Compound for inhibiting VicK protein phosphatase activity and screening method

Abstract

The invention provides a compound for inhibiting VicK protein phosphatase activity and a screening method, and belongs to the technical field of biological medicine. The compound provided by the invention can specifically block the phosphatase activity by targeting a non-ATP binding site of VicK protein, the inhibition efficiency is obviously higher than that of a traditional ATP site inhibitor, and interference on other kinases of host cells is avoided. By defining novel active sites and combining multiple rounds of molecular docking, the screening efficiency and accuracy are greatly improved, a candidate compound with high binding energy is successfully obtained, and a new direction is provided for development of anti-infective drugs. The compound can be further developed into oral care products (such as anti-caries toothpaste) or systematic anti-infection drugs, and has definite clinical application value and market potential.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a compound for inhibiting VicK protein phosphatase activity and a screening method. Background Art

[0002] In recent years, the problem of drug resistance of pathogenic microorganisms has become increasingly serious. The development of new inhibitors targeting key virulence factors has become an important direction in the research of anti-infective drugs. Among them, histidine kinase VicK, as a core component of the two-component signal transduction system, plays a key role in the survival and pathogenicity of many pathogens. For example, Streptococcus mutans ( Streptococcus mutans ) regulates the synthesis of extracellular water-insoluble polysaccharides (such as glucan) through VicK-mediated phosphorylation cascade reactions. These polysaccharides are the core virulence factors for bacteria to adhere to the enamel surface and form caries-causing biofilms; in Streptococcus pneumoniae ( Streptococcus pneumoniae ), the activity of VicK is closely related to capsule synthesis, biofilm formation, and host immune evasion. Therefore, targeted inhibition of the phosphatase activity of the VicK protein could effectively weaken the pathogenicity of the pathogen, providing a new strategy for the development of drugs against dental caries and Streptococcus pneumoniae infection.

[0003] In traditional drug development, the design of inhibitors targeting kinases often focuses on their ATP-binding sites. However, the ATP-binding domain is highly conserved across the kinase family, making such inhibitors prone to off-target effects, affecting the normal kinase function of host cells and leading to potential toxic side effects. Furthermore, pathogens can rapidly develop drug resistance through mutations in the ATP-binding site, further limiting the clinical application of such drugs. Therefore, exploring novel, non-ATP-dependent binding sites and developing inhibitors with high selectivity and low cross-reactivity have become technical challenges that urgently need to be overcome in this field.

[0004] With the rapid development of computational biology and structural biology, protein-ligand molecular docking has become a core tool for virtual drug screening. This technology uses computer simulations of the binding patterns of small molecules with the three-dimensional structure of target proteins to predict their binding affinity and mechanism of action, thereby efficiently screening for potentially active molecules. Molecular docking is typically based on force field models, conformational search algorithms, and scoring functions. Combined with multiple rounds of increasingly precise screening (such as high-throughput primary screening, standard precision optimization, and ultra-high precision verification), it can significantly improve the hit rate of candidate compounds. For example, the Glide module in the Schrödinger software package uses a hierarchical filtering strategy to gradually optimize ligand conformations and estimate binding free energies, making it a common platform for kinase inhibitor screening. However, the effectiveness of such techniques is highly dependent on the accurate definition of the active site. If the target selection is biased or the site characteristics are ambiguous, the screening results may deviate from the actual biological function.

[0005] Currently, research on the VicK protein remains focused on its kinase domain, while its phosphatase active site remains underdeveloped. Existing crystal structure data largely reveal features of the ATP-binding site, but non-ATP-dependent functional sites (such as key regions regulating phosphatase activity) remain under-explored. Furthermore, traditional virtual screening often relies on site information from known co-crystallized ligands, making it difficult to discover novel binding modes and thus limiting the development of innovative inhibitors. Therefore, combining bioinformatics analysis with molecular docking techniques to precisely define novel active sites of the VicK protein and design highly specific inhibitors based on these sites is a key approach to overcoming existing technological bottlenecks.

[0006] In summary, the development of a non-ATP site-based VicK protein inhibitor screening method can not only circumvent the limitations of traditional drugs, but also provide safer and more effective new candidate molecules for anti-infection treatment, which has important scientific value and application prospects. Summary of the Invention

[0007] The purpose of the present invention is to provide a compound and a screening method for inhibiting VicK protein phosphatase activity, which has high inhibitory activity.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a compound for inhibiting VicK protein phosphatase activity, wherein the compound is obtained by a virtual screening method comprising the following steps: a) Based on the structure of VicK protein (PDB ID 415S), the non-ATP binding site consisting of amino acid residues P222 and T221 was defined as the active site; b) Four rounds of molecular docking screening were performed using the Glide module of the Schrödinger software package, including HTVS coarse screening, SP standard precision screening, XP ultra-high precision screening, and binding energy analysis; c) Compounds with docking scores lower than -8.0 were screened out.

[0009] Preferably, the compound is ZINC000242498440.

[0010] The present invention also provides a pharmaceutical composition comprising the above compound and a pharmaceutically acceptable carrier.

[0011] The present invention also provides the use of the above compound in preparing a drug for inhibiting the pathogenicity of Streptococcus mutans or Streptococcus pneumoniae.

[0012] Preferably, the medicament is used for preventing dental caries or treating Streptococcus pneumoniae infection.

[0013] The present invention also provides a computer-implemented method for virtual screening of VicK protein inhibitors, comprising: a) Based on the protein structure of PDB number 415S, the active site is defined as consisting of amino acid residues P222 and T221; b) Perform multiple rounds of screening of the compound database using a molecular docking program, with the screening criteria including docking score and binding mode analysis; c) Output compounds with docking scores lower than -8.0 as candidate inhibitors.

[0014] Beneficial effects of the present invention: Highly efficient and specific inhibition: The compounds provided by the present invention can specifically block the phosphatase activity of VicK protein by targeting the non-ATP binding site (P222 / T221) of the protein. The inhibition efficiency is significantly higher than that of traditional ATP site inhibitors, avoiding interference with other kinases in the host cell.

[0015] Significantly reduces pathogenicity: The compound can effectively reduce the synthesis of extracellular water-insoluble polysaccharides of Streptococcus mutans, blocking its key virulence factors that adhere to the tooth surface, thereby preventing the formation of dental caries; at the same time, it also has a potential inhibitory effect on the pathogenicity of Streptococcus pneumoniae.

[0016] Innovative screening strategy: By defining novel active sites and combining multiple rounds of molecular docking (HTVS / SP / XP), the screening efficiency and accuracy were greatly improved, and high-binding energy candidate compounds were successfully obtained, providing a new direction for the development of anti-infective drugs.

[0017] Broad application prospects: The compound can be further developed into oral care products (such as anti-caries toothpaste) or systemic anti-infective drugs, with clear clinical application value and market potential.

[0018] Safety advantage: The virtual screening method reduces the cost of experimental trial and error, and the selected compounds have low cytotoxicity characteristics, laying a safe foundation for subsequent pharmacological studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 2 is the structural diagram of the compound of the present invention. DETAILED DESCRIPTION

[0020] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Example 1

[0021] 1. Protein target information PDB ID: 4I5S GenBank: AAN59167.1

[0022] 2. Molecular Docking Molecular Docking Program: Glide Active Site Definition Process: The currently solved structures of histidine kinase (target structure and its homologous proteins), including co-crystallized compounds (ATP, ADP, and non-adenosine structures), are all concentrated in the same site (ATP site), so compounds at this site will affect kinase activity.

[0023] To screen for compounds that specifically block its phosphatase activity, it is necessary to identify new sites. Currently, there are no crystal structures of co-crystallized compounds of homologous proteins that bind to other sites, so active site prediction is necessary, and non-ATP sites are selected for virtual screening. Identifying key amino acids involved in phosphatase activity from the literature and using them to define the active site will improve accuracy.

[0024] P222 and T221 both affect phosphatase activity. It appears that these two amino acids are somewhat distant from the ATP site, potentially forming a new site. Therefore, using these two amino acids to define the active site allows for screening.

[0025] Screening Methods 2.1 Structure Preparation 2.1.2 Protein Structure Preparation Download the PDB structure number 4I5S from the Protein Crystal Structure Database (http: / / www.rcsb.org / pdb / home / home.do). Prepare the protein structure using the Protein Preparation Wizard module in the Schrodinger software package. This includes adjusting the protonation state of amino acid residues according to the specified pH conditions, filling in hydrogen atoms, and potentially missing protein structures. The prepared structure is saved as a pdb file for subsequent molecular modeling studies.

[0026] 2.1.2 Small Molecule Structure Preparation: The LigPrep module in the Schrodinger software package was used to prepare the structures for screening. 3D conformations were generated and set to a protonated state of pH 7. These conformations were then optimized using the OPLS-3e force field. 2.2 Molecular Docking Software: Glide Molecular docking was performed using the Glide module in the Schrodinger software package. The prepared protein structures were imported into the software. The Receptor Grid Generation function in the Glide module was used to define the active site center using the location of the ligand small molecule, generating the corresponding docking grid file. Virtual Screening Process: The molecular docking process consisted of four rounds of screening: the first round was high-throughput rough screening (HTVS), which retained 1% of the hit structures in the database; the second round was standard precision screening (SP), which retained 10% of the hit structures. Both HTVS and SP used a series of hierarchical filters to search for possible ligand positions in the receptor binding site. The receptor's shape and properties are represented on the grid using different field sets, which provide progressively more accurate scoring of the ligand poses. Exhaustive counting of ligand torsions generates a collection of ligand conformations, which are examined during the docking process. Given these ligand conformations, a deterministic initial screening is performed across the entire available ligand space to identify promising ligand poses. Poses selected through the initial screening are refined in torsional space within the receptor domain using OPLS3 4 (Glide SP & XP) or OPLS2005 (GLIDE HTVS) with a distance-dependent dielectric model. Finally, a small number of poses are minimized within the receptor domain with full ligand flexibility (post-docking minimization, or PDM). In the third round, XP ultra-high-precision screening is performed, retaining 10% of the hit structures. In the fourth round, the final hit structures are analyzed for mode of action and binding energies.

[0027] 3. Results Analysis like Figure 1 The compound shown (docking score -12.856, demonstrating the highest binding ability) efficiently binds to the protein target and effectively inhibits protein activity. Blocking VicK protein phosphatase activity significantly reduces the production of extracellular water-insoluble polysaccharides by Streptococcus mutans, which are important virulence factors that allow Streptococcus mutans to adhere to tooth surfaces. Therefore, one of its applications is in preventing dental caries. Blocking VicK phosphatase activity in Streptococcus pneumoniae can also reduce its pathogenicity.

[0028] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A compound for inhibiting VicK protein phosphatase activity, characterized in that The compound was obtained by a virtual screening method comprising the following steps: a) Based on the structure of VicK protein (PDB ID 415S), the non-ATP binding site consisting of amino acid residues P222 and T221 was defined as the active site; b) Four rounds of molecular docking screening were performed using the Glide module of the Schrödinger software package, including HTVS coarse screening, SP standard precision screening, XP ultra-high precision screening, and binding energy analysis; c) Compounds with docking scores lower than -8.0 were screened out.

2. The compound according to claim 1, characterized in that The compound is ZINC000242498440.

3. A pharmaceutical composition, characterized in that Comprising the compound according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. Use of the compound according to claim 1 or 2 in the preparation of a medicament for inhibiting the pathogenicity of Streptococcus mutans or Streptococcus pneumoniae.

5. The use according to claim 4, characterized in that The medicine is used for preventing dental caries or treating Streptococcus pneumoniae infection.

6. A computer-implemented method for virtual screening of VicK protein inhibitors, characterized in that: include: a) Based on the protein structure of PDB number 415S, the active site is defined as consisting of amino acid residues P222 and T221; b) Perform multiple rounds of screening of the compound database using a molecular docking program, with the screening criteria including docking score and binding mode analysis; c) Output compounds with docking scores lower than -8.0 as candidate inhibitors.

Images