阅读说明：本技术 固相识别雌激素受体的雌二醇衍生物筛选方法 (Estradiol derivative screening method for solid-phase recognition of estrogen receptor ) 是由 周小红 朱茜 谭集穗 于 2021-07-23 设计创作，主要内容包括：本发明公开了一种固相识别雌激素受体的雌二醇衍生物筛选方法。该方法包括：以雌二醇-雌激素受体LBD复合物晶体构象为基础,运用分子对接对不同结构雌二醇衍生物与雌激素受体LBD的结合能力进行计算机模拟分析,筛选出满足预设要求的雌二醇衍生物；对筛选出的雌二醇衍生物,采用分子动力学模拟进行结构稳定性和能量变化的分析,筛选出与雌激素受体LBD具有特异性高亲和力的衍生物。本发明可以筛选出与雌激素受体具有高特异性高亲和力的衍生物,进而可以缩短固相雌二醇与雌激素受体结合的时间,增加体外环境下的结合稳定性。(The invention discloses a method for screening estradiol derivatives of an estrogen receptor by solid-phase recognition. The method comprises the following steps: based on the crystal conformation of the estradiol-estrogen receptor LBD compound, carrying out computer simulation analysis on the binding capacity of estradiol derivatives with different structures and the estrogen receptor LBD by using molecular docking, and screening out the estradiol derivatives meeting the preset requirements; and (3) analyzing the structural stability and energy change of the screened estradiol derivatives by adopting molecular dynamics simulation, and screening the derivatives with specificity and high affinity with an estrogen receptor LBD. The invention can screen out the derivative with high specificity and high affinity with the estrogen receptor, thereby shortening the time for combining the solid-phase estradiol with the estrogen receptor and increasing the combination stability in an in vitro environment.)

1. A method for screening estradiol derivatives capable of recognizing estrogen receptors in solid phase, which comprises the following steps:

based on the crystal conformation of the estradiol-estrogen receptor LBD compound, carrying out computer simulation analysis on the binding capacity of estradiol derivatives with different structures and the estrogen receptor LBD by using molecular docking, and screening out the estradiol derivatives meeting the preset requirements;

and (3) analyzing the structural stability and energy change of the screened estradiol derivatives by adopting molecular dynamics simulation, and screening the derivatives with high specificity and high affinity with an estrogen receptor LBD.

2. The method for screening estradiol derivatives that recognize estrogen receptors in solid phase according to claim 1, wherein the step of analyzing the structural stability and energy changes using molecular dynamics simulation is preceded by the step of:

establishing a compound initial structure of molecular dynamics simulation according to the screened estradiol derivative and the estradiol-estrogen receptor LBD crystal structure;

the initial structure of the complex was examined using the gromas program to exclude the estradiol derivative-estrogen receptor LBD complexes that had atomic-positional conflicts.

3. The method for screening estradiol derivatives that recognize estrogen receptors in solid phase according to claim 2, wherein the step of analyzing the structural stability and energy changes using molecular dynamics simulation is preceded by the step of:

screening was performed based on the exposure of the linker tail of the estradiol derivative in the initial structure of the complex outside the estrogen receptor LBD binding pocket.

4. The method for screening estradiol derivatives that recognize estrogen receptors in solid phase according to claim 1, wherein the analysis of structural stability and energy change is performed by molecular dynamics simulation, and the screening is performed based on the simulated trajectory and conformational change obtained by the molecular dynamics simulation analysis.

5. The method for screening estradiol derivatives that recognize estrogen receptors in solid phase according to claim 4, wherein the step of screening based on the simulated trajectory and conformational change obtained by the molecular dynamics simulation analysis comprises:

extracting key indexes according to the molecular dynamics simulation analysis result, and screening according to the key indexes; wherein the key indicators include root mean square deviation, root mean square fluctuation, binding energy, and number of hydrogen bonds.

6. The method for screening estradiol derivatives that recognize estrogen receptors according to claim 5, wherein the step of screening according to the key criteria comprises:

screening out derivatives which reach stable structures in molecular dynamics simulation according to the root-mean-square deviation and the root-mean-square fluctuation;

and according to the binding energy and the number of hydrogen bonds, selecting the derivative with the strongest binding energy and the largest number of hydrogen bonds from the derivatives reaching the stable structure.

7. The method for screening estradiol derivatives that recognize estrogen receptors in solid phase according to claim 4, wherein the step of screening based on the simulated trajectory and conformational change obtained by the molecular dynamics simulation analysis comprises:

according to the influence of the derivative on the conformation of the estrogen receptor LBD, the derivative with small influence on the conformation of the estrogen receptor LBD is screened.

8. The method for screening estradiol derivatives capable of solid-phase recognition of estrogen receptor according to claim 7, wherein the step of screening the derivatives having small influence on the conformation of estrogen receptor LBD according to their influence on the conformation of estrogen receptor LBD comprises:

the H11 and H12 parts of the final conformation of the complex obtained by molecular dynamics simulation are aligned, and the H12 deflection condition of the complex is compared.

9. The method for screening estradiol derivatives for solid-phase identification of estrogen receptors according to claim 1, wherein the step of screening estradiol derivatives satisfying the predetermined requirements by performing computer simulation analysis on the binding capacity of estradiol derivatives with different structures and estrogen receptors LBD by molecular docking based on the crystal conformation of estradiol-estrogen receptor LBD complex comprises:

carrying out three-dimensional structure establishment and energy minimization on estradiol derivatives with different structures by adopting an Autodock program;

based on the binding site of estradiol and estrogen receptor LBD, establishing an initial structure of a estradiol derivative and estrogen receptor LBD compound;

carrying out molecular docking on the estradiol derivative and the estrogen receptor LBD to obtain a docking conformation condition and a docking score, and screening according to the docking conformation condition and the docking score.

10. The method for screening estradiol derivatives that recognize estrogen receptors according to claim 9, wherein the molecular docking is performed by flexible molecular docking.

Technical Field

The invention relates to the technical field of biological materials, in particular to a method for screening estradiol derivatives by solid-phase recognition of estrogen receptors.

Background

The nuclear estrogen receptor, also often referred to simply as the estrogen receptor, is an important site of action for endocrine disrupting effects caused by estrogens. Classical estrogen genome signaling pathway studies have shown that estrogen (i.e., a Ligand for an estrogen receptor) allosterically binds to the estrogen receptor by binding to the LBD (Ligand binding domain) region of the estrogen receptor, and that the allosterics induce or inhibit the expression of specific genes by binding to estrogen-responsive elements in the target gene sequence or not under the combined action of the relevant transcription co-regulators (co-activators or co-repressors). The binding of estrogen to estrogen receptor protein is a molecular initiation event that regulates response to gene expression to produce effects, and the process is highly specific and theoretically can bind to all environmental estrogens that exert adverse effects via nuclear receptor pathways. The in vitro biological detection technology developed according to the interaction between estrogen and estrogen receptor has the advantages of high sensitivity, low operation cost, short experimental period, ethical acceptance and the like.

In the construction of in vitro biological detection technology, estradiol derivatives are often required to be designed, estradiol-protein conjugates are synthesized by the estradiol derivatives, and the estradiol derivatives are fixed on solid phase interfaces such as glass by utilizing rich active groups of protein motifs or are directly fixed on the solid phase interfaces such as glass. Both schemes need to expose an estradiol structure capable of being combined with an estrogen receptor with specific high affinity through the design of a derivative connecting arm. The structure and length of the linker arm can influence the binding property of estradiol and estrogen receptor in solid phase recognition, and the currently common design strategy of the derivative linker arm is as follows. In most studies, the connecting arm is connected at the 3, 6, 7 or 17 position of the estradiol, so that a key group of the estradiol is exposed, and the binding site of the estradiol and a key amino acid residue of an estrogen receptor protein is avoided to be occupied, so that high specificity and affinity are maintained. The length of the connecting arm needs to ensure that the estradiol can successfully enter the estrogen receptor protein binding pocket in the solid phase recognition process and does not cause the change of the receptor protein structure as much as possible.

However, no uniform screening method exists in the related art at present. Therefore, it is necessary to select a structure of the estradiol derivative linker arm and select derivatives having a high affinity for the nuclear receptor in order to further shorten the time for binding of solid-phase estradiol to the estrogen receptor and to increase the binding stability in an in vitro environment.

Disclosure of Invention

The invention aims to provide a method for screening estradiol derivatives capable of identifying estrogen receptors in a solid phase, which can screen estradiol derivatives capable of binding estrogen receptors with high specificity and high affinity, further shorten the binding time of solid-phase estradiol and estrogen receptors and increase the binding stability in an in vitro environment.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a method for screening estradiol derivatives capable of identifying estrogen receptors in a solid phase, which comprises the following steps: based on the crystal conformation of the estradiol-estrogen receptor LBD compound, carrying out computer simulation analysis on the binding capacity of estradiol derivatives with different structures and the estrogen receptor LBD by using molecular docking, and screening out the estradiol derivatives meeting the preset requirements; and (3) analyzing the structural stability and energy change of the screened estradiol derivatives by adopting molecular dynamics simulation, and screening the derivatives with specificity and high affinity with an estrogen receptor LBD.

Optionally, before the step of analyzing the structural stability and the energy change by using molecular dynamics simulation, the method further includes: establishing a compound initial structure of molecular dynamics simulation according to the screened estradiol derivative and the estradiol-estrogen receptor LBD crystal structure; the initial structure of the complex was examined using the gromas program to exclude the estradiol derivative-estrogen receptor LBD complexes that had atomic-positional conflicts.

Optionally, before the step of analyzing the structural stability and the energy change by using molecular dynamics simulation, the method further includes: screening was performed based on the exposure of the linker tail of the estradiol derivative in the initial structure of the complex outside the estrogen receptor LBD binding pocket.

Optionally, the analysis of structural stability and energy change is performed by molecular dynamics simulation, and the screening is performed according to the simulated trajectory and conformational change obtained by the molecular dynamics simulation analysis.

Optionally, the step of screening according to the simulated trajectory and the conformational change obtained by the molecular dynamics simulation analysis includes: extracting key indexes according to the molecular dynamics simulation analysis result, and screening according to the key indexes; wherein the key indicators include root mean square deviation, root mean square fluctuation, binding energy, and number of hydrogen bonds.

Optionally, the step of screening according to the key index includes: screening out derivatives which reach stable structures in molecular dynamics simulation according to the root-mean-square deviation and the root-mean-square fluctuation; and according to the binding energy and the number of hydrogen bonds, selecting the derivative with the strongest binding energy and the largest number of hydrogen bonds from the derivatives reaching the stable structure.

Optionally, the step of screening according to the simulated trajectory and the conformational change obtained by the molecular dynamics simulation analysis includes: according to the influence of the derivative on the conformation of the estrogen receptor LBD, the derivative with small influence on the conformation of the estrogen receptor LBD is screened.

Optionally, the step of screening the derivative having a small influence on the conformation of the estrogen receptor LBD according to the influence of the derivative on the conformation of the estrogen receptor LBD comprises: the H11 and H12 parts of the final conformation of the complex obtained by molecular dynamics simulation are aligned, and the H12 deflection condition of the complex is compared.

Optionally, the step of performing computer simulation analysis on the binding capacity of estradiol derivatives with different structures and estrogen receptor LBD by using molecular docking based on the crystal conformation of the estradiol-estrogen receptor LBD complex and screening estradiol derivatives meeting preset requirements includes: carrying out three-dimensional structure establishment and energy minimization on estradiol derivatives with different structures by adopting an Autodock program; based on the binding site of estradiol and estrogen receptor LBD, establishing an initial structure of a estradiol derivative and estrogen receptor LBD compound; carrying out molecular docking on the estradiol derivative and the estrogen receptor LBD to obtain a docking conformation condition and a docking score, and screening according to the docking conformation condition and the docking score.

Optionally, the molecular docking is by flexible molecular docking.

Compared with the prior art, the estradiol derivative screening method for solid-phase recognition of the estrogen receptor can screen out the derivative with specificity and high affinity with the estrogen receptor, further shorten the binding time of the solid-phase estradiol and the estrogen receptor and increase the binding stability in an in vitro environment.

The derivative virtual screening method is economical and efficient, can provide theoretical basis and detailed technical support for the design of other solid-phase affinity reaction small molecule derivatives, and the screened small molecule derivatives have application prospects in the fields of biosensing, nano material preparation and application thereof.

Drawings

FIGS. 1a and 1b are schematic illustrations of the docking interaction of estradiol derivatives with human estrogen receptor LBD molecules in an embodiment of the present invention, wherein FIG. 1a is a flexible derivative; FIG. 1b is a rigid derivative, with the portion of the ligand that leaks out of the binding pocket shown as circled and shaded, and the hydrogen bonding indicated by the arrows;

FIG. 2 is a schematic representation of the molecular dynamics of a rigid estradiol derivative and human estrogen receptor LBD in accordance with an embodiment of the present invention in its simulated conformation, wherein the left side is a whole and the right side is a partial enlargement of the conflicting positions;

FIG. 3 is a schematic illustration of the docking conformation of estradiol derivative numbered 4 with human estrogen receptor LBD molecules according to example of the present invention;

fig. 4a to 4d are results of molecular dynamics simulation of complexes of four flexible estradiol derivatives numbered 8, 11, 16 and 20 with human estrogen receptor LBD according to the examples of the present invention, wherein fig. 4 a: root mean square deviation RMSD; FIG. 4 b: root mean square fluctuation RMSF; FIG. 4 c: binding energy calculated using the MM/PBSA method; FIG. 4 d: the number of hydrogen bonds;

FIGS. 5a to 5d are schematic diagrams of the molecular dynamics-simulated interaction of a flexible estradiol derivative with a human estrogen receptor LBD in an embodiment of the present invention; the corresponding ligands are respectively: FIG. 5 a: derivative 8; FIG. 5 b: derivative 11; FIG. 5 c: derivative 16; FIG. 5 d: derivative 20; the portion of the ligand that leaks out of the binding pocket is indicated by the circled shaded circle;

FIG. 6 shows the positions of the human estrogen receptors LBD-H12 at the end of the MDS simulation in the examples of the present invention, wherein the ligands are derivative 8, derivative 11, derivative 16, derivative 20, respectively, reference structures ER-aginst, ER-apo.

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. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. 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.

The invention provides a estradiol derivative screening method for solid-phase recognition of estrogen receptors, which is characterized in that the method is based on the crystal conformation of a estradiol-estrogen receptor LBD compound, and computer simulation analysis is carried out on the binding capacity of different estradiol derivatives and the estrogen receptor LBD by using molecular docking; and then further analyzing the structural stability and energy change by adopting molecular dynamics simulation, and screening out the derivative of the connecting arm structure with high specificity and high affinity with the estrogen receptor LBD. The screening method is formed by developing the design idea and the screening principle of the estradiol derivative connecting arms by using computational biology.

In an alternative embodiment, in the molecular dynamics simulation analysis, for a compound structure with a reasonable structure selected by molecular docking simulation, vacuum energy minimization, solvent box addition, charge balance, constant atomic number, volume and temperature simulation, constant atomic number, pressure and temperature simulation and final molecular dynamics simulation of 10ns are performed; and then, extracting key indexes according to molecular dynamics simulation tracks and conformation changes, and comprehensively screening out the optimal derivative.

The method can comprise the following steps: establishing a compound initial structure of molecular dynamics simulation according to the estradiol derivative screened after molecular docking simulation analysis and an estradiol-estrogen receptor LBD crystal structure; and then, checking the initial structure of the compound by adopting a Gromacs program, and eliminating the estradiol derivative-estrogen receptor LBD compound with atom position conflict to obtain the compound with a reasonable structure. Further, the method can also comprise the following steps: screening was performed based on the exposure of the linker tail of the estradiol derivative in the initial structure of the complex outside the estrogen receptor LBD binding pocket.

In the molecular dynamics simulation analysis, the derivative of the connecting arm structure with high specificity and high affinity with the estrogen receptor LBD can be screened according to the simulation track and conformation change obtained by the molecular dynamics simulation analysis.

Specifically, the method may include: extracting key indexes according to the molecular dynamics simulation analysis result, and screening according to the key indexes; wherein the key indicators include root mean square deviation, root mean square fluctuation, binding energy, and number of hydrogen bonds. Further, the screening according to the key index may include: screening out derivatives which reach stable structures in molecular dynamics simulation according to the root-mean-square deviation and the root-mean-square fluctuation; and according to the binding energy and the number of hydrogen bonds, selecting the derivative with the strongest binding energy and the largest number of hydrogen bonds from the derivatives reaching the stable structure.

Further, the method can also comprise the following steps: according to the influence of the derivative on the conformation of the estrogen receptor LBD, the derivative with small influence on the conformation of the estrogen receptor LBD is screened. For example, the H11 and H12 portions of the final conformation of the complex obtained from molecular dynamics simulations can be aligned and compared for H12 deflection.

In an optional embodiment, in the molecular docking computer simulation analysis, molecular docking is adopted to perform preliminary evaluation on the binding capacity of estradiol derivatives with different structures and estrogen receptor LBD, so as to obtain docking scores and docking conformations; and (4) primarily screening the flexible estradiol derivative as a further research object according to the docking conformation rationality and the docking score.

Specifically, the method may include: carrying out three-dimensional structure establishment and energy minimization on estradiol derivatives with different structures by adopting an Autodock program; based on the binding site of estradiol and estrogen receptor LBD, establishing an initial structure of a estradiol derivative and estrogen receptor LBD compound; carrying out molecular docking on the estradiol derivative and the estrogen receptor LBD to obtain a docking conformation condition and a docking score, and screening according to the docking conformation condition and the docking score. Wherein, a flexible analysis butt joint mode can be adopted.

The technical solution of the present invention is exemplarily described below with reference to a specific embodiment:

five estradiol derivatives with flexible structures (4, 8, 11, 16 and 20) and six estradiol derivatives with rigid structures (r3, r5, r8, r11, r16 and r20) with different connecting arms are selected as objects to be investigated, and the molecular structures are shown in the following table 1.

TABLE 1 molecular Structure of estradiol derivatives of different linker arms

Taking the crystal structure (PDB ID:1GWR) of estradiol and human estrogen receptor LBD as an example, the automatic dock program is adopted to carry out three-dimensional structure establishment and molecular docking simulation after energy minimization on the estradiol derivatives with the structures, and the structure more suitable for ligand binding pocket LBP is preliminarily screened.

According to the simulation result, the following results are obtained: the flexible estradiol derivative structure (docking score Dscore-6.5) is more adapted to the structure of the ligand binding pocket LBP, and the docking score and the producible conformation are superior to the rigid estradiol derivative structure (docking score Dscore-5.0). Specifically, the molecular docking program outputs a plurality of docking conformations with the highest reasonableness and corresponding docking scores according to the algorithm, in the embodiment, the flexible structure can generate more docking conformations, and the docking scores are all better than those of the rigid structure, which indicates that the flexible structure is better in molecular docking with the human estrogen receptor LBD.

Fig. 1 schematically shows the interaction of estradiol derivative ligands with human estrogen receptor LBD, and as shown in fig. 1, analysis of the interaction of estradiol derivative ligands with estrogen receptors shows that in the docking conformation of the flexible derivative of fig. 1a, the rigid derivative of fig. 1b and the human estrogen receptor LBD, the cyclic structure of estradiol is partially exposed outside the binding pocket, and is significantly different from the normal structure of estradiol recognized by estrogen receptors. Meanwhile, considering that the tail end of the connecting arm is exposed due to a reasonable derivative structure, the molecular dynamics simulation analysis is further carried out on the flexible and rigid derivatives with different carbon chain lengths.

During molecular dynamics simulation analysis, the structures of the six rigid derivatives (r3, r5, r8, r11, r16 and r20) combined with the human estrogen receptor LBD are found to have error report in molecular dynamics simulation. Analysis shows that the rigid structure of the complex and the amino acid residues of the human estrogen receptor LBD have position conflict, so the position conflict complexes are excluded, and the complex with more reasonable structure is screened. Wherein, the position conflict is that the distance between the atoms is smaller than the normal atom distance (indicating that the positions of the atoms are approximately overlapped), the judgment is based on the atom distance, and the intuitive judgment is that the rigid structure and the LBD part of the human estrogen receptor have position conflict in the conformation chart.

FIG. 2 schematically shows a schematic representation of the molecular dynamics of the simulated conformation of a rigid estradiol derivative with the human estrogen receptor LBD, as evident from the right partial magnification, the tail end structure of the derivative crosses the protein helix of the receptor LBD, and positional conflicts arise. Therefore, these structures do not allow molecular dynamics simulation results, and this positional conflict also suggests that it may interfere with the normal structure of human estrogen receptor LBD and the allosteric conformation of H12 in the experiments. Therefore, experiments prove that flexible connecting arms are used or only rigid derivatives with similar carbon chain lengths to those of the flexible derivatives which have better performance in simulation are selected.

Fig. 3 schematically shows a schematic diagram of the docking conformation of estradiol derivative 4 (derivative numbered 4 in table 1) with the human estrogen receptor LBD molecule. As shown in FIG. 3, it can be found that the carbon chain length of the linking arm is too short to meet the design requirement of the subsequent linking protein for forming the derivative capable of specifically binding with high affinity to estrogen receptor protein, so that no further simulation is performed.

That is, too short a linker arm will affect the binding of estradiol to estrogen receptor in solid phase recognition, and therefore, the solid phase recognition needs to be screened according to whether the design requirement is satisfied, that is, whether the design requirement is satisfied is determined according to the length of the carbon chain of the linker arm in the molecular docking conformation. In addition, as shown in fig. 3, the structure is completely enclosed in the binding pocket of the receptor LBD, the tail end of the linker arm cannot be exposed, and the estradiol derivative 4 is not simulated in consideration of the reason that the reasonable derivative structure should expose the tail end of the linker arm, so far, the remaining derivatives screened out are flexible estradiol derivatives (8, 11, 16, 20).

The four selected flexible estradiol derivatives (8, 11, 16 and 20) are subjected to 10ns molecular dynamics simulation with human estrogen receptor LBD respectively. Molecular dynamics simulations were performed in gromacs2016.4 under a GROMOS 9643 a1 force field. In the simulation, the complexes were first solvated in a dodecahedral cartridge using single-point charged water molecules, with a minimum distance of 1.5nm between the complex and the solvent cartridge. Na + and Cl-ions were then added to the solvent cartridge for charge balancing. Before the final simulation of 10ns, energy minimization, temperature coupling (300k), pressure coupling (1bar) were performed.

FIGS. 4a to 4d schematically show the results of molecular dynamics simulation of the four flexible estradiol derivatives described above with respect to the human estrogen receptor LBD complex. Wherein fig. 4a schematically shows the root mean square deviation RMSD of the four derivatives; FIG. 4b schematically shows the root mean square fluctuation RMSF of four derivatives; FIG. 4c schematically shows the calculated binding energies of the four derivatives using the MM/PBSA method; figure 4d schematically shows the number of hydrogen bonds for the four derivatives.

Wherein, the variation of the root mean square deviation RMSD of the skeleton atoms of the ligand-receptor protein and the root mean square fluctuation RMSF of each amino acid of the ligand-receptor protein reflect the structural variation of the estrogen receptor in the simulation process; changes in ligand-receptor protein binding capacity reflect changes in non-binding capacity; the change in the number of hydrogen bonds of the ligand-receptor protein (Num _ Hbond) reflects the change in the number of hydrogen bonds mimicking the process.

In FIG. 4a, the RMSD structure shows that at least 0.5ns before the end of the simulation, each composite structure is substantially stable, indicating that the simulation time is reasonably selected.

In FIG. 4b, the portion of the RMSF data reflecting the major structural changes in the human estrogen receptor LBD is the amino acid sequence from position 520 onwards, i.e., portion H12.

In fig. 4c, the binding energy results show that the energy of the complex formed by the flexible estradiol derivative 8 and the human estrogen receptor LBD is changed more dramatically, which indicates that the structural change of the complex changes the acting force between the ligand and the receptor more obviously.

In fig. 4d, Hbond results indicate that the complex of estradiol derivatives 11, 16, 20 as the ligand has a weak ability to form hydrogen bonds with the ligand-receptor, reflecting the weak binding ability, whereas estradiol derivative 8 always has a strong ability to form hydrogen bonds, and most of the brackets in the upper part of fig. 4d are estradiol derivatives 8.

In conclusion, the RMSD and RMSF characteristics are used for judging whether the structure is stable in the simulation, and further, the binding energy and the number of hydrogen bonds of the stable structure are analyzed, and the derivative of the connecting arm structure with the strongest binding energy and the largest number of hydrogen bonds is screened out.

In the present invention, the final judgment of the derivative needs to be made based on conformational rationality in addition to the binding energy and the number of hydrogen bonds. That is, at the end of the molecular dynamics simulation, the human estrogen receptor LBD conformation is also analyzed.

Fig. 5a to 5d schematically show the molecular dynamics simulated interaction of the above four flexible estradiol derivatives with the human estrogen receptor LBD, wherein fig. 5a is derivative 8; FIG. 5b is derivative 11; FIG. 5c is derivative 16; figure 5d is derivative 20.

As can be seen in fig. 5a, derivative 8 is better encapsulated by the ligand binding pocket, between key amino acid residues (e.g., Glu353, Arg394, His524, etc.). While the other three derivatives change the position of LBD as shown in FIGS. 5b to 5d, the binding with human estrogen receptor LBD may be unstable and have weak binding ability. In the present invention, the filled circles in fig. 1a to 1b and fig. 5a to 5d are all amino acids.

Further, H11 and H12 portions, modeled to give the final conformation of each complex, were aligned and compared for their H12 deflections.

Figure 6 schematically shows the position of the human estrogen receptor LBD-H12 at the end of MDS simulation, as shown in figure 6, where the ligands are derivative 8, derivative 11, derivative 16, derivative 20, respectively, reference structures ER-aginst, ER-apo. As can be seen in fig. 6, binding to derivative 8 resulted in H12 undergoing a more nearly neutral (apo) to excited (agonst) position change in H12 induced by E2.

Finally, combined with the analysis of the simulation process and the simulation results, it is believed that derivative 8 has better binding ability to human estrogen receptor LBD and less allosteric influence on human estrogen receptor LBD, whereas too long carbon chains may lead to disruption of the binding pocket and weaker binding stability.

The description of the present invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to practitioners skilled in this art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.