A protein-protein interaction regulator and its application and evaluation method

Abstract

The present invention provides a protein-protein interaction regulator, its application and evaluation method. A targeting p53-MDM2 peptide regulator (referred to as CPAP) is subjected to homodimer ligation. The homodimerization of CPAP can increase its affinity by two orders of magnitude. The present invention establishes a brand-new method guided by statistical mechanics theory to quantify the affinity of ligands with different topological structures by comprehensively analyzing the change of binding enthalpy and the loss of translational entropy and rotational entropy. The present invention can overcome the pharmacological obstacles of cell internalization and tumor accumulation by means of a goldophilic interaction-driven self-assembly strategy, more effectively inhibit tumor growth while maintaining good safety.

Description

Technical Field

[0001] The present invention belongs to the medical field, and in particular relates to the technical field of protein-protein interaction (PPI) regulators. Background Art

[0002] Proteins, as fundamental building blocks of life, exert regulatory effects primarily through the 650,000 protein-protein interaction sites (PPIs) in the human protein interactome. Emerging PPI modulators have the exciting ability to serve as specific probes to modulate complex biological signals and have great therapeutic potential to correct aberrant PPIs in a variety of human diseases, including but not limited to: cancer, infectious diseases, and neurodegenerative diseases. Compared with traditional enzyme and receptor targets, PPI targets always have lower affinity because the protein surface is very flat and discontinuous, lacking discrete binding pockets. To address this issue, antibody molecules have been used to target extracellular PPIs, some of which have been approved for clinical use, such as Nivolumab for blocking PD-1 / PD-L1 interaction and Ipilimumab for interfering with CTLA-4 / B7 interaction. However, antibodies are ineffective inside cells due to the loss of topological structure caused by the intracellular reducing environment.

[0003] In addition, the prior art generally attempts to calculate the contribution of affinity through atomic molecular dynamics (MD) simulation. However, the traditional MD method cannot meet the requirements of distinguishing topology. Summary of the invention

[0004] In view of one of the technical problems existing in the above-mentioned prior art, the present invention provides a protein-protein interaction regulator, application and evaluation method, wherein a p53-MDM2 targeted peptide regulator (called CPAP) is homodimerized, and a new statistical mechanics theory-guided method is established to quantify the affinity of ligands with different topological structures by comprehensively analyzing the change in binding enthalpy and the loss of translational entropy and rotational entropy. It is concluded that CPAP homodimerization can increase its affinity by two orders of magnitude; at the same time, with the help of a gold-philic interaction-driven self-assembly strategy, Dimer CPAP and Mono CPAP was nanoengineered into gold(I)-CPAP supramolecules, further overcoming the pharmacological barriers of cellular internalization and tumor accumulation.

[0005] In one aspect, the present invention provides a protein-protein interaction regulator, wherein the regulator is a homodimer.

[0006] Furthermore, the regulator is a p53-MDM2 targeted peptide regulator linked via homodimers.

[0007] In the present invention, the regulator uses the gold affinity between Au(I)-peptide thiol precursors to drive self-assembly.

[0008] Preferably, the polypeptide modulator of p53-MDM2 is used to homodimerize via a linker consisting of 6-aminohexanoic acid and two PEGs.

[0009] In another aspect, the present invention provides an application of a protein-protein interaction regulator in the treatment of cancer, infectious diseases, and neurodegenerative diseases.

[0010] In a third aspect, the present invention provides a method for evaluating the affinity of ligands with different topologies, wherein the method quantifies the affinity of ligands with different topological structures by comprehensively analyzing the change in binding enthalpy and the loss of translational entropy and rotational entropy.

[0011] The expression of the receptor-ligand bond dissociation constant in the present invention is as follows

[0012]

[0013] where V is the volume of the solution, ΔG represents the free energy difference upon binding, kB is the Boltzmann constant, and T is the temperature; the dissociation constant is proportional to the rotational phase space volume of the receptor or ligand. For the same receptor and ligand, the binding enthalpy of monomer and dimer is U b and the combined translation volume V b will not change, which results in their translational entropy ΔS trans The loss is the same. Therefore, and The difference between the rotational entropy ΔS rot The rotational entropy is related to the rotational phase space volume. Since the rotational phase space volume of the same protein does not change, it is determined by the other three rotational phase space volumes (Ω R ,Ω L , and Ω RL )Sure.

[0014] For MDM2 and Mono The binding between PAP proteins, receptor (i.e., MDM2) and ligand (i.e., Mono PAP) can rotate freely, one of which has θ, and φ are Euler angles. The rotation phase space can be quantitatively described by these three Euler angles. MDM2 and Dimer The binding between CPAP proteins, MDM2 can still rotate freely, which will lead to However, since the linker is very short (~1 nm), Dimer There is a significant steric effect between the two binding domains of CPAP, which would strongly limitDimer The CPAP binding domain is free to rotate. This means Dimer CPAP has a higher rotational entropy loss when combined than Mono The rotational entropy loss of PAP during binding is much smaller. Therefore, the evaluation method of the present invention shows that Dimer The affinity of CPAP will be much higher than Mono CPAP.

[0015] In the present invention, the free energy difference ΔG is calculated by the following formula:

[0016]

[0017] Among them U b is the binding enthalpy of receptor and ligand, ΔS trans =-k B ln(V b / V) is the translational entropy loss when binding to Vb, Vb is the binding translational volume of receptor and ligand, ΔS rot =-k B ln(Ω RL Ω b / Ω R Ω L ) is related to Ω RL Rotational entropy loss during binding, Ω RL is the rotational volume of the receptor-ligand bond, Ω b is the rotational spatial volume of the ligand binding domain relative to the receptor binding domain in the bond, Ω R and Ω L is the rotational spatial volume of the free receptor and ligand.

[0018] By implementing the technical solution of the present invention, the following beneficial effects can be achieved:

[0019] (1) The present invention not only proposes a physicomechanical method to calculate the affinity of PPI modulators with different topological structures, but also provides a general and simple homodimerization strategy to optimize the binding affinity of targeted intracellular PPI modulators.

[0020] (2) The CPAP homodimerization of the present invention can increase affinity by two orders of magnitude, and the affinity quantification measured by isothermal titration calorimetry (ITC) can fully prove the above conclusion.

[0021] (3) The present invention utilizes the self-assembly driven by the gold-philic interaction to Dimer CPAP and Mono CPAP was nanoengineered into gold(I)-CPAP supramolecules to overcome the pharmacological barriers of cellular internalization and tumor accumulation. As expected, Nano- Dimer CPAP is better than Nano-Mono CPAP more effectively restored the p53 signaling pathway. In addition, the superiority of CPAP homodimerization in terms of efficacy was fully verified in LUAD allograft models and LUAD patient-derived xenograft (PDX) mouse models, while maintaining a good drug safety profile. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the statistical mechanics theory of the binding affinity of the dimer strategy of the present invention in intervening in disease PPI.

[0023] in Figure 1 (A) Explanation of the theory of statistical mechanics; Figure 1 (B) Dimer CPAP and Mono Schematic diagram of the function of CPAP; Figure 1 (C&D) Analysis by HPLC and electrospray ionization mass spectrometry (ESI-MS) Dimer CPAP and Mono CPAP, at 40°C, reversed phase C18 column (Waters XBridge TM 3.5μm, 4.6×150mm); Figure 1 (E&F) Isothermal titration calorimetry (ITC) was used to quantify the effect of synMDM2 on Dimer CPAP and Mono Interaction of CPAP. Each curve is the average of 3 independent measurements at 25 °C, 10 mM Hepes, 150 mM NaCl, pH 7.4; Figure 1 (G) Dimer CPAP and Mono Schematic diagram of nanoengineering of CPAP; Figure 1 (H)Nano- Dimer CPAP and Nano- Mono TEM image of CPAP; Figure 1 (I&J) Nano- Dimer CPAP and Nano- Mono Hydrodynamic diameter (I) and zeta potential (J) during CPAP.

[0024] Figure 2 For the Nano- Dimer CPAP in vitro compared with Nano- Mono CPAP can more effectively activate the p53 signaling cascade.

[0025] Figure 2 middle, Figure 2 (A) Using PBS, Nano-Dimer CPAP (10 μg / ml) or Nano- Mono NCI-1650 cells were incubated with CPAP (10 μg / ml) for 48 h, and cell apoptosis and necrosis were analyzed by flow cytometry (n=3, mean±sd); Figure 2 (B) Flow cytometric analysis of NCI-1650 cells after Nano- Dimer CPAP, Nano- Mono Cell cycle after 48 h of CPAP or control treatment (n=3, mean±sd); Figure 2 (C) NCI-H1650 cell mRNA in Nano- Dimer RNA-Seq heat map analysis of differential expression between CPAP and control groups (n=3); Figure 2 (D) GSEA results of p53 signaling pathway and p53 downstream pathway; Figure 2 (E and F) GSEA results of REACTOME cell cycle checkpoints, REACTOME cell cycle mitosis (E), and KEGG apoptosis (F); Figure 2 (G) NCI-H1650 cells were exposed to Nano- Dimer CPAP vs. Nano- Mono Hierarchical clustering of differentially expressed genes after CPAP for 24 h (n=3); Figure 2 (H)Nano- Dimer CPAP vs Nano- Mono GSEA analysis of CPAP showed enhanced p53 and downstream signaling pathways; Figure 2 (I) GSEA shows Nano- Dimer CPAP has a stronger ability to induce apoptosis than Nano- Mono CPAP.

[0026] Figure 3 In the C57 / B6 mouse LUAD xenograft model, Nano- Dimer CPAP is better than Nano- Mono CPAP was more effective in inhibiting tumor growth.

[0027] Figure 3 middle, Figure 3 (A) LLC isograft model in C57 / B6 mice treated with control (PBS), Nano- Dimer CPAP (2.5 mg / kg) and Nano- Mono Growth curve after CPAP (2.5 mg / kg) treatment (n=5); Figure 3 (C&D) Representative photos (C) and weight (D) of excised tumor tissues at the end of the experiment; Figure 3 (E) Analysis of whole blood red blood cells (RBC), white blood cells (WBC), platelets (PLT), neutrophils, and hemoglobin (HGB) of mice after treatment; Figure 3 (F&G) Representative images of H&E and TUNEL staining of mouse tumor sections (scale bar: 50 μm); Figure 3 (HK) Immunohistochemical (IHC) staining of MDM2 (H), MDMX (I), p53 (J), and p73 (K) in mouse tumor sections, scale bar: 50 μm.

[0028] Figure 4 For the embodiment of the present invention, Nano- Dimer CPAP enhanced the antitumor activity of LUAD patient-derived xenografts in NOD / SCID mice.

[0029] Figure 4 middle, Figure 4 (A) Schematic diagram of the LUAD-PDX mouse model; Figure 4 (B) Control group (PBS), Nano- Dimer CPAP (2.5 mg / kg), Nano- Mono Growth curve of LUAD-PDX mouse model after CPAP (2.5 mg / kg) administration (mean ± sd, n = 5 / group); Figure 4 (C&D): Images (C) and weight (D) of the resected tumor at the end of treatment, (p values ​​were analyzed using t-test (*, p<0.05; **, p<0.01; ***, p<0.001); Figure 4 (E&F) H&E (E) and TUNEL (F) staining of mouse tumors (scale bar 200 μm); Figure 4 (GK) Representative images of mouse tumor sections immunohistochemically stained for Ki67 (G), MDM2 (H), MDMX (I), p53 (J), and p73 (K) (scale bar: 200 μm). DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings of the specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Embodiment 1:

[0032] A peptide modulator of p53-MDM2 called CPAP was used to homodimerize via a short, semiflexible linker consisting of 6-aminohexanoic acid and two PEGs.

[0033] Furthermore, in order to solve the pharmacological barriers of cellular internalization and tumor accumulation, the present invention drives self-assembly through the gold affinity between Au(I)-peptide thiol precursors. Dimer CPAP and Mono CPAPs are nano-engineered into spherical nanostructures. Transmission electron microscopy (TEM) images confirm this clear nanostructure, in which Nano- Dimer CPAP and Nano- Mono CPAP presents a spherical morphology with a diameter of about 20 nm ( Figure 1 H). Consistent with the TEM images, Nano- Dimer CPAP has the same Mono CPAP has a similar hydrodynamic diameter of about 20 nm, indicating that the two spherical supramolecular molecules have similar hydrodynamic properties. Dimer CPAP and Nano- Mono The Zeta potential of CPAP was -18.52mV and -14.21mV respectively. Figure 1 J), indicating that the surface charges are similar and the colloidal stability is also similar. These results prompted the present invention to Nano- Dimer CPAP and Nano- Mono The cellular internalization and tumor accumulation of CPAP were compared. As expected, both supramolecules exhibited similar and satisfactory internalization into cancer cells, in stark contrast to the two monomolecular peptides. Dimer CPAP and Nano- Mono CPAP showed proximal and preferential accumulation at tumor sites.

[0034] In summary, the present invention Dimer CPAP and Mono CPAPs were all nanoengineered into spherical supramolecules with similar physicochemical and pharmaceutical properties.

[0035] Example 2

[0036] The present invention calculates the contribution of affinity through atomic molecular dynamics (MD) simulation. However, the traditional MD method cannot meet the requirements of distinguishing topologies, so the present invention establishes a method for evaluating the affinity of ligands with different topologies from scratch, and quantifies the affinity of ligands with different topological structures by comprehensively analyzing the changes in binding enthalpy and the losses of translational entropy and rotational entropy.

[0037] The present invention uses the established statistical mechanics method to compare and calculate the CPAP dimer ( Dimer CPAP) and CPAP alone ( Mono The binding affinity of CPAP to MDM2 was investigated and it was found that CPAP homodimerization increased the affinity by two orders of magnitude. The results of the present invention were confirmed by the affinity measured by isothermal titration calorimetry (ITC), which increased MDM2 binding by 128-fold. Dimer CPAP is better than Nano- Mono CPAP is more effective in restoring the p53 signaling pathway. In addition, the superiority of CPAP homodimerization in terms of efficacy was systematically studied in LUAD allograft models and LUAD patient-derived xenograft (PDX) mouse models. In summary, the present invention not only provides a physicomechanical method for calculating the affinity of PPI modulators with different topological structures, but also provides a general and simple homodimerization strategy for optimizing the binding affinity of PPI modulators in cells.

[0038] Specifically, first, the present invention establishes a method guided by statistical mechanics theory to quantify the affinity of different topological ligands by comprehensively analyzing the change of binding enthalpy and the loss of translational entropy and rotational entropy. According to statistical mechanics, the expression of receptor-ligand bond dissociation constant can be derived:

[0039]

[0040] Where V is the volume of the solution, ΔG is the free energy difference when binding, and k B is the Boltzmann constant and T is the temperature.

[0041] The free energy difference ΔG is given by

[0042]

[0043] Among them U b is the binding enthalpy of receptor and ligand, ΔS trans =-k B ln(V b / V) is related to V b The translational entropy loss upon binding, Vb is the binding translational volume of the receptor and ligand, ΔS rot =-k B ln(Ω RL Ω b / Ω R Ω L ) is related to Ω RL Rotational entropy loss during binding, Ω RL is the rotational volume of the receptor-ligand bond, Ω bis the rotational spatial volume of the ligand binding domain relative to the receptor binding domain in the bond, Ω R and Ω L is the rotational volume of the free receptor and ligand. Finally, the dissociation constant is Figure 1 As shown in A.

[0044]

[0045] According to equation (3), the dissociation constant is proportional to the rotational phase space volume of the receptor or ligand. and are the dissociation constants of monomers and dimers, respectively. For the same receptor and ligand, the binding enthalpy of monomers and dimers is U b and the combined translation volume V b will not change, which results in their translational entropy ΔS trans The loss is the same. Therefore, and The difference between the rotational entropy ΔS rot The rotational entropy is related to the rotational phase space volume. Since the rotational phase space volume of the same protein does not change, it is determined by the other three rotational phase space volumes (Ω R ,Ω L , and Ω RL )Sure.

[0046] For MDM2 and Mono The binding between PAP proteins, receptor (i.e., MDM2) and ligand (i.e., Mono PAP) can rotate freely, one of which has θ, and φ are Euler angles. The rotation phase space can be quantitatively described by these three Euler angles. MDM2 and Dimer The binding between CPAP proteins, MDM2 can still rotate freely, which will lead to However, since the linker is very short (~1 nm), Dimer There is a significant steric effect between the two binding domains of CPAP, which would strongly limit Dimer The CPAP binding domain is free to rotate. This means Dimer CPAP has a higher rotational entropy loss when combined than Mono The rotational entropy loss of PAP during binding is much smaller. Therefore, the present study shows that Dimer The affinity of CPAP will be much higher than Mono CPAP.

[0047] Depending on the geometric size of the dimer, the rotational motion in the constrained environment of the present invention differs In the range of 60° to 90°. Then, the present invention obtains the ligand The rotational phase volume is π 2 / 8~π 2 / 18. From the above, we can see that Mono CPAP and Dimer The ratio of the dissociation constants of CPAP and MDM2 is between 64 and 144. Based on the above analysis, the present invention finds that the dissociation constants can be significantly reduced by connecting monomers to form dimers, and their ratio can reach two orders of magnitude.

[0048] Example 3

[0049] In order to verify the conclusion of Example 2, the present invention synthesized the Dimer CPAP and Mono CPAP and purified by reverse phase HPLC ( Figure 1 B). Electrospray ionization mass spectrometry (ESI-MS) identification Dimer CPAP and Mono The molecular weight of CPAP was determined by HPLC and the purity of the peptide was over 95% ( Figure 1 C and D). More importantly, if ( Figure 1 E) and ( Figure 1 F), isothermal titration calorimetry (ITC) analysis revealed Dimer CPAP, Mono The association constant of CPAP binding to MDM2 yielded a quantitative relationship, which is very consistent with the prediction in Example 2. Therefore, the present invention can significantly improve the affinity of the monomers by physically connecting the monomers into dimers through a short linker.

[0050] The enhancement of affinity brought about by the homodimerization of the present invention will lead to a significant improvement in drug efficacy.

[0051] Example 4

[0052] Flow cytometry was used to detect cell apoptosis and cell cycle analysis to compare Nano- Dimer CPAP and Nano- Mono In vitro efficacy of CPAP in the NCI-H1650 lung adenocarcinoma cell line. As expected, Nano- Dimer CPAP is better than Nano- Mono CPAP can significantly induce cancer cell apoptosis and cell cycle arrest. To further explore the mechanism, cells were treated with PBS, Nano- Dimer CPAP or Nano- Mono Proteomic analysis was performed after 24 h of CPAP incubation. Figure 2 As shown in C, compared with the control group, Nano- DimerThe CPAP treatment group induced a significant increase in the p53 signaling pathway. More importantly, gene set enrichment analysis (GSEA) showed that Nano- Dimer CPAP treatment resulted in changes in upstream pathways, including upregulation of p53 and its downstream signaling pathways ( Figure 1 D) downregulate cell cycle ( Figure 1 E) and apoptotic pathways ( Figure 2 F) By comparing Nano- Dimer CPAP and Nano- Mono CPAP, 98 differentially expressed proteins were found ( Figure 2 G), most of which were enriched in the p53 signaling pathway ( Figure 2 H), p53 downstream signaling pathway ( Figure 2 H) and apoptosis pathways ( Figure 2 I), indicating that Nano- Dimer p53 activity is enhanced after CPAP treatment. Overall, the data of the present invention convincingly demonstrate that Nano- Dimer CPAP restored p53 activity in vitro compared with Nano- Mono CPAP has a higher activation efficiency.

[0053] Example 6 Comparison of Nano- Dimer CPAP vs. Nano- Mono Therapeutic effects of CPAP

[0054] The present invention established a mouse LUAD subcutaneous transplantation model. First, 1×10 6 LLC cells were subcutaneously inoculated into the iliac fossa of C57BL / 6 mice. Ten days after inoculation, LLC tumor-bearing mice were randomly divided into three groups (n=5 / group) and received intravenous injections every other day for 12 days as follows: Nano- Dimer CPAP (2.5 mg / kg), Nano- Mono CPAP (2.5 mg / kg) and saline (control). Compared with the PBS control, Nano- Dimer CPAP and Nano- Mono The CPAP group showed a tumor suppressive therapeutic effect ( Figure 3 A), weight ( Figure 1 B) Cell count ( Figure 1 C) and pathological section data confirmed that the drug has good safety. More importantly, Nano- Dimer The tumor inhibition rate of CPAP was 66.7%, which was better than that of Nano- Mono 21.0% of CPAP. In addition, Nano- Mono The average weight of tumors removed from mice in the CPAP treatment group was Nano-Dimer CPAP, which again demonstrated the superiority of the homodimerization strategy. Dimer CPAP and Nano- Mono In order to investigate the in vivo antitumor activity of CPAP, the present invention used H&E staining and TUNEL technique to analyze tumor tissues. Figure 3 As shown in F&G, with Nano- Mono Compared with the CPAP group, Nano- Dimer CPAP significantly increased the level of apoptosis in tumors. In addition, to explore its mechanism, the present invention performed immunohistochemical staining to study the levels of MDM2, MDMX, p53 and p73 in paraffin sections of tumor tissues. The results showed that Nano- Dimer CPAP and Nano- Mono CPAP significantly downregulated the expression of MDM2 in the nuclei of tumor cells. Figure 3 H) and MDMX( Figure 3 I) levels, leading to the p53 ( Figure 3 J) and p73( Figure 3 K) activity. It is noteworthy that in the regulation of these protein activities, Nano- Dimer CPAP is better than Nano- Mono CPAP is more active ( Figure 3 HK), providing additional evidence for the superiority of homodimerization. Dimer CPAP effectively and safely inhibits lung cancer progression by inhibiting MDM2 / MDMX and activating p53 / p73, and is superior to Nano- Mono CPAP group.

[0055] Example 7 Comparison of Nano- Dimer CPAP vs. Nano- Mono Therapeutic effects of CPAP

[0056] The present invention establishes a patient-derived xenograft (PDX) model of lung adenocarcinoma, which can more powerfully express the molecular and biological characteristics of the parental tumor. To analyze the effectiveness of the Dimer or Mono strategy, the present invention compares their respective effects on the growth of NOD / SCID mice carrying surgically resected LUAD patient tumors. When the mouse tumor volume grows to 50±25mm 3 At 14:00, mice were randomly assigned to receive intravenous injections of PBS (control), Nano- Dimer CPAP (2.5 mg / kg) or Nano- Mono CPAP (2.5 mg / kg). During the 15-day dosing period, the details of the dosing schedule are as follows Figure 4 A. Notably, tumor volume analysis showed that Nano- Dimer CPAP treatment was the most effective at the end, producing a 73.6% tumor growth inhibition (TGI) rate ( Figure 4 B) Nano- Mono CPAP treatment is not as effective as Nano- Dimer CPAP treatment, the TGI rate was 44.3%. These data suggest that Nano- Dimer CPAP is about as effective as Nano- Mono CPAP was 1.7 times higher. The trend of tumor suppression was consistent with the tumor images ( Figure 4 C), tumor weight ( Figure 4 D) and histological H&E staining images ( Figure 4 In addition, these results were again confirmed by TUNEL staining ( Figure 4 F) and immunohistochemical staining of Ki67 ( Figure 4 G) was confirmed. In order to explore the mechanism of tumor suppression in the above LUAD-PDX mouse model, the present invention used immunohistochemical staining in paraffin sections of tumor tissue to explore the levels of MDM2, MDMX, p53 and p73. Figure 4 H&I shows that with Nano- Mono Compared with CPAP and control, Nano- Dimer CPAP significantly downregulated the levels of MDM2 and MDMX in the nuclei of tumor cells. The restoration of p53 and p73 activities further confirmed that Nano- Dimer CPAP and Nano- Mono CPAP's ability and mechanism of suppressing cancer Figure 4 J&K). The above results show that Nano- Dimer CPAP is better than Nano- Mono In addition, the mice did not lose significant weight during treatment, suggesting that Nano- Dimer CPAP and Nano- Mono The safety and non-cytotoxicity of CPAP were verified in detail by H&E staining of liver, kidney, spleen, lung and heart. Dimer CPAP and Nano- Mono Safety of CPAP. In short, Nano- Dimer CPAP has more advantages than Nano-MonoCPAP in clinical translation.

[0057] As described above, the present invention can be well implemented. The above embodiments are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A method for evaluating the affinity of ligands with different topologies. It is characterized in that The method described quantifies the affinity of ligands of different topological structures by comprehensively analyzing the change in binding enthalpy and the loss of translational and rotational entropy; The method for quantifying the affinity of ligands of different topological structures is specifically implemented by the expression of the receptor-ligand bond dissociation constant, which is as follows Where V is the volume of the solution, ΔG is the free energy difference when binding, and k B is the Boltzmann constant, T is the temperature; The dissociation constant is proportional to the rotational phase space volume of the receptor or ligand.

2. The method for evaluating the affinity of ligands with different topologies according to claim 1, wherein the free energy difference ΔG is calculated by the following formula: Among them U b is the binding enthalpy of receptor and ligand, ΔS trans =-k B ln(V b / V) is related to V b Translational entropy loss during binding, V b is the binding translation volume of receptor and ligand, is with Ω RL Rotational entropy loss during binding, Ω RL is the rotational volume of the receptor-ligand bond, Ω b is the rotational spatial volume of the ligand binding domain relative to the receptor binding domain in the bond, Ω R and Ω L is the rotational spatial volume of the free receptor and ligand.

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