CN111653322A - Screening method for rapidly discovering Topo1 inhibitor molecules - Google Patents
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Abstract
The invention discloses a screening method for rapidly discovering Topo1 inhibitor molecules, which comprises the following steps: (1) obtaining and processing protein crystals; (2) calibrating the prediction capability of the virtual screening model and establishing the screening model; (3) biological validation/cell proliferation assay; (4) the ADMET properties of the molecules obtained by screening are calculated by utilizing online open source software, and the molecules which do not meet the properties are filtered out, compared with the prior art, the method has the beneficial effects that: the invention relates to a screening method for rapidly finding Topo1 inhibitor molecules, which utilizes a model to virtually screen an SPCES database, establishes a virtual screening method of Topo1 for the first time, can obtain clues of active compounds in a short time through virtual screening of drugs, and concentrates research targets from millions of compounds to dozens of compounds.
Description
Technical Field
The invention relates to the field of drug screening and prediction methods, in particular to a screening method for rapidly discovering Topo1 inhibitor molecules.
Background
Leukemia is a malignant clonal disease of hematopoietic stem cells, is clinically manifested by fever, hemorrhage, anemia and leukemia cell infiltration, and is listed as one of ten high-grade tumors in China. In China, the morbidity rate is in the sixth or seventh position of the mortality rate of various malignant tumors, but in children and young adults, leukemia ranks the first position.
At present, the leukemia treatment drugs mainly comprise the following drugs: (1) the research shows that leukemia stem cells mainly express myeloid antigens including CD13, CD33 and the like, and Gemtuzumab Ozogamicin (GO), which is one of CD33 antibody preparations, is a coupling compound of antitumor antibiotic calicheamicin and humanized CD33 monoclonal antibody, so that the clinical application of GO is controversial, and experiments prove that GO is combined with other traditional chemotherapeutic drugs to treat the leukemia of children to have a certain curative effect, but later researches prove that the inhibitor cannot prevent the relapse of the leukemia and cannot reduce the death rate; (2) the tyrosine kinase inhibitor Bcr-abl is an oncogene produced by fusing a Bcr gene and a c-abl proto-oncogene. The Bcr-Abl oncoprotein expressed by the gene is the pathological basis of chronic granulocytic leukemia. Bcr-abl inhibitors are a group of tyrosine kinase inhibitory drugs including imatinib, nilotinib, dasatinib. Wherein imatinib is a tyrosine kinase inhibitor with strong specificity. However, mutation of the Abl kinase domain or other causes leading to the emergence of tumor resistance greatly reduce the efficacy of the drug; (3) ubiquitin-proteasome inhibitors, the ubiquitin-proteasome pathway, are one of the important pathways for carrying out protein selective degradation, and regulate and control multiple proteins such as cell cycle dependent proteins P21, P27, cancer suppressor gene P53 and nuclear transcription factor NF-KB in cells. When the proteasome goes beyond normal physiological roles, it can affect a variety of regulatory proteins, thus promoting tumorigenesis by attenuating growth inhibition and reducing apoptosis, etc. Bortezomib is a synthetic high-selectivity proteasome inhibitor, and multiple studies prove that the bortezomib also has a treatment effect on leukemia, but the treatment effect on recurrent leukemia is poor.
The traditional drug development method is time-consuming and labor-consuming, and in recent years, the rapid development of computer chemistry and the virtual screening method based on molecular docking become an important means in the drug discovery process. As a practical technology in computer-aided drug design, virtual screening is good at screening different types of small molecule compound databases (10-100 ten thousand) to obtain compounds with novel structures, thereby greatly shortening the research time and saving the research expenditure through virtual screening. The successful application of virtual screening in the aspects of drug design and the like opens up a new thought and method for screening novel and efficient drug molecules in the fields of molecular simulation and drug design, and has important research significance.
In recent years, certain achievements have been achieved by using computer-aided leukemia drug design, so that the study of the computer-aided leukemia drug design can better develop the treatment of leukemia. A few scholars at home and abroad start to try a combined scheme based on a topoisomerase I (TopoI) inhibitor to treat chronic leukemia to obtain a good effect, and topotecan serving as a drug taking the topoisomerase I as a target can promote the rapid apoptosis of acute lymphocytic leukemia and can induce effective leukemia treatment activity in three different Severe Combined Immunodeficiency (SCID) mouse models. Therefore, the Topo1 inhibitor based on virtual screening has good application prospect in treating leukemia.
Disclosure of Invention
The invention aims to provide a screening method for rapidly finding a Topo1 inhibitor molecule, which adopts a computer-aided drug design method to carry out drug research on leukemia for the first time, saves time and labor cost, and provides a certain reference value for the design and development of leukemia drugs through theoretical combination experiments.
The design idea of the invention is as follows: the method comprises the steps of firstly obtaining and processing protein crystals, then calibrating the prediction capability of a virtual screening model and establishing a screening model, then utilizing molecular docking software to dock and score compounds, then utilizing online open source software to calculate ADMET properties of screened molecules, and filtering out molecules which do not meet the properties.
In order to achieve the purpose, the invention provides the following technical scheme: a screening method for rapidly discovering Topo1 inhibitor molecules comprises the following steps:
(1) obtaining and processing protein crystals;
(2) calibrating the prediction capability of the virtual screening model and establishing the screening model;
(3) biological validation/cell proliferation assay;
(4) and calculating the ADMET property of the screened molecules by utilizing online open source software, and filtering out the molecules which do not meet the property.
Compared with the prior art, the invention has the beneficial effects that: the invention relates to a screening method for rapidly finding Topo1 inhibitor molecules, which utilizes a model to virtually screen an SPCES database, establishes a virtual screening method of Topo1 for the first time, can obtain clues of active compounds in a short time through virtual screening of drugs, and concentrates research targets from millions of compounds to dozens of compounds.
Drawings
Fig. 1 is a graph of a Receiver Operating Characteristic (ROC) curve (AUC 0.705) in accordance with the present invention;
FIG. 2 is a complex histogram of the scores of the active and inducible molecules of the present invention;
FIG. 3 is a graph of the results of the enrichment factor calculation in the present invention;
FIG. 4 is a molecular list of 23 TopoI inhibitor molecules of the present invention;
FIG. 5 is a table of information on 5 active molecules of the present invention;
FIG. 6 is a table of chemical structural formulas of 5 molecules in the present invention;
FIG. 7 is a list of 5 molecules newly designed in the present invention;
FIG. 8 is a table showing the effect of 5 molecules inhibition rate in the present invention;
FIG. 9 is a 5 molecule-based drug-like property scoring list in accordance with the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in FIGS. 1-9, a screening method for rapid discovery of Topo1 inhibitor molecules is described in detail below for the production and use thereof:
1. a screening method for rapidly discovering Topo1 inhibitor molecules, comprising the steps of:
(1) obtaining and processing protein crystals;
(2) calibrating the prediction capability of the virtual screening model and establishing the screening model;
(3) biological validation/cell proliferation assay;
(4) and calculating the ADMET property of the screened molecules by utilizing online open source software, and filtering out the molecules which do not meet the property.
2. The screening method for rapidly discovering Topo1 inhibitor molecule according to claim 1, wherein the protein crystal is obtained and processed in step (1) by the following steps: obtaining a plurality of crystal structures of the Topo1 target point from an RCSB PDB database website (www.rcsb.org), obtaining IDs of Irr8, Ik4t and It8i, and the resolutions of the IDs are respectively
In general, the higher the resolution, the more precise the docking results and docking sites, and therefore the choice of the TopoI crystal structure PDBcode:1t8i, with a resolution of
3. The screening method for rapidly discovering Topo1 inhibitor molecule according to claim 1, wherein the calibration method for the prediction ability of the virtual screening model in step (2) comprises: constructing a pharmacophore model of a target TopoI, and virtually screening an SPECS compound library based on pharmacophore matching; utilizing molecular docking software to perform docking scoring on the compounds; plotting Receiver Operating Characteristic (ROC) curve, A of target pointThe active molecule is obtained from Binding database, Decoy inducing molecule is obtained through http:// dude.docking.org/website, and docking is carried out by active molecule 40 and inducing molecule 2000. The false positive rate (1-specificity) is taken as a horizontal axis, the true positive rate (sensitivity) is taken as a vertical axis, all points are connected for drawing, and the area (area under curve) under the ROC curve, namely the ROC AUC value is calculated and can reflect the effect of the virtual screening method. It is considered that the screening effect is exhibited when the value is 0.7 to 0.9. The specificity is considered next, looking at the lower left hand portion of the ROC curve. The closer the ROC curve of the part is to the longitudinal axis, the more effective the corresponding virtual screening method can distinguish active compounds from decoy compounds, the more accurate the active compounds are selected from the massive database, and the butt joint method and parameters can be verified to be suitable for the system; drawing a complex histogram, wherein active molecules and induced molecules can be effectively separated, and the established docking model can be used for large-scale molecular docking in the later period, wherein the green part is the induced molecules, and the red part is the active molecules; and (4) calculating an Enrichment Factor (EF), wherein the enrichment factor is an important index for evaluating the docking performance of the molecules and is mainly used for investigating whether parameters used in docking calculation can screen the active molecules from a database containing the active molecules and the bait molecules in a scoring value mode. The larger the EF value, the higher the enrichment degree, which means that the proportion of active molecules in the same proportion is higher. Which has the formula of
Wherein Nsampled is the number of the first n% molecules of the scored result (set to 0.5%, 1%, 5% and 10%), and Hitssampled is the number of active molecules; ntotal is the number of molecules in the dataset used for the docking calculations, and Hitstotal is the number of active molecules. Nsampled is the number of the first n% molecules of the scored result. For the characterization of the magnitude of EF value, when EF is>1, the process has a significant active compound enrichment capacity. Its enrichment capacity increases with increasing EF. Generally considered as EF>1, ROC area of Curve AUC>At 0.5, the screening model is applicable to the protein; selecting 23 TopoI inhibitor molecules with binding energy value S of protoligand docking as threshold (TopoI is-95.0290), and pairingThe observation of molecular efficacy pocket and the combination action mode with target point finally discovers 5 active molecules (M1-M5) which can be used as lead compounds, and carries out new molecular design and activity prediction by taking the active molecule M5 as a template, and finally obtains 5 molecules (N1-N5) with high activity.
4. The screening method for rapidly discovering Topo1 inhibitor molecule according to claim 1, wherein the biological verification/cell proliferation assay method in step (3) is: because the CCK-8 reagent contains WST-8, the WST-8 is reduced into a yellow Formazan product (Formazan dye) with high water solubility by dehydrogenase in cells under the action of an electron carrier 1-Methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS). The amount of the formazan substance generated is proportional to the number of living cells, so that rapid and high-sensitivity analysis of cell proliferation and toxicity is performed by utilizing the characteristic. Biological activity test is carried out on 5 active molecules (M1-M5) through a CCK-8 experiment, the inhibition rate is tested, and the result shows that the 5 active molecules have certain inhibition effect.
5. The screening method of claim 1, wherein the ADMET properties of the screened molecules are calculated by using online open source software in step (4), and the method for filtering out molecules not satisfying the properties comprises: by using a FAF-Drugs4 screening method, Openband software is used to convert 7 small molecules into sdf format and submit the sdf format to FAF-Drugs4 database platform (http:// fafdugs 3.mti. univ-paris-root. fr /) and default parameters. The calculation result shows that the 5 small molecules meet the requirements and have higher possible performance to become the medicine.
It should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in the process, method, article, or apparatus that comprises the element.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (5)
1. A screening method for rapidly discovering Topo1 inhibitor molecules, comprising the steps of:
(1) obtaining and processing protein crystals;
(2) calibrating the prediction capability of the virtual screening model and establishing the screening model;
(3) biological validation/cell proliferation assay;
(4) and calculating the ADMET property of the screened molecules by utilizing online open source software, and filtering out the molecules which do not meet the property.
2. The screening method for rapidly discovering Topo1 inhibitor molecule according to claim 1, wherein the protein crystal is obtained and processed in step (1) by the following steps: obtaining a plurality of crystal structures of the Topo1 target point from an RCSB PDB database website (www.rcsb.org), obtaining IDs of Irr8, Ik4t and It8i, and the resolutions of the IDs are respectively
In general, the higher the resolution, the more precise the docking results and docking sites, and therefore the choice of the TopoI crystal structure PDBcode:1t8i, with a resolution of
3. The screening method of claim 1, wherein the virtual screening model of step (2) is calibrated for predictive powerThe method comprises the following steps: constructing a pharmacophore model of a target TopoI, and virtually screening an SPECS compound library based on pharmacophore matching; utilizing molecular docking software to perform docking scoring on the compounds; and drawing a Receiver Operating Characteristic (ROC) curve, obtaining Active molecules of a target spot by Binding database, obtaining Decoy induction molecules through http:// dude. The false positive rate (1-specificity) is taken as a horizontal axis, the true positive rate (sensitivity) is taken as a vertical axis, all points are connected and drawn, the area (area under curve) under the ROC curve, namely the ROC AUC value is calculated, the value can reflect the effect of the virtual screening method, and the value is generally considered to have a certain screening effect when being 0.7-0.9. Secondly, considering the specificity, namely looking at the lower left corner part of an ROC curve, the closer the ROC curve of the part is to a longitudinal axis, the more effective the corresponding virtual screening method can be to distinguish active compounds from decoy compounds, the active compounds can be accurately selected from a massive database, and the butt joint method and parameters can be verified to be suitable for the system; drawing a complex histogram, wherein active molecules and induced molecules can be effectively separated, and the established docking model can be used for large-scale molecular docking in the later period, wherein the green part is the induced molecules, and the red part is the active molecules; and (4) calculating an Enrichment Factor (EF), wherein the enrichment factor is an important index for evaluating the docking performance of the molecules and is mainly used for investigating whether parameters used in docking calculation can screen the active molecules from a database containing the active molecules and the bait molecules in a scoring value mode. The larger the EF value, the higher the enrichment degree, which means that the proportion of active molecules in the same proportion is higher. Which has the formula of
Wherein Nsampled is the number of the first n% molecules of the scored result (set to 0.5%, 1%, 5% and 10%), and Hitssampled is the number of active molecules; ntotal is the number of molecules in the dataset used for the docking calculations, and Hitstotal is the number of active molecules. Nsampled is the number of the first n% molecules of the scored result. For the characterization of the magnitude of EF value, when EF is>1, the process has a remarkable enriching power of the active compound. Its enrichment capacity increases with increasing EF. Generally considered as EF>1, ROC area of Curve AUC>At 0.5, the screening model is applicable to the protein; by taking the binding energy value S of the butt joint of the original ligand as a threshold value (TopoI is-95.0290), 23 TopoI inhibitor molecules are screened, 5 active molecules (M1-M5) which can be used as lead compounds are finally discovered by observing the molecular efficacy pocket and the binding action mode with a target, and new molecular design and activity prediction are carried out by taking the active molecule M5 as a template, so that 5 molecules (N1-N5) with high activity are finally obtained.
4. The screening method for rapidly discovering Topo1 inhibitor molecule according to claim 1, wherein the biological verification/cell proliferation assay method in step (3) is: because the CCK-8 reagent contains WST-8, the WST-8 is reduced into a yellow Formazan product (Formazan dye) with high water solubility by dehydrogenase in cells under the action of an electron carrier 1-Methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS). The amount of the formazan substance generated is proportional to the number of living cells, so that rapid and high-sensitivity analysis of cell proliferation and toxicity is performed by utilizing the characteristic. Biological activity test is carried out on 5 active molecules (M1-M5) through a CCK-8 experiment, the inhibition rate is tested, and the result shows that the 5 active molecules have certain inhibition effect.
5. The screening method of claim 1, wherein the ADMET properties of the screened molecules are calculated by using online open source software in step (4), and the method for filtering out molecules not satisfying the properties comprises: by using a FAF-Drugs4 screening method, Openband software is used to convert 7 small molecules into sdf format and submit the sdf format to FAF-Drugs4 database platform (http:// fafdugs 3.mti. univ-paris-root. fr /) and default parameters. The calculation result shows that the 5 small molecules meet the requirements and have higher possible performance to become the medicine.
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