CN114099680A

CN114099680A - Application of AKT inhibitor IV, gram-positive bacterium inhibitor and gram-positive bacterium in-vitro inhibition method

Info

Publication number: CN114099680A
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: 2022-03-01
Anticipated expiration: 2041-12-06
Also published as: CN114099680B

Abstract

The invention discloses an AKT inhibitor IV application, a gram-positive bacterium inhibitor and a gram-positive bacterium in-vitro inhibition method, and belongs to the technical field of pharmaceutical application. The invention discloses application of AKT inhibitor IV in inhibiting the growth of gram-positive bacteria, and provides a new scheme for developing anti-gram-positive bacteria medicaments. Accordingly, the invention also provides a gram-positive bacteria inhibitor, which comprises at least one of AKT inhibitor IV, pharmaceutically acceptable solvates thereof, pharmaceutically acceptable salts thereof, polymorphs thereof and tautomers thereof, and has a wide application prospect. In addition, the invention also provides an in-vitro inhibition method of gram-positive bacteria, and provides technical support for relevant in-vitro research.

Description

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

Technical Field

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

Background

Antibiotic resistance is a common phenomenon that has begun since the first introduction of penicillin in the 40's of the 20 th century. The problem of resistance is often solved by modifying the existing antibiotic structure, however this has limited resolution of cross-resistance. Thus, the development of new antibiotics has become very important.

In view of this, the invention is particularly proposed.

Disclosure of Invention

One of the purposes of the invention is to provide an application of AKT inhibitor IV, and provide a new scheme for developing anti-gram-positive bacteria drugs.

The invention also aims to provide a gram-positive bacteria inhibitor.

The invention also aims 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 an 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 in the preparation of a gram-positive bacteria inhibitor.

In a second aspect, the present application provides a gram-positive bacteria 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 bacteria 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 bacteria inhibitor is a medicament for treating inflammation.

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

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

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

In a third aspect, the present application provides a method for in vitro inhibition of gram-positive bacteria, 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 alternative embodiments, the concentration of AKT inhibitor IV in the culture medium is 0.001-10 μmol/L.

In alternative embodiments, the concentration of AKT inhibitor IV in the culture medium is 5-10 μmol/L.

The beneficial effects of the invention include:

the invention discloses application of AKT inhibitor IV (also called as AKTi IV) in inhibiting the growth of gram-positive bacteria, firstly discloses the gram-positive bacteria resisting effect of the AKT inhibitor IV, and provides a new scheme for developing gram-positive bacteria resisting medicaments. 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 can be developed as an antibiotic, so that the gram-positive bacteria inhibitor has a wide application prospect. In addition, the invention also provides an in-vitro inhibition method of gram-positive bacteria, and provides technical support for relevant in-vitro research.

Drawings

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

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

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

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

FIG. 4 is a graph showing the results of absorbance values 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 clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The application of AKT inhibitor iv (akti iv), the gram-positive bacteria inhibitor and the method for inhibiting gram-positive bacteria in vitro provided by the present application will be specifically described below.

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

The inventor creatively provides that the AKT inhibitor IV can be used for inhibiting the growth of gram-positive bacteria through long-term research, reveals the gram-positive bacteria resisting effect of the AKT inhibitor IV for the first time, and provides a new scheme for the development of gram-positive bacteria resisting medicaments.

The growth and reproduction cycle of the bacteria comprises: in a slow phase, a logarithmic phase, a stationary phase and a decline phase, the AKT inhibitor IV can inhibit the growth of gram-positive bacteria in each phase, and particularly has obvious inhibition effect on the growth of the gram-positive bacteria in the logarithmic phase.

In terms of the application, the AKT inhibitor IV can be specifically used for preparing a gram-positive bacterium inhibitor for clinical medicine, and can also be used for in-vitro inhibition research of gram-positive bacteria, so as to provide conditions for related scientific research or experiment development.

Accordingly, the present application provides a gram-positive bacteria 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, the pharmaceutically acceptable solvate refers to a stable solvate formed by AKT inhibitor IV and an organic solvent. The organic solvent used includes, by reference, alcohol, ketone, water or the like.

By "pharmaceutically acceptable salt" is meant one or more salts of AKT inhibitor IV that possess the desired pharmacological activity of the free compound and that do not adversely affect either biologically or otherwise.

"polymorph" refers to a crystal formed by the arrangement of AKT inhibitor IV molecules in different ways. There is no difference in chemical properties between polymorphs, in that the physical properties (e.g., density, melting point, solubility or dissolution rate, etc.) of each particular polymorph differ.

"tautomer" refers to a functional isomer resulting from the rapid movement of an atom in the AKT inhibitor IV molecule at two positions.

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

In an alternative embodiment, the above-mentioned carrier may be selected, by way of example but not limitation, from at least one of a solvent, an excipient, a diluent, and an adjuvant.

The solvent may include alcohol, ketone, water, etc. Excipients may include binders, fillers, disintegrants, or lubricants in the tablet; wine, vinegar, medicinal juice, etc. in the Chinese medicinal pill; base portion in semisolid formulations ointments, creams; preservative, antioxidant, correctant, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, colorant, etc. in the 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-based adjuvants, or molecular adjuvants, among others. It should be noted that some of the fillers used in the pharmaceutical carrier may also be considered diluents.

By way of reference, the gram-positive bacteria inhibitor of the present application may be exemplified by, but not limited to, a medicament for treating 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. The respiratory tract infection also includes 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 redundant description is not repeated herein. It should be noted that the above-mentioned "drug" may be referred to as "antibiotic" or other type of drug, and that the type of drug that can inhibit the growth of gram-positive bacteria is within the scope of the present application.

In some preferred embodiments, the gram positive bacteria 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 bacteria 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.

For reference, the dosage form of the gram-positive bacteria inhibitor in the present application may be selected from any one of tablets, pills, dripping pills, capsules, granules, powders, suppositories, powders, ointments, patches, injections, solutions, suspensions, sprays, lotions, drops, liniments and emulsions, by way of example and without 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 the gram-positive bacteria and provides conditions for development of related scientific researches or experiments.

For reference, the above-mentioned 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 LB liquid culture medium formula comprises: 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride. The formulation of the LB solid medium may include: 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-mentioned medium is 0.001-10. mu. mol/L, preferably 5-10. mu. mol/L.

In the inhibition process, AKT inhibitor IV is diluted from 10 mu mol/L in stages (the final concentration is not less than 0.001 mu mol/L), and gram-positive bacteria are inhibited respectively.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

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

The effect of AKTi IV at different concentrations on Staphylococcus Aureus (SA) and Bacillus Subtilis (BS) was investigated.

Respectively inoculating gram-positive Staphylococcus Aureus (SA) and Bacillus Subtilis (BS) in logarithmic growth phase into a plurality of LB liquid culture media, respectively adding AKTi IV with different concentrations into a plurality of LB liquid culture media of the same strain, and after culturing for 24h, reading the light absorption value of 600 nm.

This example is exemplified by Staphylococcus aureus 21336 and Bacillus subtilis 25904. The LB liquid culture medium has the formula: 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 1:100To 15mL of LB liquid medium, cultured at 37 ℃ and 220rmp to OD₆₀₀Absorbance was about 0.5 and the bacteria were diluted to OD₆₀₀Absorbance was about 0.04 and placed in 96 well plates, and gradient dilutions of AKTi IV were added separately and incubated at 37 ℃ for 24 hours, and finally scanned at OD₆₀₀And detecting the growth condition of the bacteria.

The results are shown in FIG. 1, which shows: AKTi IV has inhibitory effects on Staphylococcus aureus 21336 and Bacillus subtilis 25904. And, with the increase of AKTi IV concentration, the inhibition effect on Staphylococcus aureus 21336 is gradually improved; the inhibition effect on Bacillus subtilis 25904 gradually increases with the increase of the concentration of AKTi IV, but gradually stabilizes after the concentration of AKTi IV is more than 5 mu mol/L.

Example 2

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

Respectively inoculating gram-positive Staphylococcus Aureus (SA) and Bacillus Subtilis (BS) on 2 LB solid culture media (LB gel plates), wherein each bacterium corresponds to 1 LB gel plate, respectively adding AKTi IV with different concentrations at different positions in each LB gel plate, and observing the death condition of bacterial colonies after culturing for 24 h. Meanwhile, DMSO was used as a blank control.

This example also exemplifies 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 M9 medium is described in the prior art and will not be described in detail herein.

The specific method comprises the following steps: bacteria 1:100, which were amplified overnight at 37 ℃, were diluted to 15mL of M9 medium and cultured to OD at 37 ℃ under 220rmp₆₀₀The light absorption value is about 0.5, 100 μ L of the gel is placed on a LB gel plate of 10cm (thickness of the formula after solidification), the gel is evenly spread and dried, 1 μ L of the gel is diluted by DMSO gradientThe released AKTi IV with different concentrations are respectively spotted on each specific position in the gel tray, and the gel tray is put into an incubator at 37 ℃ for 24 hours after being dried. Meanwhile, DMSO was used as a blank control.

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

Example 3

Bacteria 1:100, which were amplified overnight at 37 ℃, were diluted to 15mL M9 medium and cultured to OD 220rmp at 37 ℃₆₀₀The absorbance was about 0.5, 10. mu. mol/L AKTi IV was added for suspension culture at 37 ℃, 1mL of the suspension was taken out at a specific time point, centrifuged and washed with PBS, diluted with PBS in 10-fold gradients, 5. mu.L of the suspension was taken out at each gradient, placed on an LB gel tray, and cultured in a 37 ℃ incubator for 24 hours, and the survival of the bacteria was observed. The formulation of each medium was the same as in examples 1 and 2. This example is intended 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 Staphylococcus aureus 21336 and Bacillus subtilis 25904. When the bacteria are diluted by 10x, the number of surviving bacteria at the time of spotting can be calculated from the number of colonies at low concentration, and thus used for quantitative studies on antibiotic efficacy. When 10. mu. mol/L AKTi IV was used to treat the bacteria for 6 hours, the number of colonies formed on LB gel plates spotted showed that almost no bacteria survived in Bacillus subtilis 25904 without dilution, showing strong bactericidal action. At the same time, the difference between the number of colonies of Staphylococcus aureus 21336 and the control was 1 dilution by 10, indicating that 90% of the bacteria may be killed by AKTi IV during this time.

Example 4

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

And (2) inoculating drug-resistant gram-positive staphylococcus aureus (MRSA) in a logarithmic growth phase into a plurality of LB liquid culture mediums, wherein each bacterium corresponds to the plurality of LB liquid culture mediums respectively, adding AKTi IV with different concentrations into the plurality of LB liquid culture mediums of the same bacterium respectively, culturing for 24h, and reading a light absorption value of 600 nm. 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 1:100, which were amplified overnight at 37 ℃, were diluted to 15mL of LB medium and cultured at 37 ℃ under 220rmp to OD₆₀₀Absorbance was about 0.5 and the bacteria were diluted to OD₆₀₀Absorbance was about 0.04 and placed in 96 well plates, and gradient dilutions of AKTi IV were added separately and incubated at 37 ℃ for 24 hours, and finally scanned at OD₆₀₀And detecting the growth condition of the 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, with increasing concentration of AKTi IV, its inhibitory effect on drug-resistant gram-positive Staphylococcus aureus (MRSA) is gradually improved; and AKTi IV with concentration of 10 mu mol/L can achieve the inhibiting effect of 277 mu mol/L ofloxacin on drug-resistant gram-positive staphylococcus aureus (MRSA).

In conclusion, the invention discloses the application of the AKT inhibitor IV in inhibiting the growth of gram-positive bacteria, discloses the gram-positive bacteria resisting effect of the AKT inhibitor IV for the first time, and provides a new scheme for developing a gram-positive bacteria resisting medicament. 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 can be developed as an antibiotic, so that the gram-positive bacteria inhibitor has a wide application prospect. In addition, the invention also provides an in-vitro inhibition method of gram-positive bacteria, and provides technical support for relevant in-vitro research.

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

Claims

1. The use of an AKT inhibitor IV for inhibiting the growth of gram-positive bacteria;

preferably, the AKT inhibitor IV is used to inhibit the growth of gram positive bacteria in the logarithmic growth phase;

preferably, the AKT inhibitor IV is used for the preparation of a gram-positive bacteria inhibitor.

2. A gram-positive bacteria 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.

3. A gram-positive bacteria inhibitor according to claim 2, further comprising a pharmaceutically acceptable carrier;

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

4. A gram-positive bacteria inhibitor according to claim 2 or 3, wherein the gram-positive bacteria inhibitor is a medicament for the treatment of inflammation.

5. A gram-positive bacteria inhibitor according to claim 2 or 3, wherein the gram-positive bacteria inhibitor is a staphylococcus aureus inhibitor.

6. A gram-positive bacteria inhibitor according to claim 2 or 3, wherein the gram-positive bacteria inhibitor is a bacillus subtilis inhibitor.

7. The gram-positive bacteria inhibitor according to claim 2 or 3, wherein the gram-positive bacteria inhibitor is in a dosage form selected from any one of tablets, pills, dripping pills, capsules, granules, powders, suppositories, powders, ointments, patches, injections, solutions, suspensions, sprays, lotions, drops, liniments and emulsions.

8. An in vitro inhibition method of gram-positive bacteria, comprising the steps of: adding AKT inhibitor IV to a culture medium containing gram positive bacteria;

preferably, the medium is LB medium.

9. The method of claim 8, wherein the concentration of AKT inhibitor IV in the medium is 0.001-10 μmol/L.

10. The method of claim 9, wherein the concentration of AKT inhibitor IV in the culture medium is 5-10 μmol/L.