METHOD TO DISTINGUISH PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA FULL AGONIST, PARTIAL AGONIST AND ANTAGONIST WITH DIFFERENT ACTIVITIES AND IDENTIFICATION THEREOF

Abstract

The present invention discloses a method of constructing a pharmacophore to determine whether a molecule is a peroxisome proliferator-activated receptor full agonist, partial agonist or antagonist in terms of a binding energy or a free energy surface comprising: providing a protein receptor mimicking said peroxisome proliferator-activated receptor and a corresponding ligand; docking the corresponding ligand and the protein receptor to form a docked conformation; performing at least two rounds of molecular dynamic simulation to obtain at least one trajectory and at least one free energy surface; inputting the trajectory to construct at least one pharmacophore and obtaining the binding energy of the corresponding ligand; comparing the molecule with the corresponding ligand in terms of the binding energy thereof to the protein receptor in order to determine whether the molecule is the peroxisome proliferator-activated receptor full agonist, partial agonist or antagonist.

Claims

1. A method of constructing a pharmacophore to determine whether a molecule is a peroxisome proliferator-activated receptor full agonist, partial agonist or antagonist in terms of a binding energy or a free energy surface thereof to said peroxisome proliferator-activated receptor , the method comprising: providing a protein receptor mimicking said peroxisome proliferator-activated receptor and a corresponding ligand; docking the corresponding ligand and the protein receptor to form a docked conformation; performing at least two rounds of molecular dynamic simulation to obtain at least one trajectory and at least one free energy surface, wherein one of the at least two rounds of molecular dynamic simulation is a Well-Tempered Meta-dynamic molecular dynamic simulation, Inputting the trajectory to construct at least one pharmacophore and obtaining the binding energy of the corresponding ligand of the peroxisome proliferator-activated receptor full agonist, partial agonist or antagonist. comparing the molecule with the corresponding ligand in terms of the binding energy thereof to the protein receptor in order to determine whether the molecule is the peroxisome proliferator-activated receptor full agonist, partial agonist or antagonist.

2. The method of claim 1, wherein said providing the protein receptor comprises removing at least one small molecule mekt21 in a A chain and a B chain from said protein receptor; removing at least one water molecule to form a remaining B chain protein, wherein the remaining B chain protein after removal of the at least one small molecule mekt21 and the at least one water molecule is saved as a template for a homology modeling prior to said performing at least two rounds of simulation, and wherein a receptor sequence for comparing with the remaining B chain protein sequence in the homology modeling is obtained from Uniprot.

3. The method of claim 2, wherein said providing the corresponding ligand comprises constructing a structure of the ligand by Sketch Molecule module in sybyl7.3 and optimizing the structure with Minimize module in sybyl7.3 including using Powell method to optimize, giving Gasteiger-Hckel charge, using standard Tripos molecular force field, performing energy optimization, wherein the standard restrained energy is 0.001 kcal/(mol ), and the maximum number of iterations are 1000 times.

4. The method of claim 1, wherein said docking the corresponding ligand and the protein receptor to form the docked conformation comprises applying Surflex-Dock module in sybyl7.3 to dock the corresponding ligand to the protein receptor.

5. The method of claim 2, wherein said docking the corresponding ligand and the protein receptor to form the docked conformation comprises applying Surflex-Dock module in sybyl7.3 to dock the corresponding ligand to the protein receptor.

6. The method of claim 3, wherein said docking the corresponding ligand and the protein receptor to form the docked conformation comprises applying Surflex-Dock module in sybyl7.3 to dock the corresponding ligand to the protein receptor.

7. The method of claim 4, wherein said docking the corresponding ligand and the protein receptor to form the docked conformation comprises applying Automatic mode to search a binding pocket with a threshold value of 0.5 and a bloat value of 0.

8. The method of claim 5, wherein said docking the corresponding ligand and the protein receptor to form the docked conformation includes applying Automatic mode to search a binding pocket with a threshold value of 0.5 and a bloat value of 0.

9. The method of claim 6, wherein said docking the corresponding ligand and the protein receptor to form the docked conformation includes applying Automatic mode to search a binding pocket, wherein a threshold value is 0.5, and a bloat value is 0.

10. The method of claim 1, wherein one of the at least two rounds of molecular dynamic simulation is a molecular dynamic simulation comprising: applying Amber GAFF force field to the corresponding ligand and at least one small molecules in the protein receptor including: applying Gaussian09 D01 to optimize the structure of the corresponding ligand and the at least one small molecules; calculating an electrostatic potential thereof, fitting RESP charge, generating RESP file via Antechamber module in Ambertools; generating a Gromacs-compatible molecular topology file using a acpype.py script; generating a protein topology file using pdb2gmx using Amber99sb.ff as force field; filling TIP3P water molecule in at least 2 nm away from the surface of the protein receptor-ligand complex to construct a box; and adding at least one sodium ion to neutralize the charge in the system.

11. The method of claim 10, wherein the molecular dynamic simulation further comprises: undergoing 5000 steps of energy minimization before performing the molecular dynamic simulation, equilibrating the water molecule around the protein receptor by NVT and NPT including simulating as 500 ps in the NVT ensemble, heating gradually from 0 to 300K under minimum energy, equilibrating under 500 ps at 300K in the NPT ensemble, carrying out the molecular dynamic simulation under 20 ns and recording the trajectory every 2 ps.

12. The method of claim 1, wherein the Well-Tempered Meta-dynamic molecular dynamic simulation includes applying Gromacs-5.1.4 patched by Pumed2 to perform Well-Tempered Meta-dynamic simulation, wherein CV1 is a first distance between the C of Tyr473 on H12 and C of Leu453 on H11, and CV2 is a second distance between C of Tyr473 on H12 and C of Tys319 on H4.

13. The method of claim 12, wherein Gaussian width is 0.4 kcal/mol, Gaussian width is 0.025 nm, interval is 2 ps, and each simulation lasts for 80 ns in the Well-Tempered Meta-dynamic molecular dynamic simulation.

14. The method of claim 1, wherein constructing said pharmacophore includes extracting an activated conformation of binding full agonist and partial agonist from the trajectory, inputting the activated conformation of binding full agonist and partial agonist into the Discovery Studio as a receptor pharmacophore model of full agonist and partial agonist respectively, inputting the full and partial agonists into the receptor pharmacophore model of full agonist and partial agonist to obtain the binding energy, and fitting at least one experimental values of EC15 or EC20 and the binding energy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is the Ramachandran plot of the modeling protein structure.

[0012] FIG. 2A to FIG. 2Z are the free energy surfaces of the full agonists, the partial agonists and the antagonists obtained by the WT-metadynamic simulation; wherein FIG. 2A to FIG. 2E show the free energy surface of PPAR after binding to the agonists; FIG. 2F shows the free energy surface of PPAR after binding to the antagonists; FIG. 2G to FIG. 2Z show the free energy surface of PPAR after binding to the partial agonists.

[0013] FIG. 3A to FIG. 3C are the PPAR conformation, wherein FIG. 3A shows the corresponding conformation of the PPAR H12 binding to the full agonist ROSI of the free energy surface and the conformation of holo-PPAR; FIG. 3B shows the corresponding conformation of PPAR binding to the antagonist 9P and the antagonist conformation of PPAR; FIG. 3C shows the corresponding conformation of PPAR binding to the partial agonist DiBP and the antagonist conformation of PPAR.

[0014] FIG. 4A to FIG. 4B are the correlation diagram between the experimental values and predicted values in the present invention; FIG. 4A is shown as agonist; FIG. 4B is shown as partial agonist.

DETAILED DESCRIPTION

Embodiment 1

[0015] Materials and Data Collection

[0016] The substances used in the present invention are from the dust in the background room by Mingliang Fang et al in 2015. The researchers analysed a variety of substances from indoor dust in Beijing, including a variety of flame retardants and their metabolites. According to the previous activation and binding experiments of PPAR, it has been shown that most of these substances are full or partial agonists to PPAR. The researchers used rosiglitazone and PPAR endogenous ligand 15d-PJG2 as positive controls and reporter gene assay to study the PPAR activity of these substances. The activity data in the present invention were from this research. Before using the data, the EC15 of each substance is first converted to the equivalent of rosiglitazone, and then the logarithm is taken.

[0017] Receptor and Ligand Modeling

[0018] The template for the molecular protein structure of PPAR (PDB ID: 3VSO) is from the RCSB protein data bank. Before docking, remove the small molecules mekt21 in the A chain and B chain, all the water molecules to obtain the remaining B chain protein, then the remaining B chain protein is used as the template for the homology modeling in the next step. The receptor sequence for homology modeling is from Uniprot (Uniprot ID: P37231), then the homology modeling is performed on the Swiss-model platform, and the modeling protein structure is verified by Ramachandran plot (see FIG. 1). The results show 92.1% of all the amino acid residues are in the favored region, 6.6% are in the allowed region, and 0.8% are in generously allowed region, which means the constructed PPAR structure in the present invention is reasonable. All ligand small molecules are constructed by Sketch Molecule module in sybyl7.3 (Tripos Inc., St. Louis, Mo., USA) and optimize the structure with Minimize module in sybyl7.3. The process is as follows: Use Powell method to optimize, give Gasteiger-Hckel charge, use standard Tripos molecular force field, perform energy optimization, the standard restrained energy is 0.001 kcal/(mol ), and the maximum number of iterations are 1000 times.

[0019] Molecules Docking

[0020] Surflex-Dock module in sybyl7.3 is used to dock the ligand small molecules to the receptor protein. When docking, use the Automatic mode to search the binding pocket with the threshold value is 0.5, and the bloat value is 0. The flexibility of the ring is also considered during the docking, and each compound generates 20 binding conformations. The highest scoring conformation is the most likely bioactive conformation for the molecular dynamic simulation in the next step.

[0021] Molecular Dynamic Simulation

[0022] Gromacs-5.14 is used to perform MD simulation. During the simulation process, the Amber GAFF force field is used for the ligand small molecules and the small molecules in the receptors. The process is as follows: Gaussian09 D01 is firstly used to optimize the structure of small molecules, calculates the electrostatic potential, fit the RESP charge, then use the Antechamber module in Ambertools to generate RESP file, and use the acpype.py script to generate Gromacs available molecular topology file. The protein topological file is generated by pdb2gmx, and Amber99sb.ff is used as force field. The box is constructed at least 2 nm away from the surface of proteinsligand small molecules, and then TIP3P water molecules is filled in, i.e. there is at least 2 nm at the margin of protein from solvent, and sodium ion is added to neutralize the charge in the system.

[0023] Before performing dynamic simulation, the system undergoes 5000 steps of energy minimization. The system is then equilibrated by following two stages: Canonical ensemble (NVT) and Isothermal-isobaric (NPT) ensemble. Each system is simulated as 500 ps in the NVT ensemble, heating gradually from 0 to 300K under minimum energy, and equilibrating under 500 ps at 300K in the NPT ensemble. Finally, the MD simulation is carried out under 20 ns and recorded the trajectory every 2 ps.

[0024] Well-Tempered Meta-Dynamic Molecular Dynamic Simulation

[0025] Under regular temperature and pressure, protein molecules with nanosecond(ns) level from MD simulation are often limited to a local potential energy well. In order to achieve ideal results, it is preferred to select Well-Tempered Meta-dynamic simulation method in the present invention to deal with the sampling efficiency problem in the molecular dynamics simulation by adding artificial Gaussian bias potential to the energy of manually selected collective variable (CV) to accelerate the motions of the system along these variabilities, resulting in jumping out of the local potential well and obtain the overall free energy surface.

[0026] Gromacs-5.1.4 patched by Pumed2 is used to perform Well-Tempered Meta-dynamic simulation, the force field and other simulation parameters are the same as unbiased sampling, and the selection of collective variable (CV) is an important step in the process of meta-dynamic simulation. In this simulation, CV1 is the distance between the C of Tyr473 on helix 12 (H12) to C of Leu453 on helix 11 (H11), and CV2 is the distance between C of Tyr473 on H12 to C of Tys319 on H4. The rest of the other parameters are as follows: Gaussian width 0.4 kcal/mol, Gaussian width 0.025 nm, interval 2 ps, and each substance simulation lasts for 80 ns. At the end of the simulation, the energy deviation of CV is used to check whether the simulation converges.

[0027] Pharmacophore Construction

[0028] After competing molecular dynamics simulation and identifying full agonists and partial full agonists according to the free energy surface, the pharmacophore is further analyzed and predicted quantitatively. First of all, the activated conformation of binding full agonist (for example, ROSI) and partial agonist (for example, DiBP) are extracted from the simulation trajectory and input them into the Discovery Studio as the receptor pharmacophore model of the full agonists and the partial agonists respectively (the pharmacophore characteristics used in the model are H-bond donor, H-bond acceptor, Hydrophobic and Exclusion Sphere). Then, the full agonists and partial agonists are input respectively to obtain the predicted binding energy. Finally, fit the data from the experimental values of EC15 or EC20 and predicted binding energy.

Embodiment 2

Embodiment 1 Experimental Results

[0029] The substances used in the present invention are those from the dust in the background room analysed by Mingliang Fang et al in 2015. The activity data of PPAR and ligand binding affinity data of PPAR in the present invention are from this research. The results were shown in table 1:

TABLE-US-00001 TABLE 1 The substance activity Compounds EC15 (M) TPP 2.12 TPPi NA mono-ITP 3.6 Di-ITP 3.25 Tri-ITP 5.7 TBuP 5.86 2,4,6-TIP 8.72 2,4,6-TBP 5.89 TCBPA 0.23 TBBPA 0.32 TBPP NA TBOEP NA TBBA 8.16 BDE47 5.2 3-OH-BDE47 2.01 6-OH-BDE47 NA TCS NA DiBP 4.47 DBP 6.73 BzBP 2.94 TBMEHP 0.53 MEHP 1.26 15d-PJG2 0.51 rosiglitazone (positive control) 0.00132 NA: non-available

[0030] The H12 Conformation of PPAR and Activity Differentiation

[0031] The free energy surfaces of the full agonists, the partial agonists and the antagonists from the WT-metadynamic simulation are shown in FIG. 2A to FIG. 2Z. There are obvious differences between these two free energy surfaces. A large low energy area and one minimum energy area were shown in the free energy surface of PPAR (FIG. 2A) after binding to the full agonist ROSI. The corresponding conformation of this area is shown in FIG. 3A, and after performing the superposition and comparing to the holo-PPAR, it is found there is only slightly different between the simulated conformation of PPAR H12 binding to the agonist ROSI and the holo-PPAR, which indicates the activated conformation is the only possible conformation after the binding to the agonist.

[0032] After binding to the antagonists, there are more low energy area of PPAR and a multiple minimum energy area shown in the free energy surface (FIG. 2F), which indicates that there is more than one PPAR conformation, and a number of low energy conformations exist after binding to the antagonists. This result demonstrates why the activated conformation of PPAR is easy to be resolved, but the crystal structure of the antagonist conformation has not been resolved so far (the antagonist conformation of PPAR is first resolved in the present invention). Moreover, it is shown that the low free energy area after binding to the agonist is not matched to the low free energy area after binding to the antagonist, which indicates the conformation of PPAR H12 binding to the antagonist is completely different to the activated conformation. The corresponding conformation of this low energy area is shown in FIG. 3B. When overlapping this conformation to the conformation of the PPAR (PDB ID: 1kkq) binding to the antagonist, it was found that comparing to the activated conformation, the stable structure of H12 PPAR binding to the antagonist was far away from the binding pocket of the receptor. One of the conformations is similar to the antagonist conformation of PPAR with the disappearance of H12 helix and the cofactor AF-2 binding region is occupied, leading to the antagonist effect of PPAR.

[0033] As for the conformation of PPAR binding to the partial agonist, for example, DiBP (its maximum effect is only 25% of the maximum effect of ROSI) (FIG. 3C) also has multiple low free energy area (FIG. 2P). Compared to the full agonist/antagonist profiles (FIG. 2A or 3A; FIG. 2F or 3B) it is found there are similarities between these low free energy areas. For example, the low free energy area produced by binding the full agonist ROSI also exists in the free energy surface of DiBP, and the low free energy area produced by binding the antagonist 9P also exists in the free energy surface produced by binding to the DiBP. By analysing the corresponding conformation in the corresponding low free energy area, it can be found that there are many stable low energy structures of PPAR after binding to the DiBP: one of them is similar to the holo-PPAR, and the other is similar to the antagonist conformation of PPAR. This shows that while binding to the partial agonists, the activated and antagonistic conformations can be generated at the same time, and the energy barrier between these two conformations is relatively low, which is easy to transform to each other. Therefore, binding to the partial agonist is not able to perform stronger activation as binding to the full agonist. The classification of interferences is shown in table 2.

TABLE-US-00002 TABLE 2 Full agonist Partial agonist Antagonist ROSI, 15d-PJG2, TBMEHP, 3-OH-BDE47, 6-OH- 9P MEHP, TCBPA, BDE47, BDE47, TBuP, 2,4,6- BPDP TBP, 2,4,6-TIP, BEHF, DBP, DiBP, di- ITP, tri-FTP, mono-ITP, TBBA, TBBPA, TBOEP, TBPP, TCS, TPP, TPPi

[0034] FIG. 2A to FIG. 2Z show the free energy surfaces of conformations of PPAR H12 binding to different ligands, among which, FIG. 2A to 2E are PPAR binding to the full agonists ROSI, 15d-PJG2, MEHP, TCBPA, BPDP, respectively; FIG. 2F shows PPAR binding to the antagonist 9P; FIG. 2G to 2Z are PPAR binding to the partial agonist TBMEHP, 3-OH-BDE47, 6-OH-BDE47, BDE47, TBuP, 2,4,6-TBP, 2,4,6-TIP, BEHF, DBP, DiBP, di-ITP, tri-ITP, mono-ITP, TBBA, TBBPA, TBOEP, TBPP, TCS, TPP, TPPi, respectively; FIG. 3A to FIG. 3C show the corresponding conformation of the PPAR H12 binding to the full agonist ROSI of the free energy surfaces and the conformation of holo-PPAR (FIG. 3A); FIG. 3B shows the corresponding conformation of PPAR binding to the antagonist 9P and the antagonist conformation of PPAR; FIG. 3C shows the corresponding conformation of PPAR binding to the partial agonist DiBP and the antagonist conformation of PPAR.

[0035] Quantitative Prediction

[0036] According to the above methods, the ligands were divided into the full agonists, the partial agonists and the antagonists, and the pharmacophore characteristics generated by ROSI and DiBP were used to perform prediction. FIG. 4A to FIG. 4B demonstrate the correlation between the experimental data and the prediction data by the method in the present invention (FIG. 4A is for full agonists and FIG. 4B is for partial agonists). As shown in FIGS. 4A and 4B, the predicted values by the method in the present invention fit well to the experimental values. The R.sup.2 of the full agonists was 0.93, and 0.81 for the partial agonists.

[0037] Emax is usually used to distinguish the full agonist and the partial agonist of PPAR, but there is no universal standard. Some researcher uses 60% Emax as the threshold to divide the full agonist and the partial agonist. In the present invention, those selected substances are divided according to the free energy surfaces. It is found that some substances with lower maximal activity, such as TCBPA, belong to the full agonist. Although the binding energy of these substances to PPAR is low, they would still activate after binding and belong to the full agonist. However, the partial agonists cannot activate the receptor completely after binding to the receptor. According to the free energy surface, the activated and antagonistic conformations exist at the same time.

TABLE-US-00003 TABLE 3 the experimental values of the PPAR binding energy and predicted values for each substance EC15 Predicted binding energy Compound (M) (KJ/mol) TPP 2.12 70.52 mono-ITP 3.6 83.15 Di-ITP 3.25 95.72 Tri-ITP 5.7 96.30 TBuP 5.86 19.66 2,4,6-TIP 8.72 3.82 2,4,6-TBP 5.89 3.65 TCBPA 0.23 63.56 TBBPA 0.32 120.30 TBBA 8.16 30.42 BDE47 5.2 61.87 3-OH-BDE47 2.01 56.89 DiBP 4.47 30.31 DBP 6.73 38.84 BzBP 2.94 61.18 TBMEHP 0.53 73.27 MEHP 1.26 50.18 15d-PJG2 0.51 63.64 rosiglitazone (positive control) 0.00132 148.86

[0038] The present invention firstly uses the molecular dynamics method to simulate and study the conformation changes of PPAR after binding to the full agonist, the antagonist, and the partial agonist, and finds it is still difficult to distinguish the full agonist and the partial agonist, which shows the molecular dynamics simulation is limited by its sampling efficiency and cannot have the low energy conformation of PPAR after binding to the partial agonist. After using Well-Tempered Meta-dynamics molecular dynamics simulation, the present invention obtains the free energy surfaces of PPAR after binding to the full agonist, the partial agonist and the antagonist. The present invention can qualitatively distinguish PPAR activity through free energy surfaces, wherein PPAR only has activated conformation after binding to the full agonists and has activated conformation and antagonist conformation at the same time after binding to the partial agonists. The present invention can quickly distinguish PPAR activity of different structural compounds and greatly reduce the use of chemicals and cells in the laboratory in the process of traditional toxicological experiment, the workload of the laboratory and save the expenses of the laboratory; Therefore, PPAR activity of compounds is distinguished before QSAR modeling, which makes the results of QSAR modeling close to the reality, such that the conventional QSAR can be widely used.

[0039] It should be noted that the above embodiments are only used to explain the technical scheme of the invention, not the limitation. Although the invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical scheme of the invention can be modified or replaced equally without departing from the spirit and scope of the technical scheme of the invention which should be covered in the right of the invention.