Application and method of gastrodin in preparation of MT1 agonist with selectivity
By using Gastroentin to prepare drugs that selectively activate MT1 receptors, the problem of drugs lacking selective MT1 receptors in the prior art has been solved, and a variety of physiological effects such as efficient activation of MT1 receptors and sleep promotion have been achieved, while reducing non-targeted effects.
Patent Information
- Application Number
- CN202411727087.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-03-18
AI Technical Summary
The lack of drugs that selectively activate pineal MT1 receptors in the prior art leads to non-targeting effects in regulating sleep and other physiological functions.
By using Gastrodiatin as the active ingredient, a drug that selectively agonizes the MT1 receptor was prepared. Gastrodiatin verifies its high selectivity and stability through radioligand binding experiments, ERK phosphorylation level detection, molecular modeling and molecular dynamics simulation.
Gastrointestin significantly activates MT1 receptors, inhibits cAMP production, activates cAMP/ERK signaling pathways, thereby promoting sleep, relieves pain, anti-inflammatory and anti-tumor, and reducing non-targeting effects on MT2-related.
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Figure CN119633001A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of pharmacology, and in particular to an application and method of gastrodin in preparing a selective MT1 agonist. Background Art
[0002] The pineal gland and the melatonin it secretes are at the center of sleep regulation, regulating the circadian rhythm and sleep-wake system. Melatonin exerts its major physiological functions through its two receptors, MT1 and MT2. Activation of melatonin receptors is mainly coupled to Gαi / o proteins, inhibiting the cyclic adenosine monophosphate (cAMP) / protein kinase A (PKA) pathway, further triggering the activation of mitogen-activated protein kinase (MAPKK, also known as MEK) / extracellular regulated protein kinase (ERK) kinases and the recruitment of β-arrestins. Melatonin receptors can be activated not only by endogenous melatonin ligands, but also by clinically used drugs. These drugs include ramelteon for the treatment of insomnia, agomelatine for the treatment of major depressive disorder, and tasimelteon for the treatment of non-24-hour sleep-wake disorders in the blind. Ramelteon has high affinity for MT1 and MT2 receptors (Ki values of 14.0 ± 0.5 and 112 ± 5 pM, respectively) compared to melatonin (Ki values of 80.7 ± 2.1 and 383 ± 5 pM, respectively), agomelatine is a non-selective agonist of MT1 / MT2 receptors and an antagonist of serotonin receptors (5-ht2A / 2B / 2C), and tasimelteon has a relatively higher affinity for MT2 (Ki = 69.2 pM) than for MT1 (Ki = 304 pM). These receptors play a key role in the circadian regulation of physiological functions, and further evidence supports the view that the MT1 receptor can be considered a key target for the treatment of sleep disorders. However, the regulation of melatonin receptors has been mainly studied in the context of melatonin and synthetic agonists, which stimulate both MT1 and MT2 receptors, highlighting the lack of MT1 selective agonists in this field. In addition, few natural compounds have been reported as ligands for melatonin receptors. Summary of the invention
[0003] The purpose of the present invention is to provide an application and method of gastrodin in the preparation of a selective MT1 agonist. Gastrodin selectively activates MT1, inhibits cAMP production, and activates the cAMP / ERK signaling pathway, thereby exerting sleep-promoting, pain-relieving, anti-inflammatory, anti-tumor and other effects, and reduces non-target effects related to MT2.
[0004] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:
[0005] Application of gastrodin in the preparation of selective MT1 agonists.
[0006] Furthermore, the application includes: the field of melatonin agonists lacks MT1 receptor selective drugs, most melatonin ligands are structural analogs of melatonin, and melatonin ligands with a 10-50-fold preference for one of the MT1 or MT2 receptors are considered to be ligands with certain selectivity. The results of radioligand experiments show that the affinity of gastrodin for MT1 receptors is more than 30 times that of MT2 receptors. Gastrodin selectively activates MT1, inhibits cAMP production, and activates the cAMP / ERK signaling pathway to play a role including sleep promotion, pain suppression, and resistance, and reduces non-target effects related to MT2.
[0007] Furthermore, the agonist comprises an effective dose of gastrodin and a pharmaceutically acceptable drug adjuvant.
[0008] Furthermore, the effective dose is 0.1-10 μM (in vitro cultured cells, including: cell lines transiently transfected and stably overexpressing MT1, primary and subcultured neural cells and tumor cells endogenously expressing MT1, etc.), 1-200 mg / kg (in vivo administration, including: intraperitoneal injection, oral gavage, subcutaneous injection, intravenous injection, nasal cavity and other administration routes)
[0009] In another aspect, the present invention provides a method for selectively stimulating MT1, comprising the following steps:
[0010] S1: The binding ability of gastrodin to purified MT1 receptor membrane protein was determined by radioligand binding experiment, its effects on MT1 and MT2 were distinguished, and competition binding experiment was performed to determine its Ki value.
[0011] S2: The high functional selectivity of gastrodin was verified by detecting the ERK phosphorylation level in cell lines overexpressing MT1 receptor and MT2 receptor.
[0012] S3: Through molecular modeling and docking simulation, the optimal binding site and binding mode of gastrodin to MT1 receptor were determined, and its binding free energy was calculated to evaluate the affinity.
[0013] S4: Molecular dynamics simulation software was used to verify the binding stability and kinetic behavior of the gastrodin-MT1 complex and to observe the interactions and conformational changes between the molecules.
[0014] S5: In cells expressing MT1, the cAMP level was detected to verify that gastrodin inhibited the expression of cAMP.
[0015] S6: Fluorescence resonance energy transfer technology was used to confirm the selective effect of gastrodin on MT1.
[0016] Further, the step S1 specifically comprises: preparing a membrane from Flp-InTMT-RExTM 293 cells stably expressing human MT1 or MT2 receptors. Using 2-[ 125 I]-iodomelatonin (2-[ 125 The membrane was subjected to saturation binding experiments by 10 μM 2-[ 125 Specific binding was determined by simultaneous incubation of 2-[ 125 Competition experiments were performed with different concentrations of ligands using [I]-MLT (40 pM in MT1 experiments and 120 pM in MT2 experiments). Binding was incubated for 120 min at 27°C and rapidly passed through GF / B glass fiber filters, and the radioactivity retained on the filters was quantified using a gamma counter.
[0017] Further, the step S2 specifically includes: ERK is a key node protein in the GPCRs signal transduction pathway, and the signal transduction of GPCR leads to the phosphorylation of ERK1 / 2 MAP kinase (p-ERK), which can be used as an indicator of agonist or antagonist activity associated with upstream GPCRs. Cells overexpressing MT1 and MT2 receptors were inoculated in 6-well plates, and the cells were deprived of serum for 12 hours to achieve rest. The cells were stimulated with gastrodin for 5 minutes. A positive control of fetal bovine serum (FBS) and a negative control of deionized water were also established. The cells were then placed on ice and collected using ice-cold RIPA buffer. In order to evaluate the activation of ERK1 / 2 MAP kinase, Western blotting was performed using p-ERK antibody. Immunoglobulins were then removed from the PVDF membrane, and the protein loading equivalence was confirmed by re-detection with anti-erk1 / 2 antibodies. The activation effect of gastrodin on the two receptors was compared to confirm its high selectivity.
[0018] Further, the step S3 specifically includes: retrieving the crystal structure of the complex of human MT1 and 2-phenylmelatonin (PDB code: 6ME3) from the protein database to prepare for molecular modeling research, wherein parameters (method: X-ray diffraction, resolution: Free R value: 0.229, working R value: 0.203, observed R value: 0.205). The molecular 3D structure and properties of gastrodin were obtained from the PubChem database. PyMol 2.4.0 software was used to remove all fusion proteins and buffer molecules, as well as to repair missing atoms and add polar hydrogen atoms before docking. The docking process was performed using AutoDockVina software. The docking box was set to define the docking center according to the position of the crystal ligand 2-phenylmelatonin, and the box side length was The best docking conformation is finally visualized in MOE software, and the original crystal ligand is used as a positive control. The software outputs the binding energy and binding site information, focusing on the hydrogen bonds and hydrophobic interactions of the binding site, and identifying the most stable binding conformation and its binding free energy.
[0019] Further, the step S4 specifically includes: by carrying out molecular dynamics simulation, using Gromacs 2023.2 software to perform molecular dynamics (MD) simulation in the CHARMM 36m force field to analyze the stability of the MT1R protein-ligand complex. A cubic box was built and the structure was placed in the center, with water as the solvent. The steepest descent algorithm was used to minimize and balance the energy of each system. Under the conditions of temperature of 310K and pressure of 1bar, the Leap-frog algorithm was used to simulate the molecular dynamics calculations of the constant temperature (NVT) ensemble and the constant pressure and constant temperature (NPT) ensemble for 1ns. Subsequently, a 40ns molecular dynamics simulation was performed to analyze the molecular motion trajectory and calculate the root mean square deviation (RMSD) and root mean square fluctuation (RMSF).
[0020] Furthermore, the step S5 specifically includes: inoculating cells overexpressing MT1 receptor in a 96-well plate (1×10 4 10 cells). 0.5 mM IBMX was added to the cells and incubated for 30 minutes, followed by 15 μM Forskolin for 15 minutes to increase the intracellular cAMP content. -13 -10 -6 M) for 5 minutes, and washed twice with 200 μL pre-cooled PBS. Finally, 100 μL of cell lysis buffer was added to each well to lyse the cells on ice for 5-10 minutes. The changes in intracellular cAMP concentration before and after gastrodin treatment were detected using a cAMP special kit.
[0021] Further, step S6 specifically includes: placing cells expressing the MT1 FRET sensor in a confocal dish and adding physiological HEPES buffered saline. Subsequently, an inverted Nikon TE2000-E microscope is used to localize the cell surface of cyan fluorescent protein (CFP) and enhanced yellow fluorescent protein (eYFP). Gastrodin is added to simultaneously capture FRET and donor emission images at intervals of 40 milliseconds by flow acquisition. At the beginning of each experiment, the FRET ratio is initially set to 1.0 and then monitored over time.
[0022] Beneficial effects of the present invention:
[0023] The discovery that gastrodin has the function of selectively activating MT1 is the molecule with the highest selectivity for melatonin receptor MT1 reported so far. Through radioligand binding test (affinity experiment) and ERK1 / 2 phosphorylation level detection (functional experiment), the present invention determines that the affinity of gastrodin for MT1 receptor is 30 times higher than that for MT2 receptor, and the activation function of ERK1 / 2 mediated by gastrodin for MT1 is 3 times that for MT2, indicating that gastrodin is a melatonin receptor agonist with selectivity for MT1 receptor. This will provide evidence to support the long-term use of gastrodin as a targeted therapeutic drug. By treating sleep disorders for MT1 receptor, gastrodin may have advantages over other melatonin receptor agonists (such as melatonin, ramelteon) as a sleep inducing agent because it has higher drug stability and longer half-life. Gastrodin is very stable under short-term, long-term and freeze-thaw conditions, and is not affected by light, which degrades melatonin. The elimination half-life of gastrodin is about 6 hours, melatonin 48 minutes, and ramelteon about 1.36 hours. At the same time, Gastrodin can also be used to alleviate pathological symptoms related to abnormal reduction of the MT1 signaling pathway, such as pain, inflammation, tumors, etc.
[0024] As a natural phenolic glycoside compound, gastrodin is significantly different from traditional melatonin analogs in chemical structure. Gastrodin can not only broaden the structural diversity of melatonin ligands, but also provide a promising scaffold for the design and development of more selective MT1 receptor compounds. Gastrodin may be used as a tool to study the fine functions of MT1 receptors in various biological processes and cell signal transduction.
[0025] Since gastrodin is a natural product that has been approved as a drug, it has potential advantages in biocompatibility, toxicity and metabolic stability. The introduction of natural compounds provides a safer alternative for the development of therapeutic agonists, especially in the long-term treatment of chronic diseases such as insomnia and circadian rhythm disorders. With the help of the bioactive properties of natural compounds, scientists can design drugs with improved pharmacological properties and lower risk of side effects, thereby improving patient compliance and therapeutic efficacy.
[0026] The high selectivity of gastrodin makes it a powerful tool for studying the function of MT1 receptors. By applying gastrodin in in vivo and in vitro models, the specific role of MT1 receptors in circadian rhythm regulation, sleep-wake cycle, and related behaviors can be more accurately interpreted. This kind of research not only helps to understand the basic biological functions of MT1 receptors, but also reveals its changes in pathological physiological states, such as depression and neurodegenerative diseases. In addition, this in-depth understanding will provide a theoretical basis for the development of drugs targeting MT1 receptors and promote the formation of more targeted treatment strategies.
[0027] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.
[0029] Figure 1 The radioligand 2-[ 125 I]-MLT saturation curve and Scatchard regression diagram; A.2-[ 125 I]-Saturation curve of MLT specific binding to MT1 receptor; B.2-[ 125 Scatchard regression plot of the saturation curve of MLT on MT1 receptor; C.2-[ 125 I]-MLT specific binding to MT2 receptor saturation curve; D.2-[ 125 Scatchard regression plot of the saturation curve of I]-MLT on MT2 receptor;
[0030] Figure 2 Schematic diagram of the level of ERK phosphorylation mediated by gastrodin on overexpressed MT1 receptor and MT2 receptor; AB. ERK phosphorylation level mediated by gastrodin on MT1 receptor and its quantification diagram; BD. ERK phosphorylation level mediated by gastrodin on MT2 receptor and its quantification diagram;
[0031] Figure 3 Schematic diagram of the molecular docking of gastrodin and MT1 receptor; A. 3D structure diagram of gastrodin acting in the active pocket of MT1 receptor; B. A magnified 2D view of the details of the interaction between gastrodin and MT1 receptor; C. An explanatory diagram of the binding chemical bonds;
[0032] Figure 4 A. Schematic diagram of the differences in RMSD and RMSF over time during the molecular dynamics simulation of gastrodin and MT1 receptor; B. Schematic diagram of residual analysis;
[0033] Figure 5 Schematic diagram of the effect of gastrodin on cAMP production in MT1 receptor cells;
[0034] Figure 6 Schematic diagram of observing the fluorescence signal of gastrodin on MT1 sensor through FRET imaging. DETAILED DESCRIPTION
[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.
[0036] Example 1
[0037] This example describes the use of gastrodin in the preparation of a selective MT1 agonist.
[0038] In this embodiment, the application includes: the field of melatonin agonists lacks MT1 receptor selective drugs, most melatonin ligands are structural analogs of melatonin, and melatonin ligands with a 10-50-fold preference for one of the MT1 or MT2 receptors are considered to be ligands with certain selectivity. The results of radioligand experiments show that the affinity of gastrodin for MT1 receptors is more than 30 times that of MT2 receptors. Gastrodin selectively activates MT1, inhibits cAMP production, activates the cAMP / ERK signaling pathway, and thus exerts effects including sleep promotion, pain inhibition, and resistance, and reduces non-target effects related to MT2.
[0039] In this embodiment, the agonist comprises an effective dose of gastrodin and a pharmaceutically acceptable drug adjuvant.
[0040] In this embodiment, the effective dose is 0.1-10 μM (in vitro cultured cells, including: cell lines transiently transfected and stably overexpressing MT1, primary and subcultured neural cells and tumor cells with endogenous expression of MT1, etc.), 1-200 mg / kg (in vivo administration, including: intraperitoneal injection, gavage, subcutaneous, intravenous injection, nasal administration, etc.)
[0041] On the other hand, this embodiment provides a method for selectively stimulating MT1, comprising the following steps:
[0042] S1: The binding ability of gastrodin to purified MT1 receptor membrane protein was determined by radioligand binding experiment, its effects on MT1 and MT2 were distinguished, and competition binding experiment was performed to determine its Ki value.
[0043] S2: The high functional selectivity of gastrodin was verified by detecting the ERK phosphorylation level in cell lines overexpressing MT1 receptor and MT2 receptor.
[0044] S3: Through molecular modeling and docking simulation, the optimal binding site and binding mode of gastrodin to MT1 receptor were determined, and its binding free energy was calculated to evaluate the affinity.
[0045] S4: Molecular dynamics simulation software was used to verify the binding stability and kinetic behavior of the gastrodin-MT1 complex and to observe the interactions and conformational changes between the molecules.
[0046] S5: In cells expressing MT1, the cAMP level was detected to verify that gastrodin inhibited the expression of cAMP.
[0047] S6: Fluorescence resonance energy transfer technology was used to confirm the selective effect of gastrodin on MT1.
[0048] Example 2
[0049] Experimental Methods - Radioligand Binding Assay (RBA)
[0050] Membranes from Flp-In™ T-REx™ 293 cells stably expressing human MT1 or MT2 receptors were prepared. 2-[ 125 I]-iodomelatonin (2-[ 125 The membrane was subjected to saturation binding experiments by 10 μM 2-[ 125 Specific binding was determined by simultaneous incubation of 2-[ 125 I]-MLT (40pM in MT1 experiment and 120pM in MT2 experiment) was used for competition experiment with different concentrations of ligand. The binding was incubated at 27°C for 120 minutes and quickly passed through GF / B glass fiber filter, and the radioactivity retained on the filter was quantified using a gamma counter. The Ki value was calculated based on the IC50 value, and the formula was Ki = IC50 / (1+L / Kd), where L is the ligand concentration and Kd is the dissociation constant.
[0051] Experimental Results
[0052] Table 1 Affinity of Gastrodin and other compounds for MT1 and MT2 receptors in radiocompetitive binding assay
[0053]
[0054] The cell membranes stably expressing human MT1 or MT2 receptors were subjected to saturation binding experiments with 2-[125I]-MLT ( Figure 1 ). 1 μg of cell membranes stably overexpressing MT1 or MT2 receptors and 2-[ 125I]-MLT was tested for competitive binding at 40 pM in the MT1 receptor and 120 pM in the MT2 binding assay. The Ki values of gastrodin and melatonin receptor agonists used in this example study are shown in Table 1. Melatonin ligands with a 10-50-fold preference for one of the melatonin receptor subtypes MT1 or MT2 are considered selective ligands. Gastrodin showed picomolar affinity for the MT1 receptor (Ki=20.88, pKi=10.74±0.14), which exceeded the MT2 receptor by 30 times (Ki=759.44 pM, pKi=9.12±0.02), indicating that gastrodin is a selective MT1 agonist rather than MT2. In addition, ramelteon, melatonin, and 2-iodomelatonin all showed picomolar affinity for both MT1 and MT2 receptors, consistent with previous studies.
[0055] The discovery of gastrodin as an MT1 agonist represents an important advance in melatonin receptor pharmacology. This discovery expands the pool of available ligands that can target the MT1 receptor and highlights the potential of natural compounds as therapeutic agents. The field of melatonin agonists lacks MT1-selective drugs, and most melatonergic ligands are structural analogs of melatonin. Gastrodin as a phenolic glycoside can broaden the structural diversity of melatonergic ligands and provide structural insights into the study of MT1-selective ligands. Gastrodin may serve as a tool to study the physiological and pathophysiological roles of MT1 in various biological processes and cell signaling.
[0056] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.
Claims
1. Application of gastrodin in the preparation of selective MT1 agonists.
2. The use according to claim 1, characterized in that: Gastrodin is a melatonin receptor agonist with certain selectivity for MT1 receptor. The affinity of gastrodin for MT1 receptor is 30 times that of MT2 receptor, and the activation function of gastrodin on MT1-mediated ERK1 / 2 is 3 times that of MT2. After selectively activating MT1, gastrodin inhibits cAMP production and reduces non-target effects related to MT2.
3. The use according to claim 1, characterized in that: The agonist comprises an effective dose of gastrodin and pharmaceutically acceptable adjuvants.
4. The use according to claim 3, characterized in that: The effective dose for in vitro culture is 0.1-10 μM, including but not limited to: transiently transfected and stably over-expressed MT1 cell lines, primary and passaged neural cells and tumor cells with endogenous expression of MT1.
5. The use according to claim 3, characterized in that: The effective dose for in vivo administration is 1-200 mg / kg, including but not limited to: intraperitoneal injection, oral gavage, subcutaneous injection, intravenous injection, and nasal administration.
6. A method based on the selective stimulation of MT1 by gastrodin, comprising the following steps: S1: The binding ability of gastrodin to purified MT1 receptor membrane protein was determined by radioligand binding experiment, its effects on MT1 and MT2 were distinguished, and competition binding experiment was performed to determine its Ki value; S2: In cells overexpressing MT1 and MT2 receptors, the functional selectivity of gastrodin for MT1 was verified by detecting the ERK phosphorylation level; S3: Through molecular modeling and docking simulation, the optimal binding site and binding mode of gastrodin to MT1 receptor were determined, and its binding free energy was calculated to evaluate the affinity; S4: Use molecular dynamics simulation software to verify the binding stability and kinetic behavior of the complex between gastrodin and MT1, and observe the interaction and conformational changes between molecules; S5: In the cell lines expressing MT1, the cAMP level was detected to verify that gastrodin inhibited the expression of cAMP; S6: Use fluorescence resonance energy transfer technology to confirm the effect of gastrodin on the structural changes of MT1 molecules.
7. The method according to claim 6, characterized in that: The step S1 specifically comprises: preparing a membrane from Flp-InTM T-RExTM 293 cells stably expressing human MT1 or MT2 receptors; using 2-[ 125 I]-iodomelatonin 2-[ 125 I]-MLT was used for saturation binding experiments; 10 μM 2-[ 125 Specific binding was determined by displacement of 2-[ 125 I]-MLT was subjected to competition experiments with varying concentrations of ligand; binding was incubated for 120 min at 27°C and rapidly passed through GF / B glass fiber filters, and radioactivity retained on the filters was quantified using a gamma counter.
8. The method according to claim 6, characterized in that: The step S2 specifically includes: ERK is a key node protein in the GPCRs signal transduction pathway, and the signal transduction of GPCR leads to the phosphorylation of ERK1 / 2MAP kinase p-ERK, which serves as an indicator of agonist or antagonist activity associated with upstream GPCRs; cells overexpressing MT1 and MT2 receptors are seeded in a 6-well plate, and the cells are deprived of serum for 12 hours to achieve quiescence; the cells are stimulated with gastrodin for 5 minutes; a fetal bovine serum positive control and a deionized water negative control are also established; the cells are then placed on ice and collected using ice-cold RIPA buffer; p-ERK antibody is used for Western blotting to evaluate the activation of ERK1 / 2MAP kinase; immunoglobulins are then removed from the PVDF membrane, and the protein loading equivalence is confirmed by re-testing with anti-erk1 / 2 antibody, and the activation effect of gastrodin on the two receptors is compared to confirm its high selectivity.
9. The method according to claim 6, characterized in that: The step S3 specifically includes: retrieving the crystal structure of the complex of human MT1 and 2-phenylmelatonin from the protein database to prepare for molecular modeling research, X-ray diffraction, resolution: Free R value: 0.229, working R value: 0.203, observed R value: 0.205; the molecular 3D structure and properties of gastrodin were obtained from the PubChem database; PyMol 2.4.0 software was used to remove all fusion proteins and buffer molecules, as well as to repair missing atoms and add polar hydrogen atoms before docking; AutoDockVina software was used for docking; the docking box was set to define the docking center according to the position of the crystal ligand 2-phenylmelatonin, and the box side length was The best docking conformation was finally visualized in MOE software, and the original crystal ligand was used as a positive control; the software output the binding energy and binding site information, focusing on the hydrogen bonds and hydrophobic interactions of the binding site, and identifying the most stable binding conformation and its binding free energy.