A FtsZ protein inhibitor with antibacterial effect and its application

Abstract

The present invention discloses an FtsZ protein inhibitor with antibacterial effect and its application, belonging to the field of biotechnology. The FtsZ protein targeted inhibitor of the present invention includes compound BP210517 and compound BP210914; the structural formulas of the compound BP210517 and the compound BP210914 are shown in formula (1) and (2), respectively. The present invention also provides the application of the compound in the preparation of a drug for inhibiting FtsZ protein, and the application of the compound in enhancing the effective dose of antibiotics and enhancing the antibacterial activity of antibiotics. The disclosure of the BP210517 and BP210914 compounds of the present invention enriches the research field of targeted FtsZ antibacterial agents, can be used as lead compounds of potential antibacterial drugs, realize clinical applications, and have the value of further transformation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an FtsZ protein inhibitor with antibacterial effect and application thereof. Background Art

[0002] The problem of bacterial resistance has become extremely prominent and acute. Drug-resistant bacteria are rapidly emerging around the world. With the emergence of bacterial resistance, the demand for exploring and developing new antibacterial drugs is increasing. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA) has caused traditional antibiotics to lose their high antibacterial effect, seriously endangering human health. The development of antibacterial drugs with new mechanisms of action is imminent.

[0003] The cell division protein FtsZ in bacteria is a homologous protein of microtubule proteins in higher organisms and has become an important antibacterial target. FtsZ can form a dynamic Z ring in the center of the cell, and then recruit downstream proteins to form a fission complex to mediate cell division. Therefore, disrupting the assembly dynamics of FtsZ will affect the normal growth and proliferation of bacteria, which can disrupt the bacterial division process, leading to abnormal bacterial division, morphological changes, and inability to adapt to individual life activities or living environment, and then death, achieving the purpose of antibacterial.

[0004] Most of the existing antibiotics are derivatives of the same parent core structure. The resulting bacterial cross-resistance has led to the emergence of bacteria resistant to traditional antibiotics much faster than the development of new antibiotics. Therefore, an inhibitor with a new target or a completely new structure is the fundamental solution to the problem of bacterial resistance. Many studies on inhibitors targeting FtsZ as a drug target have been reported, but most of these compounds are modified from existing substances and have similar parent cores, and the degree of structural modification is limited. Therefore, although the research on targeting FtsZ has a long history, no new antibacterial drugs of the FtsZ inhibitor type have been approved for marketing so far. It is still necessary to screen more compounds, enrich the lead compound library targeting FtsZ, and realize clinical application. Summary of the invention

[0005] The purpose of the present invention is to provide an FtsZ protein inhibitor with antibacterial effect and its application to solve the problems existing in the above-mentioned prior art. The discovery of the BP210517 and BP210914 compounds of the present invention enriches the research field of targeted FtsZ antibacterial agents and can be used as lead compounds of potential antibacterial drugs, which are worth further modification.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A compound capable of inhibiting FtsZ protein, the compound comprising compound BP210517 and compound BP210914; the structural formula of the compound BP210517 is shown in formula (1); the structural formula of the compound BP210914 is shown in formula (2);

[0008]

[0009] Formula (1);

[0010]

[0011] Formula (2).

[0012] Technical solution 2: Use of the compound as an FtsZ protein inhibitor.

[0013] Technical solution three: A FtsZ protein inhibitor with antibacterial effect, wherein the FtsZ protein inhibitor includes the compound.

[0014] Furthermore, the antibacterial effect includes inhibiting the activity of Staphylococcus aureus and Bacillus subtilis.

[0015] Technical solution 4: Use of the compound in enhancing the effective dose of antibiotics.

[0016] Technical Solution 5: Application of the compound in enhancing the antibacterial activity of antibiotics.

[0017] Furthermore, the antibiotics include chloramphenicol, gentamicin and erythromycin.

[0018] Furthermore, the antibacterial activity includes inhibiting the activity of Staphylococcus aureus and Bacillus subtilis.

[0019] Technical Solution 6: Application of the compound in inhibiting the normal division of bacterial cells and inhibiting the in vitro aggregation of FtsZ protein.

[0020] Technical Solution 7: Use of the compound in inhibiting the GTPase activity of FtsZ protein.

[0021] The present invention discloses the following technical effects:

[0022] The molecular dynamics simulation results of the present invention show that: BP210517, BP210914 and PC190723 have similar binding stability, the binding site is the PC pocket, and both tend to form hydrogen bond interactions with LEU209 in the FtsZ protein; the BP compounds (BP210517, BP210914) have good antibacterial activity against Staphylococcus aureus and Bacillus subtilis; the BP compounds have a strong synergistic effect on chloramphenicol, gentamicin and erythromycin, can reduce the effective concentration of antibiotics and enhance their antibacterial efficacy; the BP compounds can significantly inhibit the normal division of bacterial cells, and will cause the volume of Staphylococcus aureus to increase and the radial length of Bacillus subtilis to increase; the BP compounds have lower cytotoxicity and good drugability; BP can inhibit the in vitro aggregation of FtsZ protein, and the inhibitory effect is positively correlated with the concentration; the BP compounds can inhibit the GTPase activity of FtsZ protein. The discovery of the compounds BP210517 and BP210914 of the present invention enriches the research field of antibacterial agents targeting FtsZ, can be used as lead compounds of potential antibacterial drugs, and have the value of further modification.

[0023] The present invention uses virtual screening technology to obtain potential antibacterial compounds BP210517 and BP210914, and conducts preliminary exploration of their antibacterial efficacy, synergistic efficacy and mechanism of action, develops a potential antibacterial compound targeting FtsZ, and enriches the research on FtsZ targeting compounds. Subsequent research can take these two compounds as the starting point, retain their structure of interaction with the FtsZ protein LEU209 amino acid, and structurally modify the remaining parts, fully considering the lipid-water partition coefficient, charge distribution and other factors of the compound, and develop and research new antibacterial agents with stronger antibacterial efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the structural formula of compounds BP210517 and BP210914;

[0025] Figure 2A is a schematic diagram of molecular docking; Poisson-Boltzmann distribution of the surface charge of the FtsZ molecule. In the surface charge distribution diagram of BP210517 (B) and BP210914 (C) binding to FtsZ, blue represents positive charge, red represents negative charge, the gray stripe model represents the FtsZ protein, and the smooth surface model represents the surface charge distribution of the ligand; blue represents positive charge, and red represents negative charge; D. Hydrophilicity and hydrophobicity distribution of the FtsZ molecule surface; purple represents hydrophilicity, and green represents hydrophobicity; In the surface hydrophilicity and hydrophobicity distribution diagram of the molecules of BP210517 (E) and BP210914 (F) binding to FtsZ, the gray stripe model represents the FtsZ protein, purple represents hydrophilicity, green represents hydrophobicity, and the smooth surface model represents the hydrophilicity and hydrophobicity distribution of the ligand; G and H show the detailed display of the position of compounds BP210517 and BP210914 in the pocket;

[0026] Figure 3 Molecular dynamics analysis of the complex of BP210517 and BP210914 with FtsZ. Curves showing the change of RMSD of backbone atoms of BP210517, BP210914 and PC190723 over time in molecular dynamics simulation. Curves showing the change of interaction energy of BP210517, BP210914 and PC190723 over time in molecular dynamics simulation.

[0027] Figure 4 Analysis of hydrogen bonds between BP210517 and BP210914 and FtsZ. The hydrogen bond length (A), bond angle (B), and bond angle distribution (C) formed by BP210517-FtsZ during molecular dynamics analysis. The hydrogen bond length (D), bond angle (E), and bond angle distribution (F) formed by BP210914-FtsZ during molecular dynamics analysis.

[0028] Figure 5 is the inhibitory activity of the compound against Staphylococcus aureus and Bacillus subtilis;

[0029] Figure 6Figure 2 is the growth curve of Staphylococcus aureus under the treatment of BP210517 and BP210914 at different concentrations; A. 24h growth curve of bacteria under the treatment of BP210517 at different concentrations; B. 24h growth curve of bacteria under the treatment of BP210914 at different concentrations; C. Inhibitory activity of BP210517 and BP210914 against Staphylococcus aureus at 500μΜ, 250μΜ, 125μΜ, 72.5μΜ, and 37.5μΜ; D. Inhibitory activity of BP210517 and BP210914 against Bacillus subtilis at 500μΜ, 250μΜ, 125μΜ, 72.5μΜ, and 37.5μΜ, errorbar indicates mean±SD, and each treatment group was subjected to one-way analysis of variance with the corresponding Control group, **p<0.01, *p<0.05;

[0030] Figure 7 The synergistic effect of BP210517, BP210914 and erythromycin on Staphylococcus aureus was studied; the bacterial growth status after the combined use of BP210517 (A), BP210914 (B) and Erythromycin; the bacterial growth inhibition rate of the combined use of BP210517 (A), BP210914 (B) and Erythromycin;

[0031] Figure 8 The synergistic effect of BP210517, BP210914 and erythromycin on Bacillus subtilis was studied; (A) The bacterial growth status after BP210914 (B) and Erythromycin were used in combination; The growth inhibition rate of bacteria after BP210517 (A), BP210914 (B) and Erythromycin were used in combination;

[0032] Fig. 9 The synergistic effect of BP210517, BP210914 and chloramphenicol on Staphylococcus aureus was studied; the bacterial growth status after the combined use of BP210517 (A), BP210914 (B) and Chloramphenicol; the bacterial growth inhibition rate of the combined use of BP210517 (A), BP210914 (B) and Chloramphenicol;

[0033] Fig.10 The synergistic effect of BP210517 and BP210914 with gentamicin on Staphylococcus aureus was studied; the bacterial growth status after the combined use of BP210517 (A), BP210914 (B) and Gentamicin; the bacterial growth inhibition rate of the combined use of BP210517 (A), BP210914 (B) and Gentamicin;

[0034] Fig.11 The synergistic effect of BP210517, BP210914 and gentamicin on Bacillus subtilis was studied; the bacterial growth status after the combination of BP210517 (A), BP210914 (B) and Gentamicin; the bacterial growth inhibition rate of the combination of BP210517 (A), BP210914 (B) and Gentamicin;

[0035] Fig.12 Bacterial morphology of Staphylococcus aureus (A, B, C) and Bacillus subtilis (D, E, F) after treatment by Control, BP210517, and BP210914, respectively; the ratio is 8000 times and 1000 times;

[0036] Fig.13 For BP210517 and BP210914 cytotoxicity assay;

[0037] Fig.14 PCR diagram of FtsZ gene amplification; Lanes 1 and 2: FtsZ: (Annealing Temperature: 56°C); Lanes 3 and 4: FtsZ: (Annealing Temperature: 58°C; M: DL 2000 DNA Marker;

[0038] Fig.15 The comparison results of gene sequence and protein sequence; A is the comparison result of gene sequence, the upper row is the standard sequence, and the lower row is the sequencing result; B is the comparison between the sequencing result translated into protein and the standard protein sequence;

[0039] Fig.16 This is the map of the pET-28a-FtsZ recombinant eukaryotic expression plasmid;

[0040] Fig.17 The electrophoresis diagram for exploring the imidazole concentration for elution of FtsZ protein; the lane annotations are M: Marker, 1: Remaining protein after sufficient column hanging, 2: 10mM imidazole, 3: 20mM imidazole, 4: 30mM imidazole, 5: 40mM imidazole, 6: 50mM imidazole, 7: 100mM imidazole, 8: 150mM imidazole, 9: 200mM imidazole eluted respectively;

[0041] Fig.18 This is the electrophoresis diagram of FtsZ protein extraction and purification, and the lane annotations are M: Marker; 1: 20mM imidazole five times column volume washing; 2: 30mM imidazole five times column volume elution; 3: 35mM imidazole three times column volume elution; 4-8: 1.5mL 40mM imidazole elution; 9 and 10: 200mM imidazole elution;

[0042] Fig.19 is the protein concentration standard curve;

[0043] Fig. 20 The effects of BP210517 and BP210914 on the in vitro polymerization of FtsZ; A is the effects of BP210517 (yellow) and BP210914 (green) on the in vitro polymerization of FtsZ protein; B is the effects of BP210517 at various concentrations on the in vitro polymerization of FtsZ protein; C is the effects of BP210914 at various concentrations on the in vitro polymerization of FtsZ protein;

[0044] Fig.21 Figure 2 is the effect of BP210517 and BP210914 on the GTPase activity of FtsZ; A is the inhibition rate of BP210517 and BP210914 on the protease activity of FtsZ; B is the inhibition rate of BP210517 on the enzyme activity of FtsZ protein at each concentration; C is the inhibition rate of BP210914 on the enzyme activity of FtsZ protein at each concentration, and error bar represents mean±SD. DETAILED DESCRIPTION

[0045] Example 1

[0046] 1 Experimental methods

[0047] 1.1 Molecular docking

[0048] The crystal structure of Staphylococcus aureus FtsZ protein (PDB#4DXD) was downloaded from the Protein Data Bank (PDB) (Tan, Therien, Lu, et al., 2012). The Chemscript script was written in Python. Considering the screening time and amino acid polarity, the 700,000 compounds in the Zinc Database were used as library V, and the processed FtsZ protein crystal structure was used as the receptor for molecular docking. Before the molecular docking began, the protein was hydrogenated, and the Amber99 force field was used to calibrate the charge of the protein, and then docking was performed. The processed protein was used as the receptor structure, and the 5 constructed databases were docked separately. The docking mode was set to induced fit, allowing the ligand to dock with different fit conformations. The molecular docking results were evaluated using the London dG and GBVI / WSA△G methods (Sun, Wang, Pei, et al., 2022). GBVI / WSA△G calculates the binding free energy of interaction between ligand and receptor using formula (1), where △Eele, △Esol, △EvdW are electrostatic, solvent and van der Waals interactions, respectively, △SA is the solvent exposure area, K is the average entropy change, and γ and δ are parameters calculated based on the molecular force field.

[0049] △G bind ≈γ[23(△Eele+△Esol)+△EvdW+δ△SA]+K(1)

[0050] After the docking is completed, the ligand with the highest score is selected, and its PDB structure bound to the protein is saved. The interaction between the protein and the ligand is analyzed using the GROMACS (2020.03) program installed on the Ubuntu (18.06) Linux operating system. The pdb2gmx command in GROMACS is used to generate the GROMACS format structure file and topology file of the FtsZ and ligand complex; the force field is selected as the AMBER99 SB all-atom force field; a dodecahedral calculation unit is created, and the protein complex is placed in the center of the unit box, with a distance of 1.0 nm between the protein complex and the box boundary; the TIP 3P intuitive water molecule model is selected; the unit box is filled with water molecules, and Na + and Cl - The system was made electroneutral. The energy minimization used the steepest descent algorithm with the end threshold set to 1000 kJ / mol / nm. After energy minimization, the standard Berendsen thermostat was adjusted using equation (2) to obtain the correct kinetic energy distribution.

[0051]

[0052] Where K is kinetic energy, N f is the number of degrees of freedom, dW is the variation value of the Wiener random process, τ T is close to the time constant τ of the temperature coupling and is given by equation (3):

[0053] τ=2C V τ T / N df k(3)

[0054] After the system temperature is balanced, the Parrinello-Rahman pressure coupling algorithm is used to balance the system and obtain the final isothermal-isobaric system. The volume (V) and pressure (P) of the D unit box are calculated by formula (4):

[0055]

[0056] Where P is pressure and W is pressure coupling strength, which can be calculated using formula (5):

[0057]

[0058] Where τP is the pressure time constant, β is the isothermal compressibility coefficient (4.5e -5 / bar), L is the largest box-shaped matrix unit. After the system pressure is balanced, MD analysis is performed with 250,000,000 time steps, generating a data size of about 160Gb. In each comprehensive operation, the short-range van der Waals force and short-range electrostatic force are derived from the Lennard-Jones (6) and Coulombic equations (7). The process adopts the Verlet truncation scheme and the buffer concentration is 5×10 -3 kJ / mol / ps.

[0059]

[0060]

[0061] where F ij is the force exerted by atom j on atom i, r is the position vector, r is the vector length, and q is the elementary charge, which is equal to 1.602176565×10 -19 C, f is the electrical conversion factor, its value is equal to 1 / 4πε0 or 138.935458kJ mol-1nme -2 Long-range electrostatic interactions are calculated using the smoothed particle-mesh Ewald summation method. Charges are assigned to the mesh using cardinal B-spline interpolation, followed by a 3D fast Fourier transform. The electrostatic force is given by the position vector r in Fourier space. αi The interaction force at (α=1, 2, 3) is inversely deduced to be the differential of the reciprocal energy equation (8).

[0062]

[0063] Where m is the inverse lattice vector, Q is the vector array, and θrec is the potential between atoms. Calculation of the root mean square deviation (RMSD) value of the heavy atom position change in the molecule: The above calculation system was used to analyze using the AMBER99SB force field. A dodecahedral box was defined, and the FtsZ complex with a small molecule ligand was placed in the center of the box, then filled with water and Na + and Cl - Ion neutralization. After the system energy is minimized, the temperature is stabilized by NVT equilibrium, and the pressure is balanced under the NPT ensemble. The root mean square deviation of the heavy atom position in the ligand skeleton is calculated by formula (9) to evaluate the binding stability of the ligand to FtsZ:

[0064]

[0065] where r i is the position of atom i at time t, and M is the sum of the atomic masses (m).

[0066] The interaction energy is the sum of the Lennard-Jones energy (10) and the Columbic energy (11). The calculation formulas for these two potential energies are as follows:

[0067]

[0068]

[0069] where r is the position vector length and q is the base charge equal to 1.602176565×10 -19 C, f is the electrical conversion factor, (1 / 4πε0 or 138.935458 kJ mol-1nm e -2 ).

[0070] 1.2 Construction of prokaryotic expression vector of Staphylococcus aureus FtsZ protein

[0071] First extract the whole genome from Staphylococcus aureus, and then amplify the FtsZ gene fragment by designing specific primers. The genome extraction operation procedure refers to the bacterial genomic DNA extraction kit (Tian Gen DP302). PCR is performed on the extracted Staphylococcus aureus whole genome, and by adding synthetic FtsZ specific primers with NdeI and XhoI, and adding protective bases next to the restriction sites, the FtsZ gene fragment carrying the above two restriction sites is obtained. The SaFtsZ gene fragment is shown in SEQ ID NO.1.

[0072]

[0073] The sequences of primer fragments designed according to the requirements are shown in Table 1 and synthesized by Qingke Biotechnology. The PCR system is shown in Table 2.

[0074] Table 1 Primer sequences

[0075]

[0076] Table 2 PCR reaction system

[0077]

[0078] After the PCR reaction, add 10x loading buffer, add 1μL loading buffer for every 10μL sample, dissolve 0.3g agarose in 30mL 1x TAE, and then heat in a microwave for 1min. Add 1.5μL nucleic acid dye to make a 3% agarose gel. Use the DL2000 marker as a reference, and run the electrophoresis at 100V for 15min to separate the target fragment.

[0079] (3) Gel recovery and purification of FtsZ gene fragments. Please refer to the instructions of the Bio-Tech Gel Recovery Kit for the operation.

[0080] (4) Purification: The DNA fragments recovered from the gel were purified using the EasyPure PCR Purification Kit.

[0081] (5) The concentration of the FtsZ gene fragment recovered by the gel recovery kit was measured by UV spectrophotometer, and the fragment was added to the ligation buffer at a ratio of vector: fragment = 1:7 (molar ratio) and incubated at 16° C. for 9 h to obtain the pMD19-T-FtsZ recombinant prokaryotic expression plasmid. The ligation system is shown in Table 3.

[0082] Table 3 pMD19-T-Fts vector T4 ligase ligation system

[0083] Reagents Sampling volume (μL) Carrier 0.5 Vector 5.0 T4buffer 2.0 T4-DNALigase (400U / μL) 1.0 <![CDATA[ddH2O]]> 1.5 total 10.0

[0084] (6) Transformation: Take the competent DH5α E. coli and the ligation system product from the -80°C ultra-low temperature refrigerator and place them in an ice box. Place them for 10 min until the competent DH5α thaws. Then add the entire ligation system to the DH5α and place them on ice for 30 min. Then place the mixed system in a 42°C metal bath, heat shock for 45 s, place them on ice for 1 min, add 750 μL of antibiotic-free LB medium, shake at 37°C, 180 rpm for 1 h, and take 100 μL of the bacterial solution and apply it to the Amp using a coating rod. + The cells were plated on LB solid medium with resistance and cultured at 37°C overnight.

[0085] (7) Bacterial liquid PCR and sequence alignment: Take out the plate, pick up several single colonies with distinct morphology and clear distribution with a small gun tip, blow and beat several times in tubes containing 1 mL Amp + resistance LB medium, and culture overnight at 37°C and 180 rpm in a shaking incubator. Observe the turbidity of the bacterial liquid in the tube, and select the turbid tube for bacterial liquid PCR. The specific PCR reaction system is shown in Table 4. The obtained PCR product was sent to Qingke Biotechnology Company for sequencing. The obtained results were compared with DNAman software, and the samples with correct sequence alignment were selected to extract plasmids for the construction of pET-28a-FtsZ vector.

[0086] Table 4 Bacterial liquid PCR reaction system

[0087] Reagents Sampling volume (μL) Bacterial liquid 1.0 TaqSuperMix 10.0 Primer-F 0.4 Primer-R 0.4 <![CDATA[ddH2O]]> 8.2 total 20.0

[0088] (8) Double digestion of plasmid and T-vector plasmid: pET-28a(+) plasmid and pMD19-T-FtsZ plasmid were digested with NdeI and XhoI restriction endonucleases, respectively. The digestion system and conditions of pMD19-T vector and pET-28a(+) digestion system are shown in Table 5. After digestion, 3% agarose gel electrophoresis was used for detection and recovery. For specific operations, refer to the instructions of the gel recovery kit. The recovered product was subjected to DNA purification, ligation, transformation, bacterial liquid PCR, and plasmid extraction. The constructed PET-28a(+)-FtsZ plasmid was double digested to verify that the introduced fragment was the target fragment. Since pET-28a(+) has its own His tag sequence, there is no need to add the His sequence artificially, and a protein with a His tag can be obtained by later expression. Then, the extracted plasmid was introduced into Escherichia coli BL21 using the same steps, and glycerol was added to the bacterial liquid at a ratio of 1:1 (V / V) and stored in a refrigerator.

[0089] Table 5 Enzyme digestion system of 50 μL pET-28a-FtsZ vector and pMD19-T-FtsZ vector

[0090] Reagents Sampling volume (μL) Carrier 15.0 N i 0.5 XOt 0.5 NEbuffer 5.0 <![CDATA[ddH2O]]> 29.0 total 50.0

[0091] 1.3 Prokaryotic expression, extraction and purification of FtsZ protein

[0092] (1) Cell disruption: Take out the transformed expression competent cells BL21 and add Kana + The resistant liquid LB medium and bacterial solution were added to a 10 mL centrifuge tube and cultured overnight at 180 rpm at 37 °C to activate the strain. Then, the overnight cultured strain was added to 250 mL kana +In a conical flask of LB medium with resistance, shake for 4 hours, add IPTG and then culture for another 4 hours. Centrifuge the cultured bacterial solution, pour off the supernatant, retain the precipitate, add PBS reagent to the precipitate, collect all the precipitates obtained into a centrifuge tube, and fill it up to 25mL with PBS (during the addition of PBS, use a pipette to blow the precipitate until it is completely dissolved in the solution). Resuspend the bacterial solution and perform ultrasonic disruption (set time 30min, disruption time 5s, stop time 10s), centrifuge at 10000rpm at 4℃, collect the supernatant, and inject it through a 0.22μm aqueous filter membrane.

[0093] (2) Protein purification and concentration: The histidine (His) residue carries an imidazole group, which can react with Ni 2+ 、Co 2+Transition metal ions such as chelate ligands can be fixed on the chromatographic medium with His tags, so the protein with His tag can be selectively combined on the medium when the chromatographic medium with metal ions is assembled, and other impurity proteins can not be combined or can only be weakly combined. The His tag protein combined on the medium can be competitively eluted by increasing the imidazole concentration in the buffer, so as to obtain a His tag protein of higher purity, so determining a suitable elution concentration has a great impact on the efficiency and protein purity of the eluted protein. In the present invention, in order to first explore the optimal concentration of protein elution, PBS solutions with 20mM, 30mM, 40mM, 60mM, 100mM, 150mM, and 200mM imidazole concentrations are prepared for elution and standby. (After determining the optimal concentration, subsequent protein purification is based on the optimal elution step, and a protein with higher purity can be obtained). The operation steps are as follows: drain the liquid in the Ni affinity chromatography column, add 10mL ddH2O to pass through the column twice, wash the ethanol, add 10mL PBS solution to pass through the column twice, balance the Ni column, add the bacterial lysate to the Ni column and hang it at 4℃ for half an hour, so that the protein and the filler are fully combined. After half an hour, take out the Ni column, collect the first eluate, add 10mL of 20mM, 30mM, 40mM, 60mM, 100mM, 150mM, 200mM imidazole PBS solution to the column in turn, collect the eluate respectively, then filter and wash twice with PBS, wash twice with ddH2O, and seal the Ni column with 30% ethanol. The protein solution purified under the optimal elution conditions needs to be dialyzed to remove imidazole and the solution is concentrated to increase the protein concentration. The specific operation is to add the collected protein solution to a dialysis bag that has been boiled in boiling water for 10 minutes, clamp the two ends, and place the dialysis bag in a replacement solution 100 times the volume of the protein solution. Dialyze at 4°C for 4 hours, repeat three times, and then transfer the solution in the dialysis bag to a 15mL 30kD purification column. Centrifuge at 4000rpm for 1 hour, discard the column solution, and divide the solution on the upper layer of the purification column into 200μL / tubes. Keep a small amount of solution to determine the concentration of the protein solution for subsequent experimental calculations. The protein concentration is determined by the BCA method, the principle of which is that proteins will convert Cu under alkaline conditions. 2+ reduction, and BCA will react with Cu 2+ Chelation forms a purple-blue complex, and the absorbance of the solution at 562nm is proportional to the amount of protein. The mass concentration of the protein can be calculated by measuring A562. The sample addition operation is shown in Table 6.

[0094] Table 6 BCA method protein concentration measurement sample addition table

[0095]

[0096] (4) Coomassie Brilliant Blue Test

[0097] Add 6xprotein loading buffer to the collected eluate according to the concentration ratio, set the metal bath to 99°C, place in the metal bath for 5 minutes to denature the protein, add 50 μL of sample per well to the protein electrophoresis gel well, and the electrophoresis condition is a constant voltage of 90 V. The protein electrophoresis gel is Bis-Tris gel (10%), the formula is shown in Table 7. The electrophoresis buffer is diluted to 1x using 5x low molecular weight buffer, and the electrophoresis buffer formula is shown in Table 8.

[0098] Table 7 10% Bis-Tris gel formula

[0099] Reagents Separation gel Concentrated Glue 40% Acrylamide 2.7mL 0.75mL 1.0M Bis-Tris (PH 6.5) 3.0mL 2.0mL <![CDATA[ddH2O]]> 3.0mL 3.0mL 10% ammonium persulfate 100.0μL 40.0μL TEMED 4.0μL 4.00μL

[0100] Table 85× Low Molecular Weight Electrophoresis Buffer

[0101] Reagents Sampling volume 250mM MES 53.313g 250mM Tris 30.285g 5mM EDTA 1.861g 5% SDS 5.000g <![CDATA[ddH2O]]> Add to 1L

[0102] 1.4 Antimicrobial activity assay

[0103] The present invention uses the LB liquid culture medium dilution method to determine the antibacterial activity of the compound. After sterilizing an appropriate amount of LB liquid culture medium in a high-pressure steam sterilizer, place it in a clean bench for ultraviolet sterilization for standby use, add LB culture medium to a sterile 96-well plate, dissolve the target compound solution (0.01 mol / L) and the control group, add them to the first column of the 96-well plate, dilute them twice to the 10th column, and finally inoculate the prepared bacterial solution. After culturing in a 37°C constant temperature incubator for 24 hours, use an enzyme marker to measure the OD of each well. 600 The data of each well is processed by formula (11) to obtain the antibacterial activity of each compound. In order to observe the different effects of the compounds on the bacterial growth stage, the present invention also uses the kinetic program of the microplate reader to culture the bacteria treated with the compound on the plate and continuously measure its OD at 1 hour intervals. 600 The growth curve was drawn based on the values. Each experiment was repeated three times. The specific determination method is shown in Table 9.

[0104]

[0105] Table 9 Antibacterial activity determination experimental design table

[0106]

[0107]

[0108] Combination of 1.5BP compounds with antibiotics.

[0109] The combined drug experiment is divided into two parts: the combined drug use against Staphylococcus aureus and the combined drug use against Bacillus subtilis. Each part can be divided into three small parts: combined use with erythromycin, chloramphenicol, and gentamicin respectively.

[0110] The specific steps are as follows: Determine the antibacterial efficacy of each antibiotic against Staphylococcus aureus and Bacillus subtilis, and design a combined drug test based on its MIC value (if the MIC value is too large, the antibacterial rate is determined with 500 μM as the upper limit, and the MIC is assumed to be greater than 500 μM and the MIC value of subsequent experiments is 500 μM). Plate and administer the drug according to the measured MIC value (the antibiotic MIC multiple is outside the brackets, and the BP compound concentration is in μM, see Table 10).

[0111] Table 10: Drug administration on 96-well plates according to the measured MIC values

[0112]

[0113] Dilute the overnight culture to 10 6 CFU / mL, added to each well. After plating and adding bacteria, the 96-well plate was sealed with plastic film and placed in a 37°C incubator for 24 hours. The absorbance of each well at 600nm was measured using an ELISA reader to calculate the inhibitory effect of the combined drug use on bacteria in each well.

[0114] 1.6 Observation of bacterial cell morphology

[0115] After the bacterial solution was treated with compounds of different concentrations, the diluted Bacillus subtilis bacterial solution was spread on a glass slide with an inoculation loop, methanol was added to fix the bacteria, and the bacteria were dried. The bacterial division state was observed under an optical microscope. Staphylococcus aureus is smaller in size and its deformation is not as obvious as that of Bacillus subtilis. In order to more clearly observe the morphological changes of Staphylococcus aureus, a scanning electron microscope was used for observation.

[0116] 1.7 MTT cytotoxicity assay

[0117] Weigh 0.5g of MTT in a dark place, dissolve it in 100mL of PBS solution, and filter it with a 0.22μm filter membrane to sterilize it to obtain a 5mg / mL MTT solution. After aliquoting, wrap it in tin foil and store it at -20℃. Collect HEK293 cells in the logarithmic growth phase, adjust the concentration of the cell suspension, add 100μL to each well of a 96-well cell culture plate, plate it, and incubate it under 5% CO2 and 37℃ conditions. When the cell monolayer covers the bottom of the well, that is, after the cells are attached, add the corresponding concentration of drugs and continue incubation. Add 10μL of the pre-prepared MTT solution to each well and continue culturing for 4h. Terminate the culture, aspirate the culture medium, add 100μL of DMSO, and shake it on a shaker at low speed for 10s to fully dissolve the crystals, and use an enzyme reader to detect the OD490 Data and analysis.

[0118] 1.8 GTPase activity assay

[0119] The assay was performed using the Phosphate Assay Kit (Abnova, Taiwan, China), which can measure the concentration of inorganic phosphorus released during the hydrolysis of GTP. The Malachite Green Phosphate Assay Kit can sensitively detect the amount of free phosphate ions in the solution, and FtsZ, as a GTPase, can catalyze the decomposition of GTP into GDP and produce This simple assay is based on the complex that malachite green molybdate forms with free orthophosphate under acidic conditions. The formation of the green phosphomolybdic acid complex measured at 620-640 nm is directly related to the concentration of free organic phosphorus, and applications of the assay include quantification of phosphorylation and phosphate release of protein phosphatase substrates (D'Angelo, Crutchfield and Vandiviere, 2001, O'Toole, Lau, Shepherd, et al., 2007). Before determining the protein concentration, a phosphate standard curve should be prepared as a reference. Take 8 clean test tubes and number them D2 and D3 (dilution tubes) and S1 to S6 (standard tubes). Divide 990 microliters of assay buffer (or water) into tube D2, 500 μL into tubes D3 and S1 to S6. Transfer 10 μL of bulk standard material (tube D1, 10 mM) to tube D2 and mix thoroughly; the phosphate concentration will be 100 μM. Transfer 500 μL of tube D2 to tube D3 and mix thoroughly; the phosphate concentration will be 50 μM. Next, take 500 μL from tube S1 and place it into tube S2; mix thoroughly. Repeat this process for tubes S3-S5, leaving S6 as a blank. The resulting tube standard sample phosphate concentration is shown in Table 11. A standard curve is established based on the measured data to provide a reference standard for subsequent enzyme activity determination.

[0120] Table 11 Phosphate standard concentration

[0121] PhosphateStandard PhosphateμΜConcentration Nmoles / 50μL S1 25 1.25 S2 12.5 0.63 S3 6.25 0.31 S4 3.13 0.16 S5 1.56 0.08 S6 0 0

[0122] 1.9 In vitro polymerization experiment of FtsZ protein

[0123] The degree of FtsZ polymerization was characterized by measuring the absorption signal intensity of the FtsZ protein solution at 360 nm (Kaul, Parhi, Zhang, et al., 2012).

[0124] 2 Results and analysis

[0125] 2.1 Molecular docking and compound screening

[0126] Molecular docking screening was performed on 700,000 compounds in the Zinc Database, and two compounds, BP210517 and BP210914, were hit. Their respective structures are shown in Figure 1 , molecular docking schematic diagram see Figure 2 The details of the hydrogen bond interactions formed by the two compounds with FtsZ are shown in Table 12. It can be seen from the docking details that both compounds tend to form hydrogen bonds with Leu209 of FtsZ. In order to better analyze their binding stability with FtsZ, the present invention also analyzes the bond length and bond angle of the hydrogen bonds formed on the basis of calculating RMSD and interaction energy.

[0127] Table 12 Hydrogen bond interactions between compounds and proteins

[0128]

[0129] 2.2 Analysis of the interaction between compounds and FtsZ monomer

[0130] The RMSD values ​​of compounds BP210517 and BP210914 were calculated by comparing with PC190723, and the calculation time was extended to 50ns. The two compounds have similar stability to PC190723, and their RMSD values ​​are basically stable between 0.05-0.1, and the binding is relatively stable, but BP210914 has a large fluctuation around 26ns. Observing its stretching animation, it is considered to be caused by pocket space steric hindrance. The comparison of the ligand position at 26ns and the ligand position at 50ns is shown in the appendix. By comparing the position information of BP210914 at the two time nodes, it can be seen that its main position has not changed much, but there is a small bond angle rotation. By calculating the interaction energy of the complex system ( Figure 3 ) It can be seen that the interaction energy of BP210914 is around -200 kJ / mol in the first 6 seconds, but its interaction energy increases slightly at 6s and then stabilizes at -150 kJ / mol, indicating that its binding stability weakens after 6s and then tends to stabilize; BP210517 and PC190723 have similar interaction energy curves, and their interaction energies are both stable at around -250 kJ / mol. In order to further study the docking stability of the compound with FtsZ, the present invention calculates the bond length, bond angle, and bond angle distribution of the hydrogen bonds formed during the molecular dynamics simulation. The calculation results are shown in Figure 4 It was shown that the hydrogen bonds formed by BP210517 and BP210914 with FtsZ were stable during the dynamics simulation. The calculation results show that the hydrogen bond angle is between 120° and 160°. Therefore, most of the hydrogen bond angles during the 50ns simulation are distributed at 150° and above, indicating that the hydrogen bonds formed between BP compounds and proteins are stable and reliable.

[0131] 2.3 In vitro pharmacology validation

[0132] 2.3.1 Determination of antibacterial activity of compounds

[0133] The antibacterial activity of the screened compounds was tested by broth microdilution method ( Figure 5 ), it can be seen that the BP series compounds have a strong growth inhibition on Staphylococcus aureus under the treatment of 500 μM compound concentration, and the antibacterial activity is positively correlated with the inhibitor concentration. Similarly, it also shows the same inhibitory activity on Bacillus subtilis; its growth curve is shown in Figure 6 As shown, the bacterial OD values ​​of the experimental groups added with the two compounds were significantly lower than those of the control group, the overall growth of bacteria was inhibited, and the logarithmic growth phase was greatly delayed.

[0134] 2.3.2 Synergistic effects of compounds and antibiotics

[0135] BP210517 and BP210914 were used in combination with Staphylococcus aureus and Bacillus subtilis using the chessboard method. Figure 7As shown, the inhibition rate of erythromycin against Staphylococcus aureus at a concentration of 500 μM was about 75%, but after the addition of gradient concentrations of BP210517, the bacterial growth was greatly inhibited and its MIC was reduced to 62.5 μM. At the same time, the MIC of BP210517 was also reduced to 15.6 μM. It can be seen that after the operation of reducing the concentrations of BP210517 and erythromycin by two-fold dilution, the growth of S. aureus in each well was also greatly inhibited, and this result showed a good synergistic relationship between the two; similarly, the inhibition rate of BP210517 compound on S. aureus at 500 μM was about 50%, but after combined use with different concentrations of erythromycin, its antibacterial activity was greatly improved, the MIC value of erythromycin was reduced to 125 μM, and the MIC value of BP210914 was reduced to 62.5 μM. It can be seen from the chessboard that the synergistic effect of BP compound and erythromycin is positively correlated with their respective concentrations, and the synergistic effect of BP210517 on erythromycin at lower concentrations is stronger than that of BP210914. The synergistic effect study on Bacillus subtilis showed that the combined use of BP compound and erythromycin has a stronger synergistic ability against Bacillus subtilis. The MIC value of erythromycin against Bacillus subtilis is 2μM. After combined use, the MIC value of BP210517 decreased to 15.6μM, and the MIC value of erythromycin decreased to 0.5μM; the MIC of BP210914 decreased to 15.6μM, and the MIC of erythromycin decreased to 0.06μM. The synergistic chessboard is shown in Figure 2. Figure 8 .

[0136] The synergistic effect of chloramphenicol and gentamicin on inhibiting Staphylococcus aureus and Bacillus subtilis was also studied. The results showed that for Staphylococcus aureus, the combined use of chloramphenicol and BP210517 reduced its MIC values ​​to 500μM and 125μM, respectively, and the combined use of chloramphenicol and BP210914 reduced its MIC values ​​to 125μM and 250μM, respectively; the combined use of gentamicin and BP210517 reduced its MIC values ​​to 2μM and 15.6μM, respectively, and the combined use of gentamicin and BP210914 reduced its MIC values ​​to The MIC values ​​of chloramphenicol and BP210517 were reduced to 2μM and 15.6μM respectively; for Bacillus subtilis, the combined use of chloramphenicol and BP210517 reduced its MIC values ​​to 500μM and 500μM respectively, and the combined use of chloramphenicol and BP210914 reduced its MIC values ​​to 15.6μM and 500μM respectively; the combined use of gentamicin and BP210517 reduced its MIC values ​​to 1μM and 15.6μM respectively, and the combined use of gentamicin and BP210914 reduced its MIC values ​​to 2μM and 62.5μM respectively. Among them, the growth degree and combined antibacterial rate of Staphylococcus aureus after gradient concentration treatment of gentamicin, chloramphenicol and BP compounds are shown in Fig. 9 and Fig.10 The growth degree and combined inhibition rate of Bacillus subtilis after gradient concentration treatment are shown in Fig.11 The results of the synergistic effect study are shown in Tables 14 and 15. The results show that, whether for Staphylococcus aureus or Bacillus subtilis, BP compounds can play a strong synergistic role with erythromycin and gentamicin, greatly reducing their effective dosage and improving their antibacterial effect.

[0137] Table 14 Synergistic effect of BP210517 and antibiotics on Staphylococcus aureus and Bacillus subtilis

[0138]

[0139] Table 15 Synergistic effect of BP210914 and antibiotics on Staphylococcus aureus and Bacillus subtilis

[0140]

[0141] 2.3.3 Effects of compounds on bacterial morphology

[0142] The morphological observation of bacteria in the compound treatment group was as follows: Fig.12 As shown, Figures A, B, and C show the effects of the two compounds on Staphylococcus aureus. Compared with the Control treatment group in Group A, the Staphylococcus aureus in the compound treatment group grew significantly larger and showed abnormal division. Fig.12 D, E, and F in the figure are the effects of the two compounds on Bacillus subtilis, which are similar to the effects on Staphylococcus aureus. Compared with the Control group, the length of Bacillus subtilis cells is extended by dozens of times. The observation results of bacterial morphology can prove that the compounds do cause abnormal division of bacteria, and their antibacterial target is FtsZ protein.

[0143] 2.3.4MTT cytotoxicity assay

[0144] The compound toxicity test was conducted on human embryonic kidney cells HEK293. According to the analysis of cell growth, the relative cell survival rate was obtained through data processing. It was found that except for the difference in natural cell growth, there was no correlation between cell survival rate and compound concentration, which proved that the compounds at all concentrations were non-toxic to cells ( Fig.13 ).

[0145] 2.3.5 Construction of pET-28a-FtsZ recombinant plasmid

[0146] First, the whole genome of Staphylococcus aureus was extracted, and then the FtsZ gene fragment was obtained by PCR amplification. The amplification results are as follows: Fig.14As shown, the gel results show that the band position and size are correct. According to the experimental conditions, the optimal renaturation temperature is 56°C. The FtsZ gene fragment extracted in the previous step was first T-linked, and after double enzyme digestion, gel recovery, ligation, and transformation, the plasmid was successfully constructed and sent to the company for sequencing. The results were compared with the NCBI database of Staphylococcus aureus FtsZ. The comparison results are shown in Fig.15 After confirmation, the pMD19-T-FtsZ recombinant plasmid was double-digested to obtain the FtsZ fragment with clear sequence, and the pET-28a-FtsZ vector was reconstructed. The map of the pET-28a-FtsZ recombinant plasmid is shown in Fig.16 The plasmid was subsequently transferred into DH5α E. coli competent cells to allow the plasmid to be amplified in large quantities, and then the plasmid was extracted and transferred into BL21 E. coli competent cells for subsequent expression of FtsZ protein.

[0147] 2.3.6 Expression, purification and identification of FtsZ protein

[0148] After constructing the E. coli expression vector of Staphylococcus aureus FtsZ protein (SaFtsZ), SaFtsZ was efficiently expressed by induction with IPTG, and the protein purification and elution conditions were explored at 4°C, such as Fig.17 As shown in the figure, a large amount of SaFtsZ began to elute at 40mM imidazole, so a large amount of 20mM and 30mM imidazole was used to remove impurities, and then high concentration imidazole was used to ensure that as much SaFtsZ as possible was eluted and collected. The test results of subsequent protein extraction and purification are shown in Fig.18 , collect all the protein eluates after lane 5, replace them with the buffer required for subsequent experiments, remove imidazole by ultrafiltration, and achieve the purpose of concentrating the protein. The concentrated protein solution is measured by using the BCA protein concentration test kit. First, according to the kit operation manual, a standard curve between the standard and the absorbance is established. Since the upper limit of the kit detection is low, the protein concentrate is diluted 20 times and reacted and measured with the standard test group in the same batch to avoid objective errors. The standard curve is as follows Fig.19 As shown, by substituting the sample absorbance value into the formula, the diluted protein concentration was calculated to be 337.90 μg / mL, so the concentration of the concentrate was 6758 μg / mL. According to the molecular weight of the FtsZ protein, the molar concentration of the concentrated FtsZ protein was converted to 160.9 μM.

[0149] 2.3.7 Effects of compounds on FtsZ protein polymerization

[0150] By determining A 360 The absorbance strength of the two compounds was used to indirectly explore the effect of the two compounds on the in vitro polymerization of FtsZ. First, the effect of the two compounds on FtsZ polymerization at 500 μM was measured. The results are as follows Fig. 20 As shown in the figure, compared with the control group added with DMSO, BP210517 and BP210914 can inhibit the in vitro polymerization of FtsZ to a certain extent. The present invention studied the polymerization inhibition effect of the two compounds on FtsZ protein at various concentrations. The results are shown in the figure. Fig. 20 As shown in Figures B and C, as the concentration of the compound gradually decreases, the absorbance intensity of the solution at 360 nm gradually increases, which means that the in vitro inhibitory effect on the protein is weakened. The same situation occurs in the two compound treatment groups with different concentrations, indicating that the inhibitory effect of the compound on the protein is proportional to its concentration, which also proves that the compound can indeed act on the FtsZ protein, and the drug target of its antibacterial effect is the FtsZ protein.

[0151] 2.3.8 Effects of compounds on FtsZ GTPase

[0152] The present invention further explores the effect of the compound on the enzyme activity of FtsZ protein. The test results are as follows: Fig.21 As shown, compared with the Control group, both compounds can inhibit the enzyme activity of the protein, among which BP210517 has an inhibition rate of about 66% on the GTPase activity of FtsZ protein, while BP210914 has an inhibition rate of about 40% on the enzyme activity. The experimental results show that the mechanism of action of the compound may be to affect the GTPase activity of the protein and then affect the normal kinetic function of the protein. In order to clarify the inhibitory effect of the compound on the enzyme activity of FtsZ protein, the present invention further explores the inhibitory effect of the compound on the enzyme activity of FtsZ protein at different concentrations, as shown in FIG. Fig.21 As shown in the figure, its inhibitory effect is positively correlated with the concentration range, and its inhibitory effect weakens as the concentration decreases.

[0153] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a compound in the preparation of an FtsZ protein inhibitor, characterized in that: The compounds are compound BP210517 and compound BP210914; the structural formula of compound BP210517 is shown in formula (1); the structural formula of compound BP210914 is shown in formula (2); Formula (1); Formula (2).

2. Use of a compound in the preparation of an FtsZ protein inhibitor with antibacterial effect, characterized in that: The FtsZ protein inhibitor components are compound BP210517 and compound BP210914; the structural formula of the compound BP210517 is shown in formula (1); the structural formula of the compound BP210914 is shown in formula (2); Formula (1); Formula (2); The antibacterial effect is the activity of inhibiting Staphylococcus aureus and Bacillus subtilis.

3. Use of a compound in the preparation of a drug for enhancing the effective dose of an antibiotic, characterized in that: The compounds are compound BP210517 and compound BP210914; the structural formula of compound BP210517 is shown in formula (1); the structural formula of compound BP210914 is shown in formula (2); Formula (1); Formula (2); the antibiotics are chloramphenicol, gentamicin and erythromycin.

4. Use of a compound in the preparation of a drug for enhancing the antibacterial activity of an antibiotic, characterized in that: The compounds are compound BP210517 and compound BP210914; the structural formula of compound BP210517 is shown in formula (1); the structural formula of compound BP210914 is shown in formula (2); Formula (1); Formula (2); the antibiotics are chloramphenicol, gentamicin and erythromycin; the antibacterial activity is the inhibition of the activity of Staphylococcus aureus and Bacillus subtilis.

5. Use of a compound in the preparation of an inhibitor for inhibiting normal division of bacterial cells and inhibiting in vitro aggregation of FtsZ protein, characterized in that: The compounds are compound BP210517 and compound BP210914; the structural formula of compound BP210517 is shown in formula (1); the structural formula of compound BP210914 is shown in formula (2); Formula (1); Formula (2).

6. Use of a compound in the preparation of an inhibitor for inhibiting the GTPase activity of FtsZ protein, characterized in that: The compounds are compound BP210517 and compound BP210914; the structural formula of compound BP210517 is shown in formula (1); the structural formula of compound BP210914 is shown in formula (2); Formula (1); Formula (2).

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