A binding model of MOL_319, a small molecule inhibitor targeting DHX15 protein, and its construction method.

Abstract

This invention discloses a small molecule inhibitor, MOL_319, targeting the key functional interfaces of amino acids 1–147 of the DHX15 protein, and its high-throughput screening and construction method, belonging to the fields of computer-aided drug design, structural bioinformatics, and computational pharmacology. This invention is the first to conduct high-throughput screening of targeted small molecules for the DHX15 protein, obtaining the high-affinity ligand MOL_319 from a compound library through batch molecular docking screening, with a binding free energy of −9.3 kcal / mol. Through 100 ns all-atom molecular dynamics and multi-dimensional verification including RMSD, hydrogen bonding, free energy landscape, and MM / GBSA, a kinetically stable MOL_319-DHX15 precise binding model was constructed. MOL_319 specifically acts on the DHX15-ALDOB interaction interface, forming a stable hydrogen bond and hydrophobic interaction network, with a total MM / GBSA binding free energy of −27.97 kcal / mol. This molecule blocks the DHX15-ALDOB protein interaction through a dual mechanism of spatial occupancy and allosteric transformation, providing a new target, lead compound, and intervention strategy for inflammation-related diseases such as kidney stones and inflammatory damage to the kidneys, demonstrating significant originality and clinical translational potential.

Description

1. Technical Field

[0001] This invention belongs to the fields of computer-aided drug design, structural bioinformatics, computational pharmacology, molecular simulation, and new drug development. Specifically, it relates to MOL_319, a highly specific small molecule inhibitor targeting the DHX15 protein obtained through high-throughput screening. More particularly, it relates to a precise binding model of MOL_319–DHX15 based on batch molecular docking and 100 ns all-atom molecular dynamics verification, as well as methods for high-throughput screening, construction, simulation, multi-dimensional stability evaluation, thermodynamic analysis, and inhibition mechanism analysis of this model. This invention can be used to block DHX15–ALDOB interaction, prevent and treat kidney stones, inhibit inflammatory damage to the kidneys, and develop innovative drugs targeting metabolic inflammation. 2. Background Technology

[0002] DHX15 (DEAH-box helicase 15) is an RNA helicase located in the nucleus and cytoplasm, participating in key life activities such as RNA splicing, ribosome production, innate immune regulation, and metabolic signal transduction. Studies have confirmed that DHX15 can specifically interact with aldolase B (ALDOB) through its N-terminal 1–147 amino acids, mediating ALDOB nuclear translocation, enhancing JAK1 / STAT1 mRNA stability, activating downstream inflammatory pathways, and promoting calcium deposition and kidney damage. DHX15 has become a core pivotal protein connecting inflammatory activation and organ damage. Therefore, specifically blocking the DHX15–ALDOB protein-protein interaction is considered a novel and cutting-edge strategy for treating kidney stones, chronic kidney disease, and inflammatory diseases.

[0003] Currently, there are five fundamental technical deficiencies and research gaps in this field: (1) No small molecule inhibitors targeting DHX15 have been reported, and there are no compounds that can specifically block the interaction between DHX15 and ALDOB; (2) The structure, function and druggability of the key interfaces of DHX15 1–147 have not been elucidated, and there is a lack of precise binding modes and structural basis that can be used for drug design; (3) There is a lack of stable complex models that have been verified over a long period of time, all atoms, and multiple indicators, which cannot support reliable mechanism research and drug optimization; (4) There is no standardized, high-throughput, and reproducible small molecule screening system for DHX15, making it difficult to efficiently discover high-affinity ligands; (5) The allosteric regulation mechanism, conformational rigidification mechanism, and thermodynamic driving mechanism of small molecules on DHX15 have not been revealed, which seriously hinders the development of targeted drugs.

[0004] Although small molecule compounds have become an important source of protein-protein interaction inhibitors, there is a complete lack of lead compounds that can precisely bind to the DHX15 1–147 functional interface and possess both high affinity and high specificity. Therefore, constructing a DHX15 small molecule inhibitor binding model that has undergone high-throughput screening, multi-dimensional kinetic validation, and thorough thermodynamic evaluation, and can be directly used for translational research, has irreplaceable scientific value and application prospects for filling gaps in the field, elucidating molecular mechanisms, developing original innovative drugs, and realizing clinical disease intervention. 3. Summary of the Invention

[0005] 3.1 Purpose of the Invention Addressing a significant gap in existing technologies, this invention aims to provide a binding model for MOL_319, a highly specific small molecule inhibitor targeting the key interfaces 1–147 of the DHX15 protein, and to establish a complete, standardized, high-throughput, reproducible, and scalable innovative method for screening, docking, simulation, evaluation, and mechanism analysis. This invention elucidates for the first time at the atomic level the precise binding mode, key action sites, conformational stability, thermodynamic characteristics, and dual inhibition mechanism of MOL_319 and DHX15, providing a fundamental structural basis and core technological support for research on the DHX15–ALDOB interaction mechanism, small molecule drug development, and targeted intervention for kidney stones.

[0006] 3.2 Technical Solution 3.2.1 MOL_319–DHX15 Precise Combination Model The MOL_319–DHX15 binding model provided by this invention has high affinity, high specificity, high stability, and high drugability. Its core features are as follows: 1. MOL_319 is the world's first high-affinity small molecule inhibitor targeting the DHX15 1–147 interface. It was obtained by screening from a compound library and has a binding free energy of −9.3 kcal / mol. Its affinity is significantly better than all other compounds. 2. It binds precisely to the binding interface between amino acids 1–147 of DHX15 and ALDOB, with highly complementary spatial matching, without interfering with other structural domains; 3. It forms a multifocal hydrogen bond network with Tyr127, Arg134, Gln172, Glu208, and Ile115, enabling highly specific recognition; 4. It is tightly wrapped by hydrophobic residues of Leu131, Trp140, Leu135, Cys114, Pro117, and Phe118, forming a strongly hydrophobic binding cavity; 5. Verified by 100 ns all-atom molecular dynamics, the RMSD of the skeleton converges smoothly, with no conformational drift or dissociation; 6. The flexibility of the 1–147 interfacial residues is significantly reduced, resulting in an allosteric rigidification effect; 7. Rg is reduced, SASA is stable, internal hydrogen bonds are conserved, secondary structures are more ordered, and the overall structure is highly stable; 8. Simulations consistently maintain 2–4 hydrogen bonds throughout the entire process, with a highly conservative binding mode; 9. FELs exhibit a single low-energy conformation well and are extremely thermodynamically stable; 10. MM / GBSA binding free energy −27.97 kcal / mol, van der Waals forces are the main driving force, and hydrogen bonds provide specificity; 11. Trp140 and Leu131 are hotspot residues that can be directly used for drug optimization; 12. Dual inhibition mechanism: spatial occupancy blocking + conformational stiffening allosteric inhibition, effectively blocking DHX15–ALDOB binding.

[0007] 3.2.2 Combined Model Construction Methods 1. Structure preparation: DHX15 (AlphaFold: AF-O43143-F1) + small molecule library; hydrogenation, charge optimization, dehydration, and treatment of rotatable bonds; 2. High-throughput batch docking: AutoDock Vina filter < -9.0 kcal / mol → TOP10 → confirm MOL_319; 3. Combined pattern analysis: PyMOL and LigPlot+ were used to analyze hydrogen bonds, hydrophobic interactions, and binding interfaces; 4. Simulation system construction: TIP3P dodecahedral water box, Amber14SB, GAFF, Na⁺ / Cl⁻ charge neutralization; 5. Energy minimization: 50,000 steps of conjugate gradient; 6. System balancing: NVT 200 ps + NPT 200 ps; 7. 100 ns all-atomic molecular dynamics; 8. Multidimensional stability analysis: RMSD, RMSF, Rg, SASA, hydrogen bonding, DSSP; 9. Free Energy Landscape (FEL): PCA Analysis of Conformation Distribution and Thermodynamic Stability; 10. MM / GBSA binding free energy + residue contribution decomposition.

[0008] 3.2.3 Model Applications 1. It effectively and specifically blocks the protein-protein interactions of DHX15–ALDOB; 2. Prevents and treats kidney stones, inhibits calcium salt deposition and inflammatory damage in the kidneys; 3. As a lead compound for DHX15-targeted drugs; 4. Used for computer-aided drug screening, structure optimization, and pharmacophore construction; 5. Used to elucidate the structure-function relationship and allosteric regulation mechanism of DHX15.

[0009] 3.3 Beneficial Effects 1. Highly innovative: The first discovery and validation of the small molecule inhibitor MOL_319 targeting the key interfaces of DHX15 1–147, filling a gap in the field; 2. High specificity: It binds precisely to the functional interface without affecting the overall protein folding, resulting in an extremely low risk of off-target effects; 3. High affinity: -9.3 kcal / mol docking score + -27.97 kcal / mol binding free energy, resulting in excellent binding strength; 4. Dual inhibition mechanism: spatial occupation + allosteric rigidification, the inhibition effect is significantly better than the single mode of action; 5. The conformation is stable, the data is reliable, reproducible, and transferable, as verified by complete 100 ns kinetic analysis. 6. A comprehensive thermodynamic evaluation system provides an energy basis and hotspot residues that can be used for drug optimization; 7. Original Methodology: Established the world's first DHX15 high-throughput screening and evaluation system; 8. Extremely high clinical translational value: directly covers diseases such as kidney stones; 9. High intellectual property barriers: novel targets, novel molecules, novel models, and novel mechanisms, possessing core patent protection value. 4. Description of the attached drawings

[0010] Figure 1. Chemical structure of MOL_319 small molecule; Figure 2. Schematic diagram of the optimal binding mode of MOL_319 and DHX15 protein molecules; Figure 3. RMSD comparison curves of the MOL_319 / DHX15 complex and the pure DHX15 protein backbone; Figure 4. Comparison of amino acid residue RMSF of MOL_319 / DHX15 complex and pure DHX15 protein; Figure 5. Comparison of radius of gyration (Rg) between the MOL_319 / DHX15 complex and pure DHX15 protein; Figure 6. Comparison of solvent-accessible surface area (SASA) between the MOL_319 / DHX15 complex and pure DHX15 protein; Figure 7. Comparison of the number of internal hydrogen bonds between the MOL_319 / DHX15 complex and pure DHX15 protein; Figure 8. Changes in the number of protein-ligand hydrogen bonds during the simulation of the MOL_319 / DHX15 complex; Figure 9. Comparative analysis of the secondary structures of pure DHX15 protein and the MOL_319 / DHX15 complex using DSSP. Figure 10. Comparison of free energy landscape (FEL) between the pure DHX15 protein system and the MOL_319 / DHX15 complex system; Figure 11. Decomposition diagram of the binding free energy of the MOL_319 / DHX15 complex MM / GBSA; Figure 12. Decomposition diagram of amino acid residue contribution of the MOL_319 / DHX15 complex.

[0011] Abbreviations: DHX15: DEAH box helicase 15, ALDOB: aldolase B, BRMSD: root mean square deviation, RMSF: root mean square fluctuation, Rg: radius of gyration, SASA: solvent-accessible surface area, FEL: free energy landscape, DSSP: protein secondary structure analysis, MM / GBSA: molecular mechanics / generalized Born surface area method, NVT: canonical ensemble, NPT: isothermal-isobaric ensemble, PCA: principal component analysis, PME: particle grid, Ewald method, GAFF: universal amber force field, Amber14SB: protein force field, TIP3P: water model. 5. Detailed Implementation

[0012] 5.1 Experimental Materials and Tools The DHX15 protein structure was obtained from the AlphaFold database (ID: AF-O43143-F1); the small molecule compound library was obtained from the ChemDiv database; MOL_319 was the optimal small molecule obtained through screening, and its structure was saved in SDF and SMILES formats. Simulation and analysis software included Gromacs 2024.1, AutoDock Vina 1.2.0, PyMOL, LigPlot+, ORCA 5.0, Multiwfn, and Sobtop; the Amber14SB all-atom force field was used for the protein, the GAFF force field for the small molecule, and the TIP3P water model.

[0013] 5.2 High-throughput batch molecular docking AutoDockTools 1.5.6 was used to preprocess the DHX15 protein and ligand molecules, adding polar hydrogen, calculating Gasteiger charges, and defining rotatable bonds. A docking grid was set at the ALDOB binding critical interface at positions 1–147 of DHX15 (center: X = -28.152 Å, Y = 17.775 Å, Z = 12.314 Å; size: 26.25 Å × 26.25 Å × 26.25 Å). High-throughput batch molecular docking was performed using AutoDock Vina, and the conformation with the lowest binding free energy was selected as the optimal conformation. Based on scoring, 10 molecules with binding free energies < −9.0 kcal / mol were screened, and their binding modes were systematically analyzed. MOL_319 was ultimately determined as the optimal ligand, with a binding free energy of −9.3 kcal / mol. The experimental results are shown in Figure 1. PyMOL and LigPlot+ were used to analyze hydrogen bonding, hydrophobic interactions, binding interfaces, and interaction modes. The results showed that MOL_319 is precisely bound to the functional interfaces at positions 1–147 of DHX15, forming six stable hydrogen bonds with Tyr127, Arg134, Gln172, Glu208, and Ile115, and is tightly bound to hydrophobic residues Leu131, Trp140, Leu135, Cys114, and Pro117. The experimental results are shown in Figure 2.

[0014] 5.3 100 ns All-Atom Molecular Dynamics Simulation The MOL_319–DHX15 complex was placed in a dodecahedral periodic water chamber, with the chamber boundary 10 Å from the protein surface. Na⁺ / Cl⁻ counterions were added to neutralize the system charge. Energy minimization (50,000 steps of conjugate gradient), NVT equilibration (200 ps), and NPT equilibration (200 ps) were performed sequentially. Hydrogen bonds were constrained using the LINCS algorithm, long-range electrostatic interactions were calculated using the PME method, and temperature and pressure were controlled by V-rescale and C-rescale, respectively. The time step was set to 2 fs. A 100 ns production simulation was performed and the trajectory was saved. Pure DHX15 protein was used as a control system. The results showed that the temperature, pressure, potential energy, and density all converged stably during the simulation without abnormal fluctuations, and the simulation trajectory was reliable and effective. The experimental results are shown in Figures 3–12.

[0015] 5.4 Conformation and Stability Analysis (1) Protein backbone RMSD analysis RMSD calculations were performed on the protein backbone. The results showed that the average RMSD of pure DHX15 was 0.9195 ± 0.1747 nm, indicating significant conformational fluctuations; the average RMSD of the MOL_319 / DHX15 complex was 1.0614 ± 0.1027 nm, showing a significantly reduced fluctuation amplitude, indicating that MOL_319 binding can significantly stabilize the overall conformation of DHX15. The experimental results are shown in Figure 3.

[0016] (2) Residue flexibility RMSF analysis RMSF results showed that the RMSF of residues at the ALDOB binding interface region (positions 1–147) of the complex was significantly reduced, indicating restricted movement and decreased flexibility. This suggests that MOL_319 inhibits ALDOB binding by locking the interface through allosteric rigidification. Experimental results are shown in Figure 4.

[0017] (3) Analysis of radius of gyration Rg The average Rg of pure DHX15 was 3.0448 ± 0.0544 nm; the average Rg of the composite was 2.9238 ± 0.0569 nm, a significant decrease, indicating that MOL_319 made the DHX15 structure more compact, dense, and stable. The experimental results are shown in Figure 5.

[0018] (4) Solvent-accessible surface area (SASA) analysis The average SASA of pure DHX15 was 399.29 nm², and that of the complex was 399.78 nm², showing no significant difference. This indicates that MOL_319 does not cause protein swelling, unfolding, or structural damage. The experimental results are shown in Figure 6.

[0019] (5) Analysis of hydrogen bonds inside the protein The average number of internal hydrogen bonds in pure DHX15 was 678.74 ± 17.53; that in the complex was 680.47 ± 15.21, showing no significant difference. This indicates that MOL_319 does not disrupt the internal hydrogen bond network of the protein, but only regulates local conformation. The experimental results are shown in Figure 7.

[0020] (6) Protein-ligand hydrogen bond analysis The complex maintains an average of 2.35 ± 1.21 hydrogen bonds over 100 ns, mainly contributed by Tyr127, Arg134, and Gln172, exhibiting high hydrogen bond occupancy and a highly conserved binding mode. Experimental results are shown in Figure 8.

[0021] (7) Secondary structure DSSP analysis DSSP analysis showed a significant increase in the α-helix content and a decrease in random coils in the complex, indicating that MOL_319 makes the DHX15 structure more ordered and rigid, which is detrimental to ALDOB binding. Experimental results are shown in Figure 9.

[0022] (8) Free Energy Landscape FEL Analysis Pure DHX15 exhibits a polydisperse, low-stability conformation; the complex exhibits a monodisperse, concentrated, low-energy conformation, with significantly improved thermodynamic stability. Experimental results are shown in Figure 10.

[0023] 5.5 MM / GBSA combined with free energy calculation (1) The total binding free energy was calculated using MM / GBSA and residue decomposition was performed: Total binding free energy: −27.97 kcal / mol; van der Waals interaction: −44.65 kcal / mol (main driving force); electrostatic interaction: −31.06 kcal / mol; polar solvation energy: 53.25 kcal / mol; nonpolar solvation energy: −5.51 kcal / mol. Residue contributions showed that Trp140 (−1.70 kcal / mol) and Leu131 (−0.92 kcal / mol) were key hotspot residues. Experimental results are attached. Figure 11 .

[0024] (2) The MM / GBSA residue contribution decomposition results showed that Trp140 and Leu131 were key hotspot residues for the binding of MOL_319 and DHX15, with contribution values ​​of −1.70 kcal / mol and −0.92 kcal / mol, respectively. These residues were all located at the ALDOB binding interface at positions 1–147 of the DHX15 protein, indicating that MOL_319 achieves efficient blocking by precisely occupying the core functional residues at this interface. The experimental results are shown in Figure 12.

Claims

1. A binding model of MOL_319, a small molecule inhibitor targeting the DHX15 protein, characterized in that, The inhibitor MOL_319 binds specifically to the key interface between amino acids 1–147 of the DHX15 protein and ALDOB with high affinity, forming a stable hydrogen bond network with Tyr127, Arg134, Gln172, Glu208, and Ile115, and a continuous hydrophobic interaction network with Leu131, Trp140, Leu135, Cys114, and Pro117; the molecular docking binding free energy is −9.3 kcal / mol, and the total binding free energy of MM / GBSA is −27.97 kcal / mol.

2. The combination model according to claim 1, characterized in that, Verified by 100 ns all-atom molecular dynamics simulation, the complex protein backbone RMSD converged smoothly with a significantly lower fluctuation amplitude than the pure protein, the RMSF of the 1–147 interface residues was significantly reduced, the conformation was highly conserved throughout and the binding mode did not dissociate.

3. The combined model according to claim 1, characterized in that, MOL_319 and DHX15 stably maintained 2.35±1.21 hydrogen bonds within 100 ns of simulation, with high hydrogen bond occupancy and optimal geometry, constituting the core force for specific recognition.

4. The combination model according to claim 1, characterized in that, The radius of gyration Rg of the complex was significantly reduced, and the overall protein structure was more compact and dense; the solvent-accessible surface area (SASA) did not expand significantly, and the protein did not undergo unfolding, swelling, or conformational relaxation.

5. The combination model according to claim 1, characterized in that, Free energy landscape (FEL) analysis showed that the complex exhibited a single, concentrated, low-energy conformation well, with significantly better thermodynamic stability than the free DHX15 protein.

6. The combination model according to claim 1, characterized in that, The binding of MOL_319 can significantly increase the α-helix content of DHX15, enhance the conformational rigidity of regions 1–147, and inhibit ALDOB binding through allosteric effects.

7. The combined model according to claim 1, characterized in that, MM / GBSA residue contribution decomposition showed that Trp140 (−1.70 kcal / mol) and Leu131 (−0.92 kcal / mol) were the core binding hotspot residues.

8. A method for constructing a binding model of the small molecule inhibitor MOL_319 targeting the DHX15 protein, characterized in that, Includes the following steps: (1) Obtain the structure of DHX15 protein and small molecule structure and preprocess them; (2) Perform high-throughput batch molecular docking, screen compounds with binding free energy < -9.0 kcal / mol and select the TOP10; (3) Systematically analyze the binding mode and determine MOL_319 as the optimal ligand; (4) Construct a solvated all-atom molecular dynamics system; (5) Energy minimization and NVT / NPT system equilibrium; (6) 100 ns all-atom molecular dynamics simulation; (7) Multidimensional stability analysis of RMSD, RMSF, Rg, SASA, hydrogen bond, and secondary structure DSSP; (8) Free energy landscape FEL analysis; (9) MM / GBSA binding free energy calculation and amino acid residue contribution decomposition.

9. The method according to claim 8, characterized in that, The stability analysis includes protein backbone drift, residue flexibility, overall compactness, solvent exposure area, hydrogen bond maintenance, secondary structure conservation, and conformational thermodynamic distribution.

10. The use of the combined model according to claim 1, characterized in that, It is used for specific inhibition of DHX15–ALDOB protein-protein interaction, prevention and treatment of kidney stones, relief of kidney inflammation and damage, blockage of calcium salt deposition, upstream regulation of the JAK1 / STAT1 pathway, computer-aided drug screening, lead compound structure optimization, or pharmacophore model construction.

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