Be3 auto degradation inducing agent and therapeutic drug for infectious disease containing same

Abstract

The purpose of the present invention is to provide a new anti-virulence agent that is not directly related to the growth of pathogenic microbes, but is capable of suppressing infection by inhibiting a molecular mechanism (such as suppression of host immune response) utilized by microbes to infect a host.　The present invention pertains to a bE3 auto degradation inducing agent for use in an infected host, the bE3 auto degradation inducing agent comprising a conjugate of: a first compound having affinity to an E3 ubiquitin ligase (bE3) of a pathogenic microbe; and a second compound having affinity to the E3 ubiquitin ligase (bE3) of the pathogenic microbe.

Description

bE3 self-degrading inducer and infectious disease therapeutic agent containing the same

[0001] The present invention relates to a bE3 self-degrading inducer and an infectious disease therapeutic agent containing the same.

[0002] Infectious diseases are always among the leading causes of death, and their emergence, re-emergence, and epidemics are unpredictable. Moreover, drug-resistant bacteria are on the rise, and the World Health Organization predicts that by 2050, the number of deaths from infectious diseases will exceed the number of deaths from cancer. Existing antibiotics affect the growth of bacteria such as bacterial cell wall synthesis inhibitors and protein synthesis inhibitors, so resistance is likely to occur, and there are also problems such as the emergence of persister cells and the disruption of the gut microbiota. In recent years, in order to solve the problems of existing antibiotics, the development of Anti-Virulence agents that do not act directly on bacteria but only inhibit the pathogenicity of bacteria and delay the emergence of drug resistance has been proposed (Non-Patent Document 1: Germs. 2022 Jun 30;12(2):262-275). In fact, there are also reports that the emergence of drug-resistant bacteria can be suppressed by the combined use of Anti-Virulence agents and antibiotics in the treatment of Pseudomonas aeruginosa (Non-Patent Document 2: PLoS Biol. 2020 Aug 18;18(8):e3000805). In particular, bacteria that have invaded the host often acquire drug resistance and deteriorate, so there is a need for "antibiotics that act in the host" such as Anti-Virulence agents. However, Anti-Virulence therapy aims to suppress infection by inhibiting the molecular mechanisms (such as the suppression of the host immune response) used by bacteria to infect the host, which is not directly related to the growth of pathogenic bacteria. Therefore, detailed research on the molecular mechanism of bacterial infection is also necessary at the same time, and there are few successful examples of development.

[0003] Enteropathogenic bacteria, such as Salmonella and Shigella, secrete a group of virulence factors (effectors) into host cells via an injection device called a type III secretion system in order to neutralize the host's defense system. Through analysis of Shigella virulence factors, the inventors have elucidated that these factors cleverly control and hijack the host's signaling pathway, thereby neutralizing the host's biological defense mechanisms (Non-patent Literature 3: Cell Host Microbe. 2010 Jul 22;8(1):20-35). In particular, we elucidated the molecular mechanisms by which effectors of enteropathogenic bacteria such as Shigella and E. coli O157 (IpaB, OspE, IpaH, Cif, Ospl) target host mucosal epithelial cells to establish infection (Non-patent literature 4-8: Cell. 2007 Aug; 130 (4):611-623, Nature. 2012 Mar; 483(7391):623-626, Nat Cell Biol. 2010 Jan; 12 (1):66-73, Nature. 2009 May; 459 (7246):578-582, Biochem Biophys Res Commun. 2010 Oct 15;401(2):268-74).

[0004] On the other hand, proteolysis targeting chimera (PROTAC) technology is a modality that has attracted the attention of major pharmaceutical companies and is being rapidly developed. PROTAC compounds are chimeric molecules in which a compound that binds to ubiquitin ligase (E3) and a compound that binds to a target protein to be degraded (POI: for example, a molecule that causes cancer or neurodegeneration) are linked together by a linker, and the degradation of POI is induced via the intracellular ubiquitination degradation pathway (Non-patent Literature 9: Cell. 2020 Apr 2;181(1):102-114). In infectious diseases, BacPROTAC and Homo-BacPROTAC have recently been developed as basic research for the treatment of Mycobacterium tuberculosis, which has its own unique proteolysis system. Results have been reported in which they act directly on bacteria, disrupt proteolysis within Mycobacterium tuberculosis, and suppress its growth, demonstrating for the first time the potential of PROTAC as a treatment for bacterial infections (Non-patent Literature 10: The innovation. 2023 Mar 15;4(3):100413). However, because the protein degradation systems of individual bacteria are largely unknown, the development of PROTACs that can suppress bacterial pathogenicity without directly affecting the bacteria has yet to be successful.

[0005] Germs. 2022 Jun 30;12(2):262-275PLoS Biol. 2020 Aug 18;18(8):e3000805Cell Host Microbe. 2010 Jul 22;8(1):20-35Cell. 2007 Aug; 130 (4):611-623Nature. 2012 Mar; 483(7391):623-626Nat Cell Biol. 2010 Jan; 12 (1):66-73Nature. 2009 May; 459 (7246):578-582Biochem Biophys Res Commun. 2010 Oct 15;401(2):268-74Cell. 2020 Apr 2;181(1):102-114The innovation. 2023 Mar 15;4(3):100413

[0006] The present invention aims to provide a novel anti-virulence agent that can suppress infection by inhibiting molecular mechanisms (such as suppression of the host immune response) that bacteria use to infect a host, even though these mechanisms are not directly related to the growth of pathogenic bacteria.

[0007] As a result of diligent research to solve the above problems, the inventors have succeeded in developing a novel PROTAC compound that can degrade pathogenic bacterial effectors possessing ubiquitin ligase (E3) activity within host cells infected with pathogenic bacteria. This novel PROTAC compound represents a novel modality that suppresses bacterial pathogenicity by removing effector proteins within infected cells.

[0008] The inventors have shown that effectors possessing ubiquitin ligase (E3) activity exist in various pathogenic bacteria (Kim et. al, Cells. 2014 Aug 18;3(3):848-64). When these effectors are secreted into host cells, they utilize the host's ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin to ubiquitinate host proteins (substrates) important for the infection response, causing them to be degraded by the host's proteasome, thereby suppressing the infection defense response. Currently, more than 38 types of effectors have been reported as bacterial E3 (bE3), and in addition to known E3 types (HECT type and RING / U-box type), new types not found in mammals (Novel E3 Ligase: NEL type) have also been reported. The NEL family, which the inventors are focusing their research on, is highly conserved among many pathogenic bacteria, including Shigella (10 species), Salmonella (4 species), and Pseudomonas (5 species). Although the E3 domains of these NEL families differ in three-dimensional structure from human E3 domains (HECT and RING types), they exhibit high structural and functional similarities within the family. Our research has shown that the NEL family is an important virulence factor that degrades host target proteins, suppresses the host immune response (antigen presentation suppression, inflammatory response suppression, etc.), and promotes the spread of bacterial infection (Int J Mol Sci. 2022 Jul 13;23(14):7725). As described above, given that NEL-type E3 is widely present in pathogenic bacteria, has no homology to human E3 but exhibits high homology among NEL family members, and suppresses the host immune response, the inventors hypothesized that inducing the degradation of the NEL family could lead to the development of new therapeutic methods and drugs capable of suppressing infections by a wide range of pathogenic bacteria.

[0009] Therefore, the inventors adopted PROTAC technology to uniquely identify compounds that commonly bind to the NEL family of Salmonella and Shigella, and created an autodegradation inducer by linking two molecules of this compound with a linker. They revealed that this autodegradation inducer can degrade the NEL family of Shigella and Salmonella expressed in mammalian cells, and that although it does not directly act on the bacteria to inhibit their growth, it can suppress the growth of the bacteria within host cells. Conventional PROTAC technology uses a chimeric molecule in which a compound that binds to ubiquitin ligase (E3) and a compound that binds to the target protein to be degraded are linked by a linker to induce the degradation of the target protein via the intracellular ubiquitination degradation pathway. In contrast, the technology according to the present invention uses a molecule (the self-degradation inducer of the present invention) in which two compounds that bind to ubiquitin ligase (E3), an effector of pathogenic bacteria, are linked by a linker, and the technology induces the self-degradation of the ubiquitin ligase (E3) derived from pathogenic bacteria itself within the infected host. The inventors have named this self-degradation inducer "Pathogenic microbe suicide inducing chimera: PamSic". In other words, the gist of the present invention is as follows.

[0010] [1] A bE3 autodegradation inducer in an infected host, comprising a conjugate of a first compound having affinity for the E3 ubiquitin ligase (bE3) of a pathogenic bacterium and a second compound having affinity for the E3 ubiquitin ligase (bE3) of the pathogenic bacterium. [2] The bE3 autodegradation inducer according to [1], wherein the first compound and the second compound are linked by a linker. [3] The bE3 autodegradation inducer according to [1] or [2], which induces ubiquitination of bE3 derived from a pathogenic bacterium by utilizing ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin in an infected host. [4] The bE3 autodegradation inducer according to [1] or [2], wherein the E3 ubiquitin ligase (bE3) of the pathogenic bacterium is of the NEL (Novel E3 ligase domain) type. [5] The first compound and the second compound described above do not inhibit the ubiquitin ligase activity of E3 ubiquitin ligase (bE3). The bE3 autodegradation inducer described in [1] or [2]. [6] The first compound and the second compound described above are any of the compounds 1 to 22 in the table below. The bE3 autodegradation inducer described in [1] or [2]. [7] The bE3 autodegradation inducer according to [1] or [2], wherein the first compound and the second compound are the same compound. [8] A bE3 autodegradation inducer in an infected host, represented by the following formula (I). (In the formula, E3UB1 and E3UB2 are groups that have the ability to bind to the E3 ubiquitin ligase (bE3) of pathogenic bacteria. Linker is a group that chemically binds E3UB1 and E3UB2.) [9] The bE3 autodegradation inducer according to [6], wherein E3UB1 and E3UB2 are groups that have the ability to bind to the NEL (Novel E3 ligase domain) type bE3 of pathogenic bacteria.

[10] The bE3 autodegradation inducer according to [8] or [9], wherein E3UB1 and E3UB2 do not inhibit the ubiquitin ligase activity of the E3 ubiquitin ligase (bE3).

[11] The bE3 autodegradation inducer according to [8] or [9], wherein E3UB1 and E3UB2 are groups derived from any of the compounds 1 to 22 in the table below.

[12] A bE3 autodegradation inducer according to [8] or [9], wherein E3UB1 and E3UB2 are the same group.

[13] An infectious disease treatment comprising the bE3 autodegradation inducer according to [1] or [2].

[14] An infectious disease treatment comprising the bE3 autodegradation inducer according to [8] or [9].

[15] An infectious disease treatment according to

[13] , used against Shigella, Salmonella, Pyrethrum pestis, Edwardsiella, Pseudomonas, Rhizobium, or Ralstonia.

[16] An infectious disease treatment according to

[14] , used against Shigella, Salmonella, Pyrethrum pestis, Edwardsiella, Pseudomonas, Rhizobium, or Ralstonia.

[17] An inhibitor of pathogenic bacterial growth within a pathogenic bacterial infection host, comprising the bE3 autodegradation inducer according to [1] or [2]. An inhibitor of pathogenic bacterial growth in a host infected with pathogenic bacteria, comprising the bE3 autodegradation inducer described in

[18] , [8], or [9].

[19] An inhibitor of pathogenic bacterial growth in a host infected with pathogenic bacteria, as described in

[17] , for use against Shigella, Salmonella, Pyrethrum, Edwardsiella, Pseudomonas, Rhizobium, or Ralstonia.

[20] An inhibitor of pathogenic bacterial growth in a host infected with pathogenic bacteria, as described in

[18] , for use against Shigella, Salmonella, Pyrethrum, Edwardsiella, Pseudomonas, Rhizobium, or Ralstonia.

[21] An inhibitor of cell death of host cells infected with pathogenic bacteria, comprising the bE3 autodegradation inducer described in [1], [1], or [2].

[22] An inhibitor of cell death of host cells infected with pathogenic bacteria, comprising the bE3 autodegradation inducer described in [8], or [9].

[23] A cell death inhibitor for host cells infected with pathogenic bacteria, as described in

[17] , for use against Shigella, Salmonella, Pyrethrum, Edwardsiella, Pseudomonas, Rhizobium, or Ralstonia.

[24] A cell death inhibitor for host cells infected with pathogenic bacteria, as described in

[18] , for use against Shigella, Salmonella, Pyrethrum, Edwardsiella, Pseudomonas, Rhizobium, or Ralstonia.

[0011] According to the bE3 autodegradation inducer and infectious disease treatment agent of the present invention, the autodegradation of E3 ubiquitin ligase (bE3) derived from pathogenic bacteria is induced within the host infected with pathogenic bacteria, thereby suppressing the growth of pathogenic bacteria and consequently suppressing infection. Furthermore, the bE3 autodegradation inducer of the present invention also has the effect of suppressing cell death in host cells infected with pathogenic bacteria. The bE3 autodegradation inducer and infectious disease treatment agent of the present invention also have the advantage of not directly acting on pathogenic bacteria to suppress their growth, thus making it less likely for resistant bacteria to emerge.

[0012] Figure 1 is a conceptual diagram illustrating the mechanism of action of the bE3 autodegradation inducer of the present invention. Figure 2 is a diagram showing the results of confirming the bE3 degradation ability of the bE3 autodegradation inducer of the present invention within a Shigella-expressing host. Figure 3 is a diagram showing that the degradation of bE3 by the bE3 autodegradation inducer (Cpd_M) of the present invention utilizes the ubiquitin-proteasome degradation system. Figure 4 is a diagram showing that the bE3 autodegradation inducer (Cpd_M) of the present invention also has the ability to degrade other NEL-type bE3 family members. Figure 5 is a diagram showing the effect of the bE3 autodegradation inducer (Cpd_M) of the present invention on bacterial infection. Figure 6 is a diagram showing the results of cytotoxicity tests for the bE3 autodegradation inducers (Cpd_S,M,L) of the present invention. Figure 7 is a diagram showing the inhibitory effect of the bE3 autodegradation inducers (Cpd_S,M,L) of the present invention on the growth of intracellular Salmonella in bacterial infection. Figure 8 is a cell image showing the inhibitory effect of the bE3 autodegradation inducer (Cpd_S,M,L) of the present invention on host cell death in bacterial infection. Figure 9 is a diagram showing the inhibitory effect of the bE3 autodegradation inducer (Cpd_S,M,L) of the present invention on host cell death in bacterial infection.

[0013] The present invention will now be described in detail. In this specification, molecular biological methods may be performed by methods known to those skilled in the art, or by methods similar thereto, unless otherwise specified. Furthermore, terms used herein shall be interpreted in the sense commonly used in the art unless otherwise specified.

[0014] Embodiments of the present invention will be described in detail below.

[0015] <bE3 Autodegradation Inducer> The bE3 autodegradation inducer of the present invention consists of a conjugate of a first compound having affinity for the E3 ubiquitin ligase (bE3) of pathogenic bacteria and a second compound having affinity for the E3 ubiquitin ligase (bE3) of the pathogenic bacteria, and can induce the degradation of bE3 derived from pathogenic bacteria in an infected host.

[0016] The mechanism of action of the bE3 autodegradation inducer (Pathogenic microbe suicide inducing chimera: PamSic) of the present invention is shown in Figure 1. When enteric pathogenic bacteria, such as Salmonella and Shigella, infect a host, a group of virulence factors (effectors) are injected into the host cells by an injection device called a type III secretion system in order to disable the host's defense system. It is known that various pathogenic bacteria possess ubiquitin ligase (E3) activity as one of these effectors. When these effectors are injected into host cells, they utilize the host's ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin to ubiquitinate host proteins that are important for the infection defense response, and then degrade them by the host's proteasome, thereby suppressing the host's infection defense response. This allows pathogenic bacterial infection to be established and spread. When the bE3 autodegradation inducer of the present invention is present, two molecules of ubiquitin ligase (E3) derived from pathogenic bacteria injected into the host bind to the bE3 autodegradation inducer of the present invention. These molecules then undergo autoubiquitination and degradation by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin within the host, thereby enabling the suppression of pathogenic bacterial infection.

[0017] The pathogenic bacteria in the E3 ubiquitin ligase (bE3) of the above-mentioned pathogenic bacteria are not particularly limited as long as they are pathogenic bacteria that cause infectious diseases, but examples include Shigella, Salmonella, Pyrethrum, Edwardsiella, Pseudomonas, Rhizobium, and Ralstonia. Among these, pathogenic bacteria having HECT type, RING / U-box type, or the new type NEL (Novel E3 Ligase) which is not found in mammals are preferred, and among these, Shigella, Salmonella, and Pseudomonas having NEL type E3 ubiquitin ligase (bE3) are more preferred from the viewpoint of the effects of the present invention, and Shigella and Salmonella are even more preferred.

[0018] Examples of E3 ubiquitin ligases (bE3) of the above-mentioned pathogenic bacteria include known E3 types such as HECT type (homologous to E6-associated protein C-terminus) and RING / U-box type, as well as the new type NEL type (Novel E3 ligase domain type) bE3, which is not found in mammals. More than 38 types have already been reported (Cells. 2014 Aug 18;3(3):848-64). Among these, NEL (Novel E3 ligase domain) type and HECT (homologous to E6-associated protein C-terminus) type bE3 are preferred, and NEL type bE3 (NEL family) is more preferred from the viewpoint of the effects of the present invention because it is highly conserved among many pathogenic bacteria, including Shigella, Salmonella, and Pseudomonas. Furthermore, since the NEL type does not exist in human E3, if the bE3 autodegradation inducer of the present invention has affinity for the NEL type bE3 (NEL family), it is highly likely that it will not bind to the human E3 host. Specifically, examples include IpaH4.5, IpaH9.8, and IpaH7.8, which are bE3 of Shigella, and SspH2, which is bE3 of Salmonella.

[0019] The first compound (hereinafter also simply referred to as "the first compound") and the second compound (hereinafter also simply referred to as "the second compound") that have affinity for the E3 ubiquitin ligase (bE3) of pathogenic bacteria are not particularly limited as long as they bind to any of the above-mentioned sites of bE3 and do not bind to the host's E3 ubiquitin ligase. In this specification, affinity refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., bE3) and its binding partner (e.g., the first compound, the second compound, etc.), and is synonymous with binding ability. Typically, affinity can be expressed by the equilibrium dissociation constant (KD). In the present invention, it is important that the conjugate of the first compound and the second compound binds two molecules of bE3 in the infected host, and other functions of the first compound and the second compound themselves are not relevant. Therefore, the first compound and the second compound include all compounds that bind to any of the sites of bE3.

[0020] The first and second compounds are compounds that bind to any site on bE3 and do not bind to E3 ubiquitin ligases in hosts such as humans. Screening for such compounds can be carried out by methods conventionally known in the art.

[0021] Preferred examples of the first and second compounds include the compounds listed in the table below (1-22) and their derivatives. In the bE3 self-decomposition inducer of the present invention, the first and second compounds may be the same compound or different compounds.

[0022] In the present invention, a derivative is a compound that has affinity for the E3 ubiquitin ligase (bE3) of pathogenic bacteria, and is a compound that has been modified from the original compound by substituting a part of the original compound with other atoms or functional groups, or by oxidation or reduction. In the derivatives of the first and second compounds in the present invention, it is sufficient that they contain most of the skeleton of the original compound structurally, and as long as they have affinity for the E3 ubiquitin ligase (bE3) of pathogenic bacteria, they may be structurally similar to the original compound but exhibit completely different properties.

[0023]

[0024] Antimycin A is a compound represented by the following structural formula, with MW: 548.639 and CAS No: 1397-94-0. It is an antibiotic obtained from actinomycetes and is known to inhibit respiration by acting on the electron transport chain between cytochrome 6 and cytochrome C1, thereby disrupting electron transport.

[0025]

[0026] Meclocycline sulfosalicylate is a compound represented by the following structural formula, with MW: 695.06054 and CAS No.: 73816-42-9. It is a type of tetracycline antibiotic and is known to inhibit bacterial protein synthesis.

[0027]

[0028] Pranlukast is a compound represented by the following structural formula, with MW: 481.51546 and CAS No: 103177-37-3. It is a type of leukotriene inhibitor and is known to exert anti-asthmatic and anti-rhinitis effects by inhibiting the binding of cysteinyl-leukotriene receptors.

[0029]

[0030] Acitretin is a compound represented by the following structural formula, with MW: 326.43957 and CAS No.: 55079-83-9. It is a second-generation oral retinoid marketed under the names NeoTigason and Soriatan. It is used to treat psoriasis.

[0031]

[0032] Dicumarol is a compound represented by the following structural formula, with MW: 336.299 and CAS No: 66-76-2. It is a type of anticoagulant and is used to prevent blood clotting. It is known to exert its effect by inhibiting the action of vitamin K. It is mainly used for the prevention and treatment of thrombosis.

[0033]

[0034] Tyloxapol is a compound represented by the following structural formula, with CAS No: 25301-02-4. It is a drug mainly used as a solubilizer for inhalation medications. It is a type of surfactant and is known to play a role in enhancing the solubility of other drugs and making inhalation therapy more effective.

[0035]

[0036] Methacycline hydrochrolide is a compound represented by the following structural formula, with MW: 478.88 and CAS No: 3963-95-9. It is a type of tetracycline antibiotic and is known to exhibit antibacterial activity by inhibiting bacterial protein synthesis.

[0037]

[0038] Pyrvinium pamoate is a compound represented by the following structural formula, with MW: 1151.39 and CAS No: 3546-41-6. It is mainly used for the eradication of parasites such as pinworms.

[0039]

[0040] Montelukast is a compound represented by the following structural formula, with MW: 586.19907 and CAS No: 158966-92-8. It is a leukotriene inhibitor (LTRA) and is mainly used for the treatment of various symptoms of bronchial asthma and seasonal allergic diseases. It is known to inhibit cysteinyl leukotriene receptor 1 (CysLT1) expressed in the lungs and bronchi, interfere with the action of leukotriene D4 (and C4, E4), suppress bronchoconstriction, and exhibit an anti-inflammatory effect.

[0041]

[0042] Quercetin (also known as Sophoretin) is a compound with MW: 302.24 and CAS No: 117-39-5, represented by the structural formula shown below. It is a type of flavonoid and has been reported to have antioxidant, anti-inflammatory, anti-atherosclerotic, cerebrovascular disease prevention, antitumor, antihypertensive, and strong vasodilatory effects.

[0043]

[0044] AC-220 (Quizartinib) is a compound represented by the following structural formula, with MW: 560.67 and CAS No: 950769-58-1. It is a drug used to treat acute myeloid leukemia. It is known to inhibit cell proliferation and induce apoptosis in leukemia cells with FLT3-ITD mutations (a mutation called internal tandem duplication, in which a portion of the transperimembrane region of the FLT3 gene is duplicated and repeated) by inhibiting FLT3 tyrosine kinase activity.

[0045]

[0046] Enzastaurin is a compound represented by the following structural formula, with MW: 515.62 and CAS No.: 170364-57-5. LY-317615 is the development code. It is one of the synthetic bisindolyl maleimides with anticancer activity. It is known to selectively inhibit protein kinase Cβ, an enzyme involved in the induction of angiogenesis stimulated by vascular endothelial growth factor (VEGF), by binding to its ATP binding site.

[0047]

[0048] Luteorin is a compound with the following structural formula, MW: 286.24, CAS No: 491-70-3. It is a type of flavone and, like other flavonoids, forms yellow crystals. It is found in the myrobaran plant of the Combretaceae family. It is abundant in the leaves, but is also found in the outer layer, bark, flowers of the Trifolium genus, and pollen of the Ragweed genus. It has also been isolated from the flowers of Salvia tomentosa. It has been shown to have potential effects such as antioxidant activity, promotion of hydrocarbon metabolism, regulation of the immune system, and treatment of type 2 diabetes, and many other clinical applications are being studied.

[0049]

[0050] Azelastine hydrochloride (brand name Astelin) is a compound with a molecular weight (MW) of 418.36, represented by the structural formula shown below. It is a second-generation antihistamine, and its mechanism of action involves inhibiting the release of many inflammatory mediators, including histamine. It is mainly used to treat allergic rhinitis (hay fever), allergic conjunctivitis, asthma, and skin rashes.

[0051]

[0052] AS-604850 is a compound with the structural formula shown below, MW: 285.22, and CAS No.: 648449-76-7. It is known as a PI3-Kγ inhibitor.

[0053]

[0054] A-769662 is a compound represented by the following structural formula, with MW: 360.39 and CAS No.: 844499-71-4. It is an AMP-activated protein kinase (AMPK) activator.

[0055]

[0056] Myricetin is a compound with the following structural formula, MW: 318.2351, CAS No: 529-44-2. It is a type of natural flavonol, a flavonoid found in plants such as grapes, berries, other fruits, vegetables, and herbs. It is known to have antioxidant activity.

[0057]

[0058] Tenonitrozole is a compound represented by the following structural formula, with MW: 255.27 and CAS No.: 3810-35-3. It is a nitrothiazole derivative and is used as an antiparasitic drug, insecticide, insect repellent, etc.

[0059]

[0060] Magnolol is a compound with MW: 266.340 and CAS No: 528-43-8, represented by the structural formula shown below. It is a plant component isolated from the roots and bark of Magnolia officinalis and is known to have antifungal, antimicrobial, and antioxidant properties.

[0061]

[0062] Fisetin is a compound with the structural formula shown below, MW: 286.2363, CAS No: 528-48-3. It is a type of flavonol and is found in many plants. It is a potent sirtuin-activating compound that controls sirtuins and is said to have anti-aging effects. It is also known to have antioxidant and anti-inflammatory properties.

[0063]

[0064] Emodin is a compound with the structural formula shown below, MW: 270.24, CAS No: 518-82-1. It is one of the naturally occurring anthraquinones. It is found as one of the components in plants such as cassia seeds (seeds of legumes), rhubarb (rhizomes of Polygonaceae plants), and Polygonum multiflorum (tuberous roots of Polygonaceae). It is known to have antibacterial activity.

[0065]

[0066] Colecalciferol is a compound with the following structural formula, MW: 384.65, CAS No: 67-97-0. It is a type of vitamin D, also known as vitamin D3, and is produced when the skin is exposed to sunlight. It is used to treat and prevent vitamin D deficiency, including rickets, and related diseases. It is also used for hereditary hypophosphatemia, hypoparathyroidism which causes hypocalcemia, and Fanconi syndrome.

[0067]

[0068] Furthermore, the first and second compounds can also be described as ligands for bE3.

[0069] The bE3 self-decomposition inducer of the present invention consists of a conjugate of a first compound and a second compound. Here, "conjugate" refers to a linkage between multiple compounds, such as the first compound and the second compound. "Linkage" can include covalent bonds, non-covalent bonds (not particularly limited, but such as coordination bonds, hydrophobic bonds, hydrogen bonds, electrostatic bonds, etc.), or both. It also includes cases where the first compound and the second compound are linked via a linker.

[0070] In the bE3 self-decomposition inducer of the present invention, it is preferable that the first compound and the second compound are linked by a linker. Examples of linkers include the structures listed in the table below (wherein n and m are integers from 0 to 15 or in the range between them, and k and l are integers from 0 to 10), and can be selected from these.

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] In Tables 4-1 to 4-9 above, the X, Y, and Z groups, when present in the linker, are independently O, NH, or CH. ２ Therefore, if they exist in the same linker, they do not need to be the same group.

[0081] Of the above linkers, bis-PEG1-, bis-PEG2-, and bis-PEG4- are preferred, and bis-PEG2- is more preferred from the viewpoint of the effects of the present invention.

[0082] The bE3 autodegradation inducer of the present invention can be synthesized by mixing a first compound, a second compound, and a linker compound, according to the properties of each compound, by a method known to those skilled in the art. For example, N,N-diisopropylethylamine (DIEA) is added at room temperature to a DMF mixture of the first compound, the second compound (the first and second compounds may be the same compound), the linker compound, and HBTU, and the mixture is stirred at room temperature under a nitrogen gas atmosphere for 10 to 24 hours. Then, the DMF is removed under reduced pressure, the mixture is dissolved in an organic solvent such as ethyl acetate, and transferred to a separatory funnel. The organic phase is washed with distilled water, and the aqueous phase is extracted multiple times using an organic solvent such as ethyl acetate. Na2SO4 is added to the organic phase and dried, the solids are removed by cotton plug filtration, and the solvent is removed under reduced pressure. The residue is purified by silica gel chromatography (hexane / ethyl acetate = 3:1 to 1.5:1) to obtain the target product, the bE3 autodegradation inducer.

[0083] The bE3 self-decomposition inducer of the present invention can also be represented by the following formula (I).

[0084]

[0085] In the above formula, E3UB1 and E3UB2 are groups that have the ability to bind to the E3 ubiquitin ligase (bE3) of pathogenic bacteria. Linker is a group that chemically binds E3UB1 and E3UB2.

[0086] E3UB1 and E3UB2 are groups derived from the first and second compounds described above, respectively. Furthermore, it is preferable that E3UB1 and E3UB2 are groups that have the ability to bind to the NEL (Novel E3 ligase domain) type bE3 of pathogenic bacteria. Even more preferable is that E3UB1 and E3UB2 are groups derived from any of the compounds 1 to 22 in the table below.

[0087]

[0088] <Infection Therapeutic Agent> The infection therapy of the present invention is a pharmaceutical composition containing the bE3 autodegradation inducer of the present invention described above. The infection therapy of the present invention is suitably used as a treatment for infections caused by Shigella, Salmonella, Plague bacillus, Edwardsiella, Pseudomonas, Rhizobium, or Ralstonia.

[0089] The infectious disease treatment agent of the present invention may contain, in addition to the bE3 autodegradation inducer, other active ingredients, to the extent that they do not impair the effects of the present invention. Examples of other active ingredients include conventionally known antibacterial agents, antibiotics, and the like.

[0090] In the present invention, the bE3 autodegradation inducer in the infected host is used in the form of a pharmaceutical formulation containing a pharmaceutically acceptable carrier or excipient suitable for oral or parenteral administration (including intravenous, intraperitoneal, subcutaneous, and intramuscular injection).

[0091] Suitable formulations for oral administration include solid preparations such as tablets, capsules, and sachets, as well as oral liquid preparations such as suspensions and emulsions. Suitable formulations for parenteral administration include liquid preparations such as aqueous sterile injection solutions. In either case, pharmaceutically acceptable carriers and additives may be included in accordance with conventional methods.

[0092] Solid dosage forms may contain one or more excipients such as lactose, mannitol, corn starch, potato starch, and microcrystalline cellulose; binders such as gum arabic, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; lubricants such as talc, magnesium stearate, and calcium stearate; disintegrants such as low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxymethylcellulose, and crospovidone; fluidizers; colorants; diluents; buffers; hygroscopic agents; preservatives; foaming agents; fragrances; flavoring agents; and pharmaceutically acceptable carriers.

[0093] For suspensions, suspending agents such as sodium carboxymethylcellulose, methylcellulose, and sodium alginate can be used, and for emulsions, emulsifiers such as glycerin fatty acid esters, organic acid monoglycerides, polyglycerin fatty acid esters, and lecithin can be used.

[0094] Parenteral solutions may contain antioxidants such as sodium bisulfite, dithiothreitol, and vitamin C; buffers such as phosphates and acetates; antibacterial agents such as benzalkonium chloride and parabens; isotonic agents such as sodium chloride and glycerin; and pH adjusters such as hydrochloric acid and sodium hydroxide. Parenteral solutions may also be packaged in ampoules or vials in single doses or multiple doses, freeze-dried, and prepared on demand by adding sterile water for injection.

[0095] The dosage of bE3 autolysis inducers in infected hosts may vary depending on the patient's condition (weight, age, sex, symptoms, physical condition, etc.) and / or the route of administration. However, the preferred dosage of bE3 autolysis inducers for oral administration can be selected from a range of 0.001 mg to 5000 mg per day for adults, and in some embodiments, it is in the range of 0.1 mg to 1500 mg per day. For parenteral administration, the dosage can be selected from a range of 0.001 mg to 5000 mg per day for adults, and in either case, it can be administered once or several times depending on the symptoms.

[0096] <Inhibitor of pathogenic bacterial growth within a host infected with pathogenic bacteria> As described above, the bE3 autodegradation inducer of the present invention has the effect of suppressing the growth of pathogenic bacteria within a host infected with pathogenic bacteria, and therefore can also be called an inhibitor of pathogenic bacterial growth within a host infected with pathogenic bacteria. Furthermore, a composition containing the bE3 autodegradation inducer of the present invention described above can also be called an inhibitor of pathogenic bacterial growth within a host infected with pathogenic bacteria.

[0097] The pathogenic bacteria mentioned above include Shigella, Salmonella, Pest Bacillus, Edwardsiella, Pseudomonas, Rhizobium, and Ralstonia.

[0098] The bE3 autodegradation inducer of the present invention, or the bE3 autodegradation inducer of the present invention, in the inhibitor of pathogenic bacterial growth within a pathogenic bacterial infection host, has been described in detail in the section on "bE3 autodegradation inducer," so a further explanation is omitted here.

[0099] <Inhibitor of cell death in host cells infected with pathogenic bacteria> The bE3 autodegradation inducer of the present invention described above not only suppresses the proliferation of pathogenic bacteria within host cells infected with pathogenic bacteria, but also has the effect of suppressing cell death in host cells infected with pathogenic bacteria. Therefore, the bE3 autodegradation inducer of the present invention described above can also be called an inhibitor of cell death in host cells infected with pathogenic bacteria. Furthermore, a composition containing the bE3 autodegradation inducer of the present invention described above can also be called an inhibitor of cell death in host cells infected with pathogenic bacteria.

[0100] The pathogenic bacteria mentioned above include Shigella, Salmonella, Pest Bacillus, Edwardsiella, Pseudomonas, Rhizobium, and Ralstonia.

[0101] The bE3 autodegradation inducer of the present invention, or the bE3 autodegradation inducer of the present invention, in the inhibitor of pathogenic bacterial growth within a pathogenic bacterial infection host, has been described in detail in the section on "bE3 autodegradation inducer," so a further explanation is omitted here.

[0102] <Treatment Method> The present invention also includes a method for treating infectious diseases using the above-mentioned bE3 autodegradation inducer, pathogenic bacterial growth inhibitor in a host infected with pathogenic bacteria, cell death inhibitor for host cells infected with pathogenic bacteria, or infectious disease treatment drug. Specific descriptions of the bE3 autodegradation inducer, pathogenic bacterial growth inhibitor in a host infected with pathogenic bacteria, cell death inhibitor for host cells infected with pathogenic bacteria, and infectious disease treatment drug can be found in the descriptions in their respective sections.

[0103] The present disclosure will be specifically illustrated by the following embodiments, but the present disclosure shall not be construed as being limited by these embodiments.

[0104] 1. Screening of compounds that bind to ubiquitin ligases To identify compounds that commonly bind to the ubiquitin ligase domains of the pathogenic bacteria IpaH4.5 (Shigella) and SspH2 (Salmonella), which are E3 ubiquitin ligases (bE3), a two-step process was used to screen for compounds.

[0105] (1) Step 1: Compounds to bind to the NEL domain of ubiquitin ligase: Compound library of the Medical Support Center, Kyoto University (2438 compounds) Screening system: Ubiquitin ligase domains (NEL domains) of Shigella and Salmonella bE3 (IpaH4.5 and SspH2) were prepared and purified using Escherichia coli, and a system was established to detect the interaction between the compound and the purified protein using differential scanning fluorimetry (DSF). Using the established system, high-throughput screening was performed on the compound library of the Medical Support Center, Kyoto University (2438 compounds). As a result, 102 hit compounds were obtained. The amino acid sequences of the NEL domain of IpaH4.5 (SEQ ID NO: 1) and the NEL domain of SspH2 (SEQ ID NO: 2) are shown in the sequence listing.

[0106] (2) Step 2: Target compounds that do not bind to HECT of ubiquitin ligase: 102 hit compounds obtained in Step 1 of (1) Screening system: In order to investigate the selectivity and specificity of the hit compounds obtained in Step 1, a HECT domain of HECT-type bE3 different from the NEL type was used to select binding compounds using the same DSF method as above. As a result, 22 hit compounds were obtained as candidate compounds that bind to NEL type but do not bind to HECT-type bE3 (see table below). The HECT domain of the above HECT-type bE3 was discovered by the inventors, and its amino acid sequence (SEQ ID NO: 3) is shown in the sequence listing.

[0107]

[0108] 2. Synthesis of Conjugates (1) Synthesis of Cpd_M (bis-PEG2-Antimycin A) Synthesis procedure: N,N-diisopropylethylamine (DIEA) (61 mg, 471 μmol) was added to a mixture of Antimycin A (100 mg, 182 μmol), bis-PEG2-acid (18 mg, 87 μmol), and HBTU (66 mg, 175 μmol) in DMF (1 ml) at room temperature, and the mixture was stirred at room temperature under a nitrogen atmosphere for 19 hours. Next, the DMF was removed under reduced pressure, the mixture was dissolved in ethyl acetate, and the solution was transferred to a separatory funnel. The organic phase was washed with distilled water, and the aqueous phase was extracted three times using ethyl acetate. Na2SO4 was added to the organic phase and dried, and the solids were removed by cotton plug filtration, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 3:1–1.5:1) to obtain the target product, bis-PEG2-Antimycin A (103 mg, 85 μmol, 98%), as a colorless amorphous solid.

[0109] NMR data: 1H NMR (400 MHz, CDCl3) δ 0.85-1.00 (m, 12H), 1.21-1.32 (m, 36H), 1.66-1.76 (m, 4H), 2.53 (m, 2H), 2.67 (t, J = 5.5 Hz, 4H), 3.80 (m, 4H), 3.84 (t, J = 5.5 Hz, 4H), 4.99 (m, 2H), 5.09 (m, 2H), 5.28 (dd, J = 7.6, 7.6 Hz, 2H), 5.71 (m, 2H), 6.87 (dd, J = 8.0, 8.0 Hz, 2H), 7.24 (m, 4H), 8.42 (d, J = 8.0 Hz, 2H), 8.95 (s, 2H), 12.5 (s, 2H).

[0110] The synthesis scheme for Cpd_M (bis-PEG2-Antimycin A) is shown below.

[0111]

[0112] (2) Synthesis of Cpd_S (bis-PEG1-Antimycin A) Synthesis procedure: N,N-diisopropylethylamine (DIEA) (61 mg, 471 μmol) was added to a mixture of Antimycin A (100 mg, 182 μmol), bis-PEG1-acid (18 mg, 87 μmol), and HBTU (66 mg, 175 μmol) in DMF (1 ml) at room temperature, and the mixture was stirred at room temperature under a nitrogen atmosphere for 19 hours. Next, the DMF was removed under reduced pressure, dissolved in ethyl acetate, and transferred to a separatory funnel. The organic phase was washed with distilled water, and extraction was performed three times from the aqueous phase using ethyl acetate. Na2SO4 was added to the organic phase and dried, and the solids were removed by cotton plug filtration, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 3:1 to 1.5:1) to obtain the target product, bis-PEG1-Antimycin A (103 mg, 85 μmol, 98%), as a colorless amorphous solid.

[0113] NMR data: 1H NMR (400 MHz, CDCl3) δ 0.84-1.00 (m, 12H), 1.17-1.33 (m, 36H), 1.66-1.78 (m, 4H), 2.52 (m, 2H), 2.76 (t, J = 5.9 Hz, 4H), 3.89 (m, 4H), 4.99 (m, 2H), 5.08 (m, 2H), 5.29 (dd, J = 7.6, 7.6 Hz, 2H), 5.71 (m, 2H), 6.84 (dd, J = 8.3, 8.3 Hz, 2H), 7.21 (m, 4H), 8.45 (d, J = 8.3 Hz, 2H), 8.73 (s, 2H), 12.4 (s, 2H).

[0114] The synthesis scheme for bis-PEG1-Antimycin A is shown below.

[0115]

[0116] (3) Synthesis of Cpd_L (bis-PEG4-Antimycin A) Synthesis procedure: N,N-diisopropylethylamine (DIEA) (47 mg, 365 μmol) was added to a mixture of Antimycin A (100 mg, 182 μmol), bis-PEG4-acid (26 mg, 87 μmol), and HBTU (69 mg, 182 μmol) in CH2Cl2 (1.0 ml) at room temperature, and the mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours. Next, it was dissolved in ethyl acetate and transferred to a separatory funnel. The organic phase was washed with saturated ammonium chloride aqueous solution, and extraction was performed three times from the aqueous phase using ethyl acetate. Na2SO4 was added to the organic phase and dried, and the solids were removed by cotton plug filtration, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (CH2Cl2 / hexane = 1:1 → CH2Cl2 / MeOH = 100:1 to 50:1) to obtain the target product, bis-PEG4-Antimycin A (72 mg, 56 μmol, 64%).

[0117] NMR data: 1H NMR (400 MHz, CDCl3) δ 0.84-1.00 (m, 12H), 1.17-1.32 (m, 36H), 1.70 (m, 4H), 2.52 (m, 2H), 2.70 (t, J = 5.7 Hz, 4H), 3.64 (m, 4H), 3.70 (m, 8H), 3.82 (t, J = 5.5 Hz, 4H), 4.98 (m, 2H), 5.08 (m, 2H), 5.29 (dd, J = 7.6, 7.6 Hz, 2H), 5.71 (m, 2H), 6.89 (dd, J = 8.0, 8.0 Hz, 2H), 7.26-7.33 (m, 4H), 8.40 (d, J = 8.9 Hz, 2H), 8.99 (s, 2H), 12.4 (s, 2H).

[0118] The synthesis scheme for bis-PEG4-Antimycin A is shown below.

[0119]

[0120] 3. Evaluation of Resolution Using a retrovirus expression system, MEF cell lines expressing NEL-type bE3 from Shigella and Salmonella were created, and the bE3 resolution of the three compounds synthesized in "2. Synthesis of Conjugates" was investigated.

[0121] (1) Production of NEL-type bE3-expressing MEF cells The target gene (NEL-type bE3 from Shigella or Salmonella) was incorporated into a retroviral vector, and these vectors were introduced into Plat-E cells, which are retroviral package cells, to produce retroviruses expressing each bE3. Mouse embryonic fibroblasts (MEFs) were infected with the produced viruses, and MEF cells expressing each bE3 were produced by selecting them with puromycin, a drug selection marker.

[0122] (2) Degradation of Shigella bE3 MEF cell lines expressing Shigella bE3 (FLAG-IpaH7.8) were administered Cpd_S,M,L (10,3,1,0.33 μM) for 4 hours, and the ability to induce degradation of bE3 was examined by Western blotting. Specifically, the above compounds were administered to MEF cell lines expressing FLAG-IpaH7.8, and a whole cell lysate was prepared. Western blotting was performed, and the degradation of IpaH7.8 was examined using an anti-FLAG antibody. The results are shown in Figure 2. The bands indicate the amount of IpaH7.8 protein.

[0123] As shown in Figure 2, all three compounds showed degrading activity, but when Cpd_M was administered, the degradation of bE3 was most accelerated.

[0124] (3) Confirmation of the degradation mechanism We confirmed whether the degradation confirmed in (2) above utilizes the ubiquitin-proteasome degradation system by using inhibitors. MG132 (proteasome inhibitor) and MLN7243 (E1 inhibitor) were administered simultaneously to the same cells as in Cpd_M, and the ability to suppress bE3 degradation was examined. Specifically, each compound was administered to a strain expressing FLAG-IpaH7.8, and whole cell lysates were prepared. Western blotting was performed, and the degradation of IpaH7.8 was examined using an anti-FLAG antibody. The results are shown in Figure 3. The bands indicate the amount of IpaH7.8 protein. An anti-Tubulin antibody was used to show the amount of Tuburin protein, which served as a loading control for the total amount of protein.

[0125] As shown in Figure 3, bE3 degradation was observed in cells administered with Cycloheximide (CHX; a protein synthesis inhibitor) and Cpd_M, but bE3 degradation was inhibited in cells administered with Cpd_M and MG132 simultaneously, and in cells administered with Cpd_M and MLN7243 (an E1 inhibitor). These findings indicate that Cpd_M-mediated bE3 degradation utilizes the ubiquitin-proteasome degradation system.

[0126] (4) Degradation of other NEL-type bE3s Many family molecules exist within the NEL-type bE3 family. We investigated whether Cpd_M also degrades these family molecules using a similar experimental system. There are 10 types of NEL-type bE3 (IpaH family) in Shigella and 3 types of bE3 in Salmonella. Of these, cells expressing Shigella (IpaH4.5, 9.8, 7.8) and Salmonella (SspH2) were administered each compound, and their ability to degrade bE3 was investigated. Specifically, Cpd_M (1 μM, 10 μM), Cycloheximide (CHX; protein synthesis inhibitor 20 μg / mL), or DMSO (control) were administered to each cell, and after 4 hours, a whole cell lysate was prepared. Western blotting was performed, and the degradation of IpaH4.5, 9.8, 7.8, and SspH2 was investigated using an anti-FLAG antibody. The results are shown in Figure 4. The bands indicate the protein levels at IpaH 4.5, 9.8, 7.8, and SspH 2, respectively.

[0127] As shown in Figure 4, Cpd_M decomposed all bE3 molecules. From the above, it was found that this Cpd_M decomposes the NEL-type bE3 family.

[0128] 4. Effect against bacterial infection 1x10 5 Individual MEF cells were seeded in a 24-well plate, and the following day, they were infected with cultured Salmonella (moi=500). Simultaneously with infection, Cpd_M (10 μM) was administered to the cells. One hour after infection, the cells were washed three times with killing medium containing Gentamycin and Kanamycin to remove extracellular Salmonella. Infected cells were harvested 6 and 20 hours after infection, and the harvested cells (n=4) were lysed in physiological saline containing 0.1% TritonX, serially diluted, and seeded in an LB plate. They were cultured overnight at 37°C, and the number of viable cells was counted the following day.

[0129] As a result, administration of the compound suppressed the growth of Salmonella bacteria in cells (33% suppression at 6 hours and 58% suppression at 20 hours) (Figure 5).

[0130] 5. Examination of the cytotoxicity of the compounds. Toxicity tests of the compounds were conducted using HeLa cells. Specifically, 5x10 4HeLa cells were seeded in a 24-well plate, and after 6 hours, the compound (Cpd_L, Cpd_M, Cpd_S, Antimycin, DMSO, 2.5 μM, 0.05% DMSO) was administered to the cells. Cell proliferation was monitored every 3 hours, and cell growth was observed for 60 hours.

[0131] As a result, administration of a compound known to be cytotoxic (Antimycin) prevented cell proliferation due to cytotoxicity. However, cells treated with the compounds (Cpd_L, Cpd_M, Cpd_S) proliferated similarly to control (DMSO) cells (Figure 6).

[0132] 6. Examination of the inhibitory effect on intracellular Salmonella growth in bacterial infections. Using the same infection experimental system as described in "4. Effects on bacterial infections" above, the effect of the compound on intracellular Salmonella was examined. Specifically, 1 x 10 5 Individual MEF cells were seeded in a 24-well plate, and the following day, they were infected with cultured Salmonella (moi=10). Simultaneously with infection, compounds (Cpd_L, Cpd_M, Cpd_S, DMSO) were administered to the cells. One hour after infection, the cells were washed three times with killing medium containing Gentamycin and Kanamycin to remove extracellular Salmonella. Twenty-four hours after infection, the infected cells were collected, and the collected cells (n=3) were lysed in physiological saline containing 0.1% TritonX, serially diluted, and seeded in an LB plate. They were cultured overnight at 37°C, and the number of viable cells was counted the following day.

[0133] As a result, administration of the compound suppressed the growth of Salmonella bacteria in cells (Cpd_L: 25%, Cpd_M: 99%, Cpd_S: 99.9% suppression) (Figure 7).

[0134] 7. Examination of the effect of the compound on suppressing host cell death in bacterial infections. Using the same infection experimental system as described in "4. Effects on bacterial infections" above, the effect of the compound on host cell death was examined. Specifically, 1 x 10 5Individual MEF cells were seeded in a 24-well plate, and the following day, they were infected with cultured Salmonella (moi=10). The compound was administered to the cells simultaneously with infection. One hour after infection, the cells were washed three times with a killing medium containing Gentamycin and Kanamycin to remove extracellular Salmonella. Two days after infection, the cells showed cell death due to infection in the control group, but cell death suppression was observed in the cells treated with the compound (Figure 8). To quantitatively demonstrate the cell death suppression effect, an LDH (Lactate dehydrogenase) assay was performed. LDH activity in the culture supernatant of cells two days after infection, total LDH activity in the cells, and LDH activity in the culture medium alone (as a blank) were measured using Promega's LDH-Glo® Cytotoxicity Assay. Cells treated with 0.2% Triton-X 100 were used to measure total LDH activity in the cells. The host cell death rate (%) was calculated as follows: (LDH activity in culture supernatant - blank) / (total LDH activity in cells - blank) x 100.

[0135] As a result, administration of the compound suppressed host cell death (Figure 9).

[0136] According to the bE3 autodegradation inducer and infectious disease treatment agent of the present invention, the autodegradation of E3 ubiquitin ligase (bE3) derived from pathogenic bacteria is induced within the host infected with pathogenic bacteria, thereby suppressing the growth of pathogenic bacteria and consequently suppressing infection. The bE3 autodegradation inducer and infectious disease treatment agent of the present invention also have the advantage of not directly acting on pathogenic bacteria to suppress their growth, thus making it less likely for resistant bacteria to emerge.

Claims

1. A bE3 autodegradation inducer in an infected host, comprising a conjugate of a first compound having affinity for the E3 ubiquitin ligase (bE3) of a pathogenic bacterium and a second compound having affinity for the E3 ubiquitin ligase (bE3) of the pathogenic bacterium.

2. The bE3 autodegradation inducer according to claim 1, wherein the first compound and the second compound are linked by a linker.

3. The bE3 autodegradation inducer according to claim 1 or 2, which utilizes ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin within an infected host to induce ubiquitination of bE3 derived from pathogenic bacteria.

4. The bE3 autodegradation inducer according to claim 1 or 2, wherein the E3 ubiquitin ligase (bE3) of the above-mentioned pathogenic bacteria is of the NEL (Novel E3 ligase domain) type.

5. The bE3 autodegradation inducer according to claim 1 or 2, wherein the first compound and the second compound do not inhibit the ubiquitin ligase activity of E3 ubiquitin ligase (bE3).

6. The bE3 autodegradation inducer according to claim 1 or 2, wherein the first compound and the second compound are any of the compounds 1 to 22 in the table below.

7. The bE3 autodegradation inducer according to claim 1 or 2, wherein the first compound and the second compound are the same compound.

8. A bE3 autodegradation inducer in the infected host, represented by the following formula (I). (In the formula, E3UB1 and E3UB2 are groups that can bind to the E3 ubiquitin ligase (bE3) of pathogenic bacteria. Linker is a group that chemically binds E3UB1 and E3UB2.) 9. The bE3 autodegradation inducer according to claim 8, wherein E3UB1 and E3UB2 are groups that have the ability to bind to the NEL (Novel E3 ligase domain) type bE3 of pathogenic bacteria.

10. The bE3 autodegradation inducer according to claim 8 or 9, wherein E3UB1 and E3UB2 do not inhibit the ubiquitin ligase activity of E3 ubiquitin ligase (bE3).

11. The bE3 autodegradation inducer according to claim 8 or 9, wherein E3UB1 and E3UB2 are groups derived from any of the compounds 1 to 22 in the table below.

12. The bE3 autodegradation inducer according to claim 8 or 9, wherein E3UB1 and E3UB2 are the same group.

13. A therapeutic agent for infectious diseases comprising the bE3 autodegradation inducer according to claim 1 or 2.

14. A therapeutic agent for infectious diseases comprising the bE3 autodegradation inducer according to claim 8 or 9.

15. The infectious agent according to claim 13, for use against Shigella, Salmonella, Plague bacillus, Edwardsiella, Pseudomonas, Rhizobium, or Ralstonia.

16. The infectious agent according to claim 14, for use against Shigella, Salmonella, Plague bacillus, Edwardsiella, Pseudomonas, Rhizobium, or Ralstonia.

Images