of the traditional Chinese medicine;
the screening method of the application is quick, economical and simple to operate, and can realize high-throughput screening of a plurality of compounds.
Drawings
FIG. 1 shows the homoplastic modeling structure (A) of OATP1B1 and Lawster's diagram (B); homologous modeling structure (C) of OATP1B3 and Lawster's diagram (D).
Fig. 2 shows the results of hepatotoxicity studies of the positive compound bilirubin.
FIG. 3 shows the results of examining the hepatotoxicity of physcion-8-O-glucoside.
FIG. 4 shows the results of hepatotoxicity studies of aloe-emodin-8-O-glucoside.
FIG. 5 shows the results of hepatotoxicity examination of emodin-8-O-beta-D-glucoside.
FIG. 6 shows the results of examination of hepatotoxicity of Trans-physcion dianthrone (Trans-emodin-physcion bianthrones).
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. The specification and embodiments of the application are exemplary only.
Unless otherwise indicated, all reagents used hereinafter are commercial reagents in which the chemical reagents used are not less than analytically pure.
In the application, molecular docking is to place ligand molecules at the positions of the active sites of the receptors, and then evaluate the interaction between the ligands and the receptors in real time according to the principles of geometric complementation, energy complementation and chemical environment complementation, and find the optimal combination mode between the two molecules.
The molecular docking method comprises the following steps: (1) rigid docking: in the calculation process of the rigid butt joint method, the molecular conformation participating in butt joint is not changed, only the spatial position and the gesture of the molecules are changed, the simplification degree of the rigid butt joint method is highest, the calculated amount is relatively small, and the method is suitable for processing butt joint between macromolecules; (2) semi-flexible docking: the semi-flexible butt joint method allows the conformation of small molecules to change to a certain extent in the butt joint process, but the conformation of large molecules is usually fixed, in addition, the adjustment of the conformation of small molecules can be limited to a certain extent, such as fixing the bond length, bond angle and the like of certain non-key parts, and the semi-flexible butt joint method has both calculated amount and prediction capability of a model, and is one of the butt joint methods with wider application; (3) flexible docking: the flexible butt joint method allows the conformation of a research system to change freely in the butt joint process, and as the variable grows in geometric progression along with the atomic number of the system, the calculation amount of the flexible butt joint method is very large, and the flexible butt joint method consumes a large amount of computers, thereby being suitable for accurately investigating the intermolecular recognition condition.
Software useful in molecular docking of the present application includes, but is not limited to: dock, autodock, libDock, flexX, glide, GOLD, MOE Dock, surflex-Dock, and LigandFit.
Dock is one of the most widely used molecular docking software, developed by the Kuntz group of topics. The Dock uses a semi-flexible butt joint method to fix the bond length and bond angle of small molecules, splits the small molecule ligand into a plurality of rigid fragments, and recombines the rigid fragments of the small molecules according to the geometrical property of the receptor surface to perform conformational search. In the aspect of energy calculation, dock considers non-bond interactions such as electrostatic interactions, van der Waals forces and the like, and searches potential energy surfaces of a system in the process of carrying out conformational search. The final software uses the sum of energy scoring and atomic contact penalty as the basis for evaluating the docking results.
Autodock is another widely used molecular docking program developed by the Olson scientific group. AutoDock applies a semi-flexible docking method, allowing the conformation of small molecules to change, and combining free energy is used as a basis for evaluating docking results. Since AutoDock3.0, the energy optimization adopts a Ramark genetic algorithm (LGA), the LGA combines the genetic algorithm with a local search method, the genetic algorithm is used for searching potential energy planes rapidly, and the local search method is used for carrying out fine optimization on the potential energy planes.
FlexX is molecular docking software developed by the german national information technology research center bioinformatics algorithm and scientific computing laboratory and has been commercialized as a module of the molecular design software package BioSolveIT LeadIT. FlexX uses a method of fragment growth to find the optimal conformation, which is selected according to the value of the free energy of docking. The FlexX program has fast docking speed and high efficiency, and can be used for virtual screening of a small molecule database.
LibDock is also a rapid molecular docking tool, and is suitable for rapid and accurate virtual screening of large-scale databases. LibDock rigidly couples small molecule conformations to the receptor's binding pocket according to the principle of hot-zone matching of these conformations to receptor interactions, which is most advantageous in that it is fast, can operate in parallel, and is suitable for large-scale virtual screening. LibDock interfaces based on the structural features of the receptor binding site by first calculating the hot zone characterizing the receptor site, creating a ligand multi-conformation and then interfacing. The thermal regions are divided into two types of "polar thermal regions" which denote atoms that can match hydrogen bonds in the ligand molecule and "non-polar thermal regions" which denote non-polar atoms in the ligand molecule. The method comprises the following specific steps: (1) generating a small molecule conformational set: libDock can dock multiple conformations of each ligand with the receptor, can use a variety of methods to create the conformation of the ligand for docking, or can create the ligand conformation prior to LibDock docking, if the input ligand structure already contains multiple conformations, then no conformation need be created during LibDock; (2) defining the active site of the protein: the protein structure can be directly imported from DS interface or can be locally stored to be opened from file, so as to define protein active site; (3) generating a hot zone and a butt joint result; (4) analysis of results: the docking structure of each ligand conformation can be viewed in turn, ordered according to libdock score.
The butt joint scoring is calculated by a scoring function, and the commonly used scoring functions are mainly divided into three types: one is a scoring function based on force field, which covers interactions of van der Waals force, electrostatic force, hydrogen bond force and the like, and calculates binding energy of molecules according to first sexual principle through head simulation; secondly, a scoring function based on priori knowledge, which utilizes the known structural data and the binding energy thereof in the existing database to generate some simplified coefficient terms to approximate complex physical actions, such as establishing the paired binding energy coefficients of all atom types and summing as approximation of the binding energy; thirdly, an empirical scoring function is integrated with a scoring function based on force fields and a priori knowledge, and the scoring function comprises physical parameters of force fields, and parameters such as hydrophobicity, desolvation and the like, which can be subjected to regression fitting through existing known data, are set.
EXAMPLE 1 in silico molecular docking screening for hepatotoxic Compounds
Collecting main ingredients of the polygonum multiflorum through literature and database retrieval to obtain 49 molecules, and preprocessing the molecules by adopting Discovery studio2.5, wherein the preprocessing mainly comprises the following operations: three-dimensional structure generation, structure optimization and repeated structure deletion are carried out, chemical structures of 49 molecules are obtained, a ligand library is constructed based on the structures, and subsequent virtual screening is carried out according to the structures.
First-order sequence data of OATP1B1/OATP1B3 proteins are collected from an uniprot database, sequences are input into trRossetta (https:// yanglab. Nankai. Edu. Cn/trRosetta /) to construct a homologous modeling structure of OATP1B1/OATP1B3, and a Lagrangian chart and a verity-3D algorithm are adopted to evaluate the accuracy of the structure. The structure and Laplace chart are shown in FIG. 1, and the Verify-3D score is shown in Table 1. The results suggest that the model can be used for molecular docking studies. The pretreatment operation of the protein structure mainly comprises the operations of structure optimization, protonation and the like. Subsequent molecular docking will take advantage of the processed protein structure.
TABLE 1 Verify-3D score Table
| Name of the name | Verification score | Verifying the desired high score | Verifying the expected low score |
| OATP1B1 | 215.04 | 316.539 | 142.442 |
| OATP1B3 | 217.21 | 321.618 | 144.728 |
The method of defining the active site is to record key amino acid residues in the literature as the initial positions of the active site. The active site is defined by amino acid residues recorded in the literature, and the radius of OATP1B1 isThe three-dimensional coordinate positions of the sites are 8.709612, -19.331732, -10.620168, and the key ammonia thereofThe amino acid residues are R93, H92, K90, L550 and R580; OATP1B1 has a radius of +.>The three-dimensional coordinate positions of the loci are-36.679656, 30.015304 and 21.209486, and the key amino acid residues are R93, H92, K90, T550 and R580. Select literature [1] The OATP1B1/OATP1B3 substrate described in (A) also has bilirubin reported as hepatotoxicity as a positive control.
The molecular docking procedure utilized the LibDock method in DS 2.5. Based on literature [1] The positive compound bilirubin score is described as a screening threshold, and a screening compound having a score above the threshold and similar interaction pattern to the original ligand is defined as a potential active compound, with 80%, preferably 85% of the positive compound score being the lowest criterion. To verify the accuracy and repeatability of the initially defined active site parameters, the test substance is docked with its actual hepatotoxicity IC 50 Comparing, the pearson correlation coefficient of the docking mark and the IC50 is calculated by one of the following four formulas, the correlation is larger than 0.6, and the IC is visible 50 In linear relation to the docking score, the defined active sites have accuracy.
Equation one:
formula II:
and (3) a formula III:
equation four:
the four formulas listed above are equivalent, where E is the mathematical expectation, cov represents the covariance and N represents the number of variable values.
Literature [2] As shown in the following Table 2, the substrates and inhibitors of OATP1B1 and OATP1B3 are mostly chemical monomers, little or no small molecular compound is involved in the traditional Chinese medicine (the field of protein target research, the traditional Chinese medicine is started late, most of the inhibitors are used for predicting liver toxicity when new medicines are developed for monomerized medicines), and the structural characteristics of chemical components in polygonum multiflorum are hardly reflected by the butt joint scoring of the inhibitors with the inhibition effect which are clear in the literature, so that endogenous components in bilirubin in the body are selected as positive medicines.
Table 2 substrate drugs and inhibitors of OATP1B1 and OATP1B3
After docking, the docking results of the total ingredients of polygonum multiflorum are shown in tables 3 and 4 (wherein 31 target ingredients cannot bind to OATP1B1 and 33 target ingredients cannot bind to OATP1B 3). Docking results with reference to positive compounds:
the bilirubiin butt joint scoring value in the OATP1B1 model is 107.993, the butt joint scoring value is 86.3944 when 80% of the butt joint scoring value of the positive compound is set as a standard, and the butt joint scoring value is 91.794 when 85% of the butt joint scoring value of the positive compound is set as a standard;
in the OATP1B3 model, the bilirubiin docking score is 123.953, the docking score is set to be 99.1624 by taking 80% of the docking score of the positive compound as a standard, and the docking score is set to be 105.36 by taking 85% of the docking score of the positive compound as a standard.
Taking 80% of scoring value of positive compound as the lowest standard, primarily judging that the substrate of OATP1B1 capable of competing with bilirubin also comprises chrysophanol-8-O-glucoside, emodin-8-O-beta-D-glucoside, emodin-1-O-beta-D-glucoside, aloe-emodin-8-O-glucoside, chrysophanol-8-O-beta-D-glucoside and rhein-8-O-glucoside; substrates for OATP1B3 that compete with bilirubin include physcion-8-O-glucoside, polygonumnolide A and polydatin;
taking 85% of scoring value of positive compound as the lowest standard, preliminarily judging that the substrate of OATP1B1 capable of competing with bilirubin is Polygonumnolide B, cis-physcion-dianthrone (Cis-emodin-physcion-dianthrones), polygonumnolide B2, physcion-8-beta-D- (6' -O-acetyl) -glucoside, trans-physcion-dianthrone (Trans-emodin-physcion-dianthrones), physcion-3-methyl ether-8-O-beta-D-glucoside and Polygonumnolide B; the substrate of OATP1B3 that competes with bilirubin is Trans-emodin-physcion.
TABLE 3 results of the docking of Polygoni Multiflori radix component with OATP1B1
TABLE 4 results of the docking of Polygoni Multiflori radix component with OATP1B3
EXAMPLE 2 hepatotoxicity investigation
To further verify the accuracy of the method of example 1 and the correlation between OATP1B1/3 inhibition and hepatotoxicity, the present example conducted a hepatotoxicity study.
After the HepaRG cells in logarithmic growth phase are digested, they are adjusted to 5×10 4 Approximately 5000 cells per well were seeded in 96-well plates at a rate of 18-24 h for incubation. A blank control group (without HepaRG cells), a solvent control group (0.5% DMSO), positive control bilirubin and different substances are arranged separatelyConcentration of target compound administration group. Firstly, according to the solubility of compound in solvent, making preliminary experiment to initially define concentration range of every target compound, then according to the result of preliminary experiment further making toxicity experiment to define semi-inhibition concentration (IC 50 ). The treatment concentration ranges for the different compounds are as follows:
bilirubin concentrations range from 0, 0.04, 0.1, 0.3, 1 and 3 μg/mL; aloe-emodin-8-O-glucoside concentrations range from 0, 0.3, 1, 3, 9 and 27 μg/mL; the concentration range of emodin-8-O-beta-D-glucoside is 0, 4, 13, 40, 120 and 360 mug/mL; trans-physcion dianthrones (Trans-emodin-physconbiotics) in the concentration ranges of 0, 0.3, 1, 3 and 9 μg/mL; the concentration of physcion-8-O-glucoside ranges from 0, 4, 13, 40, 120 and 360. Mu.g/mL. The system was heated to 37℃with 5% CO 2 After incubation for 24 hours under the condition, 10 mu L of cell counting kit method (CCK-8) detection reagent is added into each hole, after incubation for 2 hours at 37 ℃ in a dark place, an enzyme-labeled instrument is adopted to detect absorbance value at 450nm wavelength of each hole, and the cell survival rate is calculated according to the formula: cell viability= (a Administration group －A Blank control group )/(A Vehicle control group －A Blank control group ) X 100% and IC was calculated from cell viability 50 。
Bilirubin as OATP1B1 and OATP1B3 substrates is selected as positive control, aloe-emodin-8-O-glucoside (OATP 1B1 docking score 87.8417) which is a representative target compound with higher content in polygonum multiflorum is selected, and physcion-8-O-glucoside (OATP 1B3 docking score 104.261) is obtained.
In vitro toxicity validation experiments were performed. The results are shown in Table 4, FIGS. 2-6. From the experimental results, it was found that the positive control bilirubin IC 50 The value is 2.51 mu M, and has more obvious hepatotoxicity. Trans-physcion dianthrone with molecular docking score higher than that of positive compound docking score by 85%, aloe-emodin-8-O-glucoside with molecular docking score higher than that of positive compound docking score by 80%, and emodin-8-O-beta-D-glucoside all show obvious hepatotoxicity trend。
TABLE 4 hepatotoxicity results
| Compounds of formula (I) | IC 50 (μM) |
| Bilirubin | 2.51 |
| emodin-8-O-beta-D-glucoside | 49.43 |
| physcion-8-O-glucoside | 69.44 |
| aloe-emodin-8-O-glucoside | 16.10 |
| Trans-physcion dianthrones (Trans-emodin-physconbioanthromones) | 2.16 |
While the foregoing description illustrates and describes preferred embodiments of the present application, as aforesaid, it is to be understood that the application is not limited to the forms disclosed herein but is not to be construed as limited to other embodiments, but is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the application described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the application are intended to be within the scope of the appended claims.
Reference to the literature
1.ViragSzekelya,b,IzabelPatika,OrsolyaUngvaria,etal.Fluorescent probes for the dual investigation of MRP2 and OATP1B1function and drug interactions[J].European Journal of Pharmaceutical Sciences 151(2020).
2. Sun Xiaolin, zhao, li Junxiu, etc. the gene polymorphisms of the organic anion transport polypeptides OATP1B1, OATP1B3 and the research progress of drug interactions mediated by them [ J ]. Chinese clinical pharmacology and therapeutics, 2015,20 (11).
Claims (15)
1. A screening method of hepatotoxic compounds in polygonum multiflorum based on a computer molecule docking technology, which comprises the following steps: (1) Obtaining the three-dimensional chemical structure of at least one compound to be detected in polygonum multiflorum; (2) Constructing protein homologous modeling structures of OATP1B1 and OATP1B3, and respectively determining active sites of OATP1B1 and OATP1B 3; (3) Respectively carrying out computer molecular docking on the compound to be detected and a positive compound serving as a common substrate of OATP1B1 and OATP1B3 with the active site to obtain docking scoring; (4) The conditions under which the test compound is judged to be a hepatotoxic compound are: and the docking score of the compound to be detected reaches a docking judgment value.
2. The method of claim 1, step (1) further comprising the step of pre-treating the compound to be tested, the pre-treatment comprising three-dimensional structure generation, structure optimization, and/or deletion of repeating structures; step (2) further comprises a step of pre-treating the homomodeling structure, the pre-treating comprising structure optimization, and/or protonation.
3. The method of claim 1, wherein the chemical structure of the test compound and the homomodeling structure are pre-treated using Discovery studio2.5 software.
4. The method of claim 1, wherein the step (2) uses trRossetta to construct a protein homologous modular structure of OATP1B1, OATP1B 3.
5. The method of claim 4, wherein the step (2) uses Lawster's chart and Profile-3D to evaluate homologous modeling structures.
6. The method of claim 4, wherein in step (2), the OATP1B1 has a verification score of 215.04, a verification desired high score of 316.539, and a verification desired low score of 142.442; the validation score for OATP1B3 is 217.21, the validation desired high score is 321.618, and the validation desired low score is 144.728.
7. The method of claim 1, wherein in step (2), the OATP1B1 active site is defined by the critical amino acid residues R93, H92, K90, L550, R580; the OATP1B3 active site is defined by the key amino acid residues R93, H92, K90, T550, R580.
8. The method of claim 7, in step (2), the OATP1B1 active site is defined as: radius is ofThe three-dimensional coordinate positions of the sites are 8.709612, -19.331732, -10.620168; the OATP1B3 active site is defined as: radius is->The three-dimensional coordinate positions of the sites are-36.679656, 30.015304 and 21.209486.
9. The method of claim 1, wherein in step (3) the computer molecule docking uses LibDock software.
10. The method of claim 1, wherein the docking determination in step (4) is at least 50%, at least 60%, at least 70%, or at least 80% of the docking score for a positive compound.
11. The method of claim 1, wherein the positive compound that is a co-substrate for OATP1B1 and OATP1B3 is bilirubin.
12. The method of claim 1, wherein the docking determination value is at least 80% of a positive compound docking score when the positive compound is bilirubin.
13. The method of claim 1, wherein the docking determination value is at least 85% of a positive compound docking score when the positive compound is bilirubin.
14. The method of claim 1, wherein the docking determination value is at least 90% of a positive compound docking score when the positive compound is bilirubin.
15. The method of claim 1, wherein when molecular docking is performed using LibDock software and the positive compound is bilirubin, the test compound is determined to be a hepatotoxic compound under the following conditions: if the test compound binds to OATP1B1, a docking score of at least 86 or at least 91 is reached; if the test compound binds to OATP1B3, a docking score of at least 99 or at least 105 is achieved.
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