CN114099680B

CN114099680B - Application of AKT inhibitor IV, gram-positive bacteria inhibitor and in-vitro inhibition method of gram-positive bacteria

Info

Publication number: CN114099680B
Application number: CN202111477959.0A
Authority: CN
Inventors: 陈国凯; 任志丽
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2021-12-06
Filing date: 2021-12-06
Publication date: 2023-05-02
Anticipated expiration: 2041-12-06
Also published as: CN114099680A

Abstract

The invention discloses an application of an AKT inhibitor IV, a gram-positive bacteria inhibitor and an in-vitro inhibition method of gram-positive bacteria, and belongs to the technical field of pharmaceutical application. The invention discloses application of an AKT inhibitor IV in inhibiting growth of gram-positive bacteria, and provides a new scheme for developing medicines for resisting gram-positive bacteria. Correspondingly, the invention also provides a gram positive bacteria inhibitor which comprises at least one of AKT inhibitor IV, pharmaceutically acceptable solvate, pharmaceutically acceptable salt, polymorph and tautomer thereof, and has wide application prospect. In addition, the invention also provides an in-vitro inhibition method of gram-positive bacteria, and provides technical support for related in-vitro researches.

Description

Application of AKT inhibitor IV, gram-positive bacteria inhibitor and in-vitro inhibition method of gram-positive bacteria

Technical Field

The invention relates to the technical field of pharmaceutical application, in particular to an application of an AKT inhibitor IV, a gram positive bacteria inhibitor and an in-vitro inhibition method of gram positive bacteria.

Background

Antibiotic resistance is a common phenomenon, and the first introduction of penicillin has begun since the 40 s of the 20 th century. The problem of resistance is usually solved by modifying the existing antibiotic structure, which however has limited resolution for cross-resistance. Thus, new antibiotic development has become important.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims at providing an application of an AKT inhibitor IV and provides a new scheme for medicine development of gram-positive bacteria resistance.

It is a second object of the present invention to provide a gram positive inhibitor.

The third object of the present invention is to provide an in vitro inhibition method of gram-positive bacteria.

The application can be realized as follows:

in a first aspect, the present application provides the use of AKT inhibitor IV for inhibiting the growth of gram positive bacteria.

In an alternative embodiment, AKT inhibitor IV is used to inhibit the growth of gram positive bacteria in the logarithmic growth phase.

In an alternative embodiment, AKT inhibitor IV is used to prepare a gram positive bacterial inhibitor.

In a second aspect, the present application provides a gram positive bacterial inhibitor comprising at least one of AKT inhibitor IV, a pharmaceutically acceptable solvate thereof, a pharmaceutically acceptable salt thereof, a polymorph thereof and a tautomer thereof.

In an alternative embodiment, the gram positive inhibitor further comprises a pharmaceutically acceptable carrier.

In an alternative embodiment, the carrier is selected from at least one of a solvent, an excipient, a diluent, and an adjuvant.

In an alternative embodiment, the gram positive inhibitor is a drug for treating inflammation.

In an alternative embodiment, the gram positive bacterial inhibitor is a staphylococcus aureus inhibitor.

In an alternative embodiment, the gram positive bacterial inhibitor is a bacillus subtilis inhibitor.

In alternative embodiments, the gram positive inhibitor is in a dosage form selected from any one of a tablet, pill, drop pill, capsule, granule, powder, suppository, powder, ointment, patch, injection, solution, suspension, spray, lotion, drop, liniment, and emulsion.

In a third aspect, the present application provides a method of inhibiting gram-positive bacteria in vitro comprising the steps of: AKT inhibitor IV was added to the medium containing gram positive bacteria.

In an alternative embodiment, the medium is LB medium.

In an alternative embodiment, the concentration of AKT inhibitor IV in the medium is 0.001-10. Mu. Mol/L.

In an alternative embodiment, the concentration of AKT inhibitor IV in the medium is 5-10. Mu. Mol/L.

The beneficial effects of the invention include:

the invention discloses application of an AKT inhibitor IV (also called as 'AKTi IV') in inhibiting growth of gram-positive bacteria, firstly discloses an anti-gram-positive bacteria effect of the AKT inhibitor IV, and provides a new scheme for anti-gram-positive bacteria drug development. Correspondingly, the invention also provides a gram positive bacteria inhibitor which comprises at least one of AKT inhibitor IV, pharmaceutically acceptable solvate, pharmaceutically acceptable salt, polymorph and tautomer thereof, and the gram positive bacteria inhibitor can be used as antibiotics for development and has wide application prospect. In addition, the invention also provides an in-vitro inhibition method of gram-positive bacteria, and provides technical support for related in-vitro researches.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the absorbance results in example 1 of the present application;

FIG. 2 is a graph showing the results of the zone of inhibition in example 2 of the present application;

FIG. 3 is a graph showing the results of bacterial survival in example 3 of the present application;

FIG. 4 is a graph showing the absorbance results in example 4 of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The application of AKT inhibitor IV (AKTi IV) and the in vitro inhibition method of gram-positive bacteria provided by the present application are specifically described below.

The AKT inhibitor IV referred to herein is a specific compound, and its structural formula can be referred to in the prior art, and will not be described in detail herein.

Through long-term research, the inventor creatively proposes that the AKT inhibitor IV can be used for inhibiting the growth of gram-positive bacteria, and the anti-gram-positive bacteria effect of the AKT inhibitor IV is revealed for the first time, thereby providing a new scheme for the development of anti-gram-positive bacteria medicines.

The growth and propagation cycle of bacteria includes: the AKT inhibitor IV can inhibit the growth of gram-positive bacteria in the stages, and has obvious inhibiting effect on the growth of gram-positive bacteria in logarithmic phase.

For the application, the AKT inhibitor IV can be particularly used for preparing a gram-positive bacteria inhibitor for clinical medicine, and can also be used for in vitro inhibition research of gram-positive bacteria, thereby providing conditions for relevant scientific research or experiment development.

Accordingly, the present application provides a gram positive bacterial inhibitor comprising at least one of AKT inhibitor IV, a pharmaceutically acceptable solvate thereof, a pharmaceutically acceptable salt thereof, a polymorph thereof and a tautomer thereof.

Wherein, "pharmaceutically acceptable solvate" refers to a stable solvate of AKT inhibitor IV with an organic solvent. The organic solvents used include alcohols, ketones or water, etc.

By "pharmaceutically acceptable salt" is meant one or more salts of AKT inhibitor IV which possess the desired pharmacological activity of the free compound and which do not have a biological or other adverse effect.

"polymorph" refers to crystals formed by the various arrangements of AKT inhibitor IV molecules. Polymorphs differ chemically by the physical properties (e.g., density, melting point, solubility or dissolution rate, etc.) of each particular polymorph.

"tautomer" refers to a functional group isomer that results from the rapid movement of one atom in the AKT inhibitor IV molecule at two positions.

Further, the gram-positive bacteria inhibitor may further comprise a pharmaceutically acceptable carrier.

In alternative embodiments, the carrier may be selected from at least one of solvents, excipients, diluents, and adjuvants, by way of example and not limitation.

The solvent may include alcohol, ketone, water, or the like. Excipients may include binders, fillers, disintegrants or lubricants in the tablet; wine, vinegar, medicinal juice and the like in the traditional Chinese medicine pill; a base portion in a semisolid formulation ointment, cream; preservative, antioxidant, correctant, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, colorant, etc. in liquid preparation. Diluents may include starch, dextrin, lactose, microcrystalline cellulose, mannitol or sorbitol and the like. Adjuvants may include aluminum salt adjuvants (such as aluminum hydroxide gel or alum), oily adjuvants (such as freund's adjuvant), microbial adjuvants, surfactant-type adjuvants, molecular adjuvants, and the like. It should be noted that the filler used in part in the pharmaceutical carrier may also be regarded as a diluent.

By way of reference, the gram positive bacteria inhibitors of the present application may be, by way of example and not limitation, drugs for the treatment of inflammation. The inflammation includes inflammation caused by respiratory tract infection, inflammation caused by skin infection, nephritis, mastitis, prostatitis, cholecystitis, enteritis, otitis media, dacryocystitis or meningitis, etc. Wherein the respiratory tract infection comprises rhinitis, pharyngitis, laryngitis, bronchitis, tracheitis or pneumonia.

In addition, the gram-positive bacteria inhibitor can also be a medicament for treating other diseases caused by gram-positive bacteria, and is not described in detail herein. It should be noted that the "drug" may refer to both "antibiotics" and other types of drugs, i.e., types of drugs that can inhibit the growth of gram-positive bacteria are within the scope of the present application.

In some preferred embodiments, the gram positive bacterial inhibitor of the present application is a staphylococcus aureus inhibitor, i.e., is used to inhibit the growth of staphylococcus aureus. In other preferred embodiments, the gram positive bacterial inhibitor of the present application is a bacillus subtilis inhibitor, i.e. is used to inhibit the growth of bacillus subtilis. Correspondingly, the gram positive bacteria inhibitor can also be used for simultaneously inhibiting the growth of staphylococcus aureus and bacillus subtilis.

By way of reference, the dosage form of the gram-positive bacteria inhibitor in the present application may be selected from any one of tablets, pills, drop pills, capsules, granules, powders, suppositories, powders, ointments, patches, injections, solutions, suspensions, sprays, lotions, drops, wipes and emulsions by way of example and not limitation.

In addition, the application also provides an in vitro inhibition method of gram-positive bacteria, which is mainly used for in vitro inhibition research of gram-positive bacteria and provides conditions for development of related scientific researches or experiments.

For reference, the in vitro inhibition method may comprise the steps of: AKT inhibitor IV was added to the medium containing gram positive bacteria.

Among them, the medium is preferably an LB medium such as an LB liquid medium or an LB solid medium. The formula of the LB liquid medium can comprise: 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride. The formulation of the LB solid medium may comprise: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl and 15g/L agar.

In an alternative embodiment, the concentration of AKT inhibitor IV in the above medium is 0.001-10. Mu. Mol/L, preferably 5-10. Mu. Mol/L.

In the above inhibition, AKT inhibitor IV may be diluted stepwise from 10. Mu. Mol/L (final concentration of not less than 0.001. Mu. Mol/L) and inhibited against gram-positive bacteria, respectively.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

Effect of AKTi IV on gram-Positive bacteria (LB liquid Medium)

The effect of different concentrations of AKTi IV on staphylococcus aureus (Staphylococcus aureus, SA) and bacillus subtilis (Bacillus subtilis, BS) was studied.

Gram positive staphylococcus aureus (Staphylococcus aureus, SA) and bacillus subtilis (Bacillus subtilis, BS) in the logarithmic growth phase are respectively inoculated into a plurality of LB liquid culture media, each strain corresponds to the plurality of LB liquid culture media, AKTi IV with different concentrations is respectively added into the plurality of LB liquid culture media of the same strain, and after 24 hours of culture, the 600nm absorbance is read.

This embodiment is exemplified by Staphylococcus aureus 21336 and Bacillus subtilis 25904. The formula of the LB liquid medium is as follows: 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride.

The specific method comprises the following steps: bacteria amplified overnight at 37℃were diluted 1:100 to 15mL LB liquid medium and cultured to OD at 37℃under 220rmp ₆₀₀ The absorbance was about 0.5, and the bacteria were diluted to OD ₆₀₀ The absorbance was about 0.04 and placed in 96-well plates, and each of the plates was incubated with a gradient of diluted AKTi IV at 37℃for 24 hours, and finally with a scanner at OD ₆₀₀ And detecting the growth condition of bacteria.

The results are shown in FIG. 1, which shows: AKTi IV has inhibitory effects on both Staphylococcus aureus 21336 and Bacillus subtilis 25904. Moreover, as the concentration of AKTi IV increases, the inhibition effect on Staphylococcus aureus 21336 is gradually improved; the inhibition effect on Bacillus subtilis 25904 gradually increased with increasing AKTi IV concentration, but after AKTi IV concentration > 5. Mu. Mol/L, the inhibition effect on Bacillus subtilis 25904 gradually tended to be stable.

Example 2

Effect of AKTi IV on gram-Positive bacteria (LB solid Medium)

Gram-positive staphylococcus aureus (Staphylococcus aureus, SA) and bacillus subtilis (Bacillus subtilis, BS) are respectively inoculated on 2 LB solid media (LB gel plates), each bacterium corresponds to 1 LB gel plate, AKTi IV with different concentrations is respectively added at different positions in each LB gel plate, and bacterial colony death is observed after 24 hours of culture. Meanwhile, DMSO was used as a blank.

This embodiment is also exemplified by Staphylococcus aureus 21336 and Bacillus subtilis 25904. The formula of the LB gel plate is as follows: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl and 15g/L agar. The formulation of the M9 medium is referred to the prior art and will not be described in detail herein.

The specific method comprises the following steps: bacteria amplified overnight at 37℃were 1:100 diluted to 15mL of M9 medium and incubated at 37℃to OD at 220rmp ₆₀₀ The absorbance was about 0.5, 100. Mu.L was placed on a 10cm LB plate (thickness of the solidified formulation), spread and air-dried, 1. Mu.L of AKTi IV of different concentrations diluted with DMSO in a gradient was spotted onto each specific position in the plate, air-dried, and then placed in an incubator at 37℃for 24 hours. Meanwhile, DMSO was used as a blank.

The results are shown in FIG. 2, which shows: AKTi IV has inhibitory effects on both Staphylococcus aureus 21336 and Bacillus subtilis 25904. Moreover, as the concentration of AKTi IV increases, the larger the corresponding Staphylococcus aureus 21336 inhibition zone generated on the LB gel plate, the inhibition effect of Staphylococcus aureus 21336 is gradually improved as the concentration of AKTi IV increases; the larger the corresponding Bacillus subtilis 25904 inhibition zone on the LB gel plate is along with the increase of the AKTi IV concentration, but the size of the inhibition zone is not very different after the AKTi IV concentration is more than 1 mu mol/L, which shows that the inhibition effect on Bacillus subtilis 25904 is gradually improved along with the increase of the AKTi IV concentration, but the inhibition effect on Bacillus subtilis 25904 is gradually stabilized after the AKTi IV concentration is more than 1 mu mol/L.

Example 3

Bacteria 1:100 amplified overnight at 37℃were diluted to 15mL M9 medium at 37℃and 220rmpCulturing to OD ₆₀₀ The absorbance was about 0.5, and suspension culture was performed at 37℃with the addition of 10. Mu. Mol/L AKTi IV, 1mL of the bacterial solution was taken out at a specific time point, centrifuged and washed with PBS, diluted 10-fold with PBS, and 5. Mu.L of each gradient was taken out on LB gel plates, and incubated in an incubator at 37℃for 24 hours to observe the bacterial survival. The formulation of each medium was the same as in example 1 and example 2. This example was used to demonstrate the antibacterial effect (quantifiable) of AKT inhibitor IV, with more accurate results than example 2.

The results are shown in FIG. 3, which shows: AKTi IV has inhibitory effects on both Staphylococcus aureus 21336 and Bacillus subtilis 25904. When the bacteria were serially diluted 10x, the number of surviving bacteria at the time of spotting could be calculated from the number of colonies at low concentration, and thus used for quantitative study of antibiotic efficacy. When 10. Mu. Mol/L AKTi IV was used to treat bacteria for 6 hours, the number of colonies generated on LB gel plates spotted showed that Bacillus subtilis 25904 had almost no bacteria to survive without dilution, demonstrating a strong bactericidal effect. At the same time, the number of colonies of Staphylococcus aureus 21336 differed from the control by 1 and 10-fold dilutions, indicating that 90% of the bacteria were likely killed during this time with AKTi IV.

Example 4

Effect of AKTi IV and ofloxacin on drug resistant gram-positive Staphylococcus aureus (MRSA)

Drug-resistant gram-positive staphylococcus aureus (MRSA) in the logarithmic growth phase is inoculated into a plurality of LB liquid culture media, each strain corresponds to the plurality of LB liquid culture media, AKTi IV with different concentrations is respectively added into the plurality of LB liquid culture media of the same strain, and after 24 hours of culture, the absorbance at 600nm is read. The formulation of the medium was the same as in example 1. Meanwhile, ofloxacin was used as a control.

The specific method comprises the following steps: the above bacteria amplified overnight at 37℃were 1:100 diluted to 15mL of LB medium and cultured to OD at 37℃under 220rmp ₆₀₀ The absorbance was about 0.5, and the bacteria were diluted to OD ₆₀₀ The absorbance was about 0.04 and placed in 96-well plates, and the AKTi IV was incubated at 37 c for 24 hours with each addition of a gradient dilution,finally, at OD by scanner ₆₀₀ And detecting the growth condition of bacteria. Meanwhile, ofloxacin was used as a control.

The results are shown in FIG. 4, which shows: AKTi IV has inhibitory effect on drug resistant gram positive staphylococcus aureus (MRSA). And, as the concentration of AKTi IV increases, its inhibitory effect on drug resistant gram positive staphylococcus aureus (MRSA) increases gradually; and AKTi IV with the concentration of 10 mu mol/L can achieve the inhibition effect of ofloxacin with the concentration of 277 mu mol/L on drug-resistant gram-positive staphylococcus aureus (MRSA).

In summary, the invention discloses application of the AKT inhibitor IV in inhibiting growth of gram-positive bacteria, and discloses an anti-gram-positive bacteria effect of the AKT inhibitor IV for the first time, thereby providing a new scheme for developing anti-gram-positive bacteria medicines. Correspondingly, the invention also provides a gram positive bacteria inhibitor which comprises at least one of AKT inhibitor IV, pharmaceutically acceptable solvate, pharmaceutically acceptable salt, polymorph and tautomer thereof, and the gram positive bacteria inhibitor can be used as antibiotics for development and has wide application prospect. In addition, the invention also provides an in-vitro inhibition method of gram-positive bacteria, and provides technical support for related in-vitro researches.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of AKT inhibitor IV for inhibiting the growth of gram positive bacteria in vitro.

2. Use according to claim 1, wherein the AKT inhibitor IV is for inhibiting the growth of gram-positive bacteria in the logarithmic growth phase in vitro.

3. The use according to claim 1 or 2, wherein AKT inhibitor IV is used for the preparation of gram positive bacteria inhibitors.

4. Use of AKT inhibitor IV for the manufacture of a medicament for inhibiting the growth of gram positive bacteria, characterized in that said medicament comprises at least one of AKT inhibitor IV, a pharmaceutically acceptable solvate thereof, a pharmaceutically acceptable salt thereof, a polymorph thereof and a tautomer thereof.

5. The use according to claim 4, wherein the medicament further comprises a pharmaceutically acceptable carrier.

6. The use according to claim 5, wherein the carrier is selected from at least one of solvents, excipients, diluents and adjuvants.

7. The use according to any one of claims 4 to 6, wherein the medicament is a medicament for the treatment of inflammation.

8. The use according to any one of claims 4 to 6, wherein the medicament is a medicament for inhibiting staphylococcus aureus.

9. The use according to any one of claims 4 to 6, wherein the medicament is a medicament for inhibiting bacillus subtilis.

10. The use according to any one of claims 4 to 6, wherein the pharmaceutical dosage form is selected from any one of tablets, pills, drop pills, capsules, granules, powders, suppositories, powders, ointments, patches, injections, solutions, suspensions, sprays, lotions, drops, wipes and emulsions.

11. An in vitro method of inhibiting gram positive bacteria comprising the steps of: AKT inhibitor IV was added to the medium containing gram positive bacteria.

12. The method of in vitro inhibition of gram positive bacteria according to claim 11, wherein the culture medium is LB culture medium.

13. The method according to claim 11 or 12, wherein the concentration of AKT inhibitor IV in the medium is 0.001-10 μmol/L.

14. The method according to claim 13, wherein the concentration of AKT inhibitor IV in the medium is 5-10 μmol/L.