CN113593647B

CN113593647B - Method for screening estradiol derivatives by solid phase recognition of estrogen receptor

Info

Publication number: CN113593647B
Application number: CN202110841192.9A
Authority: CN
Inventors: 周小红; 朱茜; 谭集穗
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2024-04-19
Anticipated expiration: 2041-07-23
Also published as: CN113593647A

Abstract

The invention discloses a screening method of estradiol derivatives for solid phase recognition of estrogen receptors. 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 capacities of the estradiol derivatives with different structures and the estrogen receptor LBD by using molecular docking, and screening out the estradiol derivatives meeting preset requirements; and analyzing structural stability and energy change of the screened estradiol derivative by adopting molecular dynamics simulation, and screening out a derivative with high specificity and affinity with the estrogen receptor LBD. The invention can screen out the derivatives with high specificity and high affinity with the estrogen receptor, thereby shortening the binding time of solid-phase estradiol and the estrogen receptor and increasing the binding stability in vitro environment.

Description

Method for screening estradiol derivatives by solid phase recognition of estrogen receptor

Technical Field

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

Background

Nuclear estrogen receptors, also commonly referred to simply as estrogen receptors, are important sites of action for endocrine disrupting effects caused by estrogens. Classical estrogen genome signaling pathway studies have shown that estrogen (i.e., a ligand of an estrogen receptor) allosteric the estrogen receptor through the LBD (ligand binding domain ) region of the conjugated estrogen receptor, and that allosteric induces or inhibits the expression of specific genes by binding or not to estrogen responsive elements on the target gene sequence under the coaction of related transcriptional cofactors (coactivators or cosuppression factors). Where estrogen binding to estrogen receptor proteins is a molecular initiation event that modulates the response of gene expression to produce an effect, the process is highly specific and can theoretically bind to all environmental estrogens that exert adverse effects via nuclear receptor pathways. The in vitro biological detection technology developed according to the interaction of estrogen and estrogen receptor has the advantages of high sensitivity, low operation cost, short experimental period, ethical acceptance and the like, and is paid attention to.

In the construction of in vitro biological detection technology, it is often necessary to design an estradiol derivative, synthesize an estradiol-protein conjugate by using the estradiol derivative, and fix a solid phase interface with glass and the like by using active groups rich in protein motif, or directly fix the estradiol derivative on the solid phase interface with glass and the like. Both schemes require the design of a derivative linker arm to expose the estradiol structure that can specifically bind to estrogen receptors with high affinity. The structure and length of the linker arm can influence the binding performance of estradiol and estrogen receptor in solid phase recognition, and the design strategy of the derivative linker arm commonly used at present is as follows. In most studies, the linker arm is attached at the 3, 6, 7 or 17 position of estradiol, exposing the critical groups of estradiol, avoiding occupation of the binding site of estradiol to critical amino acid residues of the estrogen receptor protein, to maintain higher specificity and affinity. The length of the connecting arm must ensure that estradiol can successfully enter the estrogen receptor protein binding pocket in the solid phase recognition process, and the change of the receptor protein structure is not caused as much as possible.

However, there is no unified screening method in the related art. Therefore, it is necessary to select a derivative having a high affinity with the specificity of the nuclear receptor by optimizing the structure of the linking arm of the estradiol derivative, in order to further shorten the time for binding of the 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 of a solid-phase recognition estrogen receptor, which can screen out the estradiol derivatives of the high-specificity and high-affinity binding estrogen receptor, further can shorten the binding time of the solid-phase estradiol and the estrogen receptor and increase the binding stability in an in-vitro environment.

The above object of the present invention is achieved by the following technical solutions:

The invention provides a screening method of estradiol derivatives for solid phase recognition of estrogen receptors, 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 capacities of the estradiol derivatives with different structures and the estrogen receptor LBD by using molecular docking, and screening out the estradiol derivatives meeting preset requirements; and analyzing structural stability and energy change of the screened estradiol derivative by adopting molecular dynamics simulation, and screening out a derivative with high specificity and affinity with the estrogen receptor LBD.

Optionally, before the step of analyzing the structural stability and the energy change by using molecular dynamics simulation, the method further comprises: establishing a molecular dynamics simulated compound initial structure according to the screened estradiol derivative and the estradiol-estrogen receptor LBD crystal structure; checking the initial structure of the complex by using a Gromacs program, and eliminating the estradiol derivative-estrogen receptor LBD complex with atomic position conflict.

Optionally, before the step of analyzing the structural stability and the energy change by using molecular dynamics simulation, the method further comprises: screening was performed based on the extent to which the tail end of the linking arm of the estradiol derivative is exposed outside the LBD binding pocket of the estrogen receptor in the initial structure of the complex.

Optionally, analysis of structural stability and energy variation is performed using molecular dynamics simulation, and screening is performed based on simulated trajectories and conformational changes obtained from molecular dynamics simulation analysis.

Optionally, the step of screening according to the simulated trajectory and conformational change obtained by molecular dynamics simulation analysis includes: extracting key indexes according to a 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 hydrogen bond number.

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

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

Optionally, the step of screening out 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 includes: the H11 and H12 portions of the final conformation of the complex obtained by molecular dynamics simulation are aligned, and H12 deflection conditions generated by the alignment are compared.

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

Alternatively, the molecular docking is in the form of flexible molecular docking.

Compared with the prior art, the method for screening the estradiol derivatives capable of recognizing the estrogen receptor through the solid phase can screen the derivatives with high specificity and affinity with the estrogen receptor, so that the time for combining the solid phase estradiol and the estrogen receptor can be shortened, and the combination stability in an in-vitro environment can be improved.

The virtual screening method of the derivatives 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 has application prospects in the fields of biosensing, nano material preparation and application.

Drawings

FIGS. 1a and 1b are schematic diagrams showing the docking interactions of an estradiol derivative with a human estrogen receptor LBD molecule in an embodiment of the present invention, wherein FIG. 1a is a flexible derivative; FIG. 1b is a rigid derivative, the portion of the ligand that leaks out of the binding pocket is shown in circular shading, and hydrogen bonding is shown by arrows;

FIG. 2 is a schematic representation of a molecular dynamics modeling of a rigid estradiol derivative and a humanized estrogen receptor LBD in an embodiment of the present invention, wherein the left side is the whole and the right side is a partial enlarged view of a position conflicting portion;

FIG. 3 is a schematic representation of the molecular docking configuration of the estradiol derivative, numbered 4, with a human estrogen receptor LBD in accordance with an embodiment of the present invention;

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

FIGS. 5a to 5d are schematic diagrams showing simulated interaction between flexible estradiol derivatives and human estrogen receptor LBD in accordance with the present invention; the corresponding ligands are respectively as follows: fig. 5a: a derivative 8; fig. 5b: a derivative 11; fig. 5c: derivative 16; fig. 5d: a derivative 20; the portion of the ligand that leaks out of the binding pocket is shown in circular shading;

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

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention provides a screening method of estradiol derivatives for solid-phase recognition of estrogen receptors, which is based on the crystal conformation of an estradiol-estrogen receptor LBD compound, and carries out computer simulation analysis on the binding capacities of different estradiol derivatives and estrogen receptor LBDs by using molecular docking; and then further adopting molecular dynamics simulation to analyze structural stability and energy change, and screening out derivatives of the connecting arm structure with high specificity and high affinity with the estrogen receptor LBD. The screening method disclosed by the invention is formed by applying the design thought and screening principle of the estradiol derivative connecting arm developed by computing biology.

In an alternative embodiment, in the molecular dynamics simulation analysis, for the compound structure with reasonable structure screened by the 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 10ns molecular dynamics simulation are performed; then, key indexes can be extracted according to the molecular dynamics simulation track and conformational change, and the optimal derivatives can be comprehensively screened out.

May include: according to the estradiol derivative screened after molecular docking simulation analysis and the estradiol-estrogen receptor LBD crystal structure, establishing a compound initial structure of molecular dynamics simulation; and then checking the initial structure of the complex by adopting a Gromacs program, and removing the estradiol derivative-estrogen receptor LBD complex with atom position conflict to obtain the complex with reasonable structure. Further, the method may further include: screening was performed based on the extent to which the tail end of the linking arm of the estradiol derivative is exposed outside the LBD binding pocket of the estrogen receptor in the initial structure of the complex.

In the invention, in the molecular dynamics simulation analysis, the derivatives with high specificity and high affinity with the estrogen receptor LBD (LBD) of the connecting arm structure can be screened out according to the simulated track and the conformational change obtained by the molecular dynamics simulation analysis.

Specifically, it may include: extracting key indexes according to a 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 hydrogen bond number. Further, the screening according to the key index may include: screening out derivatives reaching a stable structure in molecular dynamics simulation according to the root mean square deviation and the root mean square fluctuation; and screening the derivative with the strongest binding energy and the largest hydrogen bond number from the derivatives reaching a stable structure according to the binding energy and the hydrogen bond number.

Further, the method may further include: and screening the derivatives with small influence on the conformation of the estrogen receptor LBD according to the influence of the derivatives on the conformation of the estrogen receptor LBD. For example, H11 and H12 portions of the final conformation of the complex obtained by molecular dynamics simulation can be aligned and compared for H12 deflection.

In an alternative embodiment, in the molecular docking computer simulation analysis, the binding capacity of the estradiol derivatives with different structures and the estrogen receptor LBD is primarily evaluated by adopting molecular docking to obtain docking scoring and docking conformation; and (5) primarily screening the flexible estradiol derivatives as a further study object according to the rationality of the docking conformation and the docking score.

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

The technical scheme of the invention is exemplified below with reference to a specific embodiment:

Five flexible structural estradiol derivatives (4, 8, 11, 16, 20) and six rigid structural estradiol derivatives (r 3, r5, r8, r11, r16, r 20) with different linker arms were selected as subjects, and their molecular structures are shown in table 1 below.

TABLE 1 molecular Structure of estradiol derivatives of different linker arms

Taking the LBD crystal structure (PDB ID:1 GWR) of the estradiol and the human source estrogen receptor as an example, adopting an Autodock program to carry out three-dimensional structure establishment and energy minimization on the estradiol derivatives with the above structures, then carrying out molecular docking simulation, and primarily screening the structure which is more suitable for ligand binding pocket LBP.

From the simulation results, it can be seen that: the flexible estradiol derivative structure (docking score Dscore-6.5) is more adapted to the structure of the ligand binding pocket LBP, and both the docking score and the producible conformation are superior to the rigid estradiol derivative structure (docking score Dscore-5.0). In particular, the molecular docking program will output a plurality of docking conformations and corresponding docking scores according to the algorithm that are most rational, in this embodiment the flexible structure is able to generate more docking conformations, and the docking scores are all superior to the rigid structure, indicating that the flexible structure is better in molecular docking with the human estrogen receptor LBD.

Fig. 1 schematically shows the interaction of an estradiol derivative ligand with a human estrogen receptor LBD, as shown in fig. 1, by analysis of the interaction of an estradiol derivative ligand with an estrogen receptor, which is shown in the interfacing 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, which differs considerably from the normal structure of estrogen receptor-recognizing estradiol. Meanwhile, the tail end of the connecting arm is exposed in consideration of reasonable derivative structure, so that further molecular dynamics simulation analysis is carried out on flexible and rigid derivatives with different carbon chain lengths.

During the molecular dynamics simulation analysis, six rigid derivatives (r 3, r5, r8, r11, r16, r 20) were found to have a structure that binds to the human estrogen receptor LBD that reported errors in the molecular dynamics simulation. Analysis shows that the rigid structure of the complex has position conflict with the amino acid residue of the human estrogen receptor LBD, so that the complexes with the position conflict are eliminated, and the complexes with more reasonable structures are screened. The position conflict is that the distance between atoms is smaller than the normal atomic distance (the positions with atoms are almost overlapped), the judgment basis is the atomic distance, and the visual judgment is that the position conflict occurs between the rigid structure and the LBD part of amino acid of the human estrogen receptor in the conformational diagram.

Fig. 2 schematically shows a schematic representation of the molecular dynamics simulated conformation of a rigid estradiol derivative with the human estrogen receptor LBD, from which it is evident from the right partial magnified view that the tail end structure of the derivative crosses the protein helix of the receptor LBD, with a positional conflict. Therefore, these structures cannot achieve molecular dynamics simulation results, and this position conflict also suggests that it may interfere with the normal structure of the human estrogen receptor LBD and the allosteric of H12 in the experiment. Therefore, experimental verification is carried out by using a flexible connecting arm or selecting only rigid derivatives with carbon chain lengths similar to those of flexible derivatives which are better in simulation.

Figure 3 schematically shows a schematic representation of the molecular docking conformation of estradiol derivative 4 (derivative numbered 4 in table 1) with a human estrogen receptor LBD molecule. As shown in FIG. 3, it was found that the chain length of the linker arm was too short to meet the design requirements of subsequent linker protein formation of derivatives that specifically bind to estrogen receptor proteins with high affinity, and no further simulation was performed.

That is, too short a length of the linker will affect the binding of estradiol to the estrogen receptor in the solid phase recognition, and therefore, the screening is performed according to whether the design requirement is met, i.e. whether the design requirement is met is determined according to the length of the carbon chain of the linker 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 connecting arm cannot be exposed, and the estradiol derivative 4 is not simulated again in view of the reasonable derivative structure which should expose the tail end of the connecting arm, so far, the remaining derivatives screened are flexible estradiol derivatives (8, 11, 16, 20).

The four flexible estradiol derivatives (8, 11, 16, 20) screened were each subjected to 10ns of molecular dynamics simulation with the human estrogen receptor LBD. Molecular dynamics simulations were performed in gromacss 2016.4 under the force field of GROMOS96 a 1. In the simulations, the complex was first solvated in dodecahedral boxes using single-point charged water molecules, with a minimum distance between the complex and the solvent box of 1.5nm. Na+ and Cl-ions are then added to the solvent box to allow charge balancing. Before a final simulation of 10ns, energy minimization, temperature coupling (300 k), pressure coupling (1 bar) were performed.

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

Wherein the RMSD change of the backbone atoms of the ligand-receptor protein and the RMS fluctuation RMSF of each amino acid of the ligand-receptor protein reflect the structural change of the estrogen receptor during the simulation; the change in ligand-receptor protein binding energy reflects the change in non-binding energy; the change in the number of ligand-receptor protein hydrogen bonds (num_ Hbond) reflects the change in the number of hydrogen bonds in the simulation process.

In fig. 4a, the RMSD structure can be seen to be at least 0.5ns before the end of the simulation, and each composite structure is substantially stable, indicating that the simulation time is reasonable to choose.

In FIG. 4b, the result RMSF reflects that the portion of the human estrogen receptor LBD which is mainly structurally changed is the amino acid sequence after 520 th, i.e., H12 portion.

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 more severely changed, which indicates that the structural change of the complex is more obviously changed to change the acting force between the ligand and the receptor.

In fig. 4d, hbond shows that the ligand is a complex of estradiol derivatives 11, 16, 20, the ligand-acceptor of which is weak in the formation of hydrogen bonds, reflecting that the binding capacity is weak, while estradiol derivative 8 always has strong hydrogen bond formation capacity, and most of the brackets in the upper part of fig. 4d are estradiol derivatives 8.

In summary, RMSD and RMSF are used to determine whether the structure is stable in simulation, and further, the binding energy and hydrogen bond number of the stable structure are analyzed to screen out the derivative with the strongest binding energy and the most hydrogen bond number forming the connecting arm structure.

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

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

As can be seen in FIG. 5a, derivative 8 is well encapsulated by a ligand binding pocket, between key amino acid residues (e.g., glu353, arg394, his524, etc.). The other three derivatives, which change the position of LBD as shown in FIGS. 5b to 5d, may have unstable binding to human estrogen receptor LBD and weak binding. It should be noted that, in the present invention, the solid circles in fig. 1a to 1b and fig. 5a to 5d are all amino acids.

Further, the H11 and H12 portions of the final conformations of each complex were simulated and aligned and compared for H12 deflection.

FIG. 6 schematically shows the position of the human estrogen receptor LBD-H12 at the end of MDS simulation, and FIG. 6 shows that the ligands are derivative 8, derivative 11, derivative 16, derivative 20, respectively, reference structures ER-agonist, ER-apo. As can be seen from fig. 6, binding to derivative 8 causes a closer E2-induced change in the position of H12 from neutral (apo) to excited (agonist) to occur in H12.

Finally, combining the analysis of the simulation process and simulation results, derivative 8 is believed to have better binding capacity to the human estrogen receptor LBD with less allosteric influence on the human estrogen receptor LBD, while an excessively long carbon chain may result in destruction 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 those of ordinary skill in the art. The embodiments were 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.

Claims

1. A method for screening estradiol derivatives that recognize an estrogen receptor in a solid phase, said method comprising:

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

comprising the following steps: carrying out three-dimensional structure establishment and energy minimization on the estradiol derivatives with different structures by adopting an Autodock program; based on the binding site of the estradiol and the estrogen receptor LBD, establishing an initial structure of the estradiol derivative and the estrogen receptor LBD complex; molecular docking is carried out on the estradiol derivative and the estrogen receptor LBD to obtain a docking conformation situation and docking score, and screening is carried out according to the docking conformation situation and the docking score; checking the initial structure of the complex by adopting a Gromacs program, and eliminating the estradiol derivative-estrogen receptor LBD complex with atom position conflict; screening according to the exposure degree of the tail end of the connecting arm of the estradiol derivative outside the LBD binding pocket of the estrogen receptor in the initial structure of the complex;

Analyzing structural stability and energy change of the compound corresponding to the screened estradiol derivative by adopting molecular dynamics simulation, screening according to simulation tracks and conformational changes obtained by the molecular dynamics simulation analysis, and screening out a derivative with high specificity and high affinity with an estrogen receptor LBD;

Comprising the following steps: and extracting key indexes according to a molecular dynamics simulation analysis result, wherein the key indexes comprise root mean square deviation, root mean square fluctuation, binding energy and hydrogen bond number, screening out derivatives reaching a stable structure in molecular dynamics simulation according to the root mean square deviation and root mean square fluctuation in the key indexes, and screening out the derivatives with the strongest binding energy and the largest hydrogen bond number according to the binding energy and the hydrogen bond number in the key indexes.

2. The method for screening estradiol derivatives which solid phase recognizes an estrogen receptor according to claim 1, wherein said step of screening according to a simulated trajectory and conformational change obtained by a molecular dynamics simulation analysis comprises:

And screening the derivatives with small influence on the conformation of the estrogen receptor LBD according to the influence of the derivatives on the conformation of the estrogen receptor LBD.

3. The method according to claim 2, wherein the step of screening out the derivative having a small influence on the conformation of the estrogen receptor LBD based on the influence of the derivative on the conformation of the estrogen receptor LBD comprises:

The H11 and H12 portions of the final conformation of the complex obtained by molecular dynamics simulation are aligned, and H12 deflection conditions generated by the alignment are compared.

4. The method for screening estradiol derivatives for solid phase recognition of an estrogen receptor according to claim 1, wherein said molecular docking is a flexible molecular docking.