Virtual screening method for small molecule compounds targeting tlr4
Virtual screening was performed by selecting amino acid residues at the TLR4 binding site, and compounds Z1410232649, F27210326, and HY-N0029 were discovered, which solved the problem of insufficient research on TLR4 inhibitors and achieved effective inhibition and anti-inflammatory effects on TLR4.
Patent Information
- Application Number
- CN202511241094.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2025-12-23
- Estimated Expiration
- 2045-09-02
AI Technical Summary
There is limited research on existing TLR4 inhibitors or agonists, and in-depth studies are lacking. Only a few have entered clinical trials, and there is a lack of effective TLR4-targeting small molecule compounds for the treatment of immune diseases and inflammation.
Based on the structure of Human TLR4, amino acid residues Glu42/Asp60/Arg87/Glu135/Ser183/Arg234/Arg264/Asn265/Glu266/Arg289, which interact with hydrogen and ionic bonds, were selected as binding sites. Using a computer-aided virtual screening method, small molecule compounds Z1410232649, F27210326, and HY-N0029, which have strong binding affinity to TLR4, were screened out for use in the preparation of TLR4 inhibitors and anti-inflammatory drugs.
Compounds Z1410232649, F27210326, and HY-N0029 significantly inhibited the release of LPS-induced inflammatory factors IL-6, TNF-α, and IFN-γ, exhibiting good TLR4 inhibitory effects and potential anti-inflammatory and immunotherapeutic effects.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of bioinformatics, and particularly relates to a virtual screening method for small-molecule compounds targeting TLR4. BACKGROUND
[0002] Toll-like receptors (TLRs) are the most conserved transmembrane pattern recognition receptors (PRRs) in biological evolution, mainly expressed in immune cells, and also expressed in some epithelial cells and tumor cells. Due to the excellent sensitivity and recognition ability to exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), TLRs play an extremely important role in both innate and acquired immune responses, including inducing the maturation and differentiation of immune cells, releasing cytokines and chemokines, etc.
[0003] Currently, 11 subtypes (TLR1-11) of TLRs have been found in humans, which are distributed on the cell membrane or intracellularly and recognize different PAMPs and DAMPs. TLR4 is the most studied and widely used immune receptor. Compared with other TLRs, only TLR4 can be expressed on the cell membrane and intracellularly; TLR4 is also the only receptor that can activate both MyD88-dependent and MyD88-independent / TRIF-dependent pathways. Therefore, TLR4 can recognize more PAMPs and DAMPs, recruit more adapter proteins, and produce more timely and effective immune responses. These characteristics make TLR4 play an important role in various immune diseases and tumor immune microenvironments.
[0004] With the rapid development of computer-aided technology and the increasing richness of drug databases, and under the support of artificial intelligence (AI) technology, virtual screening has become an important means to quickly obtain candidate drugs or lead compounds in new drug research and development.
[0005] Ren et al. not only identified TNIK as an anti-fibrosis drug target using AI technology combined with databases, but also screened out the TNIK inhibitor INS018_055 using AI technology. From drug target discovery to promoting the drug to enter the phase I clinical trial, only 18 months were needed, which greatly accelerated the new drug research and development process (Ren F, Aliper A, Chen J, et al. A small-molecule TNIK inhibitor targets fibrosis in preclinical and clinical models[J]. Nat. Commun., 2025, 43(1), 63-75.). Nature biotechnology
[0006] Zhang and Mishra et al. also found TLR4 inhibitors such as hit 94, C11 and C15 by using virtual screening technology ([1] Zhang T, Xing S, Du J, et al. Discovery of novel TLR4 / MD-2 inhibitors: Receptor structure-based virtual screening studies and anti-inflammatory evaluation [J]. Bioorganic chemistry, 2023, 141: 106880. [2] Mishra V, Pathak C. Structural insights into pharmacophore-assisted in silico identification of protein-protein interaction inhibitors for inhibition of human toll-like receptor 4 - myeloid differentiation factor-2 (hTLR4-MD-2) complex [J]. Journal of biomolecular structure & dynamics, 2019, 37(8): 1968-1991.), but there is no follow-up in-depth research report.
[0007] Currently, in addition to Mifamurtide and MPL, two TLR4 agonists, which are approved for marketing as components of bone sarcoma treatment drugs and vaccine adjuvants, respectively, only a few of the remaining discovered TLR4 inhibitors or agonists, such as E-5531, Eritoran, SLA, GLA-SE, have entered clinical trials.
[0008] Lipopolysaccharide (LPS) is the first discovered natural TLR4 agonist, and most of the current TLR4 agonists are based on the structure of LPS. According to the structure of TLR4-(MD-2)-LPS complex, it is composed of two symmetrical TLR4-(MD-2)-LPS complexes of m-type multi-receptors, LPS interacts with a large hydrophobic pocket close to MD-2, 5 of the 6 lipid chains of LPS are buried in the pocket, and the other lipid chain is exposed on the surface of MD-2 and forms a hydrophobic interaction with the conserved phenylalanine of TLR4. The F126 loop of MD-2 undergoes local structural changes, which supports the core hydrophobic interface by hydrophilic interaction with TLR4 (Park BS, Song DH, Kim HM, et al. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex[J]. Nature, 2009, 458(7242): 1191-1195.). The structure of the tetraacyl antagonist bound to MD-2 shows that the other two lipid chains of LPS replace the glucosamine backbone phosphorylated to the solvent region by ~5 Å (Ohto U, Fukase K, Miyake K, et al. Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa[J]. Science, 2007, 316(5831): 1632-1634.). This structural transfer allows the phosphate group of LPS to promote the multimerization of the receptor by forming ionic interactions with the cluster of positively charged residues in TLR4 and MD-2. The structure of TLR4-(MD-2)-LPS complex shows that the ligand recognition mechanism adopted by the TLR family has significant universality, which is essential for the defense against various microbial infections. SUMMARY
[0009] Based on the structure of Human TLR4, the amino acid residues interacting with A chain and C chain are predicted, and the amino acid residues Glu42 / Asp60 / Arg87 / Glu135 / Ser183 / Arg234 / Arg264 / Asn265 / Glu266 / Arg289 with hydrogen bond and ionic bond are selected as the binding sites for computer virtual screening, expecting to obtain small molecule compounds with strong binding force to TLR4.
[0010] The present application first provides a virtual screening method of small molecule compounds targeting TLR4, comprising the following steps:
[0011] Step 1, preparing a three-dimensional structure model of TLR4 protein, making a grid point file of TLR4 protein, selecting amino acid residues Glu42, Asp60, Arg87, Glu135, Ser183, Arg234, Arg264, Asn265, Glu266, Arg289 as the center to generate a grid point file, which have hydrogen bond and ionic bond interaction;
[0012] Step 2, preparing a three-dimensional structure model of the compound to be screened;
[0013] Step 3, performing molecular docking between the three-dimensional structure model of the compound to be screened in Step 2 and the receptor structure model in Step 1, and obtaining the compound to be screened with a high docking score as a candidate small molecule compound targeting TLR4.
[0014] The reason for selecting the above amino acid residues is that they contain more hydrogen bonds and ionic bonds, and are easier to bind.
[0015] The grid point file contains all the information of the protein binding site, and the grid point represents the protein in the molecular docking calculation. The grid point file can be reused, and the same grid point file can be used to dock different ligands to the same binding site. With the grid point and the prepared ligand structure, molecular docking calculation can be started. Molecular docking is to dock the specified ligand to the grid point representing the protein, and to give pose and scoring value.
[0016] Preferably, in Step 1, the crystal structure data of human TLR4 protein is used, and the protein is first hydrogenated and dehydrated using the ProteinPreparation Wizard module; then energy optimization is performed, and the energy optimization uses the OPLS2005 force field with an RMSD of 0.30 Å; and the processed protein is used to make a grid point file using the Receptor Grid Generation module.
[0017] Preferably, in Step 1, the box size of the grid point file of TLR4 protein is set to 20 Å × 20 Å × 20 Å.
[0018] Preferably, in Step 2, if the compound to be screened is a two-dimensional structure model, a three-dimensional structure model is output by hydrogenation and energy optimization processing.
[0019] Preferably, in Step 3, the molecules are docked to each other by geometric matching and energy matching. During molecular docking, first, the compounds are screened by using the high-throughput screening mode in the Glide module, and then the compounds with high scoring values are selected for the second round of screening by using the standard mode; subsequently, the compounds with high scoring values are selected for the third round of screening by using the high-precision mode, and the ranking of small molecule compounds is obtained.
[0020] The application further provides application of the compound in preparation of a TLR4 inhibitor, wherein the compound is Z1410232649, F27210326 or HY-N0029.
[0021] The structural formula of the compound Z1410232649 is as follows:
[0022] ,
[0023] The structural formula of the compound F27210326 is as follows:
[0024] ,
[0025] The structural formula of the compound HY-N0029 is as follows:
[0026] .
[0027] The application further provides application of the compound in preparation of an anti-inflammatory drug, wherein the inflammation is TLR4-mediated pneumonia, sepsis, rheumatoid arthritis or colitis, and the compound is Z1410232649, F27210326 or HY-N0029.
[0028] The application further provides application of the compound in preparation of an immunotherapy drug, wherein the immunological disease is TLR4-mediated non-alcoholic fatty liver, type 2 diabetes, atherosclerosis, myocardial ischemia-reperfusion injury or multiple sclerosis, and the compound is Z1410232649, F27210326 or HY-N0029.
[0029] In the application, based on screening of 115700 small molecule compounds in three databases of Immuno-Oncology Library, Life Chemicals Diversity Library and MCE Bioactive Compound Library, a series of potential compounds capable of binding to TLR4 are found. Through experimental verification, it is preliminarily proved that three compounds of Z1410232649, F27210326 and HY-N0029 have good TLR4 inhibiting effect. Among them, Z1410232649 performs most outstandingly, and can significantly inhibit release of inflammatory factors IL-6, TNF-alpha and IFN-gamma induced by LPS. It can be used as a new anti-inflammatory drug or immunotherapy drug targeting TLR4. BRIEF DESCRIPTION OF DRAWINGS
[0030] Figure 1 It is a virtual screening workflow based on molecular docking.
[0031] Figure 2Group action of three compounds on inhibition of inflammatory factor release induced by lipopolysaccharide LPS. Compared with the normal control group (Ctrl), P <0.001; compared with the LPS group # P <0.05, ## P <0.01, ### P <0.01; n = 3.
[0032] Figure 3 The compound acts on Human TLR4 protein.
[0033] Figure 4 It is a two-dimensional graph of the binding mode of Z1410232649 and Human TLR4 protein.
[0034] Figure 5 It is a three-dimensional graph of the binding mode of Z1410232649 and Human TLR4 protein.
[0035] Figure 6 It is a two-dimensional graph of the binding mode of F27210326 and Human TLR4 protein.
[0036] Figure 7 It is a three-dimensional graph of the binding mode of F27210326 and Human TLR4 protein.
[0037] Figure 8 It is a two-dimensional graph of the binding mode of HY-N0029 and Human TLR4 protein.
[0038] Figure 9 It is a three-dimensional graph of the binding mode of HY-N0029 and Human TLR4 protein. DETAILED DESCRIPTION
[0039] Experimental animals: SPF level Balb / C mice, 6-8 weeks old, purchased from Shanghai Slek Experimental Animal Co., Ltd., qualified certificate number: SCXK (Shanghai) 2022-0005.
[0040] Main reagents: Inflammation CBA Kit purchased from BD Company; lipopolysaccharide (LPS) purchased from Sigma-Aldrich Company; three preferred compounds (Z1410232649, F27210326, HY-N0029) purchased from MedChemExpress (MCE) Company.
[0041] Example 1
[0042] (1) Protein preparation
[0043] The crystal structure of Human TLR4 (PDB ID: 3FXI) was downloaded from RCSB PDB database. The protein was prepared by adding hydrogen and removing water using ProteinPreparation Wizard module. Then energy optimization was performed (OPLS2005 force field, RMSD 0.30 Å). When making the grid file for the prepared protein using Receptor Grid Generation module, the amino acid residues interacting with A chain and C chain were predicted first, and the amino acid residues with hydrogen bond and ionic bond interaction, Glu42 / Asp60 / Arg87 / Glu135 / Ser183 / Arg234 / Arg264 / Asn265 / Glu266 / Arg289, were selected as the center to generate the grid file, and the box size was set to 20 Å x 20 Å x 20 Å.
[0044] (2) Compound preparation
[0045] The 2D format of Immuno-Oncology Library (containing 52900 compounds), Life Chemicals Diversity Library (containing 50200 compounds), and MCE Bioactive Compound Library (containing 12600 compounds) was processed by hydrogenation, energy optimization, etc. using LigPrep Module of Schrödinger software, and the 3D structure was output for virtual screening.
[0046] (3) Molecular docking
[0047] Virtual screening was performed using the Virtual Screening Work flow module, and the work flow is shown in Figure 1 The prepared compounds were imported, and molecular docking was performed using the Glide module, i.e. the receptor and ligand molecules were docked with each other through geometric matching and energy matching.
[0048] First, the high-throughput screening (HTVS) mode in the Glide module was used to screen 52.9K prepared small molecule compounds in the Immuno-Oncology Library, and the top 20% of the small molecule compounds were selected for the second round of screening using the standard (SP) mode; then the top 20% of the scoring values were selected for the third round of screening using the high precision (XP) mode, and the ranking of the small molecule compounds was obtained.
[0049] Secondly, the same method was used to dock the compounds in Life Chemicals 50K Diversity Library and MCE Bioactive Compound Library Plus with the target protein Human TLR4 in HTVS mode, SP mode and XP mode, respectively. The binding force of the target and the compounds and the structure of the compounds were manually reviewed, and the top 200 compounds in Immuno-Oncology Library, the top 200 compounds in Life Chemicals 50K Diversity Library, and the top 200 compounds in MCE Bioactive Compound Library Plus were selected as the final results. The binding mode of the first compound in each library and the Human TLR4 protein was selected for 2D and 3D mapping.
[0050] (4) Virtual screening results
[0051] According to the molecular docking score, the top 10 compounds in Immuno-Oncology Library, Life Chemicals 50K Diversity Library, and MCE Bioactive Compound Library were screened in descending order of absolute value, and the structures of the compounds are shown below.
[0052] The top 10 compounds in Immuno-Oncology Library that bind to TLR4 are shown in Table 1.
[0053] Table 1
[0054]
[0055] The top 10 compounds in Life Chemicals Library that bind to TLR4 are shown in Table 2.
[0056] Table 2
[0057]
[0058] The top 10 compounds in MCE Bioactive Compound Library that bind to TLR4 are shown in Table 3.
[0059] Table 3
[0060]
[0061]
[0062] Example 2
[0063] Using the TLR4 agonist LPS to stimulate spleen cells to release cytokines as a model (anti-inflammatory cell model), the compound with the best affinity for TLR4 from each compound library was selected to verify TLR4 regulatory activity.
[0064] The structural formula of compound Z1410232649:
[0065] ,
[0066] The structural formula of compound F27210326:
[0067] ,
[0068] The structural formula of compound HY-N0029:
[0069] .
[0070] Mouse spleen cells were collected and prepared into a single-cell suspension using RPMI 1640 medium containing 10% newborn calf serum. 100 μL of the cell suspension was added to each well (5 × 10⁶ cells / well) of a 96-well plate. 5 (1), add the test compound and TLR4 agonist LPS (50 ng / mL) -1 50 μL of each sample was added to three wells. After culturing for 24 h, the supernatant was collected, and the levels of IL-6, IFN-γ, and TNF-α were detected using the Inflammation CBA Kit.
[0071] Statistical analysis was performed using GraphPad Prism 9.5 software. Data are expressed as mean ± standard deviation and are presented using... t Test and compare statistical differences between groups. P A value <0.05 is considered statistically significant.
[0072] The results are as follows Figure 2 As shown, compared with the blank control group, the secretion levels of IL-6, TNF-α, and IFN-γ were significantly increased after LPS stimulation. P <0.001). Compared with the LPS group, compounds F27210326 and HY-N0029 significantly reduced the content of TNF-α in the cell supernatant ( P <0.01), but had no significant effect on IL-6 and IFN-γ levels. Compound Z1410232649, at concentrations of 0.3125-5 μM, significantly inhibited LPS-induced TNF-α elevation (…). P <0.05, P <0.01 orP <0.001), and also significantly inhibited LPS-induced IFN-γ and IL-6 elevation (P < 0.05 or P <0.05 or P <0.01).
[0073] Example 3
[0074] 3 preferred compounds were subjected to 2D, 3D mapping of the binding mode with Human TLR4 protein Figure 3 ), and the results showed that Z1410232649, F27210326 and HY-N0029 could all bind to Human TLR4 protein.
[0075] Further analysis of the detailed binding mode Figure 4~Figure 9 ) showed that Z1410232649 could form 6 hydrogen bond interactions with Human TLR4 protein ASN156, ASP181, SER184, LYS186, ASN160, with specific distances of 2.0, 1.8, 2.5, 2.2, 2.2 Å, respectively; in addition, Z1410232649 could form a π-π interaction with HIS159. The multiple hydroxyl groups of F27210326 could form 3 hydrogen bond interactions with Human TLR4 protein ASP209, ASP181, SER183, with specific distances of 1.8, 1.6, 2.1 Å, respectively. HY-N0029 could form 5 hydrogen bond interactions with Human TLR4 protein SER183, ASP181, GLU154, HIS179, with distances of 1.7 Å, 2.0 Å, 1.5 Å, 2.0 Å, 2.6 Å, respectively; the hydroxyl group on the benzene ring could form three hydrogen bond interactions with SER207, ASP209, with distances of 1.7 Å, 1.7 Å, 1.6 Å, respectively; the carbonyl group of the ester group acted as a hydrogen bond acceptor to form a hydrogen bond interaction with ASN156, with a distance of 2.2 Å.
[0076] Conclusion: Due to the timely, rapid and effective characteristics of TLR4 in innate immune response, TLR4 is an important drug target for various immune diseases. Agonists of TLR4 can be used for immune therapy against pathogenic microorganisms or tumors, and as immune adjuvants for vaccines; while antagonists or inhibitors of TLR4 can be used for the treatment of various inflammatory diseases (including cytokine storm) and autoimmune diseases. In this application, based on the screening of 115700 small molecule compounds from Immuno-Oncology Library, LifeChemicals Diversity Library and MCE Bioactive Compound Library, we found a series of potential compounds that can bind to TLR4. Through experimental verification, it is preliminarily confirmed that three compounds Z1410232649, F27210326 and HY-N0029 have good TLR4 inhibitory effect. Among them, Z1410232649 performs the most outstanding, can significantly inhibit the release of inflammatory factors IL-6, TNF-α and IFN-γ induced by LPS. Therefore, this application provides a reference for the research and development of new anti-inflammatory drugs or immunotherapy drugs targeting TLR4.
Claims
1. The application of the compound in the preparation of TLR4 inhibitors, characterized in that, The compound is Z1410232649, and its structural formula is as follows: 。 2. The application of the compound in the preparation of anti-inflammatory drugs, characterized in that, Inflammation can be TLR4-mediated, such as pneumonia, sepsis, rheumatoid arthritis, or colitis. The compound is Z1410232649, and its structural formula is as follows: 。 3. The application of the compound in the preparation of therapeutic drugs for immune diseases, characterized in that, Immunological diseases include TLR4-mediated non-alcoholic fatty liver disease, type 2 diabetes, atherosclerosis, myocardial ischemia-reperfusion injury, or multiple sclerosis. The compound is Z1410232649, and its structural formula is as follows: 。