Polyphosphate kinase 1 inhibitors

Abstract

The present application provides polyphosphate kinase 1 inhibitors. The present application provides pharmaceutical compositions comprising the compounds or a collection of compounds, and the use of the compounds or a collection of compounds in the manufacture of a medicament for treating mycobacterial infections. The present application further provides pharmaceutical compositions comprising the compounds or a collection of compounds and their use in the manufacture of a medicament for inhibiting mycobacterial PPK1 activity, inhibiting mycobacterial biofilm formation, reducing polyphosphate levels in mycobacterial biofilms, killing mycobacterial persister cells, and preventing or treating mycobacterial infections.

Description

Technical Field

[0001] This application belongs to the field of medicinal chemistry. Specifically, this application provides compounds with polyphosphate kinase 1 (PPK1) inhibitory activity. Background Technology

[0002] Inorganic polyphosphates (polyPs) are linear polymers composed of tens to hundreds of phosphate residues linked by high-energy phosphate bonds, and are widely found in all organisms from bacteria to mammals. In bacteria, polyphosphate synthesis is mainly catalyzed by polyphosphate kinase 1 (PPK1). PPK1 (EC 2.7.4.1) catalyzes the reversible transfer of the terminal phosphate group of ATP to synthesize long-chain polyphosphates. PPK1 is highly conserved in bacteria and is a key enzyme in bacterial polyphosphate metabolism.

[0003] Studies have shown that PPK1 plays a crucial role in various physiological processes in bacteria. Polyphosphates, as catalytic products of PPK1, participate in regulating numerous bacterial cellular processes, including energy metabolism, stress adaptation, drug tolerance, and microbial pathogenicity. In mycobacteria, PPK1 (encoded by the PPK1 gene in Mycobacterium tuberculosis) is a key enzyme in polyphosphate synthesis. PPK1 in Mycobacterium tuberculosis (MTB) has been confirmed to be essential for bacterial growth in host tissues. Studies have shown that the PPK1-1 gene knockout mutant exhibits significantly impaired survival in macrophages, and the absence of PPK1 affects glucose utilization and lipid biosynthesis in Mycobacterium tuberculosis. Further research indicates that PPK1 is also involved in the formation, morphology, and ultrastructure of mycobacterial biofilms.

[0004] Because PPK1 plays a crucial role in the pathogenicity and drug resistance of bacteria (especially mycobacteria), and because PPK1 lacks homologous enzymes in mammalian cells, it has become an ideal target for the development of anti-infective drugs. Small molecule inhibitors targeting PPK1 hold promise for shortening chemotherapy treatment duration and effectively combating bacteria that rely on polyphosphates to maintain virulence.

[0005] Several PPK1 inhibitors have been reported. For example, raloxifene hydrochloride has been reported as a broad-spectrum PPK1-1 inhibitor, enhancing the activity of isoniazid, bedaquiline, and pretopmani against Mycobacterium tuberculosis in macrophages. Quercetin has been reported to inhibit PPK1 activity in Acinetobacter baumannii; phlorizin has been reported to inhibit PPK1 activity in Salmonella; and ebuselenline derivatives have been reported to inhibit PPK1 activity, reduce bacterial polyphosphate levels, decrease biofilm formation, and reduce bacterial motility. Summary of the Invention

[0006] This application is based on a groundbreaking research finding that theaflavin monogallic acid, 1,2,3,6-tetra-O-galloyl-β-D-glucose, salvianolic acid B, and silanol all have effective inhibitory activity against polyphosphate kinase 1 (PPK1) enzyme activity, and their inhibitory effect is not limited by the enzyme source. Regardless of whether PPK1 is derived from Mycobacterium tuberculosis (MTBPPK1) or Mycobacterium smegmatis (MS PPK1), the above compounds can significantly inhibit its catalytic activity in synthesizing polyphosphates (polyP).

[0007] This application provides a polyphosphate kinase 1 inhibitor, comprising:

[0008] I) Theaflavins monogallic acid or its pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs or isomers;

[0009] II) 1,2,3,6-tetra-O-galloyl-β-D-glucose or a pharmaceutically acceptable salt, ester, solvate, hydrate, polymorph, prodrug or isomer thereof;

[0010] III) Tanshinone B or its pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs or isomers;

[0011] IV) Silver calcinoside or its pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs or isomers;

[0012] Or a combination of multiple terms from I) to IV).

[0013] This application provides a method for inhibiting the polyphosphate synthesis activity of polyphosphate kinase 1, comprising contacting polyphosphate kinase 1 with an effective amount of the compound in (I) to (IV) or a polyphosphate kinase 1 inhibitor.

[0014] This application provides the use of the compounds in I) to IV) or polyphosphate kinase 1 inhibitors in the preparation of medicaments for inhibiting the polyphosphate synthesis activity of MTB PPK1 and / or MS PPK1.

[0015] This application provides the use of the compounds in I) to IV) or polyphosphate kinase 1 inhibitors in the preparation of medicaments for the treatment or prevention of diseases or conditions associated with MTB PPK1 and / or MS PPK1 activity.

[0016] This application provides the application of the compounds in I) to IV) or polyphosphate kinase 1 inhibitors in the preparation of mycobacterial biofilm inhibitors.

[0017] This application provides the use of the compounds in I) to IV) or polyphosphate kinase 1 inhibitors in the preparation of medicaments for enhancing the sensitivity of antimycobacterial drugs to mycobacteria.

[0018] This application provides the use of the compounds in I) to IV) or polyphosphate kinase 1 inhibitors in the preparation of a medicament for reducing polyphosphate levels in mycobacterial biofilms.

[0019] This application provides the use of the compounds in I) to IV) or polyphosphate kinase 1 inhibitors in the preparation of medicaments for killing persistent mycobacterial bacteria.

[0020] This application provides the use of the compounds in I) to IV) or polyphosphate kinase 1 inhibitors in the preparation of medicaments for the prevention or treatment of infections caused by mycobacteria.

[0021] This application provides a method for inhibiting the formation of mycobacterial biofilms in vitro, which includes the step of contacting mycobacteria with an effective amount of the compounds in (I) to (IV) or polyphosphate kinase 1 inhibitors.

[0022] This application has the following beneficial technical effects compared to the prior art:

[0023] 1. Discovery of novel PPK1 inhibitors. This application is the first to discover that theaflavins monogallic acid, 1,2,3,6-tetra-O-galloyl-β-D-glucose, salvianolic acid B, and cynomolpic acid all possess PPK1 inhibitory activity. These compounds are all known compounds from natural sources, but their PPK1 inhibitory activity has never been reported before. This application expands the new applications of these compounds.

[0024] 2. Broad-spectrum PPK1 inhibitory activity. The compounds described in this application exhibit PPK1 inhibitory activity not limited by the enzyme source, meaning they show inhibitory activity against PPK1 from different bacterial species. In particular, they demonstrate significant inhibitory activity against Mycobacterium tuberculosis (MTB) PPK1 and Mycobacterium smegma (MS) PPK1. This characteristic makes the compounds described in this application have broader application prospects and can be used to treat infectious diseases caused by various bacteria expressing PPK1.

[0025] 3. Multi-target, multi-pathway anti-infective mechanism. The compounds described in this application reduce the synthesis of polyphosphates in bacteria by inhibiting the enzymatic activity of PPK1, thereby affecting various physiological processes in bacteria, including but not limited to energy metabolism, stress adaptation, drug tolerance, biofilm formation, and pathogenicity. This multi-target, multi-pathway mechanism of action is beneficial in reducing the development of bacterial drug resistance.

[0026] 4. Good safety profile. The compounds described in this application are mostly naturally derived compounds or derivatives of natural compounds, exhibiting good safety profiles. For example, theaflavins have been clinically verified to have various health benefits without toxic side effects. Tanshinone B has been reported to be essentially non-toxic to normal cells. It has been confirmed to be non-toxic at the cellular level.

[0027] 5. Synergistic effect with existing antibacterial drugs. The compounds described in this application, by inhibiting PPK1 activity, can reduce bacterial stress adaptation and drug tolerance, thereby enhancing the sensitivity of bacteria to existing antibacterial drugs (such as isoniazid and other antimycobacterial drugs). This property allows the compounds described in this application to act as antibacterial sensitizers, used in combination with existing antibacterial drugs to improve therapeutic efficacy. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The SDS-PAGE images (A) of MTB PPK1 protein and MS PPK1, and the enzyme activity detection images (B) of both, provided for a preparation example of this application, are shown. In the images, RFI represents relative fluorescence intensity.

[0030] Figure 2 Linear fitting plot of ATP versus FRI of polyP-DAPI provided for a test example of this application.

[0031] Figure 3 IC50 of the five test compounds provided for a test example of this application on the inhibition of MTB PPK1 protease activity 50 Curves. Among them, A is TF-3'-G; B is TG; C is TF-3-G; D is DLB; and E is Tiliroside.

[0032] Figure 4IC50 values ​​of the five test compounds provided for the test example of this application on the inhibition of MS PPK1 protease activity 50 Curves. Among them, A is TF-3'-G; B is TG; C is TF-3-G; D is DLB; and E is Tiliroside.

[0033] Figure 5 The figures show the test results of an SPR test example of this application. Figure A shows the binding affinity curves of TF-3'-G to MTB PPK1 at different concentrations; Figure B shows the kinetic curves of TF-3'-G to MTB PPK1 at different concentrations; Figure C shows the binding affinity curves of TG to MTB PPK1 at different concentrations; Figure D shows the kinetic curves of TG to MTB PPK1 at different concentrations; Figure E shows the binding affinity curves of TF-3-G to MTB PPK1 at different concentrations; and Figure F shows the kinetic curves of TF-3-G to MTB PPK1 at different concentrations.

[0034] Figure 6 The effects of the test compounds provided in this application on the physiological functions of MS are shown in Figure A. Figure A shows the effects of TF-3'-G, TG, TF-3-G, and Gallein on MS biomembranes, respectively. Figure B shows the statistical results of polyP levels in MS biomembranes treated with different concentrations of TF-3'-G. Figure C shows the statistical results of polyP levels in MS biomembranes treated with different concentrations of TG. Figure D shows the statistical results of polyP levels in MS biomembranes treated with different concentrations of TF-3-G. Figure E shows the statistical results of polyP levels in MS biomembranes treated with different concentrations of Gallein. Detailed Implementation

[0035] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] the term

[0037] Unless the context clearly indicates otherwise, the singular form of a term includes the plural referent.

[0038] The terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0039] Polyphosphate kinase 1 (PPK1) is an enzyme that catalyzes the transfer of the terminal phosphate group of ATP to the polyphosphate chain to synthesize polyphosphates. Its EC designation is 2.7.4.1. PPK1 is highly conserved in bacteria and is a key enzyme in bacterial polyphosphate metabolism.

[0040] MTB PPK1: refers to polyphosphate kinase 1 derived from Mycobacterium tuberculosis.

[0041] MS PPK1: refers to polyphosphate kinase 1 derived from Mycobacterium smegmatis.

[0042] Surface plasmon resonance (SPR): a label-free, real-time technique for monitoring interactions between biomolecules, providing detailed kinetic data such as binding rate (kon), dissociation rate (koff), and affinity (KD).

[0043] Pharmaceutically acceptable salts refer to salts of the compounds of this invention that, within the bounds of proper medical judgment, are suitable for contact with tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and have a reasonable benefit / risk ratio. Pharmaceutically acceptable salts include, but are not limited to, sodium salts, potassium salts, calcium salts, magnesium salts, and ammonium salts.

[0044] Pharmaceutically acceptable esters: refer to ester derivatives of the compounds of this invention, which can be hydrolyzed in vivo to the parent compound. Pharmaceutically acceptable esters include, but are not limited to, C1-C6 alkyl esters, acetylated derivatives, galloylated derivatives, etc.

[0045] Solvent: refers to the complex formed by the combination of the compound of this invention and solvent molecules. When the solvent is water, it is called a hydrate.

[0046] Hydrate: refers to the complex formed by the combination of the compound of this invention and water molecules.

[0047] Polymorphs: refers to compounds of this invention existing in different crystalline forms. Different polymorphs of the same compound may have different physical properties (such as melting point, solubility, stability, etc.).

[0048] Prodrug: refers to a derivative of the compound of this invention, which releases the active parent compound after undergoing biotransformation (such as enzymatic hydrolysis, hydrolysis, etc.) in vivo. Prodrugs include, but are not limited to, phosphate ester derivatives, amino acid ester derivatives, etc.

[0049] Isomers: Compounds with the same molecular formula but different atomic arrangements. Isomers include, but are not limited to, positional isomers, geometric isomers (cis-trans isomers), optical isomers (enantiomers, diastereomers), and terminal isomers.

[0050] Biofilm: A structured microbial community formed by bacteria secreting extracellular polymers (including polysaccharides, proteins, nucleic acids, etc.) after attaching to living or non-living surfaces. Biofilm formation is closely related to chronic bacterial infections and drug resistance.

[0051] Minimum inhibitory concentration (MIC): The lowest drug concentration that can inhibit the visible growth of bacteria under in vitro culture conditions.

[0052] Effective dose: refers to the amount of the compound of this invention that produces the expected therapeutic effect. The effective dose can be adjusted according to factors such as route of administration, patient's age, weight, sex, and disease severity.

[0053] Subject: refers to an individual who receives administration of the compound or pharmaceutical composition of the present invention, including but not limited to humans and non-human mammals.

[0054] The term "IC50" refers to the concentration of inhibitor required to achieve 50% inhibition of enzyme activity.

[0055] The term "Kd" refers to the dissociation constant, which is used to measure the binding affinity between a compound and a protein.

[0056] The term "Km" refers to the Michaelis constant, which is the substrate concentration at which an enzyme reaction reaches half of its maximum rate.

[0057] abbreviation:

[0058] One implementation provides a polyphosphate kinase 1 inhibitor comprising one or more compounds selected from the following four classes, or pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs, or isomers thereof: I) theaflavins monogallic acid; II) 1,2,3,6-tetra-O-galloyl-β-D-glucose; III) salvianolic acid B; IV) cymoxanol; or a combination of multiples of I) to IV).

[0059] Those skilled in the art will understand that "comprising" means that the inhibitor may contain any one or more of the compounds listed above. "Selected from one or more of..." means that the inhibitor may contain only one of the listed compounds, or may contain any combination of two or more of them. "Or a combination of multiples of I) to IV) means that the inhibitor may contain any two, three, four, or a combination of all four of the above four classes of compounds.

[0060] Implementation plan for the "compound" and its "pharmaceutically acceptable salt"

[0061] The compounds described herein may exist in free form or as pharmaceutically acceptable salts. Those skilled in the art will understand that when a compound molecule contains acidic groups (such as phenolic hydroxyl groups, carboxyl groups, etc.), it can form salts with bases. These pharmaceutically acceptable salts include, but are not limited to, salts formed with inorganic bases (such as sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, etc.) and salts formed with organic bases (such as triethylamine salts, diethylamine salts, ethanolamine salts, etc.). The salts can be prepared by conventional methods, such as dissolving the free compound in a suitable solvent, adding the corresponding base, reacting, and then removing the solvent to obtain the corresponding salt.

[0062] Specifically, for theaflavins monogallic acid, its molecule contains multiple phenolic hydroxyl groups, which can react with sodium hydroxide, potassium hydroxide, calcium hydroxide, or ammonia to form sodium, potassium, calcium, or ammonium salts. For 1,2,3,6-tetra-O-galloyl-β-D-glucose, its molecule contains multiple phenolic hydroxyl groups, which can form sodium or potassium salts with alkalis. For salvianolic acid B, its molecule contains a carboxyl group and a phenolic hydroxyl group, which can form sodium, potassium, or magnesium salts with alkalis. For cylindricaloside, its molecule contains a phenolic hydroxyl group, which can form sodium or potassium salts with alkalis.

[0063] Implementation plan for "pharmaceutically acceptable esters"

[0064] When a compound molecule contains a carboxyl or hydroxyl group, it can form a pharmaceutically acceptable ester. This ester can be hydrolyzed in vivo by esterases to the active parent compound. For salvianolic acid B, which contains a carboxyl group, it can form a C1-C6 alkyl ester; a particularly preferred ester is dimethyl salvianolic acid B. For 1,2,3,6-tetra-O-galloyl-β-D-glucose, which contains a phenolic hydroxyl group, it can form a C1-C6 alkyl ester. For cypermethrin, which contains a phenolic hydroxyl group, it can form an acetylated or galloylated derivative.

[0065] Implementation plan regarding "solvents" and "hydrates"

[0066] The compounds described in the embodiments can exist as solvates, that is, complexes formed by the combination of compound molecules and solvent molecules. When the solvent is water, it is called a hydrate. The compounds described in this application can form hydrates during the preparation process, such as monohydrates, dihydrates, etc. The hydrates can be obtained by crystallizing or recrystallizing the compounds in water or aqueous solvents.

[0067] Implementation plan for "polymorphs"

[0068] The compounds described in the embodiments can exist in different crystalline forms (polymorphs). Different polymorphs of the same compound may have different physicochemical properties, such as melting point, solubility, dissolution rate, and stability. For example, theaflavins monogallic acid can exist in either polymorph I or polymorph II. Other compounds may also exist in different polymorphs, which can be obtained by those skilled in the art through screening using conventional crystallization conditions.

[0069] Implementation plan for "prodrugs"

[0070] The compounds described in the implementation plan can exist in the form of prodrugs. A prodrug is a derivative of a compound that releases the active parent compound after biotransformation (such as enzymatic hydrolysis) in vivo. The design of prodrugs can improve the pharmacokinetic properties of compounds, such as solubility and oral bioavailability. For example, phosphate or amino acid ester derivatives of theaflavins monogallic acid can serve as prodrugs. When the phenolic hydroxyl groups in the theaflavins monogallic acid molecule are phosphorylated or esterified with amino acids, the resulting prodrug can be hydrolyzed by the corresponding enzymes in vivo, releasing the active parent compound.

[0071] Implementation plan for "isomers"

[0072] The compound described in the implementation scheme may exist in different isomer forms. Isomers include positional isomers, geometric isomers (cis-trans isomers), optical isomers (enantiomers, diastereomers), and terminal isomers, etc.

[0073] Theaflavins monogallate exist as two positional isomers: theaflavins-3-gallate and theaflavins-3′-gallate. These are positional isomers of each other and both exhibit PPK1 inhibitory activity.

[0074] For 1,2,3,6-tetra-O-galloyl-β-D-glucose, there exist α-terminal isomers and β-terminal isomers. The β-terminal isomer is particularly preferred in this application.

[0075] Tanshinone B has multiple chiral centers in its molecule and exists in R,R-configuration, S,S-configuration, or mixtures thereof. This application covers all stereoisomers of Tanshinone B.

[0076] Silver glycosides contain an α,β-unsaturated ester structure and exist as two geometric isomers: trans-silver glycoside and cis-silver glycoside. This application covers all geometric isomers of silver glycosides.

[0077] Implementation plan for "multiple combinations"

[0078] The implementation scheme also covers combinations of any two or more of the four classes of compounds. For example, the inhibitor may comprise a combination of theaflavins monogallic acid and salvianolic acid B; or a combination of 1,2,3,6-tetra-O-galloyl-β-D-glucose and cynomolbital; or a combination of theaflavins monogallic acid, salvianolic acid B, and all three; or a combination of all four classes of compounds. Such combinations can produce a synergistic PPK1 inhibitory effect.

[0079] Another implementation scheme provides a polyphosphate kinase 1 inhibitor comprising at least one of the following: theaflavin monogallic acid, 1,2,3,6-tetra-O-galloyl-β-D-glucose, salvianolic acid B or cytosine, or a pharmaceutically acceptable salt, ester, solvate, hydrate, polymorph, prodrug or isomer thereof.

[0080] Those skilled in the art will understand that this embodiment covers implementations containing only one, two, three, or all four of items I through IV. In one embodiment, the inhibitor contains only theaflavins monogallic acid. In another embodiment, the inhibitor contains only 1,2,3,6-tetra-O-galloyl-β-D-glucose. In another embodiment, the inhibitor contains only salvianolic acid B. In another embodiment, the inhibitor contains only cylindricaloside. In another embodiment, the inhibitor contains a combination of theaflavins monogallic acid and salvianolic acid B. In another embodiment, the inhibitor contains a combination of 1,2,3,6-tetra-O-galloyl-β-D-glucose and cylindricaloside.

[0081] In some embodiments, the theaflavin monogallic acid is present in the form of its ester, wherein the ester is selected from theaflavin-3′-gallic acid ester, theaflavin-3-gallic acid ester, or a mixture thereof.

[0082] Those skilled in the art should understand that theaflavins monogallic acid is a collective term for theaflavins-3′-gallic acid ester and theaflavins-3-gallic acid ester. Theaflavins-3-gallic acid ester (TF-3-G) is the product of galloylation of the 3-hydroxyl group in the theaflavins molecule; theaflavins-3′-gallic acid ester (TF-3′-G) is the product of galloylation of the 3′-hydroxyl group in the theaflavins molecule. They are positional isomers of each other and both possess PPK1 inhibitory activity. In one embodiment, the theaflavins monogallic acid is theaflavins-3′-gallic acid ester. In another embodiment, the theaflavins monogallic acid is theaflavins-3-gallic acid ester. In yet another embodiment, the theaflavins monogallic acid is a mixture of theaflavins-3′-gallic acid ester and theaflavins-3-gallic acid ester. The ratio of the two components in the mixture can be any ratio, such as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 or 9:1 (w / w).

[0083] In some embodiments, the salvianolic acid B exists in the form of its ester, wherein the ester is dimethyl salvianolic acid B.

[0084] Those skilled in the art will understand that dimethyl salvianolate B (CAS No.: 875313-64-7) is a derivative in which two carboxyl groups in the salvianolate B molecule are methylated. Its molecular formula is C2. 38 H 34 O 16 The molecular weight is 746.70. Dimethyl salvianolic acid B can be converted into the active parent salvianolic acid B in vivo via ester bond hydrolysis. In one embodiment, the inhibitor comprises dimethyl salvianolic acid B. In another embodiment, the inhibitor comprises a mixture of salvianolic acid B and dimethyl salvianolic acid B.

[0085] In some embodiments, the inhibitor is used to inhibit the activity of polyphosphate kinase 1 in synthesizing polyphosphates, wherein the polyphosphate kinase 1 is Mycobacterium tuberculosis polyphosphate kinase 1 and / or Mycobacterium smegma polyphosphate kinase 1.

[0086] Those skilled in the art will understand that MTB PPK1 refers to polyphosphate kinase 1 derived from Mycobacterium tuberculosis, encoded by the PPK1 gene (Rv2984) in strain H37Rv. MS PPK1 refers to polyphosphate kinase 1 derived from Mycobacterium smegmatis. Because PPK1 is highly conserved in mycobacteria, the amino acid sequences of MTB PPK1 and MS PPK1 are highly homologous.

[0087] In one embodiment, the inhibitor is used to inhibit the activity of MTB PPK1. In another embodiment, the inhibitor is used to inhibit the activity of MS PPK1. In yet another embodiment, the inhibitor is used to simultaneously inhibit the activities of both MTB PPK1 and MS PPK1.

[0088] In some embodiments of this application, the binding mode of the compounds or polyphosphate kinase 1 inhibitors in I) to IV) to the MTBPPK1 protein includes forming hydrogen bonds or electrostatic interactions with amino acid residue E94, and forming hydrogen bond interactions with one or more amino acid residues selected from S90, N91, D93, R431, H491, Y524 and I626.

[0089] In some embodiments of this application, the compounds in I) to IV) or polyphosphate kinase 1 inhibitors have an IC50 of less than or equal to 100 μM against MTBPPK1.

[0090] In some embodiments of this application, the compounds in I) to IV) or polyphosphate kinase 1 inhibitors have an IC50 of less than or equal to 100 μM against MSPPK1.

[0091] Some implementation schemes specify that pharmaceutically acceptable salts of theaflavins monogallic acid are selected from sodium, potassium, calcium, or ammonium salts; their solvates are hydrates; their polymorphs are selected from polymorph I or polymorph II; their prodrugs are phosphate derivatives or amino acid ester derivatives; and their isomers are positional isomers of theaflavins-3′-gallic acid ester and theaflavins-3-gallic acid ester.

[0092] Some implementations specify that pharmaceutically acceptable salts of 1,2,3,6-tetra-O-galloyl-β-D-glucose are selected from sodium or potassium salts; pharmaceutically acceptable esters are C1-C6 alkyl esters; solvates are hydrates; and isomers are α-terminal or β-terminal isomers.

[0093] Some implementation schemes specify that pharmaceutically acceptable salts of salvianolic acid B are selected from sodium, potassium, or magnesium salts; pharmaceutically acceptable esters are C1-C6 alkyl esters; solvates are hydrates; and isomers are R,R-configuration, S,S-configuration, or mixtures thereof.

[0094] Some implementation schemes specify that the pharmaceutically acceptable salts of cypermethrin are selected from sodium or potassium salts; the pharmaceutically acceptable esters are acetylated derivatives; the solvates are hydrates; and the isomers are trans-cypermethrin or cis-cypermethrin.

[0095] Some implementations specify that the inhibitor also includes a pharmaceutically acceptable carrier or excipient, i.e., the compound is prepared into a pharmaceutical composition. The carrier or excipient includes, but is not limited to, diluents (such as starch, lactose, microcrystalline cellulose, etc.), binders (such as povidone, hydroxypropyl methylcellulose, etc.), disintegrants (such as croscarmellose sodium, croscarmellose, etc.), lubricants (such as magnesium stearate, talc, etc.), wetting agents (such as Tween 80, etc.), preservatives (such as parabens, etc.), antioxidants (such as vitamin C, vitamin E, etc.), buffers (such as phosphates, acetates, etc.), and isotonic adjusters (such as sodium chloride, glucose, etc.).

[0096] Some embodiments specify that the dosage form of the inhibitor is selected from: tablets, capsules, granules, powders, oral solutions, injections, lyophilized powder for injection, ointments, creams, gels, patches, sprays, aerosols, eye drops, or suppositories. Those skilled in the art will understand that different dosage forms are suitable for different routes of administration and different therapeutic needs. For example, oral solid dosage forms (tablets, capsules, granules, etc.) are suitable for oral administration; injections are suitable for intravenous, intramuscular, or subcutaneous injection; topical preparations (ointments, creams, gels, patches, etc.) are suitable for local skin administration; eye drops are suitable for ocular administration; and suppositories are suitable for rectal or vaginal administration.

[0097] In one embodiment, the inhibitor is a tablet comprising theaflavins monogallic acid and pharmaceutically acceptable excipients. In another embodiment, the inhibitor is a capsule comprising 1,2,3,6-tetra-O-galloyl-β-D-glucose and pharmaceutically acceptable excipients. In another embodiment, the inhibitor is a lyophilized powder for injection comprising salvianolic acid B and pharmaceutically acceptable excipients. In yet another embodiment, the inhibitor is an oral liquid comprising silanol and pharmaceutically acceptable excipients.

[0098] Some embodiments define the content of one or more of statements I) to IV) in the inhibitor as 5 mg to 1000 mg per unit dose. Those skilled in the art will understand that "per unit dose" refers to the amount of active ingredient contained in each smallest dosing unit (e.g., per tablet, per capsule, per injection, per sachet of granules, etc.). In one embodiment, the content is 5 mg per unit dose. In another embodiment, the content is 10 mg per unit dose. In another embodiment, the content is 25 mg per unit dose. In another embodiment, the content is 50 mg per unit dose. In another embodiment, the content is 100 mg per unit dose. In another embodiment, the content is 200 mg per unit dose. In another embodiment, the content is 500 mg per unit dose. In another embodiment, the content is 1000 mg per unit dose.

[0099] Some embodiments define the content as ranging from 100 mg to 500 mg per unit dose. In one embodiment, the content is 100 mg per unit dose. In another embodiment, the content is 200 mg per unit dose. In another embodiment, the content is 300 mg per unit dose. In another embodiment, the content is 400 mg per unit dose. In yet another embodiment, the content is 500 mg per unit dose.

[0100] Another implementation scheme provides a method for inhibiting the polyphosphate synthesis activity of polyphosphate kinase 1, comprising contacting an effective amount of the compound or a polyphosphate kinase 1 inhibitor with polyphosphate kinase 1.

[0101] Those skilled in the art will understand that "contact" refers to bringing the compound or inhibitor into spatial proximity with the PPK1 enzyme molecule, thereby enabling the compound to interact with the enzyme molecule (e.g., bind), and thus inhibit the enzyme's catalytic activity. This contact can be performed in vitro or in vivo.

[0102] In one embodiment, the method is performed in vitro. For example, in a test container such as a test tube, centrifuge tube, or microplate, the compound is mixed with purified PPK1 enzyme protein in a suitable buffer system, and the change in PPK1 enzyme activity is measured. In vitro methods can be used to screen and evaluate the PPK1 inhibitory activity of compounds, and also to study the mechanism of action of compounds.

[0103] In another embodiment, the method is performed in vivo. For example, the compound is administered to a subject infected with mycobacteria (such as an animal model or a human patient), whereby the compound contacts the bacterial PPK1 enzyme in vivo, thereby inhibiting its activity. In vivo methods can be used to treat or prevent diseases or conditions associated with PPK1 activity.

[0104] Some implementations specify that the polyphosphate kinase 1 is Mycobacterium tuberculosis polyphosphate kinase 1 (MTB PPK1). In one implementation, the method is used to inhibit the activity of MTB PPK1, which can be used to treat Mycobacterium tuberculosis infection.

[0105] Some implementations specify that the polyphosphate kinase 1 is Mycobacterium smegmatis polyphosphate kinase 1 (MSPPK1). In one implementation, the method is used to inhibit the activity of MSPPK1, which can be used to study the biological characteristics of mycobacteria and screen anti-mycobacterial drugs.

[0106] Another aspect of the implementation plan provides the use of the PPK1 inhibitor or compound in the preparation of a medicament for inhibiting the synthetic polyphosphate activity of MTB PPK1 and / or MS PPK1. Those skilled in the art will understand that "use in the preparation of a medicament" means using the compound as an active ingredient in the preparation of a pharmaceutical formulation with a specific therapeutic or preventative function.

[0107] Another aspect of the implementation plan provides the use of the PPK1 inhibitor or compound in the preparation of a medicament for the treatment or prevention of diseases or conditions associated with MTBPPK1 and / or MS PPK1 activity.

[0108] Some implementations define the diseases or conditions associated with MTB PPK1 and / or MS PPK1 activity as mycobacterial infectious diseases. Mycobacterial infectious diseases are infectious diseases caused by bacteria of the genus Mycobacterium (including Mycobacterium tuberculosis, Mycobacterium smegmatis, Mycobacterium leprae, Mycobacterium avium, etc.). In one implementation, the disease is tuberculosis (caused by Mycobacterium tuberculosis). In another implementation, the disease is Mycobacterium smegmatis infection. In another implementation, the disease is Mycobacterium avium complex infection. In yet another implementation, the disease is leprosy (caused by Mycobacterium leprae).

[0109] Some implementations specify the mycobacterial infectious disease as Mycobacterium tuberculosis infection or Mycobacterium smegmatis infection.

[0110] Some embodiments define the diseases or conditions associated with MTB PPK1 and / or MS PPK1 activity as those associated with mycobacterial biofilm formation. A biofilm is a structured microbial community formed by bacteria secreting extracellular polymers after adhering to a biological or non-biological surface. Biofilm formation can protect bacteria from antibiotics and the host immune system, leading to chronic and persistent infections. Since PPK1 is involved in mycobacterial biofilm formation, inhibiting PPK1 activity can inhibit biofilm formation, thereby preventing and treating diseases or conditions associated with biofilm formation. In one embodiment, the disease or condition is pulmonary tuberculosis associated with Mycobacterium tuberculosis biofilm formation. In another embodiment, the disease or condition is an infection associated with Mycobacterium smegmaeella biofilm formation.

[0111] Some implementations define the diseases or conditions associated with MTB PPK1 and / or MS PPK1 activity as those related to mycobacterial stress adaptation, drug resistance, or intracellular survival. PPK1 plays a key role in the synthesis of polyphosphates, which are involved in bacterial stress adaptation and drug resistance. Inhibiting PPK1 activity can reduce mycobacterial stress adaptation and drug resistance, thereby enhancing the therapeutic efficacy of existing antimycobacterial drugs. In one implementation, the disease or condition is latent infection with Mycobacterium tuberculosis. In another implementation, the disease or condition is multidrug-resistant tuberculosis. In yet another implementation, the disease or condition is extensively drug-resistant tuberculosis.

[0112] The embodiments provide the use of the PPK1 inhibitor or compound in the preparation of mycobacterial biofilm inhibitors. In one embodiment, the mycobacterial biofilm inhibitor is used to inhibit the formation of Mycobacterium tuberculosis biofilm. In another embodiment, the mycobacterial biofilm inhibitor is used to inhibit the formation of Mycobacterium smegmaeella biofilm. In yet another embodiment, the mycobacterial biofilm inhibitor is used to inhibit the formation of Mycobacterium avium biofilm.

[0113] The embodiments provide the use of the PPK1 inhibitor or compound in the preparation of a medicament for enhancing the susceptibility of antimycobacterial drugs to mycobacteria. In one embodiment, the antimycobacterial drug is isoniazid. In another embodiment, the antimycobacterial drug is rifampin. In another embodiment, the antimycobacterial drug is ethambutol. In another embodiment, the antimycobacterial drug is pyrazinamide. In another embodiment, the antimycobacterial drug is bedaquiline. In another embodiment, the antimycobacterial drug is delamani. In another embodiment, the antimycobacterial drug is clofazimine.

[0114] Those skilled in the art will understand that the compounds described in this application, by inhibiting PPK1 activity, can reduce drug tolerance in mycobacteria, making the bacteria more sensitive to existing antimycobacterial drugs. Therefore, the compounds can be used as sensitizers for antimycobacterial drugs, and can be used in combination with existing antimycobacterial drugs to improve therapeutic efficacy, reduce drug dosage, and decrease toxic side effects.

[0115] Detailed description of compounds and their derivatives

[0116] Compounds I) through VII) described in this application are all organic compounds of natural origin or that can be prepared by chemical synthesis and have a well-defined chemical structure. The following provides a detailed description of each compound and its pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs, or isomers.

[0117] (a) Theaflavins monogallic acid (I)

[0118] Theaflavin monogallate is an important component of theaflavins in black tea. Theaflavins are a class of polyphenolic hydroxy compounds with a benzophenone structure, whose basic core is formed by the benzophenone reaction of two catechin units. Theaflavin monogallate contains two isomers: theaflavin-3-gallate (TF-3-G, CAS: 30462-34-1) and theaflavin-3′-gallate (TF-3′-G, CAS: 28543-07-9). The chemical formula of theaflavin monogallate is C2. 36 H 28 O 16 With a molecular weight of 716.6, it belongs to the composition of theaflavins and catechin oxidation products. This substance was first detected in black tea in 1957. Theaflavin-3-gallate and theaflavin-3′-gallate together account for more than 50% of the theaflavins in black tea. Theaflavin monogallate is readily soluble in solvents such as water, ethanol, methanol, ethyl acetate, and n-butanol.

[0119] The molecular formula of theaflavin-3-gallate is C 36 H 28 O 16 With a molecular weight of 716.6, this compound is a major monomer of theaflavins in black tea and one of the main indicators of the color and flavor of black tea infusion. Theaflavin-3-gallate possesses antioxidant, anti-inflammatory, and antibacterial biological activities. Studies have shown that theaflavin-3-gallate can inhibit the growth of various Gram-positive and Gram-negative bacteria in vitro. Theaflavin-3-gallate also kills Staphylococcus aureus by inducing the production of reactive oxygen species, completely inhibiting the growth of standard strains of Staphylococcus aureus at a concentration of 64 μg / mL. Theaflavin-3-gallate also exhibits inhibitory activity against *Pseudomonas aeruginosa*, completely inhibiting the growth of planktonic bacteria at a concentration of 512 μg / mL.

[0120] Theaflavins-3′-gallic acid ester is a theaflavins monomer from black tea and an important biologically active component of black tea. Theaflavins-3′-gallic acid ester reacts directly with reduced glutathione in a time- and concentration-dependent manner, acting as a pro-oxidant and inducing oxidative stress in cancer cells. Theaflavins-3′-gallic acid ester has shown anti-influenza virus activity in vitro, with an IC50 of [missing information]. 50 The value was 2.53 μM. Theaflavins-3′-gallate also exhibited antibacterial activity against Bacillus anthracis. Single-crystal X-ray diffraction patterns of theaflavins-3′-gallate indicated that it belongs to the orthorhombic crystal system.

[0121] Several methods for preparing theaflavins monogallic acid have been reported. For example, it can be extracted and isolated from black tea: black tea powder is extracted by reflux with 70% ethanol, the extract is concentrated, and then extracted successively with chloroform and ethyl acetate. The ethyl acetate layer is collected, concentrated, dried, and separated by silica gel column chromatography using chloroform-methanol-water (65:35:10, lower layer) as the eluent. The fraction containing theaflavins monogallic acid is collected and then purified by preparative high-performance liquid chromatography to obtain pure theaflavins monogallic acid. Alternatively, fresh lychee peel can be used as the source of active polyphenol oxidase, and theaflavins-3-gallic acid can be prepared using epigallocatechin gallate (EGCG) and epicatechin (EC) as raw materials. This is then purified and separated using macroporous adsorption resin column chromatography to prepare high-purity theaflavins-3-gallic acid.

[0122] Theaflavins, specifically monogallic acid, have been widely used in the field of food additives. In 2010, the National Health and Family Planning Commission approved theaflavins as a new type of food additive, allowing its use in various food categories. Monogallic acid, a major component of theaflavins, has been clinically proven to possess multiple health benefits without toxic side effects.

[0123] In this application, theaflavins monogallic acid can exist in the form of a free acid, or in the form of pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs, or isomers. Pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, calcium, or ammonium salts. Theaflavins monogallic acid molecules contain multiple phenolic hydroxyl groups and can form salts with alkali metals or alkaline earth metals. Its solvates include, but are not limited to, hydrates. Its polymorphs include, but are not limited to, crystal form I or crystal form II. Its prodrugs include, but are not limited to, phosphate ester derivatives or amino acid ester derivatives. Its isomers include, but are not limited to, positional isomers of theaflavins-3′-gallic acid ester and theaflavins-3-gallic acid ester.

[0124] (II) 1,2,3,6-Tetra-O-galloyl-β-D-glucose (II)

[0125] 1,2,3,6-Tetra-O-galloyl-β-D-glucose (CAS No.: 79886-50-3) is a galloyl-β-D-glucose compound containing four galloyl groups at positions 1, 2, 3, and 6. Its molecular formula is C2. 34 H 28 O 22 The molecular weight is 788.58. This compound has a melting point of 198-200°C, a density of approximately 2.02±0.1 g / cm³, and should be stored at -20°C. It is soluble in dimethyl sulfoxide. The acidity coefficient (pKa) of this compound is 8.37±0.15.

[0126] This compound has been reported as an inhibitor of UDP-glucuronyltransferase 1 family polypeptide A1 (UGT1A1), with a Ki value of 1.68 μM. It can be extracted from various plants, including Castanopsis fissa, Euphorbia jolkini I, and Punica granatum L.

[0127] Extraction methods for 1,2,3,6-tetra-O-galloyl-β-D-glucose have been reported. For example, it can be extracted and separated from pomegranate peel: pomegranate peel is extracted with ethanol, the extract is concentrated, and then purified by macroporous adsorption resin column chromatography, silica gel column chromatography, and preparative high-performance liquid chromatography to obtain the target product. It can also be prepared by chemical synthesis: β-D-glucose and gallic acid are reacted under appropriate esterification conditions, followed by separation and purification to obtain the target product.

[0128] In this application, 1,2,3,6-tetra-O-galloyl-β-D-glucose may exist in the form of pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs, or isomers. Pharmaceutically acceptable salts include, but are not limited to, sodium or potassium salts. Pharmaceutically acceptable esters include, but are not limited to, C1-C6 alkyl esters. Solvates include, but are not limited to, hydrates. Isomers include, but are not limited to, α-terminal isomers or β-terminal isomers.

[0129] (III) Tanshinone B

[0130] Salvianolic acid B (also known as lithospermic acid B, CAS No.: 115939-25-8) is one of the main water-soluble active components of the traditional Chinese medicine Salvia miltiorrhiza. Salvianolic acid B has been reported to possess various pharmacological activities, including cardioprotective, antioxidant, anti-fibrotic, and antiviral effects. Studies have shown that salvianolic acid B can intervene in Escherichia coli-infected pyelonephritis through the JAK2 / STAT3 pathway; salvianolic acid B can inhibit the early stages of HPV infection in target cells; and salvianolic acid B also has an anti-rotavirus biosynthesis effect. Salvianolic acid B is essentially non-toxic to normal cells.

[0131] Dimethyl salvianolate B (also known as Dimethyl lithospermate B, dmLSB, CAS No.: 875313-64-7) is a dimethyl ester derivative of salvianolate B. Its molecular formula is C2. 38 H 34 O16 The molecular weight is 746.70. Dimethyl salvianolic acid B has been reported as a selective Na⁺ channel agonist, slowing sodium current (INa) inactivation and leading to an increase in early inward current of the action potential (AP). Studies have shown that salvianolic acid B is stable in human liver microsomes, while dimethyl salvianolic acid B is unstable in both human liver microsomes and blank incubation medium. Furthermore, the metabolic rate in human liver microsomes is faster than in blank incubation medium, with the main metabolic pathway being ester bond hydrolysis.

[0132] The preparation method of salvianolic acid B has been reported: Salvia miltiorrhiza is extracted with water or ethanol. After concentration, the extract is purified by macroporous adsorption resin column chromatography, polyamide column chromatography, and preparative high-performance liquid chromatography to obtain pure salvianolic acid B. Dimethyl salvianolic acid B can be prepared by esterification of salvianolic acid B with methanol in the presence of an acidic catalyst.

[0133] In this application, salvianolic acid B can exist in the form of a free acid, or in the form of a pharmaceutically acceptable salt, ester, solvate, hydrate, polymorph, prodrug, or isomer. The pharmaceutically acceptable salt includes, but is not limited to, sodium, potassium, or magnesium salts. The pharmaceutically acceptable ester includes, but is not limited to, C1-C6 alkyl esters, with dimethyl salvianolic acid B being particularly preferred. The solvate includes, but is not limited to, hydrates. The isomers include, but are not limited to, R,R-configurations, S,S-configurations, or mixtures thereof.

[0134] (iv) Silver calcinate (IV)

[0135] Tiliroside (CAS No.: 20316-62-5) is a glycoside flavonoid compound. Its molecular formula is C1. 30 H 26 O 13 It has a molecular weight of 594.52 and a melting point of 269-271°C. The chemical structure of kaempferol is 3-O-β-D-(6″-O-trans-p-hydroxycinnamoyl)glucopyranoside (6-p-coumaril ester).

[0136] Silver glycosides have been reported to possess a variety of pharmacological activities. It is a non-competitive inhibitor of α-amylase with a Ki value of 84.2 μM. Silver glycosides exhibit anti-inflammatory, antioxidant, anticancer, and hepatoprotective activities. Silver glycosides can inhibit the proliferation of pancreatic cancer PANC-1 cells, accompanied by G2 / M phase cell cycle arrest; this mechanism may be related to the inhibition of CDK1, Cyclin B1, HMGCR, and LDLR expression. Silver glycosides also possess significant anti-complement activity (IC50). 50 =5.4×10⁻5 (M), its activity is even stronger than that of rosmarinic acid, a well-known inhibitor of the complement system. Furthermore, ginsenoside has inhibitory effects on certain bacteria and fungi. Studies have shown that ginsenoside has inhibitory activity against non-pathogenic mycobacteria, with a MIC value of 5.0 mg / mL.

[0137] The preparation method of ginsenoside has been reported: it is extracted and separated from plant materials (such as raspberry, ground peach, tribulus, etc.), extracted with ethanol, concentrated, and purified by silica gel column chromatography and preparative high performance liquid chromatography to obtain pure ginsenoside.

[0138] In this application, silagenoside may exist in the form of pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs, or isomers. Pharmaceutically acceptable salts include, but are not limited to, sodium or potassium salts. Pharmaceutically acceptable esters include, but are not limited to, acetylated derivatives. Solvates include, but are not limited to, hydrates. Isomers include, but are not limited to, trans-silagenoside or cis-silagenoside.

[0139] PPK1 target and mechanism of action

[0140] The compounds described in this application exert their biological effects by inhibiting the enzymatic activity of polyphosphate kinase 1 (PPK1). PPK1 (EC 2.7.4.1) catalyzes the transfer of the terminal phosphate group of ATP to the polyphosphate chain and is a key enzyme in polyphosphate biosynthesis, playing a central regulatory role in bacterial stress adaptation, biofilm formation, virulence expression, and persistence maintenance. Notably, PPK1 lacks homologs in human and mammalian cells, making it a highly attractive selective antibacterial target. In mycobacteria, PPK1 is encoded by the PPK1 gene. Polyphosphates (polyPs) are linear polymers linked by high-energy phosphate bonds. Polyphosphates participate in the regulation of various cellular processes in bacteria, including energy metabolism, stress adaptation, drug tolerance, and pathogenicity.

[0141] In Mycobacterium tuberculosis, PPK1 has been shown to be essential for growth in host tissues. Deletion of the *PPK1-1* gene affects glucose utilization and lipid biosynthesis in Mycobacterium tuberculosis. PPK1 is also involved in the formation of biofilms in Mycobacterium smegmatis. Studies have shown that the absence of PPK1 impairs the survival of Mycobacterium smegmatis under prolonged hypoxic conditions.

[0142] This application systematically demonstrated, through multiple methods including DAPI fluorescence detection, surface plasmon resonance (SPR) technology, and enzyme kinetic analysis, that four compounds—theaflavin monogallic acid, 1,2,3,6-tetra-O-galloyl-β-D-glucose, salvianolic acid B, and cytosolic acid—all exhibit inhibitory effects on PPK1, inhibiting its catalytic activity in the synthesis of polyphosphates. More importantly, this inhibitory effect is not limited by the source of the PPK1 enzyme, including but not limited to Mycobacterium tuberculosis (MTB) PPK1 and Mycobacterium smegma (MS) PPK1.

[0143] Preparation example: Preparation of MTB PPK1 and MS PPK1 recombinant proteins

[0144] 1.1 Construction of expression plasmids

[0145] The target sequences of the PPK1 gene (NC_000962.3:3339854-3342082) of *Mycobacterium tuberculosis* and the target sequences of the PPK1 gene (CP000480.1:2473142-2475352) of *Mycobacterium smegmatis* were synthesized. After PCR amplification using primers with BamHI and HindIII linkers, the resulting molecules were double-digested with BamHI and HindIII and ligated to pET28a(+). The ligation products were transformed into *Escherichia coli* DH5α competent cells and plated on LB agar plates containing kanamycin (50 μg / mL) and incubated overnight at 37°C. Positive clones were picked, and plasmids were extracted for PCR identification and sequencing verification.

[0146] 1.2 Recombinant Expression

[0147] The correctly sequenced recombinant plasmids pET28a-MTB-ppk and pET28a-MS-ppk were transformed into Escherichia coli BL21(DE3) competent cells, plated on LB plates containing kanamycin (50 μg / mL), and incubated overnight at 37°C.

[0148] Single colonies were picked and inoculated into LB liquid medium containing kanamycin (50 μg / mL), and cultured overnight at 37°C with shaking at 200 rpm. The colonies were then transferred 1:100 to fresh LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking at 200 rpm until OD500 was reached. 600 The concentration was approximately 0.6-0.8. IPTG was added to a final concentration of 0.5 mM, and expression was induced at 16°C for 16 hours. Bacterial culture without IPTG was used as a negative control.

[0149] After induction, collect the bacterial pellet by centrifugation at 4°C and 5000 rpm for 10 minutes. Discard the supernatant and resuspend the bacterial pellet in pre-cooled lysis buffer (50 mM HEPES-KOH, pH 7.5, 100 mM NaCl and 5% glycerol). Disrupt the cells using an autoclave or sonicator, centrifuge at 4°C and 12000 rpm for 30 minutes, and collect the supernatant (soluble protein fraction).

[0150] 1.3 SDS-PAGE identification of recombinant proteins

[0151] Take samples before and after purification, add 5×SDS loading buffer, and heat at 100°C for 10 minutes to denature the proteins. Perform electrophoresis analysis using a 12% SDS-PAGE gel. The electrophoresis conditions are: 80 V constant voltage for the stacking gel, followed by 120 V constant voltage for the separating gel. After electrophoresis, stain with Coomassie Brilliant Blue R-250 for 30 minutes, then destain until the background is clear, and photograph the results.

[0152] like Figure 1 SDS-PAGE results for A showed that, after IPTG induction, the BL21(DE3) strain transformed with pET28a-MTB-ppk exhibited a distinct inducible protein band at approximately 70–100 kDa, consistent with the theoretical molecular weight of MTB PPK1 (approximately 81.6 kDa). Following Ni-NTA affinity chromatography purification, high-purity recombinant MTB PPK1 protein with a purity exceeding 90% was obtained.

[0153] like Figure 1 As shown in Figure A, the BL21(DE3) strain transformed with pET28a-MS-ppk exhibited a distinct inducible protein band at approximately 75 kDa, consistent with the theoretical molecular weight of MS PPK1 (approximately 81.0 kDa). After purification by Ni-NTA affinity chromatography, high-purity recombinant MS PPK1 protein with a purity exceeding 90% was obtained.

[0154] 1.4 Activity Verification of Recombinant Protein

[0155] To verify whether the purified recombinant protein possessed the catalytic activity of PPK1, its catalytic activity in the synthesis of polyP was detected using the DAPI fluorescence method. The reaction system (100 μL) contained: 2 mmol / L creatine phosphate, 20 μg / ml creatine kinase, 5 μg MTB PPK1 recombinant protein or MS PPK1 recombinant protein, 5 mM ATP, 400 mM ammonium sulfate, 40 mM MgCl2, and 500 mM pH 7.5 HEPES-KOH buffer. After reacting at 37°C for 30 min, the reaction was terminated by adding 40 mM EDTA, followed by adding 10 μM DAPI dye, and incubating at room temperature in the dark for 10 min. Fluorescence intensity was measured using a fluorescence microplate reader at an excitation wavelength of 415 nm and an emission wavelength of 515 nm. The control group (BLANK) consisted of either MTB PPK1 recombinant protein or MS PPK1 recombinant protein.

[0156] like Figure 1 B shows that both purified MTB PPK1 and MS PPK1 recombinant proteins can significantly catalyze the synthesis of polyP from ATP, with fluorescence intensity increasing by at least 10-fold compared to the blank control group (without PPK1). This indicates that the purified recombinant proteins have good enzyme activity and can be used for subsequent inhibitor screening and evaluation tests.

[0157] Test Example 2: Detection of the inhibitory activity of PPK1 inhibitors using PPK1 fluorescence assay

[0158] 2.1 Test Principle

[0159] DAPI (4',6-diamidinyl-2-phenylindole) is a fluorescent dye. When DAPI is bound to polyphosphate (polyP), its excitation-emission spectrum changes significantly. The DAPI-polyP complex produces a fluorescence emission peak at 515 nm under 415 nm excitation. By measuring the fluorescence intensity at 515 nm, the amount of polyP generated in the reaction system can be quantitatively reflected, thereby evaluating the enzyme activity of PPK1 and the inhibitory effect of the compound on PPK1.

[0160] 2.2 Establishment of DAPI-polyP fluorescence intensity standard curve

[0161] Prepare HEPES-KOH buffer (500 mM, pH 7.5) containing ammonium sulfate (400 mM) and MgCl2 (40 mM), and add different concentrations (0, 5, 10, 25, 50, 100, 200, 500 ng / mL) of polyP. 45Standards were prepared; after reacting at 37℃ for 30 min, the reaction was terminated by adding EDTA to a final concentration of 40 mM. Then, DAPI dye to a final concentration of 10 μM was added to each polyP solution, and the mixture was incubated at room temperature in the dark for 10 min. Fluorescence intensity was measured using a fluorescence microplate reader at an excitation wavelength of 415 nm and an emission wavelength of 515 nm. A standard curve was plotted with polyP concentration on the x-axis and fluorescence intensity on the y-axis, and the standard equation was obtained by fitting the curve.

[0162] like Figure 2 As shown, the fluorescence intensity of the DAPI-polyP complex exhibited a good linear relationship with the polyP concentration in the range of 5-500 ng / mL (R² > 0.99), and the linear regression equation was C = 7 × 10⁻⁶. -6 ×F -0.244, where F is the fluorescence intensity and C is the polyP concentration. The detection limit is approximately 5 ng / mL and the quantitation limit is approximately 25 ng / mL.

[0163] 2.3 Determination of the inhibition rate of five compounds on MTB PPK1

[0164] Test materials: MTB PPK1 recombinant protein and MS PPK1 recombinant protein (prepared from Test Example 1); Test compounds: theaflavins-3'-gallate (TF-3'-G, CAS No.: 28543-07-9), 1,2,3,6-tetra-O-galloyl-β-D-glucose (TG, CAS No.: 79886-50-3), theaflavins-3-gallate (TF-3-G), dimethyl tanshinone β-carbamate (DLB, CAS No.: 875313-64-7), tiliroside (CAS No.: 20316-62-5), all with a purity ≥98%; ATP, DAPI dye, and other reagents were all analytical grade.

[0165] Reaction system I consisted of: 2 mmol / L creatine phosphate, 20 μg / ml creatine kinase, 5 μg MTB PPK1 recombinant protein or MS PPK1 recombinant protein, 5 mM ATP, 400 mM ammonium sulfate, 40 mM MgCl2, 500 mM HEPES-KOH buffer at pH 7.5, and 100 μM of the test compound. Three replicates were prepared for each concentration.

[0166] Reaction system II contained: 2 mmol / L creatine phosphate, 20 μg / ml creatine kinase, 5 μg MTB PPK1 recombinant protein or MS PPK1 recombinant protein, 5 mM ATP, 400 mM ammonium sulfate, 40 mM MgCl2, 500 mM HEPES-KOH buffer at pH 7.5, and different concentrations of the test compound (final concentrations of 0, 0.5, 1, 2, 5, 10, 20, 50, 100, and 200 μM). Each concentration was tested in triplicate.

[0167] To rule out any inhibitory effect not due to creatine kinase, the following reaction system was provided to detect the inhibitory effect of the test compound on the enzyme activity of MTB PPK1 or MS PPK1. Reaction system III contained: 5 μg of recombinant MTB PPK1 or MS PPK1 protein, 5 mM ATP, 400 mM ammonium sulfate, 40 mM MgCl2, 500 mM HEPES-KOH buffer (pH 7.5), and 100 μM of the test compound. Three replicates were performed for each concentration.

[0168] After reacting reaction systems I through III at 37°C for 30 min, the reaction was terminated by adding EDTA to a final concentration of 40 mM, followed by adding DAPI dye to a final concentration of 10 μM, and incubating at room temperature in the dark for 10 min. Fluorescence intensity was measured using a fluorescence microplate reader at an excitation wavelength of 415 nm and an emission wavelength of 515 nm. The concentration of polyphosphate produced in the reaction was determined using a polyP standard curve. The reaction system without the test compound served as a positive control (100% enzyme activity), and the reaction system without PPK1 served as a blank control (0% enzyme activity).

[0169] The formula for calculating the inhibition rate is:

[0170] Inhibition rate (%) = [(F0 - F1) / (F0 - F_blank)] × 100%

[0171] Wherein, F0 is the fluorescence intensity value of the positive control, F1 is the fluorescence intensity value of the test compound treatment group, and F_blank is the fluorescence intensity value of the blank control.

[0172] The dose-response curve was fitted using GraphPad Prism 8.0 software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0173] 2.4 Test Results

[0174] like Figure 3 As shown, the IC50 of theaflavin-3'-gallate (TF-3'-G) on MTB PPK150 = 6.8 μM, IC50 of 1,2,3,6-tetra-O-galloyl-β-D-glucose (TG) against MTB PPK1 50 = 60.9 μM, IC50 of theaflavin-3-gallate (TF-3-G) against MTB PPK1 50 = 11.4 μM, IC50 of dimethyl tanshinone β-D (DLB) against MTB PPK1 50 = 57.0 μM, IC50 of tiliroside against MTB PPK1 50 = 94.5μM.

[0175] like Figure 4 As shown, the IC50 of theaflavin-3'-gallate (TF-3'-G) against MS PPK1 50 = 17.8 μM, IC50 of 1,2,3,6-tetra-O-galloyl-β-D-glucose (TG) against MTB PPK1 50 = 74.8 μM, IC50 of theaflavin-3-gallate (TF-3-G) against MTB PPK1 50 = 26.1 μM, IC50 of dimethyl tanshinone β-D (DLB) against MTB PPK1 50 = 24.2 μM, IC50 of tiliroside against MTB PPK1 50 = 95.1μM.

[0176] Therefore, it can be seen that the five tested compounds significantly inhibited the enzyme activity of both MTB PPK1 and MS PPK1 in a concentration-dependent manner. At a concentration of 100 μM, the inhibition rate of each compound against PPK1 from both sources reached over 79%.

[0177] It is worth noting that the IC50 of theaflavins-3'-gallate and theaflavins-3-gallate is... 50 The values ​​are very close, indicating that the two are essentially the same PPK1 inhibitory activity as positional isomers, which is consistent with the prediction that the binding conformations of the two are highly similar in molecular docking.

[0178] Compare the IC50 values ​​of each compound for MTB PPK1 and MS PPK1. 50 The values ​​show a high degree of consistency between the two (correlation coefficient R² > 0.95), which fully confirms that the inhibitory effect of these compounds on PPK1 is not limited by the enzyme source, i.e., they have broad-spectrum PPK1 inhibitory activity.

[0179] To ensure that the observed inhibitory activity was not due to the compounds inhibiting the ATP regeneration system (such as creatine kinase), the inhibitory activities of TF-3'-G, TG, DLB, TF-3-G, and Tiliroside were re-examined in reaction system III, which lacked phosphocreatine and creatine kinase (incubated at 37°C for 2 hours). The results showed that the inhibitory activities of these compounds remained unchanged in both systems, indicating that their inhibitory activity indeed stemmed from direct inhibition of the PPK1 enzyme, rather than inhibition of creatine kinase.

[0180] Test Example 3: Determination of Binding Kinetic Parameters by SPR Method

[0181] 3.1 Test Materials

[0182] The Biacore T200 SPR biomolecular interaction analyzer (GE Healthcare) is equipped with a CM5 sensor chip.

[0183] Running buffer: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4. Regeneration buffer: 10 mM Glycine-HCl, pH 2.5. Immobilization reagents: NHS (N-hydroxysuccinimide), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), ethanolamine (1 M, pH 8.5). MTB PPK1 and MS PPK1 recombinant proteins (prepared from the preparation example).

[0184] The tested compounds were theaflavins-3'-gallate (TF-3'-G, CAS No.: 28543-07-9), 1,2,3,6-tetra-O-galloyl-β-D-glucose (TG, CAS No.: 79886-50-3), and theaflavins-3-gallate (TF-3-G), all with a purity ≥98%.

[0185] 3.2 MTB PPK1 protein immobilization

[0186] Dilute the MTB PPK1 protein to a final concentration of 50 μg / mL with 10 mM sodium acetate buffer (pH 5.0). Activate the CM5 chip surface for 7 minutes using an NHS / EDC mixture (1:1) at a flow rate of 10 μL / min. Inject the diluted MTB PPK1 protein at a flow rate of 5 μL / min to achieve a protein conjugation volume of approximately 5000-8000 RU. Block unreacted activation sites for 7 minutes with 1 M ethanolamine (pH 8.5) at a flow rate of 10 μL / min.

[0187] An unfixed protein flow cell was used as a reference channel to subtract background signals and nonspecific binding.

[0188] 3.3 Kinetic Measurement

[0189] Five test compounds were diluted to different concentrations (0.156, 0.313, 0.625, 1.25, 2.5, and 5 μM) with run buffer. The solutions of each concentration were sequentially injected at a flow rate of 30 μL / min, with a binding time of 120 seconds and a dissociation time of 300 seconds. After each injection, the chip surface was regenerated with 10 mM Glycine-HCl (pH 2.5) for 30 seconds. At least three cycles were performed for each concentration gradient.

[0190] 3.4 Data Analysis

[0191] The sensor data was fitted and analyzed using Biacore Evaluation software. After subtracting the signals from the blank control and reference channels, a 1:1 Langmuir binding model was used to fit the binding rate constant (ka) and dissociation rate constant (kd). The equilibrium dissociation constant (KD) was calculated using the formula KD = kd / ka.

[0192] 3.5 Test Results

[0193] like Figure 5 As shown, theaflavin-3'-gallate, 1,2,3,6-tetra-O-galloyl-β-D-glucose, and theaflavin-3-gallate all exhibited strong binding affinity for MTB PPK1, with equilibrium dissociation constants KD of 4.28 μM, 462 μM, and 2.05 μM, respectively, indicating that the affinity measured by SPR is consistent with the enzyme activity inhibition results.

[0194] Test Example 5: Tests on the Effects of Mycobacterium smegma on Biofilm Formation

[0195] 5.1 Test Materials

[0196] Mycobacterium smegmatis strain mc²155. The tested compounds were TF-3'-G, TG, and TF-3-G.

[0197] 5.2 Test Methods

[0198] Mycobacterium smegmatis mc²155 was inoculated into Middlebrook 7H9 liquid medium (containing 0.05% Tween 80 and 10% OADC enrichment broth) containing different concentrations of the test compound (0, 25, 50, 100 μM). Initial OD 600Adjust the concentration to 0.05. Add 200 μL of bacterial culture to each well of a 96-well cell culture plate, with 6 replicates for each concentration. Incubate statically at 37°C for 48 hours. A DMSO control group without inhibitors was also included. Alizarin violet (0, 25, 50, 100 μM, Gallein) was used as a positive control group.

[0199] After the culture is completed, take photos to observe the inhibition of the biofilm.

[0200] The content of polyP in biofilms was detected using the DAPI fluorescence method.

[0201] 5.3 Test Results

[0202] The above results indicate that and Figure 6 TF-3'-G, TG, and TF-3-G all inhibited MS biofilm formation in a concentration-dependent manner. In contrast, Gallein (100 μM) had no significant effect on MS biofilm formation. This indicates that the compounds in this application have superior anti-biofilm activity compared to the known PPK1 inhibitor Gallein.

[0203] Biofilms are crucial barriers for bacterial defense against antibiotics and the host's immune system, and their formation is closely related to chronic bacterial infection and drug resistance. The compounds described in this invention inhibit biofilm formation by suppressing PPK1 activity and reducing polyphosphate synthesis, thus providing a novel strategy for treating chronic mycobacterial infections.

[0204] To verify whether TF-3'-G's inhibition of PPK1 activity affects the accumulation of polyP in biofilms, biofilms were collected, polyP was extracted, and the polyP content in MS biofilms treated with different concentrations of TF-3'-G was determined using the DAPI fluorescence method.

[0205] The results are as follows Figure 6 As shown in Figures B to E, the polyP content in MS biofilms decreased in a dose-dependent manner after treatment with TF-3'-G, TG, and TF-3-G. However, the inhibitory effect of Gallein on polyP formation in MS biofilms was not significantly altered after the concentration exceeded 25 μM, indicating that Gallein's effect on polyP formation in MS biofilms is not entirely dose-dependent.

[0206] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A polyphosphate kinase 1 inhibitor, comprising: I) Theaflavins monogallic acid or its pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs or isomers; II) 1,2,3,6-tetra-O-galloyl-β-D-glucose or a pharmaceutically acceptable salt, ester, solvate, hydrate, polymorph, prodrug or isomer thereof; III) Tanshinone B or its pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs or isomers; IV) Silver calcinoside or its pharmaceutically acceptable salts, esters, solvates, hydrates, polymorphs, prodrugs or isomers; Or a combination of multiple terms from I) to IV).

2. The polyphosphate kinase 1 inhibitor according to claim 1, wherein the theaflavin monogallic acid exists in the form of its ester, wherein the ester is selected from theaflavin-3′-gallic acid ester, theaflavin-3-gallic acid ester, or a mixture thereof; Optionally, the salvianolic acid B exists in the form of an ester, wherein the ester is dimethyl salvianolic acid B.

3. The polyphosphate kinase 1 inhibitor according to any one of claims 1 to 2, wherein the inhibitor is used to inhibit the activity of polyphosphate kinase 1 in synthesizing polyphosphates, wherein the polyphosphate kinase 1 is Mycobacterium tuberculosis polyphosphate kinase 1 and / or Mycobacterium smegmaecum polyphosphate kinase 1.

4. The polyphosphate kinase 1 inhibitor according to claim 1, wherein the inhibitor further comprises a pharmaceutically acceptable carrier or excipient.

5. The polyphosphate kinase 1 inhibitor according to claim 1, wherein the dosage form of the inhibitor is selected from: tablets, capsules, granules, powders, oral liquids, injections, lyophilized powder injections, ointments, creams, gels, patches, sprays, aerosols, eye drops, or suppositories.

6. The polyphosphate kinase 1 inhibitor according to claim 1, wherein one or more of (I) to (IV) are present in the inhibitor at a concentration of 5 mg to 1000 mg per unit dose.

7. The polyphosphate kinase 1 inhibitor according to claim 6, wherein the content is from 100 mg to 500 mg per unit dose.

8. A method for inhibiting the polyphosphate synthesis activity of polyphosphate kinase 1, comprising contacting polyphosphate kinase 1 with an effective amount of the compound or polyphosphate kinase 1 inhibitor according to any one of claims 1 to 7.

9. The method according to claim 8, wherein the polyphosphate kinase 1 is Mycobacterium tuberculosis polyphosphate kinase 1 or Mycobacterium smegmaecum polyphosphate kinase 1.

10. The use of the polyphosphate kinase 1 inhibitor or a compound thereof according to any one of claims 1 to 7, wherein the use comprises at least one of the following: Prepare drugs for inhibiting the polyphosphate synthesis activity of MTB PPK1 and / or MS PPK1; Prepare a medicament for treating or preventing diseases or conditions associated with MTB PPK1 and / or MS PPK1 activity; optionally, the disease or condition associated with MTB PPK1 and / or MS PPK1 activity is a mycobacterial infection; optionally, the mycobacterial infection is Mycobacterium tuberculosis infection or Mycobacterium smegmatis infection; optionally, the disease or condition associated with MTB PPK1 and / or MS PPK1 activity is a disease or condition associated with mycobacterial biofilm formation; optionally, the disease or condition associated with MTB PPK1 and / or MS PPK1 activity is a disease or condition associated with mycobacterial stress adaptation, drug tolerance, or intracellular survival. Preparation of mycobacterial biofilm inhibitors; Preparation of drugs to enhance the sensitivity of antimycobacterial drugs to mycobacteria.

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