A drug lead compound targeting ATP6V0A4 p.V512L mutant inhibitor and its application

Abstract

The present invention discloses a drug lead compound and application targeting an ATP6V0A4p.V512L mutant inhibitor, belonging to the field of biomedicine technology. A drug for treating or preventing renal hydrogen secretion disorders includes an inhibitor capable of targeting and inhibiting the ATP6V0A4p.V512L mutant. The present invention also relates to a potential drug screening method for inhibiting the ATP6V0A4p.V512L mutant, comprising the following steps: predicting the protein structure of the ATP6V0A4p.V512L mutant, virtually docking the protein with the compound to be screened, and virtually screening according to the binding affinity; using the RDKit fingerprint generator to generate a molecular fingerprint for the virtually screened compound, calculating the Tanimoto similarity of the fingerprint, and using hierarchical agglomerative clustering for cluster analysis; calculating the binding free energy of the compound and the mutant through molecular dynamics simulation, and further screening the compound; and performing wet experiment verification on the screened compound. This compound can be used to further design and synthesize highly active and selective inhibitors targeting the ATP6V0A4p.V512L mutant, and at the same time lay the foundation for the development of targeted drugs for patients with renal hydrogen secretion disorders caused by the ATP6V0A4p.V512L mutation.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a drug lead compound targeting an ATP6V0A4 p.V512L mutant inhibitor and an application thereof. Background Art

[0002] V-ATPase is a membrane-anchored protein complex that mediates H+ transmembrane transport through the energy obtained from the hydrolysis of ATP. It regulates a series of important life activities by acidifying various structures inside and outside the cell, such as urine acidification, cochlear endolymph pH homeostasis, protein processing, transport and degradation, etc. V-ATPase has two domains, V1 and V0. V1 is located in the cytoplasm and consists of 8 subunits (A3B3CDE3FG3H). It mainly performs the function of catalyzing the hydrolysis of ATP; V0 is anchored on the membrane and consists of a c-ring structure composed of 6 subunits (a, c, c,,,d,e, RNAseK) and two subunits ATP6AP1 and ATP6AP2 inside the c-ring. It mainly performs the function of hydrogen ion transmembrane transport. Many subunits of V-ATPase have different subtypes. The expression and localization of different subtypes of the same subunit are tissue and cell specific. The V-ATPase on the luminal membrane of the cell in the A-type intercalated cell of the kidney is responsible for transporting H+ across the membrane. + Transported to the tubule lumen to acidify urine, when the related gene encoding the V-ATPase subunit mutates abnormally, it will affect the A-type intercalated cells to H + transport function, thereby affecting the secretion of H in the distal renal tubules + function, ultimately leading to urine acidification disorder.

[0003] The ATP6V0A4 gene encodes the α4 subunit of the V0 domain. The gene is located at 7q33-34 and encodes the a4 subunit of the V0 domain of the V-ATPase composed of 840 amino acids. It is highly expressed in the kidney, epididymis and inner ear. The applicant found clinical cases with metabolic alkalosis, hypokalemia, acidic urine and sensorineural hearing loss. Whole exome sequencing showed that the patient carried a gain-of-function mutation in ATP6V0A4 (c.1534G>T, p.V512L) (such as Figure 1 In vitro experiments confirmed that the mutation increased the stability of ATP6V0A4 protein, resulting in increased abundance of ATP6V0A4 protein, which led to increased V-ATPase activity on the lumen side of type A intercalated cells in the renal tubules. + The ability to pump into the lumen is enhanced, resulting in excessive acidification of the urine. Currently, there is a lack of effective drugs in the prior art for treating the renal hydrogen secretion disorder caused by this mutation. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention proposes a drug lead compound and application of an inhibitor targeting the ATP6V0A4 p.V512L mutant.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The first aspect of the present invention relates to a drug for treating or preventing renal hydrogen secretion disorder, including an inhibitor capable of targeted inhibition of ATP6V0A p.V512L mutant.

[0007] Optionally, the inhibitor capable of targeted inhibition of ATP6V0A4 p.V512L mutant includes compound F351, having the structural formula:

[0008]

[0009] The second aspect of the present invention relates to the use of an inhibitor capable of targeted inhibition of ATP6V0A4 p.V512L mutant in the preparation of a drug for treating or preventing renal hydrogen secretion disorder.

[0010] Optionally, in the drug, the concentration of compound F351 is 20umol / L.

[0011] The third aspect of the present invention relates to a method for screening potential drugs for inhibiting the ATP6V0A4 p.V512L mutant, comprising the following steps:

[0012] The protein structure of the ATP6V0A4 p.V512L mutant was predicted.

[0013] Virtually docking the protein with the compound to be screened, and performing virtual screening based on binding affinity;

[0014] Use RDKit fingerprint generator to generate molecular fingerprints for the compounds after virtual screening, calculate Tanimoto similarity of fingerprints, and perform cluster analysis using hierarchical agglomerative clustering;

[0015] Through molecular dynamics simulation, the binding free energy of the compound and the mutant is calculated to further screen the compound;

[0016] The screened compounds were verified by wet experiment.

[0017] Optionally, the wet experiment verification includes verifying the ability of the compound to inhibit the abundance of ATP6V0A4 protein expression and V-ATPase activity.

[0018] Optionally, the ATP6V0A4 protein expression abundance is obtained by Western blot detection; and the V-ATPase activity is obtained by spectrophotometry detection.

[0019] The fourth aspect of the present invention relates to the use of a reagent capable of detecting the ATP6V0A4 p.V512L mutant in the preparation of a kit for diagnosing or screening renal hydrogen secretion disorders.

[0020] Optionally, the reagent is a Western blot related reagent.

[0021] Beneficial effects of the present invention:

[0022] The present invention explores a method for screening potential drug ingredients. The mutant protein structure is predicted by alphafold, and the structure-based virtual screening technology is used to screen and find the inhibitor F351 targeting the ATP6V0A4 p.V512L mutant. The wet experiment verifies that the compound can stably and effectively inhibit the expression abundance of the α4 subunit and the V-ATPase activity in cells containing the ATP6V0A4p.V512L mutant, and weaken the hydrogen secretion ability of the V-ATPase.

[0023] Among them, compared with other prediction tools, AlphaFold2 used in this application can improve the confidence (reliability) and coverage (measure of test effectiveness) of protein structure prediction; based on the spatial complexity of protein structure, AlphaFold2 also performs well in the prediction of multi-domain complexes; AlphaFold2 predicts proteins very quickly and can generate structures within minutes to hours.

[0024] The compounds screened by the present invention can be used to further design and synthesize highly active and highly selective inhibitors targeting the ATP6V0A4 p.V512L mutant, and at the same time lay the foundation for the development of target drugs for patients with renal hydrogen secretion disorders caused by the ATP6V0A4 p.V512L mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with the accompanying drawings.

[0026] Figure 1 Sequences before and after the mutated residues of ATP6V0A4 of wild type and mutant.

[0027] Figure 2 : In order to find compounds that inhibit the ATP6V0A4 p.V512L mutant, a structure-based virtual screening process targeting ATP6V0A4 p.V512L mutant inhibitor compounds was used.

[0028] Figure 3 : Protein structures of wild type (blue) and mutant ATP6V0A4 p.V512L (red) predicted based on Alphaofold.

[0029] Figure 4 : The docking pocket of ATP6V0A4 p.V512L mutant and ligand (A) and the front and back sides of the mutant docking with ligand (BC).

[0030] Figure 5 : Results of the virtual screening process.

[0031] Figure 6 : Binding free energy of five screened potential inhibitors of ATP6V0A4 p.V512L mutant with the mutant.

[0032] Figure 7 : Docking poses of the five screened potential inhibitors of ATP6V0A4 p.V512L mutant.

[0033] Figure 8 :Relamorelin, Forsythiaside A, PAR-1AC and F359 had no stable and significant inhibitory effect on the V-ATPase activity in ATP6V0A4p.V512L mutant cells.

[0034] Fig. 9 The most stable and effective compound for inhibiting V-ATPase activity in ATP6V0A4 p.V512L mutant cells was F351-0364 (C 25 H 26 FN 3O O2S), the drug concentration is 20umol / L when the effect is the best, and it is concentration-dependent.

[0035] Fig.10 : F351 intervention had no significant inhibition on V-ATPase activity in wild-type M1 cells, indicating that F351 targeted and inhibited the V-ATPase activity of ATP6V0A4 p.V512L mutant.

[0036] Fig.11 : After 24 hours of intervention with ATP6V0A4 p.V512L mutant M1 cells, F351 showed the strongest ability to inhibit V-ATPase activity.

[0037] Fig.12 : After F351 intervention in ATP6V0A4 p.V512L mutant M1 cells for 24 hours, the expression abundance of ATP6V0A4 was significantly downregulated.

[0038] Fig.13 : Through CCK8 screening, 50% of cells died only when the IC50 value exceeded 140.9um, while the optimal efficacy concentration of the F351 compound was only 20um, indicating that the F351 compound had low cytotoxicity.

[0039] Fig.14 : The F351 compound increased the lysosomal pH of ATP6V0A4 p.V512L mutant M1 cells in a concentration-dependent manner. When the drug concentration was 20umol / L, the lysosomal pH was closest to that of wild-type cells, reversing lysosomal overacidification. DETAILED DESCRIPTION

[0040] 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.

[0041] 1. Preparation of Receptors and Ligands

[0042] The crystal structure of the template protein was obtained from the RCSB protein data bank (PDB) as 7UNF. The structure of the a-subunit of V-ATPase was modeled using homology using the online server SWISS-MODEL, and the structure of the a-subunit V512L mutant protein of V-ATPase was predicted using AlphaFold2. The quality of the protein structure was evaluated by Ramachandran plots, ERRAT, and verify3D using the SAVES web server. The protein was preprocessed using AutoDock Tools, including the introduction of hydrogen atoms, charge assignment, and format conversion. The docking pocket was selected using the CavityPlus prediction website as well as human judgment. For virtual screening, more than 2 million molecules from ChemDiv, COCONUT, and DrugBank constituted the screening library. The ligand structure was downloaded from the official website in the format of a structural data file (SDF), in which the important chirality was specified, retaining its assigned chirality. The compounds in the screening library were screened for pains and warning structures. Gypsum-DL was used to generate the ionized states of the compounds at pH 7.0±2.0, and a low-energy conformation was generated for each ligand. Finally, the Meeko package was used to convert the compound structure files into pdbqt format.

[0043] 2. Structure-based virtual screening

[0044] A multi-stage virtual screening process was used to identify potential lead molecules targeting V512L, the a subunit of V-ATPase. Structure-based virtual screening of the compounds in the screening library was performed by using the AutoDock Vina 1.2.5 software package. Lower docking scores indicate better binding affinity between the receptor and the ligand. The top 5000 docking molecules with the highest pre-scores were selected for cluster analysis. Molecular fingerprints were generated for the compounds using the RDKit fingerprint generator, and the Tanimoto similarity of the fingerprints was calculated. Hierarchical agglomerative clustering was used to perform cluster analysis on the compounds.

[0045] 3. Molecular dynamics simulation

[0046] The docked structure was selected as the initial structure for molecular dynamics simulation. Molecular dynamics simulations were performed using the molecular dynamics simulation package GROMACS ver.2023.2 (van2005gromacs). RESP2 (0.5) charges were generated for small molecules by Multiwfn software, and the topology and gro files of GROMACS were generated by Sobtop (Lu2024) software. Amber ff14SB and GAFF force fields were used for proteins and small molecules, respectively. Each system was solvated into a dodecahedral water box with the TIP3P water model, and an appropriate number of sodium ions or chloride ions were added to neutralize each system. Periodic boundary conditions (PBC) were applied to all three directions of the system. First, the steepest descent method was used for energy minimization, and then the system was pre-equilibrated by NVT and NPT ensembles, respectively. At a stable temperature of 300K, a velocity-rescale temperature coupler was used with a time constant of 0.1ps for 100ps of NVT equilibration. A Berendsen pressure coupler was used to maintain a pressure of 1 bar, and NPT equilibrium was performed for 100 ps with a time constant of 2.0 ps. Electrostatic interactions were treated using the SPME algorithm, with a cutoff of 12 angstroms for real-space interactions. The LINCS algorithm was used to constrain all bonds involving hydrogen atoms. The molecular dynamics simulation time step was set to 2 fs, and conformations were recorded every 10 ps. For the batch-screened complexes, a 10 ns dynamics simulation was performed for each system, and the binding free energy was calculated using the last 1 ns of the simulation trajectory.

[0047] 4.MM / GBSA calculation

[0048] Molecular dynamics simulations and thermodynamic calculations, such as the Molecular Mechanics / Generalized Born Surface Area (MM / GBSA) method (genheden2015mm), can be combined to measure the free energy of protein-ligand complexes. In standard MM / GBSA, the binding free energy (ΔG bind ) can be decomposed into different energy terms as follows:

[0049] ΔG bind =ΔG bind,gas +(ΔG COM,sol -ΔG REC,sol -ΔG LIG,sol )=ΔG bind,gas +ΔG sovaltion

[0050] ΔG bind,gas =ΔH-TΔS≈ΔE MM -TΔS

[0051] ΔG bind ≈ΔE MM +ΔG sol -TΔS

[0052] ΔE MM =ΔE int +ΔE vdw +ΔE ele

[0053] ΔG sol =ΔG GB +ΔG SA

[0054] ΔG SA =γ·SASA+b

[0055] In the formula, the binding free energy of the complex can be approximated as the molecular mechanical energy ΔE MM , Solvation energy ΔG sol and conformational entropy - TΔS. ΔE MM From the internal energy (ΔE int ), van der Waals energy (ΔE vdw ) and electrostatic energy (ΔE ele ) composition. sol By polar solvation term (ΔG GB ) and the nonpolar solvation term (ΔG SA ) composition, ΔG GB Estimated by the generalized Born model, ΔG SACalculated by solvent accessible surface area. MM / GBSA calculations were done using the gmx_MMPBSA tool.

[0056] 5. Wet-experimental validation of virtual drug screening

[0057] 5.1 Cell culture:

[0058] The culture conditions of wild-type and ATP6V0A4 p.V512L mutant M1 (renal collecting duct cells) cell lines were 10% FBS, 1% P / S, puro (1 ug / ml), DMEM / F12, 37°C, 5% CO2.

[0059] 5.2 Cellular intervention

[0060] Wild-type and ATP6V0A4 p.V512L mutant M1 cells were seeded in confocal dishes at appropriate density. After the cells successfully attached to the wall, the cells were cultured until the density reached 60%. The inhibitors targeting ATP6V0A4 p.V512L mutants screened out by virtual screening were added to the cell culture medium and configured to intervene in the cells at concentrations of 0 / 5 / 10 / 20 / 40 / 80umol / L. Cell lysates were collected after 24 hours of culture, and the V-ATPase activity and the expression of α4 subunit protein encoded by the ATP6V0A4 gene were measured in turn to evaluate whether the drug was effective. Then, according to the previous experimental method, the lysates of cells at different time points of 0 / 12 / 24 / 48 hours after drug intervention were collected to evaluate the best onset time of intervention.

[0061] 5.3 V-ATPase activity assay

[0062] The process of ATPase hydrolyzing ATP to produce ADP is accompanied by the oxidation reaction of reduced nicotinamide adenine dinucleotide (NADH). Therefore, the activity of V-ATPase can be quantitatively analyzed by spectroscopic determination of the peak change (340nm) generated by the conversion of NADH to NAD (oxidized nicotinamide adenine dinucleotide). According to the instructions of the GENMED Cellular V-ATPase Activity Assay Kit (GMS50247.1), the total protein extracted from the cells was collected for protein quantitative detection. GENMED buffer, enzymatic solution, reaction solution and substrate solution were added to the ELISA plate in sequence, and the plate was placed in a 37°C incubator for 3 minutes. Then, a sample solution containing 100ug of protein was added, and the total system was 250ul. Mix well and immediately put into the multifunctional microplate reader for detection. The 340nm wavelength readings of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20min were read in sequence.

[0063] 5.4Western blot

[0064] Prepare cell lysis buffer and extract total cell protein. Determine protein concentration using BCA method and boil at 75℃ for 10 minutes to denature protein. Separate 40ug total protein by gel electrophoresis, transfer to membrane, block with western fast blocking solution for 30 minutes, add ATP6V0A4 primary antibody at 4℃ overnight, wash the membrane with TBST for 10 minutes × 3 times, add corresponding secondary antibody and incubate at room temperature for 1 hour, and develop by electrochemiluminescence exposure after washing.

[0065] 5.5 CCK8 Experiment

[0066] Cell viability and drug toxicity were detected by CCK8 (Catalog No. C0005, TargetMol) reagent. 100ul of WT and ATP6V0A4V512L M1 cell suspension was inoculated in a 96-well plate and incubated in a 37°C cell culture incubator until the cell density reached 60%-70%. The supernatant was discarded, and 1 / 10 volume of CCK-8 reagent was added to the cell culture medium, mixed thoroughly to ensure color uniformity in the wells, and continued to culture in the cell culture incubator for 4 hours. The absorbance at 450nm was read with an ELISA reader at 0.5h, 1h, 2h, and 4h after adding CCK8, and the cell viability was calculated.

[0067] 5.6 Lysosomal pH determination

[0068] For the quantitative detection of lysosomal pH, cells were seeded in 96-well plates at an appropriate density. After the cells attached successfully, LysoSensor Yellow / Blue DND-160 was diluted to 2 μmol / L with preheated culture medium, and 100 μl was added to each well. After incubation in a 37°C incubator in the dark for 30 min, it was rinsed twice with preheated PBS. 10 μmol / L monensin was added to the buffers of 5 mmol / L NaCl, 115 mmol / L KCl, 1.2 mmol / L MgSO4 and 25 mmol / L MES at pH 3.5, 4, 4.5, 5, 5.5, and 6.0, respectively. 100 μl was added to each well. Different pH buffers were used to make standard curves. The role of monensin here is to balance the pH inside and outside the cells. 100 ul of buffer without monensin (not titrated with concentrated hydrochloric acid) was added to the experimental group; incubated at 37°C for 10 min. The fluorescence intensity was measured at wavelengths of 340 / 440nm and 380 / 535nm using an enzyme-labeled instrument, and the ratio R=F535 / F440 was calculated. A double logarithmic standard curve was drawn with R as the ordinate and pH as the abscissa. The fluorescence intensity ratio R of the experimental group was substituted into the curve, and the lysosomal pH value was calculated by comparing with the standard curve.

[0069] 5.7 Statistical analysis Statistical data are given in the form of mean ± standard error. SPSS26.0 statistical software was used to process the data. The t test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the groups. P < 0.05 was considered statistically significant. The experimental results were repeated more than three times.

[0070] result:

[0071] In order to find compounds that inhibit the ATP6V0A4 p.V512L mutant, this example uses structure-based virtual screening. The workflow is as follows: Figure 2 As shown, first, the protein structures of wild-type ATP6V0A4 and ATP6V0A4 p.V512L mutant were predicted using Alphafold ( Figure 3 ), the ATP6V0A4 p.V512L mutant protein structure was used as the docked receptor protein, and the docking pocket was selected based on the V512L mutation position and the cavityplus prediction tool, e.g. Figure 4 As shown in A, the volume of the pocket is 2674.38, containing 12 H-Bond donor centers, 9 H-Bond acceptor centers and 9 hydrophobic centers, and is evaluated as strong Druggability by cavityplus. Figure 4 Medium B and Figure 4 C in the middle is the positive and negative sides of the mutant docking with the ligand compound. According to the workflow, more than 2 million compounds from the chemical database ChemDiv, the natural product database COCONUT, the active compound database MCE and the drug database DrugBank were screened. According to the docking scores, the top 5,000 compounds were selected for clustering analysis based on structural similarity, and the cluster center compounds were selected to calculate the binding free energy of the complex. The top five potential inhibitors with the highest binding free energy were screened and subjected to in vitro binding analysis ( Figure 5 ). Figure 6 It is the binding free energy of the five screened ATP6V0A4p.V512L mutant inhibitors and the mutant.

[0072] Figure 7 These are the docking poses of the five screened ATP6V0A4 p.V512L mutant inhibitors and the mutant.

[0073] In this example, five compounds, including Relamorelin, Forsythiaside A, Protease-Activated Receptor-1, PAR-1 Agonist acetate (PAR-1AC) and chem div compound library screening compounds F359-0497 (F359) and F351-0364 (F351), were verified by in vitro wet experiments. It was found that Relamorelin, Forsythiaside A, PAR-1AC and F359 had no stable and significant inhibitory effect on the V-ATPase activity in ATP6V0A4 p.V512L mutant M1 cells ( Figure 8 The compound that inhibited the V-ATPase activity of ATP6V0A4 p.V512L mutant M1 cells most stably and effectively in a concentration-dependent manner was only the F351-0364 screening compound from the chemdiv compound library ( Fig. 9 ), the drug concentration was 20umol / L, and the best effect was achieved. At the same time, it caused the mutant cells to compensate for the concentration-dependent H + -K + -ATPase activity increased. The molecular formula of F351-0364 compound is C 25 H 26 FN3O2S, molecular weight 451.6g / mol, simple molecular structure, and extremely high drug-like properties. The compound F351-0364 has no significant inhibition of V-ATPase activity in wild-type M1 cells ( Fig.10 ), indicating that F351 targeted inhibition of V-ATPase activity in ATP6V0A4 p.V512L mutant M1 cells. Then, this example further evaluated the inhibitory effect of the compound at different time points such as 0 / 6 / 12 / 24 / 48 hours, and found that the drug had the strongest ability to inhibit V-ATPase activity after F351 intervened in mutant M1 cells for 24 hours ( Fig.11 ). When F351 was used at a drug concentration of 20umol / L and the mutant cells were intervened for 24 hours, the abundance of ATP6V0A4 protein expression was significantly reduced ( Fig.12 ). Next, this example measured the IC50 value of F351 drug by CCK8 experiment to be 140.9 μm, with low cytotoxicity ( Fig.13 ). Subsequently, the effect of F351 on lysosomal pH in mutant M1 cells was evaluated, and it was found that F351 increased lysosomal pH in a concentration-dependent manner ( Fig.14 In summary, F351-0364 may be a clinical candidate drug for the treatment of renal tubular alkalosis caused by ATP6V0A4 p.V512L mutation and can be used as a potential lead compound for further structural modification and optimization.

[0074] In the examples disclosed herein, the effect of the ATP6V0A4 p.V512L mutant inhibitor F351 is mainly to reduce the abundance of ATP6V0A4 protein expression in the renal collecting duct cell model carrying the ATP6V0A4 p.V512L mutant, inhibit the activity of the V-ATPase encoded by the mutant, weaken the hydrogen secretion ability of the V-ATPase caused by the mutant, and increase the lysosomal pH. 25 H 26 FN3O2S) compounds can be used as lead compounds for further structural modification and optimization or as active ingredients of drugs, and verified in mutant cells and animal models, providing a new approach for the future development of target drugs for renal tubular acid-base imbalance caused by ATP6V0A4 gene mutations.

[0075] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0076] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Use of compound F351 in the preparation of an inhibitor targeting the inhibition of ATP6V0A4 p.V512L mutant, characterized in that: The structural formula of the compound F351 is:

2. Use of the compound F351 according to claim 1 in the preparation of an inhibitor targeting the inhibition of ATP6V0A4 p.V512L mutant, characterized in that: The concentration of the compound F351 was 20 μmol / L.

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