A NAT inhibitor and its application

Abstract

The present invention discloses a NAT inhibitor and its application. The present invention conducts virtual screening on the NAT inhibitor derived from Vibrio vulnificus, and obtains the NAT inhibitor, specifically compound AK-968-11563024 or compound AG-205-36710025, which is the first inhibitor targeting (VIBVN) NAT. The present invention also proves through experiments that the two compounds AK-968-11563024 and AG-205-36710025 have good inhibitory effects on the transfer of the acetyl group of acetyl coenzyme A catalyzed by (VIBVN) NAT to 4-acetylsalicylic acid. Among them, the IC50 of compound AK-968-11563024 reaches 16.15 μM. Therefore, the NAT inhibitor has good application value and prospects in the field of drug development targeting (VIBVN) NAT.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a NAT inhibitor and its application. Background Art

[0002] Vibrio vulnificus is a Gram-negative aquatic bacterium commonly present in the family Vibrionaceae. It has been recognized as one of the most diverse and dangerous pathogens in the world. This highly virulent pathogen accounts for approximately 95% of seafood-related death cases in the United States and about 50% of immunocompromised and high-risk populations. Therefore, it is particularly important to find an accurate and rapid method for treating this bacterium in a clinical setting. Vibrio vulnificus is usually sensitive to a variety of antibiotics, such as tetracyclines, aminoglycosides, third-generation cephalosporins, chloramphenicol, and novel fluoroquinolones. However, recent studies have shown that Vibrio vulnificus has developed resistance to various antibiotics. Therefore, it is urgent to understand the resistance mechanism of Vibrio vulnificus. Aromatic amine N-acetyltransferase (NATs, E.C. 2.3.1.5) present in Vibrio vulnificus has become a key target. NATs are a family of phase II drug-metabolizing enzymes that have an endogenous role in lipotoxins. NATs catalyze the transfer of an acetyl group from acetyl coenzyme A to the terminal nitrogen of hydrazines and arylamine drugs and carcinogens. They play a very important role in protecting bacteria from aromatic and probiotic agents, and they are xenobiotic-metabolizing enzymes responsible for the detoxification of many drugs and carcinogens. Thus, the discovery of inhibitors of NATs is crucial for understanding the resistance mechanism of Vibrio vulnificus, and inhibitors of NATs may become new drugs for treating diseases caused by Vibrio vulnificus.

[0003] The present invention expressed, purified, and identified aromatic amine N-acetyltransferase ((VIBVN) NAT) from Vibrio vulnificus, predicted the three-dimensional protein structure of (VIBVN) NAT, and finally screened new inhibitors targeting (VIBVN) NAT with IC50 values of 16.15 μM and 128.4 μM through computer-aided virtual screening. Summary of the Invention

[0004] The purpose of the present invention is to provide a NAT inhibitor and its application. The NAT inhibitor has the effect of inhibiting the activity of aromatic amine N-acetyltransferase ((VIBVN) NAT) of Vibrio vulnificus, and has good application value and prospects in the field of drug development targeting NAT.

[0005] To achieve the above-mentioned invention purpose, the technical solution of the present invention is as follows:

[0006] The present invention provides a NAT inhibitor, which contains the compound AK-968-11563024 with the structural formula shown in (Ⅰ) and / or the compound AG-205-36710025 with the structural formula shown in (Ⅱ):

[0007]

[0008] Furthermore, the NAT inhibitor is compound AK-968-11563024 or compound AG-205-36710025.

[0009] Furthermore, the NAT inhibitor can inhibit the activity of aromatic aminonitrogen acetyltransferase derived from Vibrio vulnificus.

[0010] The present invention also provides the use of the NAT inhibitor described above in the preparation of a targeted drug for treating diseases caused by Vibrio infection.

[0011] Furthermore, the drug contains at least one of compound AK-968-11563024 and compound AG-205-36710025.

[0012] Furthermore, the target of the targeted drug is (VIBVN)NAT.

[0013] Furthermore, compound AK-968-11563024 exerts an anti-Vibrio effect by forming hydrophobic interactions with PHE124 and TRP230, π-cation interactions with HIS167 and TRP230, and Pi-Pi stacking interactions with TRP230 and PHE124.

[0014] Furthermore, compound AG-205-36710025 exerts an anti-Vibrio effect by forming Pi-Pi stacking interactions with PHE205 and water bridge interactions with TYR95 through water molecules.

[0015] Furthermore, the IC50 of compound AK-968-11563024 for the inhibitory activity against (VIBVN)NAT is 16.15 uM.

[0016] Furthermore, the IC50 of compound AG-205-36710025 for the inhibitory activity against (VIBVN)NAT is 128.4 uM.

[0017] Furthermore, the Vibrio is Vibrio vulnificus.

[0018] Compared with the prior art, the advantages and beneficial technical effects of the present invention are as follows:

[0019] The present invention performs virtual screening on NAT inhibitors from Vibrio vulnificus, conducts enzyme activity assays at the protein level, obtains compounds AK-968-11563024 and AG-205-36710025, and verifies the role of these two compounds in locally inhibiting the catalytic activity of (VIBVN) NAT, with IC50 values of 16.15 uM and 128.4 uM.

[0020] The inhibition of the catalytic activity of (VIBVN) NAT by compounds AK-968-11563024 and AG-205-36710025 is determined by their molecular structural characteristics. In the binding pocket of (VIBVN) NAT, the two compounds competitively bind to the same binding pocket with acetyl coenzyme A, and both compounds AK-968-11563024 and AG-205-36710025 play a role in inhibiting catalytic activity by forming important interactions with key amino acids in the pocket. Therefore, the two compounds have good application value and prospects in the field of drug development targeting (VIBVN) NAT. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the homology modeling structure of (VIBVN) NAT;

[0022] Figure 2 It is a structural formula diagram of compound AK-968-11563024;

[0023] Figure 3 It is a structural formula diagram of compound AG-205-36710025;

[0024] Figure 4 It is a curve of the inhibitory activity of compound AK-968-11563024 at different concentrations against (VIBVN) NAT;

[0025] Figure 5 It is a curve of the inhibitory activity of compound AG-205-36710025 at different concentrations against (VIBVN) NAT;

[0026] Figure 6 It is a schematic diagram of the structural interaction between compound AK-968-11563024 and (VIBVN) NAT;

[0027] Figure 7 It is a schematic diagram of the structural interaction between compound AG-205-36710025 and (VIBVN) NAT. DETAILED DESCRIPTION OF THE INVENTION

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the new embodiments; obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1: Obtaining of inhibitors

[0030] Inhibitors were obtained by computer-aided virtual screening. First, the amino acid sequence of (VIBVN)NAT was obtained from the UniProt database (A0A4V3BB88), and the obtained amino acid sequence was used for homology modeling by I-TASSER to obtain the three-dimensional structure of (VIBVN)NAT ( Figure 1 , which was displayed as a cartoon by the open-source software pymol). Then, a preliminary virtual screening was performed on the Specs database. Based on the structure of (VIBVN)NAT, pharmacophore features matching the binding pocket were constructed, and the top 10% of small molecules with matching pharmacophore features were screened out. Then, virtual screening by molecular docking using watvina was carried out, and the top 1% of small molecules with the highest docking scores were selected. Then, the binding free energy of all molecules was calculated using the MM / GBSA method, and 20 small molecules were selected. Finally, through empirical selection of small molecules, compounds AK-968-11563024 and AG-205-36710025 were obtained, and their structural formulas are respectively as Figure 2 and Figure 3 shown, and their molecular formulas are respectively: C 18 H 11 I2N9O5 and C 23 H 27 N3O7S2, and their molecular weights are respectively: 687.15 and 521.60.

[0031] Example 2

[0032] In the evaluation of the inhibitory effect on the catalytic activity of (VIBVN)NAT, first, the amino acid sequence of (VIBVN)NAT was obtained from the UniProt database (A0A4V3BB88), and the corresponding plasmid was synthesized and the (VIBVN)NAT protein was expressed and purified. Then, using 4-acetylsalicylic acid as the catalytic substrate, in the presence of acetyl coenzyme A, in the presence of different concentrations of compounds AK-968-11563024 and AG-205-36710025, after reacting for an appropriate time, the absorbance at 405 nm was measured to determine the catalytic activity.

[0033] 1. Test method:

[0034] After obtaining the amino acid sequence of (VIBVN)NAT from the UniProt database (A0A4V3BB88), the amino acid sequence was translated into a gene sequence, which was synthesized by Personalbio (Shanghai, China) and subcloned into the NotI and BamHI restriction sites of the pET-28a(+) expression vector and transformed into Escherichia coli DH5α cells. Then the recombinant plasmid was transformed to obtain the (VIBVN)NAT protein in Escherichia coli BL21(DE3) cells. A single colony of Escherichia coli BL21(DE3) was inoculated into LB medium containing kanamycin (50 μg / mL) and incubated on a rotary shaker at 37 °C until the optical density (OD600) at 600 nm reached approximately 0.8. IPTG was added to a final concentration of 200 μM to induce enzyme expression, and the bacterial culture was further grown at 16 °C for 18 h. The resulting cells were collected and sonicated in a buffer solution containing 25 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 20 mM imidazole, 0.2 mg / mL lysozyme, 1 mM MgCl2, 1 mM PMSF, and 0.05% Triton X-. Cell debris was removed by centrifugation at 12,000 rpm for 45 min at 4 °C. The supernatant was loaded onto a Ni Sepharose column and then extensively washed with the wash buffer 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 20 mM imidazole. The (VIBVN)NAT protein was eluted with a Tris-HCl buffer gradient containing 40 - 200 mM imidazole and desalted using a pre-packed column containing 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The final protein was stored at -80 °C without further modification. The purity of (VIBVN)NAT was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a 12% polyacrylamide gel. The proteins separated after SDS-PAGE were stained with Coomassie Brilliant Blue R-250, and their molecular weights were determined by comparing their mobilities with those of standard pre-stained markers of known molecular weights (ranging from 15 - 180 kDa).

[0035] Its catalytic activity was analyzed by the DTNB method, and AcCoA-dependent acetylation was measured using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB reagent). At room temperature, 5 μL of NAT enzyme (25 μg / mL, final concentration) was mixed with 10 μL of 4-aminosalicylic acid (4-AS) (500 μM, final concentration), and then compounds with different final concentrations were added respectively. 10 μL of AcCoA (500 μM, final concentration) was added to start the reaction, and the total reaction system was 100 μL. After 10 min, the reaction was stopped by adding 50 μL of cold DTNB (2.0 mg / mL, final concentration) in 6 M guanidine chloride (GdmCl). The assay was carried out in 25 mM Tris-HCl buffer (pH 7.5), and the rate was determined by the linear change in absorbance at 405 nm.

[0036] 2. Test evaluation:

[0037] To evaluate the effect of these inhibitors on the activity of NAT, the enzyme was expressed and purified by Ni affinity chromatography. The expression of NAT was analyzed by SDS-PAGE, and most protein contaminants were removed after one-step affinity chromatography eluted with a linear gradient of imidazole. The purified (VIBVN) NAT protein migrated as a single band on the SDS-PAGE gel. The molecular weight of the purified enzyme was determined by SDS-PAGE. Compared with the standard molecular weight marker, the purified (VIBVN) NAT protein produced a distinct single band with a molecular weight of 30 kDa, which was consistent with the predicted molecular weight of the (VIBVN) NAT protein and previous reports on NAT in other species of bacteria.

[0038] As Figure 4 and Figure 5 shown, by measuring the absorbance at 405 nm, the IC50 values of AK-968-11563024 and AG-205-36710025 were calculated to be 16.15 μM and 128.4 μM respectively, and they can act as novel NAT inhibitors.

[0039] As Figure 6 and Figure 7As shown (the protein is displayed as cartoon, the small molecule is displayed as sticks, and the important interactions are drawn through the open-source software pymol), both compound AK-968-11563024 and AG-205-36710025 play a role in inhibiting catalytic activity by forming important interactions with key amino acids in the pocket. AK-968-11563024 forms hydrophobic interactions with LYS166 and LEU165, hydrogen bond interactions with ASN219, ASN127, and GLY125, and Pi-Pi stacking interactions with TRP230; AG-205-36710025 forms hydrophobic interactions with LYS166, LEU165, TRP230, PHE38, PHE205, and LEU210, and hydrogen bond interactions with ASN127.

[0040] In summary, based on the method of computer virtual screening, the present invention performs virtual screening on the Specs database, screens out compounds with good pharmacophore matching and good docking ability, and tests the inhibition rate of their catalytic activity on (VIBVN)NAT at the protein level. Compounds AK-968-11563024 and AG-205-36710025 with IC50 values of 16.15 uM and 128.4 uM for inhibiting catalytic activity are obtained, demonstrating their good application value and prospects in the field of drug development targeting (VIBVN)NAT.

[0041] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A NAT inhibitor, characterized in that, The NAT inhibitor contains the compound AK-968-11563024 with the structural formula shown in (I) and / or the compound AG-205-36710025 with the structural formula shown in (II): 。 2. The NAT inhibitor according to claim 1, wherein The NAT inhibitor can inhibit the activity of aromatic amine N-acetyltransferase derived from Vibrio vulnificus.

3. Use of the NAT inhibitor according to claim 1 in the preparation of a targeted drug for treating diseases caused by Vibrio infection, characterized in that, The vibrio is Vibrio vulnificus.

4. The application according to claim 3, wherein The drug contains at least one of the compound AK-968-11563024 and the compound AG-205-36710025.

5. The application according to claim 4, characterized in that, The target of the targeted drug is the aromatic amine N-acetyltransferase of Vibrio vulnificus.

6. The application according to claim 5, wherein The IC50 of the compound AK-968-11563024 for the inhibitory activity of aromatic amine N-acetyltransferase of Vibrio vulnificus is 16.15 uM.

7. The application according to claim 5, characterized in that, The IC50 of the compound AG-205-36710025 for the inhibitory activity of aromatic amine N-acetyltransferase of Vibrio vulnificus is 128.4 uM.

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