Inhibitors of cell envelope proteinases from lactic acid bacteria

Abstract

The present disclosure is a screening procedure allowing the identification of inhibitors of cell envelope proteases (CEP) in pathogenic bacteria by using lactic acid bacteria (LAB). The screening platform can be used to screen compound libraries, and thus generate a high throughput evaluation of drug candidates, and as exemplified below one screening of 6808 compounds has resulted in the identification of 20 compounds inhibiting the CEP of Lactococcus lactis.

Description

[0001] INHIBITORS OF CELL ENVELOPE PROTEINASES FROM LACTIC ACID BACTERIA

[0002] FIELD

[0003] The present disclosure is a screening procedure allowing the identification of inhibitors of cell envelope proteases (CEP) in pathogenic bacteria by using lactic acid bacteria (LAB). CEP enzymes can usually not be assayed using general chromogenic substrates, but the present screening platform can identify compounds capable of inhibiting cell envelope proteases (CEP) of lactic acid bacteria (LAB).

[0004] The screening platform was used to screen a compound library of 6808 compounds, and as exemplified below one screening has resulted in the identification of 20 compounds inhibiting the CEP of Lactococcus lactis.

[0005] BACKGROUND

[0006] CEP enzymes can usually not be assayed using general chromogenic substrates. Pathogenic invasive streptococci for example have very specific CEP enzymes with only one or few known substrates. These specific CEP enzymes are virulence factors inactivating chemokines of the innate immune system. Consequently, neutrophils are not recruited, and the invasive infection can progress rapidly and cause death within hours or days.

[0007] So far, there is no drugs targeting these virulence factors, for example there are no drugs targeting CEPs of group A streptococci (GAS), thus surgery is often required, if the infection has progressed.

[0008] Antibiotic treatment of GAS infections can be effective if initiated early whereas surgery is often required if the infection has progressed.

[0009] The medical need for new antibiotics is clear. Drugs able to inhibit for example proteolytic virulence factors like ScpA of Streptococcus pyogenes would prevent the bacteria’s ability to jam the immune system. Such drugs would increase the efficiency of the antibiotic treatment and slow down the progress of the infection.

[0010] Targeting virulence rather than viability has been proposed as a promising strategy for identifying future antimicrobials. This would allow the expansion of the repertoire and probably reduce the problem of development of antimicrobial resistance. Virulence factors as adhesion, toxins, and biofilm formation has been the targets for this strategy. Targeting the CEP virulence factors of Streptococcal pathogens has been proposed but due to the lack of suitable screenings procedures not reduced to practice. Thus, there is an unmet need for screening platforms that can identify potential compounds drugs with effect on pathogenic bacteria, for example by targeting the cell envelope proteases (CEP) of the pathogenic bacteria.

[0011] SUMMARY

[0012] LAB used in dairy fermentations also have CEP enzymes. The CEP of the dairy cultures allows the culture to use casein as nitrogen source. The CEP enzymes of dairy strains are also very selective in only using caseins and typically only one of the caseins, beta-casein. Also, the dairy-CEPs are difficult to assay with chromogenic substrates, and typically show only very low activity in such assays.

[0013] However, the activity of a dairy-CEP gives an easily identifiable phenotype to the bacterium - the ability to acidify deeply in milk, reaching a pH of 4.6 within 12 hours. A non-proteolytic strain will grow in milk but only reaching a lower cell density and non-proteolytic strains will not acidify below pH 5.5.

[0014] Using a simple acidification assay will allow libraries of potential inhibitors to be screened for inhibition of acidification. Cultures only inhibited due to inhibition of the protease will be able to resume growth and complete the acidification if the culture is supplied with a mixture of amino acids, whereas cultures inhibited for other reasons will stay inhibited.

[0015] As such, inhibitors of dairy-CEP enzymes may inhibit also other similar enzymes and probably inhibit the whole class of LAB-CEP, thus some inhibitors will discriminate between CEP enzymes.

[0016] In its broadest aspect, the present disclosure relates to a method for the identification of inhibitors of cell envelope proteases (CEP) in a pathogenic bacterium, the method comprising: a) providing a fermentable substrate comprising a lactic acid bacterium (LAB) strain and a pH indicator; b) adding a target molecule; c) monitoring the acidification of the fermentable substrate; d) adding a source of amino acids to the fermentable substrate; e) determining a pH of the fermentable substrate; and f) in accordance with the pH of the fermentable substrate being below 4.6, identifying the target molecule as an inhibitor. The core of this invention is to use a dairy-CEP for a high throughput screening of a large library of compounds and to use a secondary screen to identify inhibitors with activity against other CEP enzymes.

[0017] The secondary screen or verification step can be done using specific assays not suitable for high throughput screening.

[0018] Another aspect of the present disclosure relates to target inhibitors identified by the present method. The Examples section below for example show that alexidine and methylene blue can be inhibitors of cell envelope proteases (CEP) in a pathogenic bacterium.

[0019] DETAILED DESCRIPTION

[0020] The experimental data below show a screening of a library with 6808 compounds from Broad Institute. The library was received in 384 multi-well plates containing 1n mole of compounds in separate wells. A milk substrate was added to each well. The milk was inoculated with lactic acid bacteria harboring a truncated version of a CEP.

[0021] The milk also contained glucose and two pH indicators. The plates were incubated at 30°C and the evolution of acidification was followed by monitoring the color of the milk by the use of a flatbed scanner. The scans were collected and analysed by the software pH MultiScan from HNH.

[0022] The acidification curves from screening of the first 1500 compounds are shown on Figure 1 , Figure 2 shows the pH evolution on day two after the addition of extra amino acids.

[0023] The screening method

[0024] In one or more exemplary embodiments, the method for the identification of inhibitors of cell envelope proteases (CEP) in a pathogenic bacterium, the method comprising: a) providing a fermentable substrate comprising a lactic acid bacterium (LAB) strain and a pH indicator; b) adding a target molecule; c) monitoring the acidification of the fermentable substrate; d) adding a source of amino acids to the fermentable substrate; e) determining a pH of the fermentable substrate; and f) in accordance with the pH of the fermentable substrate being below 4.6, identifying the target molecule as an inhibitor. Thereby, the skilled person can identify a potential inhibitor of cell envelope proteases (CEP), as a target molecule capable of inhibiting the acidification until the fermentable substrate has been supplemented with amino acids, and after that supplementation reach a pH below 4.6.

[0025] As most target molecules do not inhibit, the pH falls for those candidates to 4.6 within the first 5-10 hours of fermentation but are not interesting candidates. Molecules neither inhibiting the cell envelope protease nor any other function needed for growth will cause no change in growth and acidification kinetics. Such non-inhibiting molecules will not be analyzed further.

[0026] Molecules showing partial inhibition of the cell envelope protease or partial inhibition of any other function needed for growth will show a delay in acidification kinetics and reach pH 4.6 with a large delay. Such molecules will be further analyzed to identify if the delay can be prevented by the addition of hydrolyzed casein indicating that the inhibited function might be the CEP enzyme. This test will have to be conducted subsequently as the acidification has already reached pH 4.6.

[0027] Molecules showing complete inhibition of the CEP enzyme will show the same growth and acidification kinetics as an isogenic non-proteolytic version of the indicator bacterium. If the reduced acidification is indeed due to inhibition of the CEP enzyme growth and acidification will resume after the addition of hydrolyzed protein to the inhibited culture.

[0028] Molecules inhibiting other functions essential for growth will neither grow nor acidify. It cannot be excluded that such molecules could also inhibit the CEP enzyme. If the number of such inhibitors are within the capacity of the verification assay this small set of molecules should be tested.

[0029] Temperature

[0030] Once the wells have been filled, we will incubate them at a temperature permissive for growth of the lactic acid bacterium used for the primary screening and follow the growth kinetics of the indicator bacterium. If the indicator bacterium is a Lactococcus lactis or Lactococcus cremoris the temperature will be in the range of 15°C to 37°C typically 30°C. If Streptococcus thermophilus or other thermophilic LAB species are used, the temperature will be in the range of 30 °C to 45°C typically 37°C, whereas the use of species belonging to the genus Leuconostoc will typically be incubated at lower temperatures, typically 25 °C. Thus, in one or more exemplary embodiments, the method for the identification of inhibitors of cell envelope proteases (CEP) in a pathogenic bacterium is a temperature permissive for growth of the lactic acid bacterium.

[0031] Thus, in one or more exemplary embodiments, the temperature is in the range of 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.

[0032] In one or more exemplary embodiments, the temperature is 37°C.

[0033] In one or more exemplary embodiments, the temperature is 30°C.

[0034] Readouts

[0035] The growth kinetics can be monitored by any method suitable to follow growth of lactic acid bacteria in a high throughput setup, such as but not limited to cell density, consumption of nutrients, production of metabolites, or acidification of the medium as markers for bacterial growth.

[0036] Turbid media as milk can interfere with some analytical procedures and reduce the available methods for growth tracking. We have followed acidification kinetics by monitoring the change of colour of a milk-based medium containing a mixture of pH indicators. The change of colour could be followed by placing the micro well plates on top of a flatbed scanner and read the bottom of the wells for example every 5 minutes during 18h.

[0037] CEP enzymes

[0038] CEP enzymes are extracellular enzymes associated to the outside surface (the cell envelope) of the bacteria. The CEP enzymes are large multi-domain enzymes. Typically, they have a length of 1000-2000 amino acids. The proteolytic domain is a serine protease with homology to the unspecific protease subtilisin. Subtilisin is highly active and unspecific, whereas CEP enzymes are selective and specific for particular substrates.

[0039] Inhibitors of the CEP of L. lactis as shown in the Examples will be useful for the development of novel starter cultures for the food industry. From the library of inhibitors of the L. lactis CEP a secondary screen has allowed the identification of inhibitors of ScpA of Streptococcus pyogenes. Such inhibitors have the potential to be developed into drugs for treatment of streptococcal infections including necrotizing fasciitis. The necrotizing fasciitis is the worst conditions. However, the relatively innocent streptococcal infections might be the most profitable business.

[0040] For some inhibitors, prophylactic use might also be considered.

[0041] CEP enzymes can usually not be assayed using general chromogenic substrates. Pathogenic invasive streptococci have very specific CEP enzymes with only one or few known substrates. These specific CEP enzymes are virulence factors inactivating chemokines of the innate immune system. Consequently, neutrophils are not recruited, and the invasive infection can progress rapidly and cause death within hours or days.

[0042] Streptococcal C5a peptidase, ScpA

[0043] In one or more exemplary embodiment, the CEP enzyme is ScpA, also known as Streptococcal C5a peptidase. ScpA is a is a subtilisin-like serine protease, containing a Ser-Asp-His catalytic triad, as well as an oxyanion hole asparagine. ScpA is known to be involved in the virulence (disease-causing ability) of Streptococcus pyogenes.

[0044] ScpA has been studied for its role in the pathogenesis of Streptococcus pyogenes infections. It has been found to contribute to the immune evasion, and modulation of the host immune response. Understanding the activity and function of ScpA can help researchers develop strategies for targeting this enzyme as a potential therapeutic target or for the development of vaccines or antibiotics against Streptococcus pyogenes infections.

[0045] In one or more exemplary embodiment, the CEP enzyme is selected from the group consisting of streptococcal CEP enzymes specific for mammalian chemotactic peptides, human peptide C5a, human peptide C3a, mammalian chemokines of the CXC family, or other mammalian immune peptides.

[0046] In one or more exemplary embodiment, the CEP enzyme is selected from the group consisting of ScpA, ScpB, ScpC (SpyCEP) from Streptococcus pyogenes.

[0047] In one or more exemplary embodiment, the CEP enzyme is selected from the group of CEP enzymes from Streptococcus pyogenes, Streptococcus agalactiae, or Streptococcus dysgalactiae shoving more than 50% homology to the ScpA enzyme of Streptococcus pyogenes.

[0048] In one or more exemplary embodiment, the CEP enzymes is from from group A streptococci (GAS). In one or more exemplary embodiment, the CEP enzymes is from from group B streptococci (GBS).

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

[0050] Cell envelope proteases (CEP) of lactic acid bacteria

[0051] Cell envelope proteases of lactic acid bacteria are a class of enzymes that are located in the lactic acid bacteria cell envelope and are involved in the degradation of proteins.

[0052] Examples of useful cell envelope proteases of lactic acid bacteria:

[0053] PrtP

[0054] PrtP: A cell wall-associated serine protease found in Lactococcus lactis that is involved in the degradation of casein proteins in milk and is important for the development of texture and flavor in cheese.

[0055] PrtS

[0056] PrtS; A cell wall-associated serine protease found in Streptococcus thermophilus that is involved in the degradation of casein proteins in milk and is important for the development of texture and flavor in cheese. PrtS is also important for degradation of soy proteins during fermentation of soy based milk analogues.

[0057] PrtB

[0058] PrtB: A cell wall-associated serine protease found in Lactobacillus delbrueckii subsp. bulgaricus that is involved in the degradation of milk proteins and is important for the development of texture and flavor in dairy products.

[0059] PrtH

[0060] PrtH: A cell wall-associated serine protease found in Lactobacillus helveticus that is involved in the degradation of casein proteins in milk and is important for the development of texture and flavor in dairy products and for the production of bioactive peptides. These are just a few examples of the many different types of cell envelope proteases found in lactic acid bacteria, each with its own specific role and substrate preferences.

[0061] CEP enzymes belonging to the family of subtilisin like serine proteases found in any lactic acid bacterium belonging to the order of Lactobacillales.

[0062] CEP enzymes belonging to the family of subtilisin like serine proteases found in any lactic acid bacterium belonging to one of the families: Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, or Streptococcaceae.

[0063] CEP enzymes belonging to the family of subtilisin like serine proteases found in any lactic acid bacterium belonging to one of the genera: Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Schleiferilactobacillus, Weissella, Lactococcus, Streptococcus.

[0064] Inhibitors of cell envelope proteases (CEP)

[0065] Inhibitors of cell envelope proteases are molecules that selectively bind to and inhibit the activity of proteases that are located in the cell envelope of bacteria. These proteases play critical roles in bacterial growth and survival, and their inhibition can disrupt key cellular processes, leading to bacterial death. Inhibitors of cell envelope proteases are therefore of great interest as potential antibacterial agents and have been the subject of extensive research in the development of new antibiotics.

[0066] In the present context, a potential inhibitor of cell envelope protease(s) in a pathogenic bacterium is a target inhibitor identified by the method according to the present disclosure.

[0067] Proteases are enzymes that catalyse the breakdown of proteins by hydrolysing peptide bonds. They play essential roles in various cellular processes, including protein degradation, signal transduction, and cell regulation. In the context of cell envelope proteases, these enzymes are typically involved in the processing and maturation of proteins important for the cell envelope's structure and function.

[0068] Inhibitors of cell envelope proteases can have different mechanisms of action. Some inhibitors may bind directly to the protease enzyme, blocking its active site and preventing substrate binding or catalytic activity. Others may modulate the protease's activity indirectly by influencing its regulatory mechanisms or affecting its interaction with other cellular components.

[0069] The use of inhibitors for cell envelope proteases can have significant implications in various fields, including medicine and biotechnology. Inhibiting specific proteases can disrupt the integrity of the cell envelope, making them potential targets for antimicrobial therapies to combat bacterial infections. Additionally, inhibitors may be employed in biotechnological applications to control or manipulate the production of specific proteins by altering protease activity.

[0070] It's important to note that the specific inhibitors and their effects can vary depending on the protease targeted and the organism or system under study.

[0071] There are several known inhibitors of cell envelope proteases. Here are a few examples:

[0072] Protease Inhibitor Cocktail: This is a mixture of multiple inhibitors targeting different proteases. It is commonly used in research laboratories to inhibit a broad range of proteases simultaneously. Examples of protease inhibitor cocktails include Complete™ and PhosSTOP™, which are commercially available.

[0073] Serine Protease Inhibitors: Serine proteases are a class of proteases that play essential roles in various cellular processes. Inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, and leupeptin can inhibit serine proteases involved in cell envelope processing.

[0074] Metalloprotease Inhibitors: Metalloproteases are proteases that require metal ions, such as zinc, for their catalytic activity. Inhibitors such as EDTA (ethylenediaminetetraacetic acid) and 1 ,10-phenanthroline can chelate metal ions and inhibit the activity of metalloproteases.

[0075] Peptidomimetic Inhibitors: These are synthetic compounds designed to mimic the structure and function of specific peptide substrates of proteases. By competitively binding to the protease active site, they can inhibit protease activity. Peptidomimetic inhibitors can be designed to target specific proteases and can have high specificity and potency.

[0076] Small-Molecule Inhibitors: Various small molecules have been identified as inhibitors of specific proteases. These molecules can be discovered through high-throughput screening or structure-based drug design approaches. Small-molecule inhibitors often target specific binding sites or allosteric sites on the protease enzyme. As disclosed in the Examples below, a chemical library was allocated to wells in which the compound is set with milk, L. lactis inoculum, and a pH indicator (Purple and green Bromocresol at pH 7). By monitoring the acidification, new inhibitors of cell envelope proteases of lactic acid bacteria were identified and verified as inhibitors of cell envelope proteases (CEP) in a pathogenic bacterium.

[0077] Thus, the present disclosure describes a range of inhibitors identified by the present screening method. In particular the screening method has identified inhibitors selected from the group consisting of acivicin, alexidine, azaguanine-8, dihydrostreptomycin, fdcyd, floxuridine, hygromycin- B, leucomethylene-blue, mercaptopurine, methylene-blue, napabucasin, nifuroxazide, nithiamide, nitrofurantoin, NSC-663284, octenidine, Ro-08-2750, tolonium, walrycin-B, YM-155.

[0078] This, in one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure can be selected from the group consisting of floxuridine, mercaptopurine, and dihydrostreptomycin.

[0079] Acivicin

[0080] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Acivicin.

[0081] Acivicin is a natural compound produced by Streptomyces sviceus. Acivicin inhibits glytamyl dependent amidotransferases and has been evaluated as a treatment for cancers. Toxicity has however prevented the use.

[0082] Thus, in one or more exemplary embodiments, the present disclosure describes Acivicin for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0083] Azaguanine-8

[0084] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Azaguanine-8.

[0085] Azaguanine-8 isa is a triazolo analog of guanine which in the 1950ies has been tested as a treatment of leukaemia, however with disappointing clinical results. Thus, in one or more exemplary embodiments, the present disclosure describes Azaguanine-8 for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0086] Hygromycin-B

[0087] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Hygromycin-B.

[0088] Hygromycin-B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus. The known mode of action of Hygromycin-B is inhibition of protein synthesis by interfering with the ribosome. Hygromycin-B is used as an anthelmintic in feed.

[0089] Thus, in one or more exemplary embodiments, the present disclosure describes Hygromycin-B for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0090] Napabucasin

[0091] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Napabucasin.

[0092] Napabucasin is Napabucasin is a natural naphthoquinone isolated from Newbouldia laevis, Ekmanianthe longiflora, and Handroanthus impetiginosus. Napabucasin is under investigation as an anti-cancer drug. Napabucasin has been identified to interfere with the signal transducer STAT3.

[0093] Thus, in one or more exemplary embodiments, the present disclosure describes Napabucasin for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0094] Nifuroxazide

[0095] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Nifuroxazide.

[0096] Nifuroxazide is a broad-spectrum antibacterial drug that for decades has been used for the treatment of infectious diarrhoea. It has recently been shown to inhibit the transcription factor STAT3. The compound is under evaluation as an anticancer drug. Thus, in one or more exemplary embodiments, the present disclosure describes Nifuroxazide for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0097] Nithiamide

[0098] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Nithiamide.

[0099] Nithiamide is an aromatic amide and a member of acetamides it is used as an antibiotic in veterinary medicine.

[0100] Thus, in one or more exemplary embodiments, the present disclosure describes Nithiamide for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0101] Nitrofurantoin

[0102] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Nitrofurantoin.

[0103] Nitrofurantoin is a nitrofuran antibiotic used for treatment of urinary tract infections since the 1950ies.

[0104] Thus, in one or more exemplary embodiments, the present disclosure describes Nitrofurantoin for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0105] NSC-663284

[0106] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is NSC-663284.

[0107] NSC-663284 (6-Chloro-7-(2-morpholin-4-yl-ethylamino)quinoline-5, 8-dione) is a phosphatase inhibitor.

[0108] Thus, in one or more exemplary embodiments, the present disclosure describes NSC-663284 for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection. Ro-08-2750

[0109] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Ro-08-2750.

[0110] 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).

[0111] Thus, in one or more exemplary embodiments, the present disclosure describes Ro-08-2750 for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0112] Tolonium

[0113] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Tolonium.

[0114] Tolonium is a blue cationic dye used in histology. The chemical structure is close to the structure of methylene blue.

[0115] Thus, in one or more exemplary embodiments, the present disclosure describes Tolonium for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0116] Walrycin-B

[0117] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Walrycin-B.

[0118] Walrycin-B (1 ,6-dimethyl-3-(4-(trifluoromethyl)phenyl)pyrimido[5,4-e][1 , 2, 4]triazine-5, 7-dione) is a compound identified as an inhibitor of the WaIR response regulator of Gram-positive bacteria.

[0119] Thus, in one or more exemplary embodiments, the present disclosure describes Walrycin-B for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection. YM-155

[0120] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is YM-155.

[0121] YM-155 (Sepantronium bromide) is an inhibitor of the survivin protein involved in apoptosis. Survivin is a member of the inhibitor of apoptosis (IAP) family.

[0122] Thus, in one or more exemplary embodiments, the present disclosure describes YM-155 for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0123] Alexidine

[0124] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is alexidine.

[0125] Alexidine is a bisguanide with broad spectrum antiseptic properties. It is use similarly to another bisguanide chlorhexidine as an irrigating solution in dental therapy.

[0126] Thus, in one or more exemplary embodiments, the present disclosure describes Alexidine for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0127] Octenidine

[0128] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Octenidine.

[0129] Octenidine is a biguanide compound with structural similarity to chlorhexidine and alexidine.

[0130] Octenidine is an antiseptic effective against Gram-positive and Gram-negative bacteria, yeasts and fungi.

[0131] Thus, in one or more exemplary embodiments, the present disclosure describes Octenidine for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection. Methylene blue

[0132] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is methylene blue.

[0133] Methylene blue is a heterocyclic aromatic compound with a long history of medical and biochemical use. It has been used as an anti-malaria treatment as well as an antidote for cyanide poisoning. Recently it has been proposed as a possible treatment of Zika virus infections through inhibition of the viral protease NS3.

[0134] Thus, in one or more exemplary embodiments, the present disclosure describes methylene blue for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0135] Leucomethylene-blue

[0136] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Leucomethylene-blue.

[0137] Leucomethylene-blue is the reduced form of methylene blue. Methylene blue and leucomethylene blue constitutes a well known redox couple.

[0138] Thus, in one or more exemplary embodiments, the present disclosure describes Leucomethylene- blue for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0139] Floxuridine

[0140] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is floxuridine.

[0141] Floxuridine, is a pyrimidine analog, and is also known by its trade name FUDR (Floxuridine for Injection). It’s presently mostly known as a chemotherapy medication used in the treatment of various types of cancer, particularly colorectal cancer. It belongs to the class of medications known as antimetabolites.

[0142] Thus, in one or more exemplary embodiments, the present disclosure describes floxuridine for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection. Antimetabolites

[0143] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is an antimetabolite.

[0144] Antimetabolites are a class of drugs used in chemotherapy that interfere with the normal metabolic processes of cells, particularly those involved in DNA and RNA synthesis. They are structurally similar to the naturally occurring molecules involved in these processes and can disrupt the function of cancer cells by acting as faulty substitutes or competitive inhibitors.

[0145] Antimetabolites work by entering cells and interfering with crucial biochemical pathways. They can inhibit the synthesis of DNA, RNA, or both, depending on the specific drug and its mechanism of action. By disrupting these processes, antimetabolites prevent cancer cells from dividing and growing, ultimately leading to cell death.

[0146] Some examples include:

[0147] 1. Pyrimidine Analogs: These antimetabolites resemble the building blocks of DNA and RNA known as pyrimidines. They can be incorporated into the growing DNA or RNA strands, leading to impaired replication and synthesis. Examples include 5-fluorouracil (5-FU) and cytarabine.

[0148] 2. Purine Analogs: These antimetabolites mimic the 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.

[0149] 3. Folic Acid Analogs: Folic acid is essential for the synthesis of nucleotides, the building blocks of DNA and RNA. Folic acid analogs inhibit the enzyme dihydrofolate reductase, which is involved in the conversion of folic acid to its active form. This disruption impairs nucleotide synthesis and cell division. Methotrexate is a common folic acid analog.

[0150] Antimetabolites are commonly used in the treatment of various types of cancer, including leukemia, lymphoma, breast cancer, and colorectal cancer. However, they can also affect normal, healthy cells that divide rapidly, leading to side effects such as hair loss, gastrointestinal disturbances, and decreased blood cell counts.

[0151] Thus, in one or more exemplary embodiments, the present disclosure describes Antimetabolites for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection. Pyrimidine analog

[0152] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is a pyrimidine analog.

[0153] A pyrimidine analog is a chemical compound that is structurally similar to the natural pyrimidine nucleotides found in DNA and RNA. Pyrimidines are one of the two types of nucleotide bases that make up the genetic code, with the other being purines.

[0154] Thus, in one or more exemplary embodiments, the present disclosure describes pyrimidine analogs for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0155] In one or more exemplary embodiments, a pyrimidine analog according to the present disclosure can be selected from the group consisting of 5-Fluorouracil (5-FU), Cytarabine (Cytosar-U), and Gemcitabine (Gemzar).

[0156] Purine analog

[0157] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is a purine analog.

[0158] A purine analog is a chemical compound that is structurally similar to the natural purine nucleotides found in DNA and RNA. Purines are one of the two types of nucleotide bases that make up the genetic code, with the other being pyrimidines.

[0159] They interfere with DNA and RNA synthesis by inhibiting specific enzymes involved in purine metabolism, disrupting the production of nucleotides and ultimately inhibiting e.g., cancer cell growth and division.

[0160] Thus, in one or more exemplary embodiments, the present disclosure describes purine analogs for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0161] In one or more exemplary embodiments, a purine analog according to the present disclosure can be selected from the group consisting of Mercaptopurine (6-MP) and Thioguanine (6-TG).

[0162] In one or more exemplary embodiments, the purine analog according to the present disclosure is Mercaptopurine (6-MP). In one or more exemplary embodiments, the purine analog according to the present disclosure is Thioguanine (6-TG).

[0163] Folic Acid Analogs

[0164] Folic acid analogs are chemical compounds that structurally resemble folic acid, a B-vitamin essential for various cellular processes, including the synthesis of DNA, RNA, and proteins.

[0165] Folic acid analogs work by inhibiting the enzyme dihydrofolate reductase (DHFR), which is involved in the conversion of di hydrofol ate (DHF) to tetra hydrofol ate (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 the production of THF, which leads to the depletion of nucleotides required for DNA and RNA synthesis.

[0166] Thus, in one or more exemplary embodiments, the present disclosure describes Folic acid analogs for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0167] One of the most well-known folic acid analogs used in cancer treatment is methotrexate.

[0168] Other folic acid analogs used in cancer treatment include pemetrexed, pralatrexate, and trimetrexate. These medications have varying mechanisms of action and may be used to target different types of cancer.

[0169] In one or more exemplary embodiments, a folic acid analog according to the present disclosure can be selected from the group consisting of methotrexate, pemetrexed, pralatrexate and trimetrexate.

[0170] In one or more exemplary embodiments, the folic acid analog according to the present disclosure is methotrexate.

[0171] In one or more exemplary embodiments, the folic acid analog according to the present disclosure is pemetrexed.

[0172] In one or more exemplary embodiments, the folic acid analog according to the present disclosure is pralatrexate. In one or more exemplary embodiments, the folic acid analog according to the present disclosure is trimetrexate.

[0173] Fdcyd

[0174] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is Fdcyd.

[0175] Fdcyd , or 5-fluoro-2'-deoxycytidine, is a fluoropyrimidine nucleoside analogue that interferes with DNA metabolism. Fdcyd is structurally similar to floxuridine.

[0176] Thus, in one or more exemplary embodiments, the present disclosure describes Fdcyd for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

[0177] Thiram

[0178] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is thiram.

[0179] Thiram is an organic sulfur compound that is widely used as a fungicide and animal repellent. It belongs to a class of chemicals called dithiocarbamates.

[0180] Fusidic acid

[0181] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is fusidic acid.

[0182] Fusidic acid belongs to a class of antibiotics known as fusidanes and is derived from the fungus Fusidium coccineum. Fusidic acid is primarily effective against Gram-positive bacteria, including Staphylococcus aureus.

[0183] Fusidic acid works by inhibiting bacterial protein synthesis, specifically targeting a protein called elongation factor G (EF-G). EF-G is involved in the movement of ribosomes along the mRNA during protein synthesis. By binding to EF-G, fusidic acid prevents the elongation of the protein chain, ultimately inhibiting bacterial growth. Surprisingly fusidic acid was found to inhibit the virulence factor ScpA of Streptococcus pyogenes although fusidic acid is known not to be an efficient inhibitor of bacterial growth for this pathogen. Fusidic acid is for Streptococcus pyogenes inhibiting virulence rather than viability.

[0184] Eprosartan

[0185] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is eprosartan.

[0186] Eprosartan is an angiotensin II receptor antagonist, also known as an angiotensin receptor blocker (ARB). It is used as an antihypertensive medication to treat high blood pressure (hypertension). Eprosartan selectively blocks the binding of angiotensin II to the angiotensin II receptor, thereby preventing its action.

[0187] Mercaptopurine

[0188] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is mercaptopurine.

[0189] Mercaptopurine, also known as 6-mercaptopurine or 6-MP, is a medication that belongs to the class of drugs called purine analogues. It is an antimetabolite and immunosuppressive agent. Mercaptopurine is primarily used in the treatment of certain types of cancer, particularly acute lymphoblastic leukemia (ALL).

[0190] 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.

[0191] Dihydrostreptomycin

[0192] In one or more exemplary embodiments, an inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium according to the present disclosure is dihydrostreptomycin.

[0193] Dihydrostreptomycin, also known as dihydrostreptomycin sulfate, is an antibiotic that belongs to the class of aminoglycosides. It is derived from the bacterium Streptomyces griseus. Dihydrostreptomycin is primarily used for the treatment of bacterial infections. As an aminoglycoside antibiotic, di hydrostreptomycin works by inhibiting bacterial protein synthesis. It binds to the bacterial ribosomes, which are responsible for protein synthesis, and disrupts the formation of functional proteins. This leads to the inhibition of bacterial growth and eventually results in the death of susceptible bacteria.

[0194] Surprisingly we find that di hydrostreptomycin is inhibiting the ScpA virulence factor of Streptococcus pyogenes and not only acting by inhibiting protein synthesis. Dihydrostreptomycin is an inhibitor of virulence and not only an inhibitor of viability.

[0195] Dihydrostreptomycin is particularly effective against Gram-negative bacteria, including certain strains of Escherichia coli and Klebsiella pneumoniae. It is commonly used to treat infections caused by these bacteria, such as urinary tract infections, respiratory tract infections, and certain types of gastrointestinal infections.

[0196] The inhibitory activity towards the CEP virulence factors of pathogenic streptococci might open new treatments for streptococcal diseases even if the causative bacterium is not inhibited.

[0197] Pathogenic bacteria

[0198] Pathogenic bacteria are microorganisms that are capable of causing disease in humans, animals, or plants. Most species of bacteria are harmless and are often beneficial, but others can cause infectious diseases. The number of these pathogenic species in humans is estimated to be fewer than a hundred. By contrast, several thousand species are part of the gut flora present in the digestive tract.

[0199] Pathogenic bacteria are specially adapted and endowed with mechanisms for overcoming the normal body defences and can thereby invade.

[0200] Typically, identification is done by growing the organism in a wide range of cultures which can take up to 48 hours. The growth is then visually or gnomically identified. The cultured organism is then subjected to various assays to observe reactions to help further identify species and strain.

[0201] These bacteria possess virulence factors, such as toxins, adhesins, and capsules, that enable them to colonize and infect host tissues, leading to a range of clinical symptoms and diseases. They can cause a wide range of diseases, from mild infections such as urinary tract infections to life-threatening conditions such as sepsis. Effective treatment of pathogenic bacterial infections typically involves the use of antibiotics or other antimicrobial agents.

[0202] Here are a few examples of well-known pathogenic bacteria: Escherichia coli (E. coli): While most strains of E. coli are harmless and even beneficial, certain pathogenic strains can cause severe gastrointestinal infections, such as food poisoning.

[0203] Staphylococcus aureus: S. aureus is a common bacterium that can cause various infections, including skin infections, pneumonia, and bloodstream infections. Methicillin-resistant Staphylococcus aureus (MRSA) is a particularly concerning strain that is resistant to many antibiotics.

[0204] Salmonella enterica: Salmonella species can cause salmonellosis, a type of foodborne illness characterized by symptoms like diarrhea, fever, and abdominal cramps. Contaminated food, especially raw or undercooked poultry and eggs, is a common source of Salmonella infection.

[0205] Streptococcus pneumoniae: S. pneumoniae is a leading 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.

[0206] Mycobacterium tuberculosis: This bacterium is responsible for tuberculosis (TB), a highly contagious respiratory disease that primarily affects the lungs but can also spread to other organs. TB remains a significant global health concern.

[0207] In the present context, Streptococcus is an important genus and group A streptococci (GAS), group B streptococci (GBS), group C streptococci (GCS), Streptococcus pyogenes, Streptococcus agalactiae, or Streptococcus dysgalactiae are all important species to develop new inhibitor of cell envelope proteases against.

[0208] These are just a few examples, and there are numerous other pathogenic bacteria that cause various infectious diseases. It's important to note that the severity and symptoms of bacterial infections can vary widely, and appropriate medical treatment is often necessary to combat these pathogens.

[0209] Lactic acid bacteria

[0210] Lactic acid bacteria (LAB) are a group of Gram-positive, non-sporulating 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 in anaerobic conditions. LAB is used in the present context are added deliberately to milk to start desirable fermentation under controlled conditions. As the bacteria culture grows, it converts lactose into lactic acid and other organic acids, reducing the pH of milk. Considerable variation has been detected among different LAB species and strains tested for their ability to reduce pH, and these differences become more marked over the course of incubation.

[0211] Here are some key characteristics and features of lactic acid bacteria in general:

[0212] 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 gives rise to the characteristic sour taste and acidic environment associated with fermented foods.

[0213] Acid Tolerance: Lactic acid bacteria are adapted to thrive in acidic conditions. They can tolerate and even thrive in environments with low pH levels, which helps them outcompete other microorganisms that cannot survive in such acidic conditions.

[0214] Probiotic Potential: Certain strains of lactic acid bacteria, such as Lactobacillus and Bifidobacterium, have been widely studied for their probiotic properties. Probiotics are live microorganisms that, when consumed in adequate amounts, can confer health benefits on the host. These LAB strains are often used in the production of fermented dairy products, like yogurt and kefir, as well as in dietary supplements.

[0215] Food Fermentation: LAB are crucial in the production of various fermented foods. They play a vital role in processes like sourdough bread fermentation, sauerkraut production, cheese-making, and pickling. The lactic acid produced by these bacteria not only contributes to flavour and preservation but also helps create an environment that inhibits the growth of harmful bacteria.

[0216] Industrial Applications: LAB have several industrial applications beyond food production. They are used in the production of certain chemicals, pharmaceuticals, and biofuels. LAB strains can produce enzymes, exopolysaccharides, and antimicrobial compounds, which find applications in various industries.

[0217] Overall, lactic acid bacteria are a diverse group of bacteria with significant importance in the food industry, probiotics, and other industrial applications. They contribute to the flavor, texture, and preservation of fermented foods and offer potential health benefits to consumers. There are numerous known species of lactic acid bacteria (LAB).

[0218] Here is a list of some well-known LAB species:

[0219] Lactobacillus acidophilus

[0220] Lacticaseibacillus casei

[0221] Lactiplantibacillus plantarum

[0222] Lacticaseibacillus rhamnosus

[0223] Lactobacillus delbrueckii

[0224] Levilactobacillus brevis

[0225] Limosilactobacillus fermentum

[0226] Lactobacillus delbrueckii subsp. bulgaricus

[0227] Lactococcus lactis

[0228] Streptococcus thermophilus

[0229] Pediococcus acidilactici

[0230] Leuconostoc mesenteroides

[0231] These are just a few examples, and there are many other species and strains of LAB that have been identified and studied. Each species within the LAB group can exhibit unique characteristics in the present context.

[0232] In one or more exemplary embodiments, the lactic acid bacteria (LAB) are member of the family Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, or Streptococcaceae.

[0233] In one or more exemplary embodiments, the lactic acid bacteria (LAB) are non-pathogenic.

[0234] CEP enzymes belonging to the family of subtilisin like serine proteases found in any lactic acid bacterium belonging to one of the genera: Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Schleiferilactobacillus, Weissella, Lactococcus, and Streptococcus.

[0235] Thus, in one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Schleiferilactobacillus, Weissella, Lactococcus, or Streptococcus. In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Lactobacillus, Lactococcus, or Streptococcus.

[0236] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Lacticaseibacillus.

[0237] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Lactiplantibacillus.

[0238] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Lactobacillus.

[0239] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Leuconostoc.

[0240] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Oenococcus.

[0241] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Pediococcus.

[0242] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Schleiferilactobacillus.

[0243] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Weissella.

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

[0245] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure is selected from the genus Streptococcus.

[0246] In one or more exemplary embodiments, the lactic acid bacterium strain is a Lactococcus lactis.

[0247] In one or more exemplary embodiments, the lactic acid bacterium strain is a Lactococcus cremoris. In one or more exemplary embodiments, the lactic acid bacterium strain is a Streptococcus thermophilus.

[0248] In one or more exemplary embodiments, the lactic acid bacteria (LAB) according to the present disclosure does not include the order Bifidobacterium.

[0249] Lactococcus lactis

[0250] Lactococcus lactis is the LAB used for the production of the largest volumes of cheese.

[0251] It is also used for the production of buttermilk and several fermented milk products.

[0252] In one or more exemplary embodiments, the lactic acid bacteria (LAB) used in the method of the present disclosure is Lactococcus lactis.

[0253] In one or more exemplary embodiments, the lactic acid bacteria (LAB) used in the method of the present disclosure is the proteolytic Lactococcus lactis strain (WG2G3).

[0254] Streptococcus thermophilus

[0255] Streptococcus thermophilus is used for the production of yoghurt; skyr, mozzarella, and several other cheese types. Streptococcus thermophilus is the LAB species produced in largest volumes. Streptococcus thermophilus is expected to be important for the production of plant based fermented food products.

[0256] In one or more exemplary embodiments, the lactic acid bacteria (LAB) used in the method of the present disclosure is Streptococcus thermophilus.

[0257] Fermentable substrate

[0258] A fermentable substrate is a carbohydrate or other organic compound that can be metabolized by microorganisms through fermentation, resulting in the production of energy and / or metabolic end products such as alcohol, lactic acid, and carbon dioxide. Fermentable substrates can be derived from a variety of sources, including plants, animals, and microbial sources, and can be present in a wide range of food and feed ingredients.

[0259] Examples of fermentable substrates include simple sugars such as glucose and fructose, as well as more complex carbohydrates such as starches, cellulose, and hemicellulose. In one or more exemplary embodiments, the fermentable substrate is a milk substrate.

[0260] In the present context, the term "milk substrate" may be any raw and / or processed milk material that can be subjected to fermentation. Thus, useful milk substrates include, but are not limited to, solutions / suspensions of any milk or milk like products comprising protein, such as whole or low fat milk, skim milk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquid from crystallization of lactose, whey protein concentrate, or cream.

[0261] Obviously, the milk substrate may originate from any mammal, e.g. being substantially pure mammalian milk, or reconstituted milk powder. Preferably, at least part of the protein in the milk substrate is proteins naturally occurring in milk, such as casein or whey protein. However, part of the protein may be proteins which are not naturally occurring in milk. Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.

[0262] The term "milk" is to be understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk. The term milk also comprises soy milk. Optionally the milk is fortified with nutrients or reagents, e.g. by addition of an acid (such as formic acid, citric, acetic or lactic acid), purines, pyrimidines, amino acids, or mixed, e.g. with water. The milk may be raw or processed, e.g. by filtering, sterilizing, pasteurizing, homogenizing etc, or it may be reconstituted dried milk. An important example of "bovine milk" according to the present invention is pasteurized cow's milk. It is understood that the milk may be acidified, mixed or processed before, during and / or after the inoculation with bacteria.

[0263] In one or more exemplary embodiments, the fermentable substrate is a medium containing protein as nitrogen source and a sugar as carbon source.

[0264] In one or more exemplary embodiments, the fermentable substrate is selected from the group consisting of soya milk, pea milk, almond milk, or other plant milks.

[0265] In one or more exemplary embodiments, the fermentable substrate is skimmed milk.

[0266] In one or more exemplary embodiments, the fermentable substrate is a milk substrate added 1% glucose.

[0267] Other carbohydrates could be used, if the bacterium used in the screening procedure is able to metabolize the carbohydrate. In this case sucrose, fructose, lactose, galactose could be relevant carbohydrates to use. Thus, in one or more exemplary embodiments, the fermentable substrate is or contains sucrose, fructose, lactose, or galactose,

[0268] Thus, in one or more exemplary embodiments, the fermentable substrate contains nutrients such as but not limited to purines or pyrimidines.

[0269] Thus, in one or more exemplary embodiments, the fermentable substrate contains formic acid or sodium formiate.

[0270] Thus, in one or more exemplary embodiments, the fermentable substrate contains proteins as N- source.

[0271] Thus, in one or more exemplary embodiments, the fermentable substrate contains peptides. pH indicator

[0272] A pH indicator is a substance that exhibits a different colour or spectral absorption depending on the acidity or basicity of the solution it is dissolved in. pH indicators are typically weak acids or bases that undergo reversible changes in their molecular structure or electronic properties in response to changes in pH. pH indicators work based on the principle of acid-base chemistry. When a pH indicator is added to a solution, the presence of hydrogen ions (H+) or hydroxide ions (OH-) causes a shift in the equilibrium of the indicator, resulting in a change in color.

[0273] The colour 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 colour at different pH values. Some indicators are more suitable for acidic solutions, while others are better suited for alkaline solutions. pH indicators are commonly used in laboratory experiments and industrial processes to monitor pH changes in solutions and are also used in various applications such as water quality testing, food and beverage production, and medical diagnostics.

[0274] 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 in acidic conditions (pH below 7) and blue in alkaline conditions (pH above 7).

[0275] Phenolphthalein: Phenolphthalein is colorless in acidic solutions and turns pink in alkaline solutions (pH above 8.2).

[0276] Bromothymol Blue: Bromothymol Blue is yellow in acidic solutions (pH below 6) and blue in alkaline solutions (pH above 7.6).

[0277] Methyl Orange: Methyl Orange is red in acidic solutions (pH below 3.1) and yellow in alkaline solutions (pH above 4.4).

[0278] Universal Indicator: Universal Indicator is a mixture of several different pH indicators that covers a wide range of pH values. It changes color across the entire pH scale, displaying a range of colors to indicate the pH of the solution. pH indicators are commonly used in various 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 a substance.

[0279] There are numerous known pH indicators, each with its own specific pH range and color changes. Here is a list of some commonly used pH indicators:

[0280] Litmus: Blue (alkaline) to red (acidic)

[0281] Phenolphthalein: Colorless (acidic) to pink (alkaline)

[0282] Bromothymol Blue: Yellow (acidic) to blue (alkaline)

[0283] Methyl Orange: Red (acidic) to yellow (alkaline)

[0284] Congo Red: Blue (acidic) to red (alkaline)

[0285] Thymol Blue: Red (acidic) to yellow (alkaline)

[0286] Bromocresol Purple: Yellow (acidic) to purple (alkaline)

[0287] Bromophenol Blue: Yellow (acidic) to blue (alkaline)

[0288] Alizarin Yellow R: Yellow (acidic) to red (alkaline)

[0289] Cresol Red: Yellow (acidic) to red (alkaline)

[0290] Neutral Red: Red (acidic) to yellow (alkaline)

[0291] Thymolphthalein: Colorless (acidic) to blue (alkaline)

[0292] Malachite Green: Green (acidic) to colorless (alkaline)

[0293] Rosolic Acid: Yellow (acidic) to red (alkaline)

[0294] Naphtholphthalein: Colorless (acidic) to pink (alkaline)

[0295] Nitrazine Yellow: Yellow (acidic) to blue (alkaline) Thymol Purple: Red (acidic) to yellow (alkaline)

[0296] Cresolphthalein: Colorless (acidic) to purple (alkaline) Xylenol Orange: Yellow (acidic) to red (alkaline) Cresol Red-Methyl Red: Red (acidic) to yellow (alkaline)

[0297] Please note that this is not an exhaustive list, as there are many other pH indicators available with varying pH ranges and colour changes. The choice of pH indicator depends on the specific pH range being measured and the desired colour change for visual detection.

[0298] The change in pH throughout fermentation is usually measured by immersion of glass electrodes into fermentation medium (e. g. defined medium or milk), but this method is time consuming and does not allow the fast screening of numerous strains. Colorimetric procedures may present an alternative, as they can be processed quickly by simply mixing reagents and employ simple, low- cost instrumentation.

[0299] In one or more exemplary embodiments, the pH indicator is selected from the group consisting of bromocresol purple and bromocresol green.

[0300] In one or more exemplary embodiments, the pH indicator is selected from the group consisting of phenolphthalein, bromothymol blue, and litmus.

[0301] In one or more exemplary embodiments, the pH indicator is a combination of bromocresol purple and bromocresol green.

[0302] A target inhibitor

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

[0304] Compound libraries can be obtained from various sources. One source is the Broad institute providing repurposing libraries with a few thousands of compounds as well as larger compound libraries. In Europe there is an initiative called “EU open screen” engaged in the creation and distribution of large compound libraries for drug screening purposes.

[0305] In one or more exemplary embodiments, a target inhibitor is a compound, which is part of a EU open screen screening library. In one or more exemplary embodiments, a target inhibitor is a compound, which is part of a Broad Institute screening library.

[0306] Monitoring the acidification

[0307] One can monitor the acidification of a solution by measuring its pH over time. There are several methods to monitor the acidification of a solution: pH Meter: The most accurate and precise method is to use a pH meter. A pH meter consists of a probe that is inserted into the solution, and it measures the voltage generated by the solution's hydrogen ions. The pH meter then converts the voltage into a pH value, providing real-time monitoring of the acidification process. pH Indicator Strips: pH indicator strips or paper are simple and cost-effective tools. These strips contain pH-sensitive chemicals that change colour according to the pH of the solution. By dipping the strip into the solution and comparing the resulting colour to a colour chart, one can estimate the approximate pH of the solution. However, it is important to note that pH indicator strips provide a less precise measurement compared to a pH meter. pH Test Kits: pH test kits typically include a pH indicator solution or paper strips and a colour chart. By adding a few drops of the indicator solution to the solution being monitored or by dipping a strip into the solution, the resulting colour change can be matched to the chart to determine the pH.

[0308] Electronic pH Probes: Like pH meters, electronic pH probes can be used for monitoring the acidification of a solution. These probes are connected to data loggers or other electronic devices that provide real-time pH measurements and data logging capabilities.

[0309] Regardless of the method chosen, it is important to ensure proper calibration and follow the manufacturer's instructions for accurate and reliable pH measurements. Monitoring the acidification of a solution is crucial in various fields, including research, industrial processes, environmental monitoring, and quality control in food and beverage production.

[0310] In one or more exemplary embodiments, the pH is monitored by a pH indicator as described above. Flatbed scanner

[0311] In one or more exemplary embodiments, the pH is monitored by monitoring the color of the milk by the use of a flatbed scanner.

[0312] In one or more exemplary embodiments, acidification monitoring is made by a flatbed scanner.

[0313] The advantage of using a flatbed scanner is that it allows the use of turbid media. Media containing proteins as N-source will often be turbid. Any other method to follow bacterial growth could be used.

[0314] Software pH MultiScan

[0315] In one or more exemplary embodiments, the scans were collected and analyzed by the software pH MultiScan from HNH.

[0316] Software to control the flatbed scanner and to capture the colour of individual wells might be acquired from other sources or it could be designed by a person skilled in software development. If bacterial growth is detected by other means than acidification alternative software must be established to capture data.

[0317] Adding a source of amino acids

[0318] Using a simple acidification assay will allow libraries of potential inhibitors to be screened for inhibition of acidification. Cultures only inhibited due to inhibition of the protease will be able to resume growth and complete the acidification, if the culture is supplied with a mixture or source of amino acids, whereas cultures inhibited for other reasons will stay inhibited.

[0319] In the present context, the term "a source of amino acids" refers to any group of mixtures, blends, supplements, compounds or nutrition source that provides a sufficient amount of amino acids.

[0320] These sources of amino acids often contain a concentrated or isolated form of amino acids derived from various protein sources. These sources also refers to a combination or blend of different amino acids. Amino acid mixtures are commonly used in cell culture media to provide the necessary nutrients for cell growth and protein synthesis.

[0321] Several commercially available amino acid mixtures are commonly used in cell culture media to provide the necessary nutrients for cell growth and protein synthesis. Here are a few examples: Eagle's Minimum Essential Medium (MEM) Amino Acids 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.

[0322] Dulbecco's Modified Eagle Medium (DMEM) Amino Acids Solution: Similar to MEM, this mixture provides all 20 standard amino acids in a balanced composition and is commonly used in cell culture applications.

[0323] RPMI 1640 Amino Acids Solution: RPMI 1640 is a widely used cell culture medium, and the amino acid solution specifically designed for it provides the necessary amino acids for supporting the growth of a variety of cell types.

[0324] Ham's F-12 Nutrient Mixture Amino Acids Solution: Ham's F-12 is another commonly used cell culture medium, and its amino acid solution is tailored to support the nutritional requirements of various cell lines.

[0325] Leibovitz's L-15 Medium Amino Acids Solution: Leibovitz's L-15 medium is used for specialized cell culture applications, and the corresponding amino acid solution provides the essential amino acids needed for cell growth.

[0326] These are just a few examples of amino acid mixtures used in cell culture media. It's important to note that different cell types and specific research applications may require different formulations or modifications of these mixtures to meet their specific nutritional requirements.

[0327] In one or more exemplary embodiments, the source of amino acids can be an amino acid mixture.

[0328] In one or more exemplary embodiments, the source of amino acids can be hydrolyzed beta casein.

[0329] In one or more exemplary embodiments, the source of amino acids can be hydrolyzed casein.

[0330] Mixtures of individual amino acids or hydrolysates of any protein might be used for this part of screening. The protein hydrolysate might be prepared by acid hydrolysis or by enzyme hydrolysis.

[0331] Completing the acidification and reach a pH below 4.6

[0332] As the skilled addressee would recognize, then there are three possible outcomes to this method: 1. The acidification curves drop at first to a max. determined as the minimum pH reached by a non-proteolytic strain of the same species. For Lactococcus lactis fermentation in skim milk of a good quality this was determined to be pH 5,8. Once the media is further supplied with amino acids it drops to a max. pH of 4,4. This would indicate that the CEP was inhibited by the compound and thus this could be a potential inhibitor. In the Examples below this was for example achieved for Lactococcus lactis WG2 PrtP

[0333] 2. The acidification curves drop to a max. pH of 4,4. Once the media is further supplied with amino acids the pH stays at 4,4. This would indicate that the CEP was not inhibited and thus the compound is not suitable as an inhibitor.

[0334] 3. The acidification curves show no pH drop before or after supplementation with amino acids. Therefore, the compound kills the bacteria and is not of interest.

[0335] In one or more exemplary embodiments, the inhibitors of interest are target inhibitors capable of completing the acidification and reach a pH below 4.6.

[0336] Verification assays

[0337] In one or more exemplary embodiments, the method comprises subjecting a target molecule identified as an inhibitor to a verification assay, wherein said verification assay is configured to verify inhibition of the cell envelope proteases (CEP) in the pathogenic bacterium.

[0338] In one or more exemplary embodiments, the verification assay is a protease assay using substrates selected from the group consisting of mammalian chemotactic peptides, human peptide C5a, human peptide C3a, mammalian chemokines of the CXC family, or other mammalian immune peptides.

[0339] In one or more exemplary embodiments, the verification assay use gel electrophoresis to verify the presence and size of the substrate.

[0340] An activity of a CEP enzyme can be detected as disappearance of the substrate molecule and possibly as appearance of a truncated molecule arising from the cleavage of the targeted peptide bond in the substrate molecule.

[0341] In one or more exemplary embodiments, the verification assay is a HPLC. The HPLC can detect the substrate and product molecules. In one or more exemplary embodiments, the verification assay is based on fluorescence and / or fluorescence quenching.

[0342] In one or more exemplary embodiments, the verification assay is based on gel electrophoresis. The substrate and product molecules can be detected by gel electrophoresis.

[0343] Verified inhibitors

[0344] As shown below in the Examples and Figures, and as described above, the present method has identified a range inhibitor of cell envelope proteases (CEP) in a pathogenic bacterium.

[0345] Twenty compounds were identified as PrtP inhibitors. The twenty compounds were: acivicin, alexidine, azaguanine-8, dihydrostreptomycin, fdcyd, floxuridine, hygromycin-B, leucomethylene- blue, mercaptopurine, methylene-blue, napabucasin, nifuroxazide, nithiamide, nitrofurantoin, NSC- 663284, octenidine, Ro-08-2750, tolonium, walrycin-B, YM-155.

[0346] These verified inhibitors include alexidine, methylene blue, floxuridine, mercaptopurine, and di hydrostreptomycin.

[0347] General

[0348] Any feature and / or aspect discussed above in connections with the compounds according to the invention apply by analogy to the methods described herein.

[0349] The following figures and examples are provided below to illustrate the present invention. They are intended to be illustrative and are not to be construed as limiting in any way.

[0350] BRIEF DESCRIPTION OF THE FIGURES

[0351] Figure 1

[0352] Acidification curves for cultures with individual potential inhibitors. Most compounds do not interfere with the acidification in milk. A fraction of compounds inhibits the acidification completely, and some compounds show intermediary inhibition. The x axis shows the time since the start of the fermentations. The time range from 0 to 1152 minutes. The Y axis shows the pH ranging from 4.0 to 6.7.

[0353] Figure 2

[0354] After addition of extra amino acids to every well we experience that some inhibited cultures are able to complete the acidification and reach 4.6 or below. The x axis shows the time since the addition of amino acids. The time range goes from 0 to 1152 minutes. The Y axis shows the pH ranging from 3.8 to 6.4.

[0355] Figure 3

[0356] Secondary acidification curves for 13 compounds showing aa reversible inhibition. The x axis shows the time in minutes since the addition of amino acids. The time range goes from 0 to 1200 minutes. The Y axis show the pH ranging from 4.1 to pH 6.6.

[0357] Figure 4

[0358] Inhibition of the CEP by di hydrostreptomycin. In milk supplemented with amino acids the acidification proceeds with normal speed and the pH reach 4.3 in less than 10 hours for both strains. Both strains are inhibited by di hydrostreptomycin in the absence of supplementation and the two proteases are inhibited to a different degree. The WG2 CEP seems to be completely inhibited whereas the SK11 CEP is only partially inhibited. The x axis shows the time in minutes since the start of the fermentation. The range is from 0 to 1200 minutes. The y axis shows the pH ranging from 4.2 to 6.7.

[0359] Figure 5

[0360] Inhibition of CEP by floxuridine. Both strains acidify down to pH 4.4 within 10 hours in milk supplemented with amino acids whereas bot strains CEP are completely inhibited floxuridine and pH does not go below 5.2 within 20 hours. The x axis shows the time in minutes since the start of the fermentation. The range is from 0 to 1200 minutes. The y axis shows the pH ranging from 4.2 to 6.7. Figure 6

[0361] ScpA assay

[0362] The Streptococcus pyogenes ScpA enzyme was assayed by Dr Kagawa in the laboratory of Dr Cooney, University of Limerick, using a gel shift assay.

[0363] The ScpA assay shown in this figure demonstrated inhibition by four compounds. The four inhibitors were:

[0364] 13 floxuridine

[0365] 18 thiram

[0366] 111 fusidic acid

[0367] 112 eprosartan

[0368] Figure 7

[0369] ScpA assay

[0370] ScpA enzyme can be assayed using a gel shift assay. Six compounds were shown to be inhibitors of ScpA and three compounds to be partial inhibitors.

[0371] The six inhibitors were: floxuridine, thiram, fusidic acid, eprosartan, mercaptopurine, and di hydrostreptomycin.

[0372] The partial inhibiting compounds were: carbenicillin, bronopol, and cetrimonium.

[0373] Figure 8

[0374] Steps in identifying ScpA inhibitors.

[0375] 1. Select an enzyme with a structure similar to the ScpA structure but able to confer a phenotype to the bacterium hosting the enzyme. One example is PrtP of Lactococcus lactis providing an acidification phenotype in milk.

[0376] 2. Assemble a screening platform for the phenotype.

[0377] 3. Prepare a compound library in a multi-well format and add inoculated indicative medium to the wells

[0378] 4. Identify the inhibitors of the selected model CEP (PrtP in this case)

[0379] 5. Conduct an enzyme assay using the real ScpA enzyme to identify the ScpA inhibitors.

[0380] Figure 9

[0381] Acidification curves for uninhibited cultures of Lactococcus cremoris strains MS22422 and MS22425 in milk and milk supplemented with hydrolysed casein. Both strains have similar acidification kinetics and both strains grow faster in milk supplemented with hydrolysed casein. Both strains require around 650 min to reach pH5.5 in unfortified skim milk whereas they reach pH5.5 in about 410 min in milk fortified with hydrolysed casein. The average standard deviation on the time to reach pH 5.5 was found to be 10.6 min, and this value was used as a scale for the delay in acidification of inhibitor compounds. The x axis shows the time in minutes since the start of the fermentation. The range is from 0 to 1200 minutes. The y axis shows the pH ranging from 3.5 to 7.0.

[0382] Figure 10

[0383] The difference between the delay (measured in standard deviations) determined in unfortified and fortified milk was calculated for eight different doses of each compound using two different Lactococcus cremoris strains MS22422 and MS22426. MS22422 carry the prtP gene of strain SK11 in a truncated version and MS22426 carry a similar prtP gene of strain Wg2. The figure shows the graphs for five of the 20 identified PrtP inhibitors.

[0384] Figure 11

[0385] ScpA assay for 39 compounds selected in example 3. The substrate cleavage reactions were conducted at 37 °C for 35 min and stopped with the addition of SDS-PAGE loading buffer and heating at 100 °C for 30 sec. The extent of substrate cleavage was analysed with SDS-PAGE using 16% gels. Control reactions for C5a alone and for ScpA activity in the absence of any compound were conducted in parallel for comparison. These are labelled ‘hC5a’ and ‘ScpA’ in figs 11. In addition, the impact of DMSO was examined by pre-incubating 10 pL of ScpA with 0.1 pL of DMSO (1 % DMSO) prior to the cleavage reaction (‘ScpADMSO’ in Fig. 11). To date, two replicate assays have been completed. Compounds with near complete inhibition of ScpA are indicated with an asterisk in Fig. 11 and in Table 1

[0386] Table 1 : Compounds tested in ScpA inhibition assays.

[0387] EXAMPLES

[0388] Example 1 - Screening of library

[0389] This idea has been reduced to practice by screening a library of 3300 compounds from Broad Institute. The library was received in 384 multi-well plates containing 1 nmole of compounds in separate wells.

[0390] We added 55 pL inoculated milk to each well. The milk was inoculated with a recombinant strain of Lactococcus lactis harbouring a truncated version of CEP from the strain WG2, PrtPwG2G3. The milk also contained 1 % glucose and two pH indicators. The plates were incubated at 30 °C and the evolution of acidification was followed by monitoring the color of the milk by the use of a flatbed scanner. The scans were collected and analysed by the software pH MultiScan from HNH.

[0391] The setup is shown on Figure 8 and the acidification curves from screening of the first 1500 compounds are shown on Figure 1 , Figure 2 shows the pH evolution on day two after the addition of extra amino acids.

[0392] After completing the screening of the entire library, we identified 12 compounds showing an inhibition which could be reversed by the addition of amino acids. Figure 3 shows the curves for the secondary acidification of those 12 compounds.

[0393] A library of 46 compounds was selected based on the primary screening. The library contained the 12 compounds identified to have an aa reversible inhibition plus 34 compounds showing a delay larger than 2.5 SD in the time to reach pH5.5.

[0394] The reason for including these partial inhibitors, was to investigate if any of those would be delayed due to a partial inhibition of the CEP enzyme.

[0395] A cherry-picking library containing the selected 46 compounds was ordered from the Broad Institute, and screened using two different, but closely related, enzymes. Both enzymes, PrtPwG2G3 and PrtPsKHG3 were expressed in the same background, Lactococcus lactis MG1363, so the two strains were identical except for the prtP genes. In the secondary screen the fermentations in milk supplemented with amino acids were conducted in parallel with the fermentations in unsupplemented milk. Graphs showing the acidification curves for two compounds are shown in Figure 4 and 5. Example 2 - Materials and method set-up

[0396] MATERIALS o Proteolytic L. lactis strain (WG2G3) o Bromocresol Purple o Bromocresol Green o NaOH o MiliQ water o Beta hydrolysed casein (casein hydrolysate CAS no. 65072-00-6) o Glucose o M17 enriched media o Erythromycin o Skimmed milk o 0,22 urn Filters o 50mL syringe o Flatbed scanner o Computer o pH Screening software o 100mL graduated cylinder o pH meter o Parafilm o 100 uL tips o 5ml tips o 1250 ul tips o 1000 ul pipet o 24 row 10Oul Eppendorf automatic pipet o 5ml pipet

[0397] METHODS

[0398] A chemical library was allocated to the wells in which the compound is set with milk, L. lactis inoculum, and a pH indicator (Purple and green Bromocresol at pH 7).

[0399] Once the wells have been filled, we will incubate them at 30°C on top of a flatbed scanner and read the bottom of the wells every 5 minutes during at least 18h. In this way, we will have recorded the pH-dependent colour change and thus, through our software, we will be able to create acidification curves. After the initial 18h incubation and reading round, we will inoculate every well with hydrolysed casein 1 ,75%, suitable for direct inclusion into L. lactis. We will then begin another round of incubation within the same parameters and observe how the pH behaves.

[0400] Experiment steps

[0401] This experiment will be conducted in a flow bench to keep the environment as sterile as possible. For the experiment I will follow these steps:

[0402] 1. Acquire a 100 ml bottle of boiled skimmed milk at 99°C during 30’.

[0403] 2. Extract 5mL of milk and add 5mL of pH indicator.

[0404] 3. Extract 5mL of milk and add 5mL of glucose 20% (this way we have a total concentration of glucose of 1%)

[0405] 4. Extract 1000 pl of milk f and inoculate with 10OOul frozen inoculum for a 1% concentration of bacteria. We will use the following strain: o MS22426: L. lactis WG2G3 (Pil23) M17 enriched with glucose 1%and 2,5mg / mil erythromycin

[0406] (This means we will take two aliquots of each strain out of the freezer)

[0407] 5. We will then add 50 pl of each inoculated milk to the 384-microtiter plate.

[0408] 6. We will cover the microtiter plate with parafilm wrap and its lid and place it on a flatbed scanner. Then, we will turn on the incubator at 30°C and program the experiment.

[0409] 7. We will run the program for 19h.

[0410] 8. The next day we will uncover the 384-microtiter plate and add 20 microliters of 1 ,75% hydrolysed casein solution to each well.

[0411] (This way we will have a 0,5% concentration of hydrolysed casein in 70 microliters of volume)

[0412] 9. We will take a picture of the plate.

[0413] 10. We will again cover the microtiter plate with parafilm and its lid and run the program overnight again.

[0414] 11 . The following day we will see the aspect of the microtiter plate, take a picture, and freeze it at -25°C.

[0415] 12. We will export and save the data and proceed to analyse it. REAGENTS PREPARATION

[0416] Boiled skimmed milk

[0417] We will follow the next steps:

[0418] 1. Buy a litre of skimmed milk.

[0419] 2. Preheat the water in the laboratory water bath up to 99°C.

[0420] 3. Divide the litre into 8 or 9 100ml sterile glass bottles (or any other volume of your choosing).

[0421] 4. Do not totally screw the lid on the bottles.

[0422] 5. Carefully put the bottles in the hot water.

[0423] 6. Wait 30 minutes.

[0424] 7. Extract the bottles screwing the lid back on when doing so.

[0425] 8. Keep at 5°C. pH indicator

[0426] Under a extractor cabin we will follow the next steps:

[0427] 1. Add 80 ml of miliQ water to a 100ml sterile glass bottle

[0428] 2. Weigh 100mg of bromocresol purple and add it to the bottle

[0429] 3. Weigh 100 mg of bromocresol purple and add it to the bottle

[0430] 4. Mix completely

[0431] 5. Measure the pH

[0432] 6. Adjust the pH to 7 using NaOH.

[0433] 7. Adjust the volume to 100mL

[0434] 8. With a 50mL syringe and a 0,22 ul filter, filter the solution into another sterile bottle.

[0435] 9. Place the proper labels including concentration and pH.

[0436] 10. Keep at 5°C

[0437] Hydrolysed casein 1, 75%

[0438] To prepare this solution we will follow the next steps:

[0439] 1. Add 80 mL of miliQ water into a sterile glass bottle

[0440] 2. Weigh 1 ,75 grams of hydrolized casein to the bottle.

[0441] 3. Adjust the volume to 100 mL

[0442] 4. Make sure everything is dissolved.

[0443] 5. Add the proper labels indicating the concentration, date and name. Duplicated the amounts to make sure one has enough beta casein; in that case these would be the steps:

[0444] 1. Add 180 mL of miliQ water into a setrile glassbottle

[0445] 2. Weigh 3,5 grams of beta hydrolized casein to the bottle.

[0446] 3. Adjust the volume to 200 mL

[0447] 4. Make sure everything is dissolved.

[0448] 5. Add the proper labels indicating the concentration, date and name. Glucose 20%

[0449] To prepare this solution we will follow the next steps:

[0450] 1. Add 80 mL of miliQ water to a sterile glass bottle.

[0451] 2. Add 20g of glucose to the bottle

[0452] 3. Adjust the volume to 100mL.

[0453] 4. Using a 50mL sterile syringe and a 0,22 urn filter, filter the solution into another sterile glass bottle.

[0454] 5. Add the proper labels indicating the concentration, date and name.

[0455] Example 3 - Screening of Full REPO Library

[0456] The full REPO compound library from Broad Institute consisting of 6808 drugs or drug candidates under development was screened by the same procedure as described in Example 1 , This REPO library consists of all three Libraries REPO1 , REPO2, and REPO3,

[0457] We added 55 pL inoculated milk to each well. The milk was inoculated with a recombinant strain of Lactococcus lactis harbouring a truncated version of CEP from the strain WG2, PrtPwG2G3. The milk also contained 1% glucose and two pH indicators. The plates were incubated at 30 °C and the evolution of acidification was followed by monitoring the color of the milk by the use of a flatbed scanner. The scans were collected and analysed by the software pH MultiScan from HNH.

[0458] After the initial 18h incubation and reading round, we inoculated every well with hydrolysed casein and followed the pH evolution.

[0459] A picking library of 39 compounds was ordered from broad Institute. The compounds are listed in Table 2.

[0460] Each compound was tested in 8 concentrations (2-fold dilutions going from 1 nmol down to 0.008 nmol in the wells. The plates were screened with two different proteolytic strains of L. cremoris: MS22422 (SK11G3) and MS22426 (Wg2G3). The fermentations were done in two media, skim milk and skim milk fortified with casein hydrolysate. In addition to the eight doses of each inhibitor candidate every plate of the picking library contains 72 wells with no compounds. The data from these wells were used to determine the uninhibited acidification kinetics and the standard deviation in the acidification activity.

[0461] The time needed to reach pH 5.5 can be used as a measure for the acidification activity for each strain in each medium

[0462] Both strains require around 650 min (11 h) to reach pH 5.5 in milk MS22422 is slightly faster than MS22426. In milk fortified with hydrolyzed casein both strains are faster and reach pH 5.5 in about 410 min (6.8 h) now with MS22426 as the fastest strain. The acidification curves for uninhibited cultures are shown in Figure 9.

[0463] The standard deviations of the time to pH 5.5 for the four plates were found to be in the range from 7.5 min to 15.1 min. There is no obvious explanation for why the SD should be different, and in the evaluation of inhibitor effect we have used the average SD of 10.6 min as a measure for the inhibiting effect.

[0464] For each well containing a potential inhibiting compound the time to pH 5.5 is determined. The delay is calculated by subtracting the average T5.5 for the uninhibited strain in the same medium and expressed in SD units by dividing the delay with 10.6. For severely inhibited strains unable to reach pH 5.5 the end time of the analysis (1080 min) was used as a measure for the delay. The max delay in milk is thus around 40 SD and in fortified milk around 60 SD.

[0465] Eight compounds from the picking library were found to be only weak inhibitors of acidification. The weakest inhibitors were found to be: acelarin, BAY-11-7082, chloroxine, eprosartan, nithiamide, rifapentine, thiram, troleandomycin.

[0466] Ten compounds were found to inhibit the acidification in amino acid supplemented milk to the same or higher extend as inhibition in the un-supplemented milk. Those inhibitors must have other targets instead of (or in addition to) inhibition of PrtP. The difference between the delay in milk and the delay in fortified milk can be used as a measure of the PrtP inhibition. The delta-delay (SD) was calculated for each dose of every compound and for both strains harboring the PrtP enzyme from either strain Wg2 or strain SK11. Graphs for five PrtP inhibitors are shown in Figure 10.

[0467] Twenty compounds were identified as PrtP inhibitors. The twenty compounds were: acivicin, alexidine, azaguanine-8, dihydrostreptomycin, fdcyd, floxuridine, hygromycin-B, leucomethylene- blue, mercaptopurine, methylene-blue, napabucasin, nifuroxazide, nithiamide, nitrofurantoin, NSC- 663284, octenidine, Ro-08-2750, tolonium, walrycin-B, YM-155.

[0468] Example 4 - ScpA Inhibition by PrtP inhibiting compounds

[0469] The compounds selected in the picking library of Example 4 were assayed for ScpA inhibition in a simple end point assay. Individual wells contained 0.1 pL of a compound at a concentration of 10 mM in DMSO. The activity of ScpA was examined by first pre-incubating 10 pL of 10 nM ScpA (50 mM HEPES pH7.5, 100 mM NaCI) added to each compound in the plate. The plate was left to incubate on ice for 20 min. After 20 min, 10 pL of 11.2 pM the substrate hC5a (PBS) was added to each well. The substrate cleavage reactions were conducted at 37 °C for 35 min and stopped with the addition of SDS-PAGE loading buffer and heating at 100 °C for 30 sec. The extent of substrate cleavage was analysed with SDS-PAGE using 16% gels. Control reactions for C5a alone and for ScpA activity in the absence of any compound were conducted in parallel for comparison. These are labelled ‘hC5a’ and ‘ScpA’ in figs 1X. In addition, the impact of DMSO was examined by preincubating 10 pL of ScpA with 0.1 pL of DMSO (1 % DMSO) prior to the cleavage reaction (‘ScpADMSO’ in Fig. 1X). To date, two replicate assays have been completed. Compounds with near complete inhibition of ScpA are indicated with an asterisk in Fig. 11 and Table 3.

[0470] Of the 20 PrtP inhibitors identified in Example 4 two compounds show a clear ScpA inhibition activity. Those compounds are: alexidine and methylene-blue.

[0471] Table 3: Compounds tested in ScpA inhibition assays.

Claims

CLAIMS1 . A method for the identification of inhibitors of cell envelope proteases (CEP) in a pathogenic bacterium, the method comprising: a) providing a fermentable substrate comprising a lactic acid bacterium (LAB) strain and a pH indicator; b) adding a target molecule; c) monitoring the acidification of the fermentable substrate; d) adding a source of amino acids to the fermentable substrate; e) determining a pH of the fermentable substrate; and f) in accordance with the pH of the fermentable substrate being below 4.6, identifying the target molecule as an inhibitor.

2. A method according to claim 1 , wherein the method comprises subjecting a target molecule identified as an inhibitor to a verification assay, wherein said verification assay is configured to verify inhibition of the cell envelope proteases (CEP) in the pathogenic bacterium.

3. A method according to any of claims 1-2, wherein the lactic acid bacterium strain is selected from the order Lactobacillales.

4. A method according to any of claims 1-3, wherein the lactic acid bacterium strain is a Lactococcus lactis.

5. A method according to any of claims 1-3, wherein the lactic acid bacterium strain is a Streptococcus thermophilus.

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

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

8. A method according to claim 7, wherein the fermentable substrate is selected from the group consisting of soya milk, pea milk, almond milk, or other plant milks.

9. A method according to claim 7-8, wherein the fermentable substrate is skimmed milk.

10. A method according to any of claims 1-9, wherein the acidification monitoring is made by a flatbed scanner.11 . A method according to claim 1 , wherein the source of amino acids is selected from the group consisting of hydrolysed casein, hydrolysed animal protein, hydrolysed plant protein, or a defined blend of the individual amino acids.

12. A method according to claim 2, wherein the verification assay is a protease assay using substrates selected from the group consisting of mammalian chemotactic peptides, 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 claim 1-12 for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

14. An inhibitor selected from the group consisting of acivicin, alexidine, azaguanine-8, di hydrostreptomycin, fdcyd, floxuridine, hygromycin-B, leucomethylene-blue, mercaptopurine, methylene-blue, napabucasin, nifuroxazide, nithiamide, nitrofurantoin, NSC-663284, octenidine, Ro-08-2750, tolonium, walrycin-B, YM-155 for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

15. An inhibitor selected from the group consisting of alexidine, methylene-blue, floxuridine, mercaptopurine, and di hydrostreptomycin for use in the treatment of a bacterial infection, preferably a Streptococcus pyogenes infection.

16. Use of the inhibitors according to claim 13-15 as virulence inhibiting agents for treatment of streptococcal infections.

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