Inhibitors of cell envelope proteases from lactic acid bacteria

Abstract

The present disclosure is a screening procedure that allows for the identification of inhibitors of cell envelope protease (CEP) in pathogenic bacteria by the use of lactic acid bacteria (LAB). In some embodiments, a screening platform can be used to screen a library of compounds, thereby generating a high throughput assessment of drug candidates, and it has been demonstrated by example that a single screening of 6808 compounds identifies 20 CEP inhibiting compounds of Lactococcus lactis.

Description

Technical Field

[0001] This disclosure describes a screening procedure that allows the identification of inhibitors of cell envelope proteases (CEPs) in pathogenic bacteria using lactic acid bacteria (LABs). CEP enzymes are typically not detectable using common chromogenic substrates, but the screening platform of this invention can identify compounds capable of inhibiting cell envelope proteases (CEPs) in lactic acid bacteria (LABs).

[0002] This screening platform was used to screen a compound library of 6808 compounds. As illustrated by the example below, a single screening identified 20 compounds that inhibit *Lactococcus lactis*. Lactococcus lactis ) of CEP compounds. Background Technology

[0003] CEP enzymes typically cannot be detected using common chromogenic substrates. For example, pathogenic invasive streptococci have highly specific CEP enzymes with only one or a few known substrates. These specific CEP enzymes are virulence factors that inactivate chemokines of the innate immune system. As a result, neutrophils cannot be recruited, and invasive infections develop rapidly, leading to death within hours or days.

[0004] To date, there are no drugs that target these virulence factors. For example, there are no drugs that target CEP of Group A Streptococcus (GAS). Therefore, if the infection has already developed, surgery is usually required.

[0005] Antibiotic treatment can be effective for GAS infection if started early; however, if the infection has progressed, surgery is usually required.

[0006] The medical need for new antibiotics is evident. These should be able to inhibit, for example, bacteria like Streptococcus pyogenes (…). Streptococcus pyogenes Drugs containing the proteolytic virulence factor SCPA will prevent bacteria from paralyzing the immune system. These drugs will improve the effectiveness of antibiotic treatment and slow the progression of infection.

[0007] Targeting virulence rather than viability has been proposed as a promising strategy for identifying future antimicrobial agents. This would allow for an expansion of the antimicrobial agent pool and potentially reduce the development of antimicrobial resistance. Virulence factors, such as adhesion, toxins, and biofilm formation, are already targets of this strategy. CEP virulence factors targeting streptococcal pathogens have been proposed, but have not yet been implemented due to a lack of suitable screening procedures.

[0008] Therefore, there is an unmet need for screening platforms that can, for example, identify potential compound drugs that are effective against pathogens via cell envelope proteases (CEPs) targeting pathogens. Summary of the Invention

[0009] LABs used for dairy fermentation also possess CEP enzymes. The CEP in dairy cultures allows them to use casein as a nitrogen source. The CEP enzymes in dairy strains are also highly selective, using only casein, and typically only one of several casein strains, β-casein. Furthermore, dairy CEP is difficult to detect using chromogenic substrates, and in such assays, it typically shows very low activity.

[0010] However, the activity of dairy CEP gives the bacteria an easily identifiable phenotype—the ability to deeply acidify milk, reaching a pH of 4.6 within 12 hours. Non-protein hydrolysate strains will grow in milk, but only at lower cell densities, and will not acidify below pH 5.5.

[0011] Using a simple acidification assay will allow for screening a library of potential inhibitors to suppress acidification. If cultures are supplied with a mixture of amino acids, cultures inhibited solely by protease inhibition will be able to resume growth and complete acidification, while cultures inhibited for other reasons will remain inhibited.

[0012] Thus, inhibitors of dairy CEP enzymes may also inhibit other similar enzymes and may inhibit the entire class of LAB-CEP, so some inhibitors will distinguish CEP enzymes.

[0013] In its broadest aspect, this disclosure relates to a method for identifying inhibitors of cell envelope proteases (CEPs) in pathogenic bacteria, the method comprising: a) Provide fermentable substrates containing lactic acid bacteria (LAB) strains and pH indicators; b) Add the target molecule; c) Monitor the acidification of fermentable substrates; d) Add an amino acid source to the fermentable substrate; e) Determine the pH of the fermentable substrate; and f) Based on the pH of the fermentable substrate being below 4.6, the target molecule is identified as an inhibitor.

[0014] The core of this invention is to use dairy-CEP for high-throughput screening of a large compound library and to use secondary screening to identify inhibitors that are active against other CEP enzymes.

[0015] Secondary screening or validation steps can be performed using specific assays that are not suitable for high-throughput screening.

[0016] Another aspect of this disclosure relates to target inhibitors identified by this method. The following examples section, for example, shows that alexidin and methylene blue can be inhibitors of cell envelope proteases (CEPs) in pathogens. Detailed Implementation

[0017] The following experimental data demonstrate the screening of a library containing 6808 compounds from the Broad Institute. The library was received in 384-well plates, with each well containing 1 nanomolar of a compound. A milk matrix was added to each well. The milk was inoculated with lactic acid bacteria containing a truncated version of CEP.

[0018] The milk also contained glucose and two pH indicators. Plates were incubated at 30°C, and a plate scanner was used to monitor the milk's color to track acidification changes. The scans were collected and analyzed using pH MultiScan software from HNH.

[0019] Figure 1 The acidification curves from the screening of the first 1500 compounds are shown. Figure 2 The pH change was shown on the second day after the addition of the extra amino acid.

[0020] Filtering methods

[0021] In one or more exemplary embodiments, a method for identifying inhibitors of cell envelope proteases (CEPs) in pathogenic bacteria, the method comprising: a) Provide fermentable substrates containing lactic acid bacteria (LAB) strains and pH indicators; b) Add the target molecule; c) Monitor the acidification of fermentable substrates; d) Add an amino acid source to the fermentable substrate; e) Determine the pH of the fermentable substrate; and f) Based on the pH of the fermentable substrate being below 4.6, the target molecule is identified as an inhibitor.

[0022] Thus, technicians are able to identify potential inhibitors of cell membrane proteases (CEPs) as target molecules that can inhibit acidification until the fermentable substrate is replenished with amino acids and reaches a pH below 4.6 after that replenishment.

[0023] Since most target molecules were not inhibited, these candidates lowered the pH to 4.6 within the first 5 to 10 hours of fermentation, but they were not candidates of interest. Molecules that neither inhibited cell membrane proteases nor any other functions required for growth would not cause changes in growth and acidification kinetics. These non-inhibitory molecules would not be analyzed further.

[0024] Molecules exhibiting partial inhibition of cell membrane proteases or any other growth-required function will show a delay in acidification kinetics, reaching pH 4.6 with a large delay. These molecules will be further analyzed to identify whether the delay can be prevented by adding hydrolyzed casein, suggesting that the inhibited function may be a CEP enzyme. Since acidification has reached pH 4.6, this test must be performed subsequently.

[0025] Molecules exhibiting complete inhibition of the CEP enzyme will display the same growth and acidification kinetics as the non-proteolytic version of the indicator bacteria. If the reduced acidification is indeed due to inhibition of CEP enzyme growth, the acidification will recover upon the addition of hydrolyzed protein to the inhibited culture.

[0026] Molecules that inhibit other functions essential for growth neither grow nor acidify. It cannot be ruled out that these molecules may also inhibit CEP enzymes. If the amount of these inhibitors is within the capability of the validation assay, these small amounts of molecules should be tested.

[0027] temperature

[0028] Once the wells are filled, the bacteria will be incubated at a temperature that allows for the growth of lactic acid bacteria suitable for initial screening, and the growth kinetics of the indicator bacteria will be monitored. If the indicator bacteria are *Lactococcus lactis* (…), the bacteria will be incubated at a temperature that allows for the growth of lactic acid bacteria suitable for initial screening. Lactococcus lactis ) or Lactococcus fatii ( Lactococcus cremoris If thermophilic streptococci or other thermophilic LAB species are used, the temperature will be between 15°C and 37°C, usually 30°C; while when using species belonging to the Leuconostoc genus, they will usually be cultured at a lower temperature, usually 25°C.

[0029] Therefore, in one or more exemplary embodiments, a method for identifying inhibitors of cell envelope proteases (CEP) in pathogenic bacteria is a temperature that allows lactic acid bacteria to grow.

[0030] Therefore, in one or more exemplary embodiments, the temperature is 10°C to 50°C, such as, but not limited to, 20°C to 40°C, 25°C to 40°C, 30°C to 40°C, 35°C to 40°C, 10°C to 20°C, 10°C to 30°C, or 10°C to 40°C.

[0031] In one or more exemplary embodiments, the temperature is 37°C.

[0032] In one or more exemplary embodiments, the temperature is 30°C.

[0033] reading

[0034] Growth kinetics can be monitored using any method suitable for tracking lactic acid bacteria growth in a high-throughput setting, such as, but not limited to, cell density, nutrient consumption, metabolite production, or culture medium acidification as markers of bacterial growth.

[0035] Milk-like turbid culture media can interfere with some analytical procedures and reduce the methods available for growth tracking. Acidification kinetics have been tracked by monitoring color changes in milk-based media containing a mixture of pH indicators. Color changes can be tracked by placing a microplate on top of a flatbed scanner, for example, reading the bottom of the well every 5 minutes over an 18-hour period.

[0036] CEP enzyme

[0037] CEP enzymes are extracellular enzymes associated with the outer surface (cell membrane) of bacteria. CEP enzymes are large, multi-domain enzymes, typically ranging from 1000 to 2000 amino acids in length. The proteolytic domain is a serine protease homologous to the nonspecific protease subtilisin. While subtilisin exhibits high activity but lacks specificity, CEP enzymes are selective and specific to particular substrates.

[0038] Inhibitors of CEP from *Lactococcus lactis*, as shown in the examples, will be useful for the development of novel starter cultures in the food industry. Secondary screening enabled the identification of *Streptococcus pyogenes* (a strain of *Lactococcus lactis*) from a library of *Lactococcus lactis* CEP inhibitors. Streptococcus pyogenes Inhibitors of scpA. These inhibitors have the potential to be developed as drugs for treating streptococcal infections, including necrotizing fasciitis, one of the most serious conditions. However, relatively harmless streptococcal infections may be the most profitable business.

[0039] For some inhibitors, prophylactic use can also be considered.

[0040] CEP enzymes cannot typically be detected using common chromogenic substrates. Pathogenic invasive streptococci possess highly specific CEP enzymes with only one or a few known substrates. These specific CEP enzymes are virulence factors that inactivate chemokines of the innate immune system. As a result, neutrophils cannot be recruited, and invasive infections develop rapidly, leading to death within hours or days.

[0041] Streptococcal C5a peptidase, SCPA

[0042] In one or more exemplary embodiments, the CEP enzyme is SnpA, also known as streptococcal C5a peptidase. SnpA is a subtilisin-like serine protease containing a Ser-Asp-His catalytic triplet and an oxyanion hole asparagine. SnpA is known to be involved in the virulence (pathogenicity) of Streptococcus pyogenes.

[0043] The role of Spirodiclofenac (ScpA) in the pathogenesis of Streptococcus pyogenes infection has been investigated. It has been found to contribute to immune evasion and modulate the host's immune response. Understanding the activity and function of Spirodiclofenac can help researchers develop strategies to target this enzyme as a potential therapeutic target, or to develop vaccines or antibiotics against Streptococcus pyogenes infection.

[0044] In one or more exemplary embodiments, the CEP enzyme is selected from the group consisting of streptococcal CEP enzymes that are specific to mammalian chemokines, human peptide C5a, human peptide C3a, mammalian chemokines of the CXC family, or other mammalian immune peptides.

[0045] In one or more exemplary embodiments, the CEP enzyme is selected from the group consisting of SnpA, SnpB, and SnpC (SpyCEP) of Streptococcus pyogenes.

[0046] In one or more exemplary embodiments, the CEP enzyme is selected from Streptococcus pyogenes, Streptococcus agalactiae (Streptococcus pyogenes, Streptococcus agalactiae) Streptococcus agalactiae ) or Streptococcus lactis ( Streptococcus dysgalactiae The CEP enzyme in the group consisting of [a group name] showed greater than 50% homology with the SCPA enzyme of Streptococcus pyogenes.

[0047] In one or more exemplary embodiments, the CEP enzyme is derived from group A streptococci (GAS).

[0048] In one or more exemplary embodiments, the CEP enzyme is derived from Group B Streptococcus (GBS).

[0049] In one or more exemplary embodiments, the CEP enzyme is derived from group C streptococci (GCS).

[0050] Cell membrane protease (CEP) of lactic acid bacteria

[0051] Lactic acid bacteria cell membrane proteases are a class of enzymes located in the cell membrane of lactic acid bacteria and involved in protein degradation.

[0052] Examples of cell membrane proteases in lactic acid bacteria: PrtP PrtP: A cell wall-associated serine protease found in Lactococcus lactis, which is involved in the degradation of casein in milk and is important for the development of cheese texture and flavor.

[0053] PrtS

[0054] PrtS: A cell wall-associated serine protease found in Streptococcus thermophilus, involved in the degradation of casein in milk and important for the development of cheese texture and flavor. PrtS is also important for the degradation of soy protein during the fermentation of soy-based milk analogs.

[0055] PrtB

[0056] PrtB: A species found in *Lactobacillus delbrueckii* subsp. bulgaricus (… Lactobacillus delbrueckii subsp. bulgaricus Cell wall-associated serine proteases in milk proteins are involved in the degradation of milk proteins and are important for the development of dairy product texture and flavor.

[0057] PrtH

[0058] PrtH: A species found in Lactobacillus helveticus ( Lactobacillus helveticus Cell wall-associated serine proteases in milk are involved in the degradation of casein in milk and are important for the development of dairy product texture and flavor as well as for the production of bioactive peptides.

[0059] These are just a few examples of the many different types of cell membrane proteases found in lactic acid bacteria, each with its own specific function and substrate preference.

[0060] CEP enzymes, belonging to the subtilisin-like serine protease family, are present in any lactic acid bacteria belonging to the order Lactobacillus.

[0061] CEP enzymes, belonging to the subtilis protease-like serine protease family, are present in any lactic acid bacteria belonging to the following family: Sarcobacteriaceae ( Carnobacteriaceae ), Enterococci ( Enterococcaceae Lactobacillus family ( Lactobacillaceae ) 、 Leuconostocaceae ( Leuconostocaceae ) or Streptococcus ( Streptococcaceae ).

[0062] CEP enzymes, belonging to the subtilis protease-like serine protease family, are present in any lactic acid bacteria belonging to the following genera: *Lactobacillus* ( Lacticaseibacillus Lactobacillus spp. Lactiplantibacillus Lactobacillus spp. Lactobacillus Leuconostoc ( ) Leuconostoc ), genus *Sacchariformis* Oenococcus ), Pediococcus ( Pediococcus ), Lactobacillus schleiferus ( Schleiferilactobacillus ), Weissella spp. WeissellaLactococcus spp. Lactococcus Streptococcus spp. Streptococcus ).

[0063] Inhibitors of cell membrane protease (CEP)

[0064] Inhibitors of cell membrane proteases are molecules that selectively bind to and inhibit the activity of proteases located in the bacterial cell membrane. These proteases play a crucial role in bacterial growth and survival, and their inhibition can disrupt key cellular processes, leading to bacterial death. Therefore, inhibitors of cell membrane proteases have attracted considerable interest as potential antibacterial agents and have been the subject of extensive research in the development of novel antibiotics.

[0065] In this context, potential inhibitors of cell membrane proteases in pathogens are targeted inhibitors identified by the methods according to this disclosure.

[0066] Proteases are enzymes that catalyze the breakdown of proteins by hydrolyzing peptide bonds. They play crucial roles in various cellular processes, including protein degradation, signal transduction, and cellular regulation. In the context of cell membrane proteases, these enzymes are typically involved in the processing and maturation of proteins important for the structure and function of the cell membrane.

[0067] Inhibitors of cell membrane proteases can have different mechanisms of action. Some inhibitors may bind directly to the protease, blocking its active site and preventing substrate binding or catalytic activity. Other inhibitors may indirectly regulate protease activity by affecting its regulatory mechanisms or its interactions with other cellular components.

[0068] The use of inhibitors of cell membrane proteases has significant implications for various fields, including medicine and biotechnology. Inhibiting specific proteases can disrupt the integrity of the cell membrane, making them potential targets for antimicrobial therapies against bacterial infections. Furthermore, inhibitors can be used in biotechnological applications to control or manipulate the production of specific proteins by altering protease activity.

[0069] It is important to note that specific inhibitors and their effects can vary depending on the targeted protease and the organism or system being studied.

[0070] Several known inhibitors of cell membrane proteases exist. Here are some examples: Protease inhibitor mixtures: These are mixtures of multiple inhibitors that target different proteases. They are commonly used in research laboratories to simultaneously inhibit a wide range of proteases. Examples of protease inhibitor mixtures include Complete™ and PhosSTOP™, both of which are commercially available.

[0071] Serine protease inhibitors: Serine proteases are a class of proteases that play a vital role in various cellular processes. For example, inhibitors of benzyl sulfonyl fluoride (PMSF), aprotinin, and leupeptin can inhibit serine proteases involved in cell membrane processing.

[0072] Metalloproteinase inhibitors: Metalloproteinases are proteases that require metal ions, such as zinc, to exert their catalytic activity. Inhibitors of metalloproteinases, such as EDTA (ethylenediaminetetraacetic acid) and 1,10-phenanthroline, can chelate metal ions and inhibit the activity of metalloproteinases.

[0073] Peptide mimic inhibitors: These are synthetic compounds designed to mimic the structure and function of specific peptide substrates of proteases. They inhibit protease activity by competitively binding to the active site of the protease. Peptide mimic inhibitors can be designed to target specific proteases and can exhibit high specificity and potency.

[0074] Small molecule inhibitors: Various small molecules have been discovered as inhibitors of specific proteases. These molecules can be identified through high-throughput screening or structure-based drug design methods. Small molecule inhibitors often target specific binding sites or allosteric sites on proteases.

[0075] As disclosed in the following examples, a chemical library was dispensed into wells, wherein the compounds were set together with *Lactococcus lactis* inoculum and pH indicators (bromocresol purple and bromocresol green at pH 7). Novel inhibitors of cell membrane proteases of lactic acid bacteria, specifically inhibitors of cell membrane proteases (CEPs) in pathogenic bacteria, were identified and validated by monitoring acidification.

[0076] Therefore, this disclosure describes a series of inhibitors identified by this screening method. In particular, the screening method has identified inhibitors selected from the group consisting of: acivicin, alexidine, azaguanine-8, dihydrostreptomycin, FDCYD, fluxuridine, hygromycin-B, leucomethylene-blue, mercaptopurine, methylene blue, napabucasin, nifuroxazide, nithiamide, nitrofurantoin, NSC-663284, octenidine, Ro-08-2750, tolonium, walrycin-B, and YM-155.

[0077] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria may be selected from the group consisting of fluorouridine, mercaptopurine and dihydrostreptomycin.

[0078] Asiwisin

[0079] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is azithromycin.

[0080] Asivoxil is a type of... Streptomyces sviceus A naturally occurring compound. Asivoxil inhibits glytamyl-dependent amidotransferase and has been evaluated as a cancer therapeutic. However, its toxicity has hindered its application.

[0081] Therefore, in one or more exemplary embodiments, this disclosure describes ascitisine for the treatment of bacterial infections, preferably streptococcal infections.

[0082] 8-Zyguanine

[0083] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is 8-nitroguanine.

[0084] 8-Azoguanine is a triazolone analog of guanine that was tested in the 1950s as a treatment for leukemia, but the clinical results were disappointing.

[0085] Therefore, in one or more exemplary embodiments, this disclosure describes the use of 8-nitroguanine for the treatment of bacterial infections, preferably streptococcal infections.

[0086] Hygromycin B

[0087] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is hygromycin B.

[0088] Hygromycin B is a fungicide produced by *Streptomyces hygroscopicus* (…). Streptomyces hygroscopicus Hygromycin B is an aminoglycoside antibiotic produced by [the production of hygromycin B]. Its known mode of action is to inhibit protein synthesis by interfering with ribosomes. Hygromycin B is used as an anthelmintic in animal feed.

[0089] Therefore, in one or more exemplary embodiments, this disclosure describes the use of hygromycin B for the treatment of bacterial infections, preferably streptococcal infections.

[0090] Napakasin

[0091] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is napkasin.

[0092] Napacasin is a species of jacaranda from Africa (… Newbouldia laevis ), Longflower Suhao Tree ( Ekmanianthe longiflora ) and purple trumpet tree ( Handroanthus impetiginosus Naphthoquinone, a natural compound isolated from [a specific source], is being investigated as an anticancer drug. It has been shown that naphthoquinone interferes with the signal transduction molecule STAT3.

[0093] Therefore, in one or more exemplary embodiments, this disclosure describes the use of napkasin for the treatment of bacterial infections, preferably streptococcal infections.

[0094] Nifurazolidone

[0095] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is nifurazolidone.

[0096] Nifurazolidone is a broad-spectrum antibacterial drug that has been used for decades to treat infectious diarrhea. It has recently been shown to inhibit the transcription factor STAT3. This compound is currently being evaluated as an anticancer drug.

[0097] Therefore, in one or more exemplary embodiments, this disclosure describes the use of nifurazolidone for the treatment of bacterial infections, preferably streptococcal infections.

[0098] Amidazole

[0099] In one or more exemplary embodiments, according to this disclosure, an inhibitor of cell envelope protease (CEP) in pathogens is acetaminophen.

[0100] Aminonitrazole is an aromatic amide, a member of the acetamide class, which is used as an antibiotic in veterinary medicine.

[0101] Therefore, in one or more exemplary embodiments, this disclosure describes the use of acetamidoazole for the treatment of bacterial infections, preferably streptococcal infections.

[0102] Nitrofurantoin

[0103] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is nitrofurantoin.

[0104] Nitrofurantoin is a nitrofuran antibiotic that has been used to treat urinary tract infections since the 1950s.

[0105] Therefore, in one or more exemplary embodiments, this disclosure describes the use of nitrofurantoin for the treatment of bacterial infections, preferably streptococcal infections.

[0106] NSC-663284

[0107] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is NSC-663284.

[0108] NSC-663284 (6-chloro-7-(2-morpholin-4-yl-ethylamino)quinoline-5,8-dione) is a phosphatase inhibitor.

[0109] Therefore, in one or more exemplary embodiments, this disclosure describes the use of NSC-663284 for treating bacterial infections, preferably streptococcal infections.

[0110] Ro-08-2750

[0111] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is Ro-08-2750.

[0112] Ro-08-2750(2,3,4,10-tetrahydro-7,10-dimethyl-2,4-dioxobenzo[] g Pteridine-8-carboxaldehyde is a non-peptide inhibitor of NGF (nerve growth factor).

[0113] Therefore, in one or more exemplary embodiments, this disclosure describes the use of Ro-08-2750 for treating bacterial infections, preferably streptococcal infections.

[0114] Toluene

[0115] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is toluene.

[0116] Tropicamine is a blue cationic dye used in histology. Its chemical structure is similar to that of methylene blue.

[0117] Therefore, in one or more exemplary embodiments, this disclosure describes the use of toluene for the treatment of bacterial infections, preferably streptococcal infections.

[0118] Walrycin-B

[0119] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is Walrycin-B.

[0120] Walrycin-B (1,6-dimethyl-3-(4-(trifluoromethyl)phenyl)pyrimidino[5,4-e][1,2,4]triazine-5,7-dione) is a compound identified as an inhibitor of WalR response modulators in Gram-positive bacteria.

[0121] Therefore, in one or more exemplary embodiments, this disclosure describes the use of Walrycin-B for the treatment of bacterial infections, preferably streptococcal infections.

[0122] YM-155

[0123] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is YM-155.

[0124] YM-155 (Sepantronium bromide) is an inhibitor of survivin proteins involved in apoptosis. Survivin proteins are members of the inhibitory apoptosis protein (IAP) family.

[0125] Therefore, in one or more exemplary embodiments, this disclosure describes the use of YM-155 for the treatment of bacterial infections, preferably streptococcal infections.

[0126] Alexidine

[0127] In one or more exemplary embodiments, according to this disclosure, an inhibitor of cell envelope protease (CEP) in pathogenic bacteria is alexidin.

[0128] Alexidin is a biguanide with broad-spectrum antibacterial properties. Similar to another biguanide, chlorhexidine, it is used as an irrigation solution in dental treatments.

[0129] Therefore, in one or more exemplary embodiments, this disclosure describes the use of alexidin for the treatment of bacterial infections, preferably streptococcal infections.

[0130] Otinididine

[0131] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is oteninidine.

[0132] Otinididine is a biguanide compound with a structure similar to chlorhexidine and alexiconazole. Otinididine is an antibacterial agent effective against Gram-positive bacteria, Gram-negative bacteria, yeasts, and fungi.

[0133] Therefore, in one or more exemplary embodiments, this disclosure describes the use of otinididine for the treatment of bacterial infections, preferably streptococcal infections.

[0134] Methylene blue

[0135] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is methylene blue.

[0136] Methylene blue is a heterocyclic aromatic compound with a long history of medicinal and biochemical applications. It has been used as an antidote for malaria and cyanide poisoning. Recently, it has been proposed as a potential treatment for Zika virus infection by inhibiting the viral protease NS3.

[0137] Therefore, in one or more exemplary embodiments, this disclosure describes the use of methylene blue for treating bacterial infections, preferably streptococcal infections.

[0138] Colorless methylene blue

[0139] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is colorless methylene blue.

[0140] Colorless methylene blue is the reduced form of methylene blue. Methylene blue and colorless methylene blue form a well-known redox pair.

[0141] Therefore, in one or more exemplary embodiments, this disclosure describes the use of colorless methylene blue for the treatment of bacterial infections, preferably streptococcal infections.

[0142] fluorouracil

[0143] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is fluorouracil.

[0144] Fluorouracil is a pyrimidine analogue, also known by its brand name FUDR (fluorouridine for injection). It is currently primarily known as a chemotherapy drug used to treat various types of cancer, particularly colorectal cancer. It belongs to a class of drugs called antimetabolites.

[0145] Therefore, in one or more exemplary embodiments, this disclosure describes the use of fluorouracil for the treatment of bacterial infections, preferably streptococcal infections.

[0146] Antimetabolites

[0147] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is an antimetabolite.

[0148] Antimetabolites are a class of drugs used in chemotherapy that interfere with normal cellular metabolic processes, particularly those involved in DNA and RNA synthesis. They are structurally similar to naturally occurring molecules involved in these processes and can disrupt cancer cell function by acting as faulty substitutes or competitive inhibitors.

[0149] Antimetabolites work by entering cells and interfering with key biochemical pathways. Depending on the specific drug and its mechanism of action, they can inhibit the synthesis of DNA, RNA, or both. By disrupting these processes, antimetabolites prevent cancer cells from dividing and growing, ultimately leading to cell death.

[0150] Some examples include: 1. Pyrimidine analogues: These antimetabolites are analogous to pyrimidines, the building blocks of DNA and RNA. They can be incorporated into growing DNA or RNA chains, leading to impaired replication and synthesis. Examples include 5-fluorouracil (5-FU) and cytarabine.

[0151] 2. Purine analogues: These antimetabolites mimic purine bases found in DNA and RNA, disrupting the production of these nucleotides. By interfering with purine metabolism, they inhibit DNA and RNA synthesis. Examples include mercaptopurine and cladribine.

[0152] 3. Folic acid analogs: Folic acid is essential for the synthesis of nucleotides, the building blocks of DNA and RNA. Folic acid analogs inhibit dihydrofolate reductase, an enzyme involved in converting folate to its active form. This interference impairs nucleotide synthesis and cell division. Methotrexate is a common folic acid analog.

[0153] Antimetabolites are commonly used to treat various types of cancer, including leukemia, lymphoma, breast cancer, and colorectal cancer. However, they can also affect rapidly dividing normal, healthy cells, leading to side effects such as hair loss, gastrointestinal disorders, and decreased blood cell counts.

[0154] Therefore, in one or more exemplary embodiments, this disclosure describes the use of antimetabolites to treat bacterial infections, preferably streptococcal infections.

[0155] Pyrimidine analogues

[0156] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is a pyrimidine analogue.

[0157] Pyrimidine analogues are chemical compounds that are structurally similar to the naturally occurring pyrimidine nucleotides found in DNA and RNA. Pyrimidine is one of the two nucleotide bases that make up the genetic code; the other is purine.

[0158] Therefore, in one or more exemplary embodiments, this disclosure describes the use of pyrimidine analogs for treating bacterial infections, preferably streptococcal infections.

[0159] In one or more exemplary embodiments, the pyrimidine analogues according to this disclosure may be selected from the group consisting of 5-fluorouracil (5-FU), cytarabine (Cytosar-U), and gemcitabine (Gemzar).

[0160] purine analogues

[0161] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is a purine analogue.

[0162] Purine analogues are chemical compounds that are structurally similar to the naturally occurring purine nucleotides found in DNA and RNA. Purines are one of the two nucleotide bases that make up the genetic code; the other is pyrimidine.

[0163] They interfere with DNA and RNA synthesis by inhibiting specific enzymes involved in purine metabolism, disrupting nucleotide production, and ultimately inhibiting, for example, the growth and division of cancer cells.

[0164] Therefore, in one or more exemplary embodiments, this disclosure describes the use of purine analogs for treating bacterial infections, preferably streptococcal infections.

[0165] In one or more exemplary embodiments, the purine analogues according to this disclosure may be selected from the group consisting of mercaptopurine (6-MP) and thioguanine (6-TG).

[0166] In one or more exemplary embodiments, the purine analogue according to this disclosure is mercaptopurine (6-MP).

[0167] In one or more exemplary embodiments, the purine analogue according to this disclosure is thioguanine (6-TG).

[0168] folic acid analogues

[0169] Folic acid analogs are chemical compounds that are structurally similar to folic acid, a B vitamin essential for a variety of cellular processes including DNA, RNA, and protein synthesis.

[0170] Folic acid analogs work by inhibiting dihydrofolate reductase (DHFR), which is involved in the conversion of dihydrofolate (DHF) to tetrahydrofolate (THF). THF is a coenzyme that plays a crucial role in the transfer of one-carbon units during nucleotide synthesis. By inhibiting DHFR, folic acid analogs disrupt THF production, leading to the depletion of nucleotides required for DNA and RNA synthesis.

[0171] Therefore, in one or more exemplary embodiments, this disclosure describes the use of folic acid analogs to treat bacterial infections, preferably streptococcal infections.

[0172] One of the most well-known folic acid analogs used in cancer treatment is methotrexate.

[0173] Other folic acid analogues used in cancer treatment include pemetrexed, pralatrexate, and trimethoprim. These drugs have different mechanisms of action and can be used to target different types of cancer.

[0174] In one or more exemplary embodiments, the folic acid analogues according to this disclosure may be selected from the group consisting of methotrexate, pemetrexed, pralatrexate and trimethoprim.

[0175] In one or more exemplary embodiments, the folic acid analogue according to this disclosure is methotrexate.

[0176] In one or more exemplary embodiments, the folic acid analogue according to this disclosure is pemetrexed.

[0177] In one or more exemplary embodiments, the folic acid analogue according to this disclosure is pralatrexate.

[0178] In one or more exemplary embodiments, the folic acid analogue according to this disclosure is trimethoprim.

[0179] Fdcyd

[0180] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is Fdcyd.

[0181] Fdcyd, or 5-fluoro-2'-deoxycytidine, is a fluoropyrimidine nucleoside analog that interferes with DNA metabolism. Fdcyd is structurally similar to fluorouridine.

[0182] Therefore, in one or more exemplary embodiments, this disclosure describes the use of Fdcyd for the treatment of bacterial infections, preferably streptococcal infections.

[0183] Fumei Shuang

[0184] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is thiram.

[0185] Thiram is an organosulfur compound widely used as a fungicide and veterinary repellent. It belongs to a class of chemicals called dithiocarbamates.

[0186] Fusidic acid

[0187] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is fusidic acid.

[0188] Fusidic acid belongs to a class of antibiotics called fusidanes, which are derived from the fungus *Streptomyces clostridium*. Fusidium coccineum Fusidic acid is primarily effective against Gram-positive bacteria, including Staphylococcus aureus.

[0189] Fusidic acid works by inhibiting bacterial protein synthesis, particularly targeting proteins called elongation factor G (EF-G). EF-G is involved in the movement of ribosomes along mRNA during protein synthesis. By binding to EF-G, fusidic acid prevents protein chain elongation, ultimately inhibiting bacterial growth.

[0190] Surprisingly, fusidic acid was found to inhibit the virulence factor SCPA of Streptococcus pyogenes, although fusidic acid is known not to be an effective inhibitor of bacterial growth in this pathogen. For Streptococcus pyogenes, fusidic acid inhibits virulence rather than viability.

[0191] Ipsartan

[0192] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogens is eprosartan.

[0193] Eprosartan is an angiotensin II receptor antagonist, also known as an angiotensin receptor blocker (ARB). It is used as an antihypertensive drug to treat hypertension (hypertension). Eprosartan selectively blocks the binding of angiotensin II to its receptors, thereby inhibiting its action.

[0194] mercaptopurine

[0195] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is mercaptopurine.

[0196] Mercaptopurine, also known as 6-mercaptopurine or 6-MP, is a drug belonging to a class of drugs called purine analogues. It is an antimetabolite and immunosuppressant. Mercaptopurine is primarily used to treat certain types of cancer, particularly acute lymphoblastic leukemia (ALL).

[0197] As an antimetabolite, mercaptopurine interferes with the synthesis of DNA and RNA in rapidly dividing cells, including cancer cells. By disrupting the production of these nucleic acids, it inhibits the growth and proliferation of cancer cells, leading to their destruction.

[0198] dihydrostreptomycin

[0199] In one or more exemplary embodiments, according to this disclosure, the inhibitor of cell envelope protease (CEP) in pathogenic bacteria is dihydrostreptomycin.

[0200] Dihydrostreptomycin, also known as dihydrostreptomycin sulfate, is a class of aminoglycoside antibiotics. It originates from the bacteria *Streptomyces griseus* (…). Streptomyces griseus Dihydrostreptomycin is primarily used to treat bacterial infections.

[0201] As an aminoglycoside antibiotic, dihydrostreptomycin works by inhibiting bacterial protein synthesis. It binds to bacterial ribosomes responsible for protein synthesis and disrupts the formation of functional proteins. This leads to inhibition of bacterial growth and ultimately the death of susceptible bacteria.

[0202] Surprisingly, dihydrostreptomycin was found to inhibit the scpA virulence factor of Streptococcus pyogenes, and not only by inhibiting protein synthesis. Dihydrostreptomycin is not only an inhibitor of viability but also an inhibitor of virulence.

[0203] Dihydrostreptomycin is particularly effective against certain strains of Gram-negative bacteria, including Escherichia coli and Klebsiella pneumoniae. It is commonly used to treat infections caused by these bacteria, such as urinary tract infections, respiratory infections, and certain types of gastrointestinal infections.

[0204] Even if the pathogenic bacteria are not suppressed, the inhibitory activity against the CEP virulence factor of pathogenic streptococci may open up new treatment options for streptococcal diseases.

[0205] Pathogens

[0206] Pathogens are microorganisms that can cause disease in humans, animals, or plants. Most bacterial species are harmless and often beneficial, but others can cause infectious diseases. It is estimated that fewer than one hundred of these pathogenic species exist in the human body. In contrast, the gut microbiota, present in the digestive tract, contains thousands of species.

[0207] Pathogens are particularly adapted to and have mechanisms that overcome normal bodily defenses, thus enabling them to invade.

[0208] Typically, identification is accomplished by culturing the organism in a wide range of cultures, which can take up to 48 hours. Growth is then visually or graphically identified. The cultured organism is then subjected to various assays to observe the responses, aiding in further identification of species and strains.

[0209] These bacteria possess virulence factors such as toxins, adhesins, and capsules, enabling them to colonize and infect host tissues, leading to a range of clinical symptoms and diseases. They can cause a wide range of illnesses, from minor infections like urinary tract infections to life-threatening conditions like sepsis. Effective treatment of pathogenic infections typically involves the use of antibiotics or other antimicrobial agents.

[0210] Here are some examples of well-known pathogens: Escherichia coli (E. coli): Although most strains of E. coli are harmless or even beneficial, some pathogenic strains can cause serious gastrointestinal infections, such as food poisoning.

[0211] Staphylococcus aureus Staphylococcus aureus ( S. aureus Staphylococcus aureus (SAA) is a common bacterium that can cause a variety of infections, including skin infections, pneumonia, and bloodstream infections. Methicillin-resistant Staphylococcus aureus (MRSA) is a particularly concerning strain, as it is resistant to many antibiotics.

[0212] Enteric Salmonella: Salmonella species can cause salmonellosis, a foodborne illness characterized by symptoms such as diarrhea, fever, and painful abdominal cramps. Contaminated food, especially raw or undercooked poultry and eggs, is a common source of Salmonella infection.

[0213] Streptococcus pneumoniae: Streptococcus pneumoniae ( S. pneumoniae It is a major cause of pneumonia, meningitis, and other respiratory infections. It can also cause infections in other parts of the body, such as the bloodstream and middle ear.

[0214] Mycobacterium tuberculosisThis bacterium is the cause of tuberculosis (TB), a highly contagious respiratory disease that primarily affects the lungs but can spread to other organs. TB remains a significant global health problem.

[0215] In this context, Streptococcus ( Streptococcus Streptococcus pyogenes is an important genus, and group A streptococci (GAS), group B streptococci (GBS), group C streptococci (GCS), pyogenic streptococci, agalactococci, or dysgalactococci are all important species for developing novel inhibitors of cell membrane proteases.

[0216] These are just a few examples, and there are many other pathogens that cause a wide range of infectious diseases. It is important to note that the severity and symptoms of bacterial infections can vary widely, and appropriate medical care is usually required to combat these pathogens.

[0217] Lactic acid bacteria

[0218] Lactic acid bacteria (LAB) are a group of Gram-positive, non-spore-forming bacteria that convert sugars into lactic acid as their primary metabolic end product. They are typically characterized by their ability to produce lactic acid through the fermentation of carbohydrates, as well as their tolerance to low pH and their ability to grow under anaerobic conditions.

[0219] In this context, LAB is intentionally added to milk to initiate the desired fermentation under controlled conditions. As the bacterial culture grows, it converts lactose into lactic acid and other organic acids, lowering the pH of the milk. Significant differences have been detected between different LAB species and strains in their ability to lower pH, and these differences become more pronounced during cultivation.

[0220] The following are some key characteristics and features of lactic acid bacteria: Fermentation: Lactic acid bacteria are known for their ability to ferment sugars. They convert carbohydrates such as glucose and lactose into lactic acid through anaerobic metabolism. This fermentation process produces the characteristic sour taste and acidic environment associated with fermented foods.

[0221] Acid tolerance: Lactic acid bacteria are adapted to thrive in acidic environments. They can tolerate and even thrive in low pH levels, which helps them outperform other microorganisms that cannot survive in such acidic conditions.

[0222] Probiotic Potential: Certain strains of lactic acid bacteria, such as Lactobacillus and Bifidobacterium, have been extensively studied for their probiotic properties. Probiotics are live microorganisms that, when ingested in sufficient quantities, can confer health benefits to the host. These LAB strains are commonly used in the production of fermented dairy products, such as yogurt and kefir, as well as dietary supplements.

[0223] Food fermentation: LABs are essential in the production of various fermented foods. They play a crucial role in sourdough fermentation, sauerkraut production, cheese making, and pickling. The lactic acid produced by these bacteria not only contributes to flavor and preservation but also helps create an environment that inhibits the growth of harmful bacteria.

[0224] Industrial Applications: Besides food production, LAB strains have a variety of industrial applications. They are used to produce certain chemicals, pharmaceuticals, and biofuels. LAB strains can produce enzymes, extracellular polysaccharides, and antimicrobial compounds, which have applications across various industries.

[0225] In general, lactic acid bacteria are a diverse group of bacteria that play an important role in the food industry, probiotics, and other industrial applications. They help improve the flavor, texture, and preservation of fermented foods and offer potential health benefits to consumers. Many known species of lactic acid bacteria (LAB) exist.

[0226] Here is a list of some well-known LAB species: Lactobacillus acidophilus

[0227] Lactobacillus casei

[0228] Lactiplantibacillus plantarum

[0229] Lactobacillus rhamnosus

[0230] Lactobacillus delbrueckii

[0231] Lactobacillus brevis

[0232] Fermenting Lactobacillus fermentum

[0233] Lactobacillus delbrueckii subsp. bulgaricus

[0234] Lactococcus lactis

[0235] Streptococcus thermophilus

[0236] Pediococcus acidilactici

[0237] Leuconostoc mesenteroides

[0238] These are just a few examples, and many other species and strains of LAB have been identified and studied. In this context, each species in the LAB group can exhibit unique characteristics.

[0239] In one or more exemplary embodiments, lactic acid bacteria (LAB) belong to the family Carnobacteriaceae (…). Carnobacteriaceae ), Enterococci ( Enterococcaceae Lactobacillus family ( Lactobacillaceae ) 、 Leuconostocaceae ( Leuconostocaceae ) or Streptococcus ( Streptococcaceae (members of)

[0240] In one or more exemplary embodiments, lactic acid bacteria (LAB) are non-pathogenic.

[0241] CEP enzymes, belonging to the subtilis protease-like serine protease family, are present in any lactic acid bacteria belonging to the following genera: *Lactobacillus* ( Lacticaseibacillus Lactobacillus spp. Lactiplantibacillus Lactobacillus spp. Lactobacillus Leuconostoc ( ) Leuconostoc ), genus *Sacchariformis* Oenococcus ), Pediococcus ( Pediococcus ), Lactobacillus schleiferus ( Schleiferilactobacillus ), Weissella spp. Weissella Lactococcus spp. Lactococcus ) and Streptococcus spp. Streptococcus ).

[0242] Therefore, in one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genera *Lactobacillus*, *Lactobacillus*, *Lactobacillus*, *Leuconostoc*, *Sacchariformis*, *Pediococcus*, *Lactobacillus schleifer*, *Weissella*, *Lactococcus*, or *Streptococcus*.

[0243] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genera Lactobacillus, Lactococcus, or Streptococcus.

[0244] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus Lactobacillus.

[0245] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus *Lactobacillus*.

[0246] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus Lactobacillus.

[0247] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus Leuconostoc.

[0248] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus *Sacchariformis*.

[0249] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus Pediococcus.

[0250] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus *Lactobacillus schleifer*.

[0251] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus Weissella.

[0252] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus *Lactococcus*.

[0253] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure are selected from the genus Streptococcus.

[0254] In one or more exemplary embodiments, the lactic acid bacteria strain is Lactococcus lactis.

[0255] In one or more exemplary embodiments, the lactic acid bacteria strain is *Lactococcus lactis*.

[0256] In one or more exemplary embodiments, the lactic acid bacteria strain is Streptococcus thermophilus.

[0257] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to this disclosure do not include the order Bifidobacteria.

[0258] Lactococcus lactis

[0259] Lactococcus lactis is a lactic acid bacteria used in the production of the largest volumes of cheese.

[0260] It is also used to produce buttermilk and a variety of fermented dairy products.

[0261] In one or more exemplary embodiments, the lactic acid bacteria (LAB) used in the methods of this disclosure are lactococci.

[0262] In one or more exemplary embodiments, the lactic acid bacteria (LAB) used in the methods of this disclosure are protein-hydrolyzed lactococcus strains (WG2G3).

[0263] Streptococcus thermophilus

[0264] Streptococcus thermophilus is used in the production of yogurt, Icelandic yogurt, mozzarella, and several other types of cheese. Streptococcus thermophilus is the most widely produced LAB strain.

[0265] Streptococcus thermophilus is expected to be important for the production of plant-based fermented foods.

[0266] In one or more exemplary embodiments, the lactic acid bacteria (LAB) used in the methods of this disclosure are Streptococcus thermophilus.

[0267] Fermentable substrate

[0268] Fermentable substrates are carbohydrates or other organic compounds that can be metabolized by microorganisms through fermentation to produce energy and / or metabolic end products (such as alcohols, lactic acid, and carbon dioxide). Fermentable substrates can be derived from a variety of sources, including plant, animal, and microbial sources, and can be found in a wide range of food and feed ingredients.

[0269] Examples of fermentable substrates include monosaccharides such as glucose and fructose, as well as more complex carbohydrates such as starch, cellulose, and hemicellulose.

[0270] In one or more exemplary embodiments, the fermentable substrate is a milk substrate.

[0271] In this context, the term "milk substrate" can refer to any raw milk and / or processed milk material that can undergo fermentation. Therefore, useful milk substrates include, but are not limited to, solutions / suspensions of any milk or milk-like product containing proteins, such as whole or low-fat milk, skim milk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, lactose crystal mother liquor, whey protein concentrate, or cream.

[0272] Obviously, the milk substrate can be derived from any mammal, such as substantially pure mammalian milk or reconstituted milk powder. Preferably, at least a portion of the proteins in the milk substrate are proteins naturally present in milk, such as casein or whey protein. However, some proteins may not be naturally present in milk. Prior to fermentation, the milk substrate can be homogenized and pasteurized according to methods known in the art.

[0273] The term "milk" is understood to refer to the milk secretion obtained by milking any mammal, such as a cow, sheep, goat, buffalo, or camel. In a preferred embodiment, milk is cow's milk. The term milk also includes soy milk. Optionally, milk is fortified with nutrients or agents, such as by adding acids (e.g., formic acid, citric acid, acetic acid, or lactic acid), purines, pyrimidines, amino acids, or by mixing with water, for example. Milk can be raw or processed, for example by filtration, sterilization, pasteurization, homogenization, etc., or it can be reconstituted dry milk. According to the invention, a key example of "cow's milk" is pasteurized cow's milk. It is understood that milk may be acidified, mixed, or processed before, during, and / or after inoculation with bacteria.

[0274] In one or more exemplary embodiments, the fermentable substrate is a culture medium containing proteins as a nitrogen source and sugars as a carbon source.

[0275] In one or more exemplary embodiments, the fermentable substrate is selected from the group consisting of soy milk, pea milk, almond milk, or other plant milk.

[0276] In one or more exemplary embodiments, the fermentable substrate is skim milk.

[0277] In one or more exemplary embodiments, the fermentable substrate is a milk substrate with 1% glucose added.

[0278] If the bacteria used in the screening process are capable of metabolizing carbohydrates, other carbohydrates can be used. In this case, sucrose, fructose, lactose, and galactose can be relevant carbohydrates to use.

[0279] Therefore, in one or more exemplary embodiments, the fermentable substrate is or contains sucrose, fructose, lactose or galactose.

[0280] Therefore, in one or more exemplary embodiments, the fermentable substrate contains nutrients, such as, but not limited to, purines or pyrimidines.

[0281] Therefore, in one or more exemplary embodiments, the fermentable substrate contains formic acid or sodium formate.

[0282] Therefore, in one or more exemplary embodiments, the fermentable substrate contains a protein as the N source.

[0283] Therefore, in one or more exemplary embodiments, the fermentable substrate contains peptides.

[0284] pH indicator

[0285] pH indicators are substances that exhibit different colors or spectral absorption based on the acidity or alkalinity of the solution in which they are dissolved.

[0286] pH indicators are typically weak acids or weak bases that respond to changes in pH by undergoing reversible changes in their molecular structure or electronic properties. pH indicators function based on the acid-base chemistry principle. When a pH indicator is added to a solution, the presence of hydrogen ions (H+) or hydroxide ions (OH-) causes a shift in the indicator's equilibrium, resulting in a color change.

[0287] The color change of a pH indicator occurs within a specific pH range, known as the indicator's "transition range." Different pH indicators have different transition ranges, meaning they change color at different pH values. Some indicators are better suited for acidic solutions, while others are better suited for alkaline solutions.

[0288] pH indicators are commonly used in laboratory experiments and industrial processes to monitor pH changes in solutions, and are also used in a variety of applications such as water quality testing, food and beverage production, and medical diagnostics.

[0289] The most common examples of pH indicators include: Litmus: Litmus paper or solution is one of the oldest and most widely used pH indicators. It turns red under acidic conditions (pH below 7) and blue under alkaline conditions (pH above 7).

[0290] Phenolphthalein: Phenolphthalein is colorless in acidic solutions and turns pink in alkaline solutions (pH above 8.2).

[0291] Bromothymol blue: Bromothymol blue is yellow in acidic solutions (pH below 6) and blue in alkaline solutions (pH above 7.6).

[0292] Methyl orange: Methyl orange is red in acidic solutions (pH below 3.1) and yellow in alkaline solutions (pH above 4.4).

[0293] Universal indicators: Universal indicators are mixtures of several different pH indicators that cover a wide range of pH values. They change color across the entire pH scale, displaying a range of colors to indicate the pH of a solution.

[0294] pH indicators are commonly used in a variety of scientific, educational, and industrial applications to determine the pH of solutions, test water quality, monitor chemical reactions, and more. They provide a simple and visual way to assess the acidity or alkalinity of substances.

[0295] There are many known pH indicators, each with its own specific pH range and color change. Below is a list of some commonly used pH indicators: Litmus: Blue (alkaline) to red (acidic) Phenolphthalein: Colorless (acidic) to pink (basic) Bromothymol Blue: Yellow (acidic) to blue (basic) Methyl orange: Red (acidic) to yellow (basic) Congo Red: Blue (acidic) to red (basic) Thymol Blue: Red (acidic) to yellow (basic) Bromocresol purple: Yellow (acidic) to purple (basic) Bromophenol blue: Yellow (acidic) to blue (basic) Alizarin Yellow R: Yellow (acidic) to red (alkaline) Cresol red: Yellow (acidic) to red (basic) Neutral Red: Red (acidic) to yellow (basic) Thymolphthalein: Colorless (acidic) to blue (basic) Malachite green: Green (acidic) to colorless (alkaline) Rosehip acid: Yellow (acidic) to red (alkaline) Naphtholphthalein: Colorless (acidic) to pink (basic) Nitrozine Yellow: Yellow (acidic) to blue (alkaline) Thymol purple: Red (acidic) to yellow (alkaline) Cresolphthalein: Colorless (acidic) to purple (basic) Xylenol Orange: Yellow (acidic) to red (basic) Cresol red-methyl red: Red (acidic) to yellow (basic) Please note that this is not an exhaustive list, as many other pH indicators exist with different pH ranges and color changes. The choice of pH indicator depends on the specific pH range being measured and the color change required for visual detection.

[0296] pH changes throughout fermentation are typically measured by immersing a glass electrode in the fermentation medium (e.g., a defined medium or milk), but this method is time-consuming and does not allow for rapid screening of numerous strains. Colorimetric procedures may be an alternative because they can be performed quickly by simply mixing reagents and using simple, low-cost instruments.

[0297] In one or more exemplary embodiments, the pH indicator is selected from the group consisting of bromocresol purple and bromocresol green.

[0298] In one or more exemplary embodiments, the pH indicator is selected from the group consisting of phenolphthalein, bromothymol blue, and litmus.

[0299] In one or more exemplary embodiments, the pH indicator is a combination of bromocresol purple and bromocresol green.

[0300] Targeted inhibitors

[0301] In one or more exemplary embodiments, the target inhibitor is a compound that is part of a screening library.

[0302] Compound libraries can be obtained from various sources. One source is the Broad Institute, which provides a reuse library with thousands of compounds, as well as larger libraries. In Europe, there is an initiative called the "EU Open Screen," which is dedicated to creating and distributing large compound libraries for drug screening purposes.

[0303] In one or more exemplary embodiments, the target inhibitor is a compound that is part of the EU openscreen screening library.

[0304] In one or more exemplary embodiments, the targeted inhibitor is a compound that is part of the Broad Institute's screening library.

[0305] Monitoring acidification

[0306] Acidity of a solution can be monitored by measuring its pH over time. Several methods exist for monitoring solution acidity: pH meter: The most accurate and precise method is to use a pH meter. A pH meter consists of a probe inserted into the solution, which measures the voltage generated by hydrogen ions in the solution. The pH meter then converts the voltage into a pH value, providing real-time monitoring of the acidification process.

[0307] pH indicator strips: pH indicator strips, or test strips, are simple and cost-effective tools. These strips contain pH-sensitive chemicals that change color according to the pH of the solution. By immersing the strip in the solution and comparing the resulting color to a colorimetric table, the approximate pH of the solution can be estimated. However, it is important to note that pH indicator strips provide measurements that are less accurate than a pH meter.

[0308] pH test kits: pH test kits typically include a pH indicator solution or test strip and a colorimetric table. The pH is determined by matching the resulting color change to the table, either by adding a few drops of the indicator solution to the solution being monitored or by immersing the strip in the solution.

[0309] Electronic pH probes: Similar to pH meters, electronic pH probes can be used to monitor the acidification of solutions. These probes are connected to a data logger or other electronic device that provides real-time pH measurement and data logging capabilities.

[0310] Regardless of the method chosen, it is important to ensure proper calibration and follow the manufacturer's instructions to obtain accurate and reliable pH measurements. Monitoring solution acidification is crucial in various fields, including research, industrial processes, environmental monitoring, and quality control in food and beverage production.

[0311] In one or more exemplary embodiments, pH is monitored using a pH indicator as described above.

[0312] Flatbed scanner

[0313] In one or more exemplary embodiments, pH is monitored by using a flatbed scanner to monitor the color of the milk.

[0314] In one or more exemplary embodiments, acidification monitoring is performed using a flatbed scanner.

[0315] The advantage of using a plate scanner is that it allows the use of turbid culture media. Protein-containing media that serve as nitrogen sources are typically turbid. Other methods can be used to track bacterial growth.

[0316] pH MultiScan software

[0317] In one or more exemplary embodiments, scans are collected and analyzed by pH MultiScan software from HNH.

[0318] The software that controls the flatbed scanner and captures the color of individual holes may be obtained from other sources, or it may be designed by someone skilled in software development.

[0319] If bacterial growth is detected using methods other than acidification, alternative software must be developed to capture the data.

[0320] Add amino acid source

[0321] Using a simple acidification assay will allow for screening a library of potential inhibitors to suppress acidification. If cultures are supplied with a mixture of amino acids or an amino acid source, cultures inhibited solely by protease inhibition will be able to resume growth and complete acidification, while cultures inhibited for other reasons will remain inhibited.

[0322] In this context, the term "amino acid source" refers to any group of mixtures, blends, supplements, compounds, or nutrient sources that provide sufficient amounts of amino acids.

[0323] These amino acid sources typically contain concentrated or isolated forms of amino acids derived from various protein sources. These sources also refer to combinations or blends of different amino acids. Amino acid mixtures are commonly used in cell culture media to provide the nutrients necessary for cell growth and protein synthesis.

[0324] A mixture of several commercially available amino acids is often used in cell culture media to provide the nutrients necessary for cell growth and protein synthesis. Here are some examples: Eagle's Minimum Essential Medium (MEM) Amino Acid Solution: This mixture contains a balanced combination of all 20 standard amino acids and is often used as a supplement to basal cell culture media.

[0325] Dalberg Modified Eagle Medium (DMEM) Amino Acid Solution: Similar to MEM, this mixture provides a balanced combination of all 20 standard amino acids and is commonly used in cell culture applications.

[0326] RPMI 1640 Amino Acid Solution: RPMI 1640 is a widely used cell culture medium, and its specially designed amino acid solution provides essential amino acids to support the growth of various cell types.

[0327] Ham's F-12 Nutrient Mixture Amino Acid Solution: Ham's F-12 is another commonly used cell culture medium, and its amino acid solution is customized to support the nutritional needs of a variety of cell lines.

[0328] Leibovitz's L-15 medium amino acid solution: Leibovitz's L-15 medium is used for specialized cell culture applications, and the corresponding amino acid solution provides the essential amino acids required for cell growth.

[0329] These are just a few examples of amino acid mixtures used in cell culture media. It is important to note that different cell types and specific research applications may require different formulations or modifications to these mixtures to meet their specific nutritional needs.

[0330] In one or more exemplary embodiments, the amino acid source may be a mixture of amino acids.

[0331] In one or more exemplary embodiments, the amino acid source may be hydrolyzed β-casein.

[0332] In one or more exemplary embodiments, the amino acid source may be hydrolyzed casein.

[0333] Mixtures of individual amino acids or any protein hydrolysate can be used for this part of the screening. Protein hydrolysates can be prepared by acid hydrolysis or by enzymatic hydrolysis.

[0334] Complete acidification and reach a pH below 4.6.

[0335] As a technician would understand, this method has three possible outcomes: 1. The acidification curve first descends to a minimum value (max), which is determined by the lowest pH reached by the same non-proteolytic strain. For Lactococcus lactis fermentation in high-quality skim milk, this was determined to be pH 5.8. Once the medium is further supplied with amino acids, it descends to a minimum pH of 4.4. This indicates that CEP is inhibited by a compound, and therefore this compound could be a potential inhibitor. In the following examples, this is achieved, for example, by Lactococcus lactis WG2 PrtP.

[0336] 2. The acidification curve decreased to a minimum pH of 4.4. Once the medium was further supplied with amino acids, the pH remained at 4.4. This indicates that CEP was not inhibited, therefore this compound is not suitable as an inhibitor.

[0337] 3. The acidification curves showed no pH decrease before or after amino acid supplementation. Therefore, this compound kills bacteria and is not of interest.

[0338] In one or more exemplary embodiments, the inhibitor of interest is a target inhibitor capable of achieving acidification and reaching a pH below 4.6.

[0339] Verification Measurement

[0340] In one or more exemplary embodiments, the method includes subjecting a target molecule identified as an inhibitor to a validation assay, wherein the validation assay is configured to validate inhibition of cell envelope protease (CEP) in pathogens.

[0341] In one or more exemplary embodiments, the validation assay is a protease assay using a substrate selected from the group consisting of mammalian chemokines, human peptide C5a, human peptide C3a, mammalian chemokines of the CXC family, or other mammalian immune peptides.

[0342] In one or more exemplary embodiments, the validation assay uses gel electrophoresis to verify the presence and size of the substrate.

[0343] The activity of CEP enzymes can be detected by the disappearance of substrate molecules, and may also be detected by the appearance of truncated molecules resulting from the breaking of targeted peptide bonds in the substrate molecule.

[0344] In one or more exemplary embodiments, the validation assay is HPLC. HPLC can detect substrate and product molecules.

[0345] In one or more exemplary embodiments, the verification assay is based on fluorescence and / or fluorescence quenching.

[0346] In one or more exemplary embodiments, the validation assay is based on gel electrophoresis. Substrate and product molecules can be detected by gel electrophoresis.

[0347] Validated inhibitors

[0348] As shown in the following examples and figures, and as described above, this method has identified a range of inhibitors of cell envelope proteases (CEPs) in pathogens.

[0349] Twenty compounds were identified as PrtP inhibitors. These twenty compounds are: acevecin, alexicin, 8-azaguanine, dihydrostreptomycin, fdcyd, fluorouridine, hygromycin B, colorless methylene blue, mercaptopurine, methylene blue, napakacin, nifurazolidone, amidazole, nitrofurantoin, NSC-663284, oteninidine, Ro-08-2750, tropane, walrycin-B, and YM-155.

[0350] These validated inhibitors include alexidin, methylene blue, fluorouridine, mercaptopurine, and dihydrostreptomycin.

[0351] General

[0352] Any features and / or aspects discussed above relating to the compounds according to the invention are similarly applicable to the methods described herein.

[0353] The following figures and embodiments are provided to illustrate the present invention. They are for illustrative purposes only and should not be construed as limiting in any way. Attached Figure Description

[0354] Figure 1

[0355] Acidification curves of cultures containing specific potential inhibitors. Most compounds do not interfere with milk acidification. Some compounds completely inhibit acidification, and some show moderate inhibition. The x-axis shows the time from the start of fermentation, ranging from 0 to 1152 minutes. The y-axis shows the pH range from 4.0 to 6.7.

[0356] Figure 2

[0357] After adding additional amino acids to each well, some of the previously inhibited cultures were found to be able to complete acidification and reach a pH of 4.6 or below. The x-axis shows the time since the addition of amino acids, ranging from 0 to 1152 minutes. The y-axis shows the pH range from 3.8 to 6.4.

[0358] Figure 3

[0359] Secondary acidification curves for 13 compounds showed reversible inhibition of amino acids (aa). The x-axis shows the time in minutes since the addition of the amino acid. The time range is 0 to 1200 minutes. The y-axis shows the pH range of 4.1 to 6.6.

[0360] Figure 4

[0361] CEP was inhibited by dihydrostreptomycin. In amino acid-supplemented milk, acidification proceeded at a normal rate, and the pH of both strains reached 4.3 in less than 10 hours. In the absence of supplementation, both strains were inhibited by dihydrostreptomycin, and the two proteases were inhibited to different degrees. WG2 CEP appeared to be completely inhibited, while SK11 CEP was only partially inhibited. The X-axis shows the time in minutes from the start of fermentation, ranging from 0 to 1200 minutes. The Y-axis shows the pH range from 4.2 to 6.7.

[0362] Figure 5

[0363] CEP was inhibited by fluorouracil. In amino acid-supplemented milk, both strains acidified to pH 4.4 within 10 hours, while CEP was completely inhibited by fluorouracil, and the pH did not fall below 5.2 within 20 hours. The X-axis shows the time in minutes from the start of fermentation, ranging from 0 to 1200 minutes. The Y-axis shows the pH range from 4.2 to 6.7.

[0364] Figure 6

[0365] ScpA assay

[0366] Dr. Kagawa in Dr. Cooney's laboratory at the University of Limerick used a gel migration assay to determine the SCPA enzyme in Streptococcus pyogenes.

[0367] The SCPA assay shown in the figure demonstrates the inhibitory effects of four compounds. The four inhibitors are: I3 fluorouracil I8 Fumei Shuang I11 Fusidic acid I12 Iprosartan Figure 7 ScpA assay Gel migration assays can be used to determine the activity of the SCPA enzyme. The results showed that six compounds were inhibitors of SCPA, and three compounds were partial inhibitors.

[0368] The six inhibitors are: fluorouridine, ferram, fusidic acid, eprosartan, mercaptopurine, and dihydrostreptomycin.

[0369] Some of the inhibitory compounds are: carbenicillin, bronopol, and cetyltrimethylammonium.

[0370] Figure 8

[0371] Steps for identifying SCPA inhibitors.

[0372] 1. Select an enzyme with a structure similar to SCPA but capable of conferring a host bacterial phenotype. An example is PrtP, which provides an acidified phenotype in lactococci in milk.

[0373] 2. Screening platform for assembled phenotypes.

[0374] 3. Prepare a compound library in a porous form and add the inoculated indicator culture medium into the wells.

[0375] 4. Identify inhibitors of the selected model CEP (PrtP in this example).

[0376] 5. Use real SCPA enzyme to perform enzyme activity assays to identify SCPA inhibitors.

[0377] Figure 9

[0378] Acidification profiles of uninhibited cultures of *Lactococcus lactis* strains MS22422 and MS22425 in milk and milk supplemented with hydrolyzed casein. Both strains exhibited similar acidification kinetics, and both strains grew faster in milk supplemented with hydrolyzed casein.

[0379] Both strains required approximately 650 minutes to reach pH 5.5 in unfortified skim milk, while in milk fortified with hydrolyzed casein they reached pH 5.5 in approximately 410 minutes. The mean standard deviation of the time to reach pH 5.5 was found to be 10.6 minutes, and this value was used as a scale for the acidification delay of the inhibitory compounds. The X-axis shows the time in minutes from the start of fermentation, ranging from 0 to 1200 minutes. The y-axis shows the pH range from 3.5 to 7.0.

[0380] Figure 10

[0381] Using two different *Lactococcus lactis* strains, MS22422 and MS22426, the differences in delays (measured as standard deviations) determined for eight different doses of each compound in unfortified and fortified milk were calculated. MS22422 carries a truncated version of the prtP gene from strain SK11, while MS22426 carries a similar prtP gene from strain Wg2. The figure shows a graph of 5 of the 20 identified PrtP inhibitors.

[0382] Figure 11

[0383] ScpA assays were performed on 39 compounds selected in Example 3. The substrate cleavage reaction was carried out at 37°C for 35 minutes, and the reaction was terminated by adding SDS-PAGE loading buffer and heating at 100°C for 30 seconds. The extent of substrate cleavage was analyzed by SDS-PAGE using a 16% gel. For comparison, separate control reactions for C5a and control reactions for ScpA activity in the absence of any compounds were performed in parallel. Figure 11 The samples were labeled "hC5a" and "ScpA". Furthermore, prior to the lysis reaction, 10 µL of ScpA was pre-cultured in 0.1 µL of DMSO (1% DMSO). Figure 11 The effect of DMSO was examined using "ScpADMSO". Two replicate assays have been completed to date. Figure 11 The compounds that exhibit near-complete inhibition of SCPA are indicated by asterisks in Table 1.

[0384] Table 1: Compounds tested in the SCPA inhibition assay.

[0385]

[0386] Example

[0387] Example 1 - Library Screening

[0388] This idea has been put into practice by screening a library of 3,300 compounds from the Broad Institute. The library was received in 384-well plates, with each well containing 1 nanomolar of compound.

[0389] Add 55 μL of inoculated milk to each well. The milk is inoculated with a recombinant strain of *Lactococcus lactis* carrying a truncated version of CEP, PrtP, from strain WG2. WG2G3 The milk also contained 1% glucose and two pH indicators. Plates were incubated at 30°C, and a plate scanner was used to monitor the milk's color to track acidification changes. Scans were collected and analyzed using pH MultiScan software from HNH.

[0390] Figure 8 The settings were displayed, and Figure 1 The acidification curves from the screening of the first 1500 compounds are shown. Figure 2 The pH change was shown on the second day after the addition of the extra amino acid.

[0391] After screening the entire library, 12 compounds were identified that showed that the inhibitory effect could be reversed by adding amino acids. Figure 3 The secondary acidification curves of these 12 compounds are shown.

[0392] A library of 46 compounds was selected based on initial screening. This library contains 12 compounds identified as having reversible inhibition of amino acids (aa), and 34 compounds showing a time lag of more than 2.5 SD in reaching pH 5.5.

[0393] The reason for including these partial inhibitors is to investigate whether partial inhibition of the CEP enzyme would cause a delay in the inhibition of any inhibitors.

[0394] A curated library containing 46 selected compounds was ordered from the Broad Institute, and screening was performed using two different but closely related enzymes. The two enzymes are PrtP. WG2G3 and PrtP SK11G3 The strains were expressed under the same background of *Lactococcus lactis* MG1363, therefore, the two strains were identical except for the *prtP* gene. In the secondary selection, fermentation in milk supplemented with amino acids was performed in parallel with fermentation in milk without supplementation. Figure 4 and Figure 5 The figure shows the acidification curves of the two compounds.

[0395] Example 2 - Materials and Methods Setup

[0396] Material

[0397] o Protein-hydrolyzed Lactococcus lactis strain (WG2G3)

[0398] o Bromocresol purple

[0399] o Bromocresol Green

[0400] o NaOH

[0401] o MiliQ Water

[0402] o β-hydrolyzed casein (casein hydrolysate CAS No. 65072-00-6)

[0403] o glucose

[0404] o M17 enrichment medium

[0405] o Erythromycin

[0406] o Skim milk

[0407] o 0.22um filter

[0408] o 50mL syringe

[0409] o Flatbed scanner

[0410] o Computer

[0411] pH screening software

[0412] o 100mL graduated measuring cylinder

[0413] pH meter

[0414] o Paraffin film

[0415] o 100uL suction head

[0416] o 5ml suction head

[0417] o 1250ul suction head

[0418] o 1000ul pipette

[0419] o 24-line 100ul Eppendorf automatic pipette

[0420] o 5ml pipette

[0421] method

[0422] The chemical library was assigned to wells, where compounds were set together with milk, lactococcus lactis inoculum and pH indicators (bromocresol purple and bromocresol green at pH 7).

[0423] Once the wells are filled, incubate them at 30°C on top of a flatbed scanner and read the bottom of the wells every 5 minutes for at least 18 hours.

[0424] In this way, pH-dependent color changes were recorded, allowing the software to create acidification profiles. After the first round of 18-hour incubation and reading, each well was inoculated with 1.75% hydrolyzed casein, suitable for direct addition to Lactococcus lactis. Another round of incubation was then started under the same parameters, and pH response was observed.

[0425] Experimental steps

[0426] The experiment will be conducted on a flow test bench to maintain a sterile environment as much as possible.

[0427] The experiment will be conducted following these steps: 1. A bottle containing 100 ml of boiled skim milk obtained by heating at 99°C for 30 minutes.

[0428] 2. Take 5 mL of milk and add 5 mL of pH indicator.

[0429] 3. Take 5 mL of milk and add 5 mL of 20% glucose solution (this will give you a glucose solution with a total concentration of 1%).

[0430] 4. Take 1000 μl of milk and inoculate it with 1000 μl of frozen inoculum to obtain a 1% bacterial concentration. The following strains will be used: o MS22426: Lactococcus lactis WG2G3(Pil23) M17 enriched with 1% glucose and 2.5 mg / ml erythromycin (This means that two equal portions of each strain will be taken from the refrigerator.) 5. Then add 50 μl of each inoculation milk to a 384-microtiter plate.

[0431] 6. Cover the microtiter plate with a paraffin film and its cap, then place it on a flatbed scanner. Next, turn on the incubator at 30°C and program the experiment.

[0432] 7. The program ran for 19 hours.

[0433] 8. On the second day, uncover the 384-micro titration plate and add 20 microliters of 1.75% hydrolyzed casein solution to each well.

[0434] (This yields 70 microliters of hydrolyzed casein at a concentration of 0.5%)

[0435] 9. A photo of the shooting board.

[0436] 10. Cover the microtiter plate again with the paraffin film and its cap, and run the procedure again overnight.

[0437] 11. The next day, observe the appearance of the microtiter plate, take a picture, and freeze it at -25°C.

[0438] 12. Export and save the data, and then analyze it.

[0439] Reagent preparation

[0440] Boiled skim milk

[0441] Follow these steps: 1. Purchase one liter of skim milk.

[0442] 2. Preheat the water in the laboratory water bath to 99°C.

[0443] 3. Divide 1 liter into 8 or 9 sterile glass vials of 100 ml each (or any other volume of your choice).

[0444] 4. Do not tighten the bottle cap completely.

[0445] 5. Carefully place the bottle into hot water.

[0446] 6. Wait 30 minutes.

[0447] 7. When removing the bottle, tighten the cap.

[0448] 8. Store at 5°C.

[0449] pH indicator

[0450] Inside the evacuation chamber, the following steps will be followed: 1. Add 80ml of miliQ water to a 100ml sterile glass bottle.

[0451] 2. Weigh 100 mg of bromocresol purple and add it to the bottle.

[0452] 3. Weigh 100 mg of bromocresol green and add it to the bottle.

[0453] 4. Mix thoroughly.

[0454] 5. Measure pH.

[0455] 6. Adjust the pH to 7 using NaOH.

[0456] 7. Adjust the volume to 100mL.

[0457] 8. Using a 50 mL syringe and a 0.22 μL filter, filter the solution into another sterile bottle.

[0458] 9. Affix the correct label, including concentration and pH.

[0459] 10. Store at 5°C.

[0460] 1.75% hydrolyzed casein

[0461] To prepare this solution, the following steps will be followed: 1. Add 80 mL of miliQ water to a sterile glass bottle.

[0462] 2. Weigh 1.75 grams of hydrolyzed casein and add it to the bottle.

[0463] 3. Adjust the volume to 100mL.

[0464] 4. Ensure all substances are dissolved.

[0465] 5. Add the correct labels indicating concentration, date, and name.

[0466] Double the dosage to ensure sufficient β-casein; in this case, the steps are as follows: 1. Add 180 mL of miliQ water to a sterile glass bottle.

[0467] 2. Weigh 3.5 grams of β-hydrolyzed casein and add it to the bottle.

[0468] 3. Adjust the volume to 200mL.

[0469] 4. Ensure all substances are dissolved.

[0470] 5. Add the correct labels indicating concentration, date, and name.

[0471] 20% glucose

[0472] To prepare this solution, the following steps will be followed: 1. Add 80 mL of miliQ water to a sterile glass bottle.

[0473] 2. Add 20g of glucose to the bottle.

[0474] 3. Adjust the volume to 100mL.

[0475] 4. Using a 50mL sterile syringe and a 0.22µm filter, filter the solution into another sterile glass vial.

[0476] 5. Add the correct labels indicating concentration, date, and name.

[0477] Example 3 - Screening of the complete REPO library

[0478] The complete REPO compound library, consisting of 6,808 drugs or drug candidates in development, from the Broad Institute, was screened using the same process as described in Example 1. This REPO library comprises all three libraries: REPO1, REPO2, and REPO3.

[0479] Add 55 μL of inoculated milk to each well. The milk is inoculated with a recombinant strain of *Lactococcus lactis* carrying a truncated version of CEP, PrtP, from strain WG2. WG2G3 The milk also contained 1% glucose and two pH indicators. Plates were incubated at 30°C, and a plate scanner was used to monitor the milk's color to track acidification changes. Scans were collected and analyzed using pH MultiScan software from HNH.

[0480] After the first round of 18-hour incubation and reading, each well was inoculated with hydrolyzed casein, and pH changes were then tracked.

[0481] A selection of 39 compounds was ordered from the Broad Institute. The compounds are listed in Table 2.

[0482]

[0483] Each compound was tested at eight concentrations (concentrations in wells were diluted 2-fold from 1 nmol to 0.008 nmol).

[0484] A selection plate was used to screen for proteolytic strains of two different *Lactococcus fat* strains: MS22422 (SK11G3) and MS22426 (Wg2G3). Fermentation was carried out in two media: skim milk and skim milk fortified with casein hydrolysate.

[0485] In addition to eight doses for each inhibitor candidate, each plate in the selected library contains 72 wells without any compound. Data from these wells were used to determine the standard deviation of uninhibited acidification kinetics and acidification activity.

[0486] The time required to reach pH 5.5 can be used as a measure of the acidification activity of each strain in each culture medium.

[0487]

[0488] Both strains required approximately 650 minutes (11 hours) to reach pH 5.5 in milk, with MS22422 reaching it slightly faster than MS22426. In milk fortified with hydrolyzed casein, both strains reached pH 5.5 even faster, within approximately 410 minutes (6.8 hours), with MS22426 being the fastest. Figure 9 The acidification curves for the uninhibited culture are shown.

[0489] The standard deviation of the time to reach pH 5.5 was found to be between 7.5 minutes and 15.1 minutes for the four plates. There is no clear explanation for why the SDs are different, and the average SD of 10.6 minutes was used as a measure of inhibitory effect when assessing the inhibitory effect.

[0490] For each well containing a potentially inhibitory compound, the time to reach pH 5.5 was determined. The delay was calculated by subtracting the average T5.5 of uninhibited strains in the same medium and expressed as SD units by dividing the delay by 10.6. For severely inhibited strains that could not reach pH 5.5, the end time of the analysis (1080 minutes) was used as a measure of delay. Therefore, the maximum delay for milk was approximately 40 SD, and the maximum delay for fortified milk was approximately 60 SD.

[0491] Eight compounds from the selected library were found to be weak inhibitors of acidification. The weakest inhibitors found were: acelarin, BAY-11-7082, chloroxacin, eprosartan, amidazole, rifapentine, thiram, and troleandomycin.

[0492] Ten compounds were found to inhibit acidification in milk supplemented with amino acids, with the same or greater inhibition levels as in unsupplemented milk. These inhibitors must have other targets besides (or other than) PrtP inhibition.

[0493] The difference between the delay in milk and the delay in fortified milk can be used as a measure of PrtP inhibition. Δ delay (SD) was calculated for each dose of each compound, and for both strains carrying PrtP enzymes from strain Wg2 or strain SK11. Figure 10 A graph showing five PrtP inhibitors is displayed.

[0494] Twenty compounds were identified as PrtP inhibitors. These twenty compounds are: acevecin, alexicin, 8-azaguanine, dihydrostreptomycin, fdcyd, fluorouridine, hygromycin B, colorless methylene blue, mercaptopurine, methylene blue, napakacin, nifurazolidone, amidazole, nitrofurantoin, NSC-663284, oteninidine, Ro-08-2750, tropane, walrycin-B, and YM-155.

[0495] Example 4 - ScpA inhibition by PrtP inhibitory compound

[0496] The inhibition of ScpA by compounds selected from the curated library in Example 4 was determined in a simple endpoint assay. Each well contained 0.1 µL of the compound at a concentration of 10 mM in DMSO. ScpA activity was pre-tested by adding 10 µL of 10 nM ScpA (50 mM HEPES pH 7.5, 100 mM NaCl) to each compound in the plate. The plate was incubated on ice for 20 min. After 20 min, 10 µL of 11.2 µM substrate hC5a (PBS) was added to each well. The substrate lysis reaction was performed at 37 °C for 35 min, and terminated by adding SDS-PAGE loading buffer and heating at 100 °C for 30 s. The extent of substrate lysis was analyzed by SDS-PAGE using a 16% gel. For comparison, separate control reactions for C5a and control reactions for ScpA activity in the absence of any compound were performed in parallel. These were... Figure 11 The samples were labeled "hC5a" and "ScpA". Furthermore, prior to the lysis reaction, 10 µL of ScpA was pre-cultured in 0.1 µL of DMSO (1% DMSO). Figure 11 The effect of DMSO was examined using "ScpADMSO". Two replicate assays have been completed to date. Figure 11 The compounds that exhibit near-complete inhibition of SCPA are indicated by asterisks in Table 3.

[0497] Of the 20 PrtP inhibitors identified in Example 4, two compounds showed significant scpA inhibitory activity. These compounds were alexidin and methylene blue.

[0498] Table 3: Compounds tested in the SCPA inhibition assay.

[0499]

Claims

1. A method for identifying inhibitors of cell envelope protease (CEP) in pathogenic bacteria, the method comprising: a) Provide fermentable substrates containing lactic acid bacteria (LAB) strains and pH indicators; b) Add the target molecule; c) Monitor the acidification of fermentable substrates; d) Add an amino acid source to the fermentable substrate; e) Determine the pH of the fermentable substrate; and f) Based on the pH of the fermentable substrate being below 4.6, the target molecule is identified as an inhibitor.

2. The method of claim 1, wherein the method comprises subjecting a target molecule identified as an inhibitor to a validation assay, wherein the validation assay is configured to validate inhibition of cell envelope protease (CEP) in pathogens.

3. The method according to any one of claims 1 to 2, wherein the lactic acid bacteria strain is selected from the order Lactobacillus.

4. The method according to any one of claims 1 to 3, wherein the lactic acid bacteria strain is *Lactococcus lactis* (…). Lactococcus lactis ).

5. The method according to any one of claims 1 to 3, wherein the lactic acid bacteria strain is Streptococcus thermophilus (Streptococcus thermophilus). Streptococcus thermophilus ).

6. The method according to any one of claims 1 to 5, wherein the pH indicator is selected from the group consisting of bromocresol purple and bromocresol green.

7. The method according to any one of claims 1 to 6, wherein the fermentable substrate is a culture medium containing protein as a nitrogen source and sugar as a carbon source.

8. The method of claim 7, wherein the fermentable substrate is selected from the group consisting of soy milk, pea milk, almond milk or other plant milk.

9. The method according to claims 7 to 8, wherein the fermentable substrate is skim milk.

10. The method according to any one of claims 1 to 9, wherein acidification monitoring is performed by a flatbed scanner.

11. The method according to claim 1, wherein the amino acid source is selected from the group consisting of hydrolyzed casein, hydrolyzed animal protein, hydrolyzed plant protein, or a specific blend of multiple individual amino acids.

12. The method of claim 2, wherein the verification assay is a protease assay using a substrate selected from the group consisting of mammalian chemokines, human peptide C5a, human peptide C3a, mammalian chemokines of the CXC family, or other mammalian immune peptides.

13. An inhibitor identified by the method according to claims 1 to 12, for treating bacterial infections, preferably Streptococcus pyogenes (… Streptococcus pyogenes )Infect.

14. An inhibitor selected from the group consisting of azithromycin, alexiconazole, 8-azaguanine, dihydrostreptomycin, fdcyd, fluorouracil, hygromycin B, colorless methylene blue, mercaptopurine, methylene blue, napakacin, nifurazolidone, amidazole, nitrofurantoin, NSC-663284, oteninidine, Ro-08-2750, tropane, walrycin-B, and YM-155, for the treatment of bacterial infections, preferably Streptococcus pyogenes (Streptococcus pyogenes). Streptococcus pyogenes )Infect.

15. An inhibitor selected from the group consisting of alexiconazole, methylene blue, fluorouridine, mercaptopurine, and dihydrostreptomycin, used for the treatment of bacterial infections, preferably Streptococcus pyogenes (Streptococcus pyogenes). Streptococcus pyogenes )Infect.

16. Use of the inhibitor according to claims 13 to 15 as a virulence inhibitor for the treatment of streptococcal infections.

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