RIPK1 kinase inhibitor and composition for prevention or treatment of RIPK1-mediated inflammatory diseases comprising same

Abstract

The present invention relates to a RIPK1 kinase inhibitor comprising phensuximide or a pharmaceutically acceptable salt thereof as an active ingredient.

Description

RIPK1 kinase inhibitor and composition for the prevention or treatment of RIPK1-mediated inflammatory diseases containing the same

[0001] The present invention relates to a RIPK1 kinase inhibitor and a composition for the prevention or treatment of RIPK1-mediated inflammatory diseases comprising the same.

[0002] Receptor-interacting serine / threonine kinase (RIPK1) acts as a central regulator controlling the decision between cell survival, apoptosis, and inflammation in response to various stimuli. While RIPK1's scaffold function participates in normal NF-κB and MAPK pathways, RIPK1 kinase activity plays a key role in determining cell fate during RIPK3-mediated necroptosis, a form of immunogenic apoptosis. RIPK1 kinase activity is known to mediate or exacerbate pathological conditions in inflammatory or degenerative diseases such as rheumatoid arthritis, sepsis, inflammatory bowel disease (IBD), ischemia, psoriasis, and multiple sclerosis. In particular, inhibition of RIPK1 kinase has been shown to provide a potent and consistent protective effect against sepsis by alleviating hypothermia and preventing lethality. This suggests the importance of RIPK1 kinase activity in sustaining post-infection immune hypersensitivity responses.

[0003] Therefore, RIPK1 is considered a promising therapeutic target, and numerous RIPK1 inhibitors have been developed to date. Necrostatin-1 (Nec-1), the first RIPK1 kinase inhibitor, binds to the allosteric pocket to disrupt the structural rearrangement of the N-lobe and C-lobe and binds to the back side of the kinase domain. Nec-1 has been widely used to study the mechanisms of necroptosis and to understand the role of RIPK1 kinase in disease models. However, this compound failed to achieve clinical relevance due to its very short in vivo half-life and poor metabolic stability. Several other RIPK1 selective small molecule inhibitors, such as GSK'2982772 and DNL747, have entered human phase I / II clinical trials. Researchers continue to develop RIPK1 inhibitors for clinical application. The presence of a unique allosteric pocket regulating kinase activity in RIPK1 enables the development of selective small molecule kinase inhibitors, which is a major factor highlighting RIPK1 as a therapeutic target. Clinical trials of RIPK1 inhibitors in patients are still in the early stages, and to date, only RIPK1 inhibitors with limited chemical structures possessing appropriate in vivo properties have been developed. Importantly, the ability to develop highly selective small molecule kinase inhibitors targeting RIPK1 is attributed to the presence of a unique allosteric pocket adjacent to RIPK1's ATP binding site. This pocket maintains a specific DLG-out / Glu-out inactive structure that regulates the kinase activity of RIPK1.

[0004] Meanwhile, there is growing interest in drug repurposing, which involves using drugs for diseases other than their original indications. Drug repurposing offers numerous advantages, and several recent success stories have demonstrated both public health benefits and commercial value. Due to its various interconnected benefits, drug repurposing significantly accelerates and lowers the initial development stages of repurposed drugs, thereby increasing their potential for market entry. Approaches used in drug repurposing can be divided into computational and experimental approaches. Various computational approaches can be used individually or in combination to systematically analyze diverse types of large-scale data, such as gene expression, chemical structure, genotype, proteomic data, or electronic health records. Among these, molecular docking is a structure-based computational strategy designed to predict binding site complementarity between a drug and a therapeutic target protein.

[0005] Therefore, there is a need to repurpose drugs that are already FDA-approved and possess RIPK1 inhibitory effects, enabling their immediate use as compositions for the prevention or treatment of RIPK1-mediated diseases without complex procedures such as clinical use approval.

[0006] Korean Published Patent No. 10-2023-0031851, which is the technology forming the background of the present invention, relates to isoxazolidine as a RIPK1 inhibitor and its use.

[0007] The present invention aims to solve the problems of the aforementioned prior art by providing a RIPK1 kinase inhibitor comprising phensuximide, which has already been FDA-approved as an epilepsy treatment, as an active ingredient.

[0008] In addition, a composition for the prevention or treatment of RIPK1-mediated inflammatory diseases is provided, comprising the above-mentioned RIPK1 kinase inhibitor as an active ingredient.

[0009] In addition, a health functional food for the prevention or improvement of RIPK1-mediated inflammatory diseases is provided, comprising the above-mentioned RIPK1 kinase inhibitor as an active ingredient.

[0010] However, the technical problems that the embodiments of the present invention aim to solve are not limited to the technical problems described above, and other technical problems may exist.

[0011] As a technical means to achieve the above-mentioned technical problem, the first aspect of the present invention provides a RIPK1 kinase inhibitor comprising phensuximide or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] According to one embodiment of the present invention, the phensuximide may specifically inhibit the activity of RIPK1 kinase, but is not limited thereto.

[0013] According to one embodiment of the present invention, the phensuximide may not affect NF-κB activation, but is not limited thereto.

[0014] According to one embodiment of the present invention, the phensuximide may not affect MAPK activation, but is not limited thereto.

[0015] According to one embodiment of the present invention, the expression of inflammatory cytokines may be inhibited by the phensuximide, but is not limited thereto.

[0016] According to one embodiment of the present invention, the inflammatory cytokine may include IL-8, but is not limited thereto.

[0017] According to one embodiment of the present invention, necroptosis may be inhibited by the phensuximide, but is not limited thereto.

[0018] In addition, the second aspect of the present invention provides a composition for the prevention or treatment of RIPK1-mediated inflammatory diseases, comprising a RIPK1 kinase inhibitor according to the first aspect of the present invention as an active ingredient.

[0019] According to one embodiment of the present invention, the RIPK1-mediated inflammatory disease may include, but is not limited to, a disease selected from the group consisting of systemic inflammatory response syndrome (SIRS), sepsis, degenerative brain disease, ischemic brain injury, liver disease, and combinations thereof.

[0020] In addition, the third aspect of the present invention provides a health functional food for the prevention or improvement of RIPK1-mediated inflammatory diseases, comprising a RIPK1 kinase inhibitor according to the first aspect of the present invention as an active ingredient.

[0021] According to one embodiment of the present invention, the RIPK1-mediated inflammatory disease may include, but is not limited to, a disease selected from the group consisting of systemic inflammatory response syndrome (SIRS), sepsis, degenerative brain disease, ischemic brain injury, liver disease, and combinations thereof.

[0022] The means for solving the problem described above are merely exemplary and should not be interpreted as intended to limit the present invention. In addition to the exemplary embodiments described above, additional embodiments may exist in the drawings and the detailed description of the invention.

[0023] The present invention identifies phensuximide, a drug previously approved as a treatment for epilepsy, as a novel inhibitor of RIPK1 kinase activity, thereby presenting a new therapeutic strategy for RIPK1-mediated diseases. Specifically, the phensuximide can effectively prevent RIPK1-dependent necroptosis, and thereby improve the pathological conditions of various inflammatory and degenerative diseases associated with RIPK1 kinase activity.

[0024] In addition, since the above-mentioned phensuximide does not affect apoptosis that does not depend on NF-κB and MAPK pathways or RIPK1 kinase activation, it can selectively act to inhibit RIPK1 kinase activity, which may be advantageous in terms of drug safety.

[0025] In addition, since the above phensuximide showed an effect of alleviating RIPK1-mediated inflammation in an LPS-induced systemic inflammatory response syndrome (SIRS) mouse model, it can be used for the prevention or treatment of serious RIPK1-mediated inflammatory diseases such as sepsis.

[0026] Furthermore, since the aforementioned fensuximide is a drug already approved by the FDA, drug repurposing could shorten the development process and reduce costs for treatments for RIPK1-related diseases, which could ultimately contribute to the rapid clinical application of the new treatment and improved patient access.

[0027] However, the effects obtainable from this invention are not limited to those described above, and other effects may exist.

[0028] Figure 1a is a schematic diagram of the process of ranking drugs that share structural similarity with Necrostatin-1 in the DrugBank dataset, Figure 1b shows the results displaying the similarity values ​​for the top 7 ranked drugs and Nec-1, Figure 1c shows the docking scores of Phensuximide (Phen), Ethotoin, and Sumatriptan for the RIPK1 kinase domain and the results of comparing their corresponding chemical structures, Figure 1d shows the results of molecular docking analysis depicting the binding patterns of Nec-1, Phen, ethotoin, and sumatriptan within the RIPK1 kinase domain, and Figure 1e shows the root-mean-square deviation (RMSD) values ​​of the backbone (backbone of the protein), protein (RIPK1 kinase domain), and ligand (Necrostatin-1 or Phensuximide) through molecular dynamics (MD) simulation analysis.

[0029] Figures 2a to 2c show the results of pre-treating HT-29 cells with Phensuximide or Nec-1 (40 μM) at indicated concentrations for 1 hour, followed by treatment with TNF-α (30 ng / mL) + SMAC (200 nM) + z-VAD (20 μM) for indicated times, and measuring cytotoxicity using LDH analysis (2a and 2c, left panel), analysis of SytoxOrange fluorescence intensity using Lionheart FX automated microscopy (2b, left panel), and phase contrast microscopy (2a to 2c, right panel); Figures 2d and 2e show the results of SytoxOrange staining used to measure cell viability of MC-38 (Figure 2d) and MEF cells (Figure 2e) detected by Lionheart FX automated microscopy. (As representative images from the indicated number of independent experiments, statistical analysis was performed using two-way ANOVA, and P-values ​​less than 0.05 were considered significant in the following manner: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.)

[0030] Figures 3a and 3b show the chemical structure of ethotoin (Figure 3a, left panel), the results of analyzing cell viability by MTT assay (Figure 3a, middle panel), phase contrast microscopy (Figure 3a, right panel), and detection of fluorescence intensity of SytoxOrange using Lionheart FX automated microscopy after pre-treating HT-29 cells with the indicated concentration of ethotoin for 1 hour followed by treatment with TSZ for the indicated time (Figure 3b), and Figures 3c and 3d show the chemical structures of Phensuximide and ethosuximide (Figure 3c, left panel), the results of analyzing cell viability by detecting LDH leakage (Figure 3c, middle panel), phase contrast microscopy (Figure 3c, right panel), and fluorescence intensity of SytoxOrange after pre-treating cells with the indicated concentration of ethosuximide for 1 hour followed by treatment with TSZ for the indicated time (Figure 3d). (Representative images from the indicated number of independent experiments; statistical analysis was performed using two-way ANOVA, and P-values ​​less than 0.05 were considered significant in the following manner: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant)

[0031] Figures 4a to 4c show the results of Western blot analysis of cell lysates after pre-treating HT-29 (Figures 4a and 4b) or MC-38 cells (Figure 4c) with indicated concentrations of Phensuximide or Nec-1 for 1 hour followed by treatment with TSZ for the indicated time; Figure 4d shows the results of visualizing HT-29 cells treated with TSZ stained with p-MLKL antibody and visualized by confocal fluorescence microscopy (green: p-MLKL, blue: DAPI); Figure 4e shows the results of treating stable HT-29 cells expressing Tamoxifen-inducible MLKL T357E / S358D with Phensuximide, Nec-1, or necrosulfonamide (NSA, 1 μM) for 3 hours and with or without Tamoxifen; and Figure 4f shows TRAIL (50) after pre-treating cells with Phen or Nec-1 for 1 hour Figure 4g shows the results of analyzing cell lysates by Western blot (top panel) and cell viability by LDH leakage (middle panel) or MTT assay (bottom panel) after treatment with ng / mL) + Smac + z-VAD (4 hours for immunoblotting, 6 hours for apoptosis assay); Figure 4g shows the results of analyzing cell lysates by immunoblotting after transfecting 293T cells with Flag-tagged RIPK1 (left panel) or Flag-tagged RIPK3 (right panel) for 18 hours and adding Phen, Nec-1, or GSK'872 4 hours after transfection; and Figure 4h shows RIPK1 - / - This is the result of transfecting / RIPK3 silenced MEF cells with Flag-tagged RIPK1 for 18 hours, and adding z-VAD, Phen, Nec-1, or GSK'872 6 hours after transfection.

[0032] Figure 5a shows the results of immunoblotting analysis of cell lysates after treating HT-29 (top panel), MC-38 (middle panel), or MEF cells (bottom panel) with TNF for the indicated times depending on the presence or absence of Phensuximide, and Figures 5b to 5e show the results of treating HT-29 cells with cycloheximide (CHX, 5 μg / mL) (Figs. 5b and 5c) or TRAIL (6 hours for immunoblotting, 12 hours for apoptosis analysis) (Figs. 5d and 5e) for 1 hour depending on the presence or absence of Phensuximide, followed by treatment with TNF (4 hours for immunoblotting, 12 hours for apoptosis analysis), and analyzing cell lysates by Western blot (Figs. 5b and 5d), and cell viability by LDH leakage (Fig. 5c top panel and Fig. 5e) or MTT assay (Fig. 5c bottom panel). This is the result.

[0033] Figures 6a to 6c show the results of immunoblotting analysis of cell lysates (Figure 6a) or HMGB1 release (Figure 6c) and qPCR measurement of relative mRNA levels of IL-8 or CXCL1 after treating HT-29 cells with TSZ for the indicated times in the presence or absence of Phensuximide or Nec-1 (Figure 6b); Figures 6d to 6f show the results of Western blot analysis of cell lysates (Figure 6d), LDH leakage analysis of cell viability (Figure 6e), and qPCR analysis of mRNA levels of Il-1β, Il-6, Cxcl1, and Tnf-α after pre-treating BMDM with Phensuximide or Nec-1 for 1 hour followed by treatment with Smac + z-VAD for 6 hours (Figure 6f); and Figures 6g and 6h show BMDM for 24 hours in the presence or absence of Phensuximide The results of analyzing cell lysates by Western blot (Fig. 6g) after treatment with LPS / IFN-γ (M1) or interleukin-4 (M2) and evaluating the expression of CD86 and CD206 using a flow cytometer (Fig. 6h), Fig. 6i shows the results of treating BMDM with LPS for the indicated times with or without the presence of Phensuximide, and Fig. 6j shows the results of analyzing the relative mRNA expression levels of BMDM by qPCR after treating BMDM with Phen for 1 hour followed by LPS for 4 hours.

[0034] Figure 7a shows the results of tracking survival for 24 hours in 8-9 week old C57BL / 6J mice after intraperitoneal pretreatment with Phen (50 mg / kg) or PBS for 1 hour followed by intraperitoneal injection of LPS (40 mg / kg); Figure 7b shows the results of ALT, AST, and BUN measurements in the serum of mice treated with LPS for 6 hours; Figure 7c shows representative images of H&E staining of lung tissue (scale bar = 50 μm); and Figures 7d and 7e show the results of measuring relative mRNA levels of cytokines in the lungs (Figure 7d), liver (Figure 7e, top panel), and kidneys (Figure 7e, bottom panel) (results are expressed as mean ± SEM, and statistical analysis was performed using an unpaired two-sided Student's t-test).

[0035] Embodiments of the present invention are described below with reference to the attached drawings so that those skilled in the art can easily implement them. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification are denoted by similar reference numerals.

[0036] Throughout this specification, when a part is described as being "connected" to another part, this includes not only cases where they are "directly connected," but also cases where they are "electrically connected" with other elements interposed between them.

[0037] Throughout the entire specification, when a component is described as being located "on," "on top," "on top," "under," "on bottom," or "on bottom" of another component, this includes not only cases where the component is in contact with the other component but also cases where another component exists between the two components.

[0038] Throughout this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0039] As used herein, terms of degree such as “about,” “substantially,” etc., are used to mean at or near the stated value when inherent manufacturing and material tolerances are presented in the stated meaning, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure in which precise or absolute values ​​are mentioned to aid in understanding the present invention. Furthermore, throughout this specification, “a step of” or “a step of” does not mean “a step for”.

[0040] Throughout this specification, the term “combination thereof” included in the Markush-type expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush-type expression, and means including one or more selected from the group consisting of said components.

[0041] Throughout the entire specification, the description "A and / or B" means "A, B, or A and B".

[0042] Hereinafter, the RIPK1 kinase inhibitor of the present invention and the composition for the prevention or treatment of RIPK1-mediated inflammatory diseases containing the same will be described in detail with reference to embodiments, examples, and drawings. However, the present invention is not limited to these embodiments, examples, and drawings.

[0043]

[0044] As a technical means to achieve the above-mentioned technical problem, the first aspect of the present invention provides a RIPK1 kinase inhibitor comprising phensuximide or a pharmaceutically acceptable salt thereof as an active ingredient.

[0045] The present invention discovers phensuximide, a drug previously approved as a treatment for epilepsy, as a novel inhibitor of RIPK1 kinase activity, thereby presenting a new therapeutic strategy for RIPK1-mediated diseases.

[0046] According to one embodiment of the present invention, the phensuximide may specifically inhibit the activity of RIPK1 kinase, but is not limited thereto.

[0047] According to one embodiment of the present invention, the phensuximide may not affect NF-κB activation, but is not limited thereto.

[0048] According to one embodiment of the present invention, the phensuximide may not affect MAPK activation, but is not limited thereto.

[0049] Since the above-mentioned phensuximide does not affect apoptosis that does not depend on NF-κB and MAPK pathways or RIPK1 kinase activation, it can selectively act to inhibit RIPK1 kinase activity, which may be advantageous in terms of drug safety.

[0050] According to one embodiment of the present invention, the expression of inflammatory cytokines may be inhibited by the phensuximide, but is not limited thereto.

[0051] According to one embodiment of the present invention, the inflammatory cytokine may include IL-8, but is not limited thereto.

[0052] IL-8 is a potent inflammatory cytokine that plays a crucial role in attracting and activating immune cells, such as neutrophils, to sites of inflammation. The aforementioned phensuximide can regulate excessive inflammatory responses and alleviate tissue damage caused by inhibiting the expression of IL-8.

[0053] In addition, the above IL-8 can interact with other inflammatory cytokines and mediators and play a role in amplifying the inflammatory response; by inhibiting the expression of IL-8, the above phensuximide can regulate the inflammatory cytokine network overall and contribute to blocking the vicious cycle of the inflammatory response.

[0054] According to one embodiment of the present invention, necroptosis may be inhibited by the phensuximide, but is not limited thereto.

[0055] The above-mentioned phensuximide can effectively prevent RIPK1-dependent necroptosis and, thereby, improve the pathological conditions of various inflammatory and degenerative diseases associated with RIPK1 kinase activity.

[0056] In addition, since the above phensuximide showed an effect of alleviating RIPK1-mediated inflammation in an LPS-induced systemic inflammatory response syndrome (SIRS) mouse model, it can be used for the prevention or treatment of serious RIPK1-mediated inflammatory diseases such as sepsis.

[0057] Furthermore, since the aforementioned fensuximide is a drug already approved by the FDA, drug repurposing could shorten the development process and reduce costs for treatments for RIPK1-related diseases, which could ultimately contribute to the rapid clinical application of the new treatment and improved patient access.

[0058]

[0059] In addition, the second aspect of the present invention provides a composition for the prevention or treatment of RIPK1-mediated inflammatory diseases, comprising a RIPK1 kinase inhibitor according to the first aspect of the present invention as an active ingredient.

[0060] Regarding the composition for the prevention or treatment of RIPK1-mediated inflammatory diseases according to the second aspect of the present invention, detailed descriptions of parts that overlap with the first aspect of the present invention have been omitted, but even if such descriptions are omitted, the contents described in the first aspect of the present invention may be applied equally to the second aspect of the present invention.

[0061] According to one embodiment of the present invention, the RIPK1-mediated inflammatory disease may include, but is not limited to, a disease selected from the group consisting of systemic inflammatory response syndrome (SIRS), sepsis, degenerative brain disease, ischemic brain injury, liver disease, and combinations thereof.

[0062]

[0063] *

[0064] In addition, the third aspect of the present invention provides a health functional food for the prevention or improvement of RIPK1-mediated inflammatory diseases, comprising a RIPK1 kinase inhibitor according to the first aspect of the present invention as an active ingredient.

[0065] Regarding the health functional food for the prevention or improvement of RIPK1-mediated inflammatory diseases according to the third aspect of the present invention, detailed descriptions of parts that overlap with the first aspect of the present invention have been omitted, but even if such descriptions are omitted, the contents described in the first aspect of the present invention may be applied equally to the third aspect of the present invention.

[0066] According to one embodiment of the present invention, the RIPK1-mediated inflammatory disease may include, but is not limited to, a disease selected from the group consisting of systemic inflammatory response syndrome (SIRS), sepsis, degenerative brain disease, ischemic brain injury, liver disease, and combinations thereof.

[0067] The present invention is to be explained in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0068]

[0069] [Example 1] Identification of Phensuximide as a potential RIPK1 inhibitor

[0070] To investigate potential inhibitors of RIPK1 kinase activity-dependent necroptosis, compounds structurally similar to Nec-1 were screened. Utilizing DrugBank, an established drug database containing 2,468 approved drugs and 8,282 experimental drugs, several candidate substances sharing structural similarity with Nec-1 were identified. (Figures 1a and 1b)

[0071] After identifying structurally similar compounds, their potential interactions with RIPK1 were analyzed in comparison to Nec-1. A comprehensive molecular docking study of candidate compounds sharing structural similarities with Nec-1 found that Phensuximide (Phen) and Ethotoin exhibited higher docking scores than Sumatriptan. (Fig. 1c)

[0072] Nec-1 was observed to form hydrogen bonds with Ser161 and Asp156 within the RIPK1 kinase domain in computer simulations using AutoDock Vina, consistent with results found in the literature (Fig. 1d).

[0073] These interactions are important for inhibiting the activation of the necroptosis pathway by stabilizing the kinase to an inactive structure. Specifically, Ser161 plays a key role in regulating the active state of RIPK1 by influencing the structural kinetics of the kinase. When Nec-1 binds to Ser161, it interferes with phosphorylation, keeping RIPK1 in an inactive state. Asp156, located near the ATP binding site, is important for preserving the structural integrity of this region. Interaction with Nec-1 further inhibits kinase activity by altering the geometry of the ATP binding pocket, thereby interfering with ATP binding and subsequent autophosphorylation.

[0074] When analyzing their binding modes within the RIPK1 kinase domain, it was found that Phen exhibits a binding mode similar to that of Nec-1. Significant π-π stacking interactions were identified between the phenyl ring of Phen and the phenylalanine residue at position 162 of RIPK1 (Phe162). This residue critically contributes to the structural stability of the activation loop, regulating the structural equilibrium between the active and inactive states of the kinase. This suggests that Phen can stabilize RIPK1 into an inactive structure in a manner similar to Nec-1 (Fig. 1d).

[0075] Ethotoin, another structurally similar compound, also showed a binding mode similar to Phen, but Sumatriptan did not form a significant binding with RIPK1 despite the structural similarity (Fig. 1d).

[0076] In addition, molecular dynamics (MD) simulations revealed that Phen forms a stable interaction with the RIPK1 binding site. The RMSD value of the ligand Phen showed less variability and a shorter initial equilibrium period compared to Nec-1, indicating the potential for a more stable interaction with RIPK1 (Fig. 1e).

[0077] In hydrogen bond (H-bond) kinetic simulations, Phen was found to form fewer hydrogen bonds compared to Nec-1. While Nec-1 exhibited stable H-bonds with Val76, Asp156, and Ser161, Phen was expected to form an H-bond with Asp156 of RIPK1. These results indicate that the stable bonding of Phen with RIPK1 is due to strong interactions with Phe162 of the RIPK1 DLG motif.

[0078]

[0079] [Example 2] Strong inhibitory effect of Phensuximide on TNF-induced necroptotic apoptosis

[0080] Phensuximide demonstrated the highest docking score in computer simulations. Phensuximide, belonging to the succinimide class, exhibited anticonvulsant properties associated with impaired consciousness in petit mal seizures by suppressing paroxysmal brainwave patterns characterized by three spikes and wave activity per second.

[0081] First, we tested whether Phensuximide affects TNF-induced necroptosis. TNF-α, the Smac analog, and the pan-caspase inhibitor z-VAD (hereinafter TSZ) are a well-established cocktail used to induce RIPK1-mediated necroptotic apoptosis. The protective effect against Nec-1 is generally understood to indicate inhibition of necroptosis at a concentration of 40 μM. HT-29 cells were treated with TSZ in the presence or absence of Phensuximide, ranging from 25 μM to 100 μM. Phensuximide concentrations below 100 μM showed a partial inhibitory effect on necroptosis.

[0082] Next, the concentration of Phensuximide was increased to 800 μM, and at this high concentration, it completely blocked necroptotic apoptosis without exhibiting cytotoxicity (Fig. 2a). This was further supported by measuring the number of SytoxOrange-positive cells over a longer period (Fig. 2b). Notably, Phensuximide protected cells from necroptosis for over 48 hours without any cytotoxicity, suggesting that Phensuximide may be a potent inhibitor comparable to Nec-1 (Fig. 2c). The inhibitory effect of Phensuximide on TNF-mediated necroptosis was also evident in other human cancer cell lines, such as RIPK3-expressing MDA-MB231, mouse MC-38 cells, and MEF, extending to various cell types and species (Figs. 2d and 2e).

[0083] Next, we tested Ethotoin, a hydantoin derivative with anticonvulsant properties. Ethotoin exhibits an antiepileptic effect without overall central nervous system inhibition, and its mechanism of action appears to be very similar to that of phenytoin. More interestingly, phenytoin has been proposed as an inhibitor of necroptosis and may be partially related to RIPK1 inhibitors. Similar to phenytoin at a concentration of 200 μM, ethotoin provided an inhibitory effect on TNF-mediated necroptosis, suggesting that the screening strategy for identifying drugs structurally related to Nec-1 and associated with high docking scores for RIPK1 was successful (Figs. 3a and 3b).

[0084] Since phenuximide was selected as a candidate drug to inhibit RIPK1-mediated necroptosis in the screening strategy, we verified whether other substances in the succinimide class exhibit the same effect on necroptosis. Ethosuximide is the most commonly used succinimide and remains a useful drug for managing seizures in pediatric medicine. While phenuximide showed a protective effect at 100 μM, ethosuximide provided no inhibitory effect on TNF-mediated necroptosis up to a concentration of 800 μM and did not exhibit cytotoxicity even at this highest concentration. Methsuximide, a succinimide-class anticonvulsant similar to phenuximide, also showed no observable effect on TNF-mediated necroptosis, suggesting that inhibition of necroptosis is specific to phenuximide (Figures 3e and 3f).

[0085]

[0086] [Example 3] Inhibition of RIPK1 kinase activity by Phensuximide treatment

[0087] As previously mentioned, given that Phensuximide exhibits a protective effect against TNF-mediated necroptosis, we found that Phensuximide at a concentration of 100 μM inhibits the phosphorylation of RIPK1 (Serine 166, autophosphorylation site) in response to TSZ treatment (Figs. 4a and 4b). As an upstream kinase of the necroptosis signaling pathway, the inhibition of RIPK1 phosphorylation led to the loss of downstream kinase activation pathways, including RIPK3 and MLKL. This inhibitory property of the drug on RIPK1 phosphorylation was evident in RIPK3-expressing MDA-MB231 cells, another human cell line, as well as in mouse cells MC-38 and MEF (Fig. 4c). We further demonstrated the inhibitory properties of Phensuximide on the RIPK1 phosphorylation sub-events in combination with the RIPK3 kinase inhibitor GSK'872 and other necroptosis inhibitors, including the MLKL inhibitor NSA (necrosulfonamide). As correlated with the apoptosis data (Figs. 3c and 3d), ethosuximide and methsuximide did not inhibit RIPK1 phosphorylation in response to TSZ. Since MLKL phosphorylation is known to be the terminal step of phosphorylation events in the necrosis signaling pathway, Phensuximide treatment eliminated TNF-induced MLKL phosphorylation through RIPK1 inhibition, similar to Nec-1 (Fig. 4d).

[0088] To further support the inhibitory properties of the drugs, tamoxifen-inducible active MLKL-expressing cells (HT-29 (shMLKL / T357E / S358D)) were used to induce necroptosis without upstream signaling such as TSZ. While Nec-1 and Phensuximide did not provide any inhibitory effects on active MLKL-mediated necroptosis, only NSA was able to protect against apoptosis under these conditions (Fig. 4e). Since Phensuximide blocks TNF-induced necroptosis, we next tested whether it inhibits TRAIL-induced necroptosis, which is known to induce RIPK1-mediated necroptosis. Notably, Phensuximide inhibited TRAIL-induced necroptosis as well as TNF-induced necroptosis through the inhibition of RIPK1 phosphorylation (Fig. 4f). Overexpression of RIPK1 or RIPK3 has been reported to induce autophosphorylation, and RIPK1 or RIPK3 were overexpressed in 293 cells, respectively. Similar to Nec-1, Phensuximide completely blocked RIPK1 autophosphorylation rather than RIPK3 autophosphorylation, which was eliminated by GSK'872, suggesting that Phensuximide is a specific inhibitor of RIPK1 (Fig. 4g). These results indicate that RIPK1 - / - / RIPK3 silenced MEF cell, RIPK1 - / - It was consistently reproduced in other cell types, including MEF cells and HeLa cells (Fig. 4h).

[0089]

[0090] [Example 4] Phensuximide does not participate in receptor-mediated complex I signaling

[0091] Given that the apparent inhibitory effect of phen on TNF-induced necroptosis across various cell types and species is characterized by a reduction in RIPK1 autophosphorylation induced by TNFα stimulation, we questioned whether phenuximide interferes with the initial activation of RIPK1 within the TNF receptor 1 (TNFR1) signaling complex I. It was observed that when TNFα binds to TNFR1, the receptor rapidly initiates the formation of complex I, thereby promoting cell survival by activating the NF-κB pathway. Therefore, we investigated whether phenuximide affects TNF-mediated NF-κB and MAPK activation. TNF treatment activated both NF-κB and MAPK signaling regardless of species, with or without the presence of phenuximide (Fig. 5a).

[0092] To rule out the possibility that phenuximide affects the recruitment of RIPK1 to TNFR1, TNFR1 immunoprecipitation experiments were performed in the presence or absence of phenuximide. No changes were observed in the recruitment or ubiquitination of RIPK1 to TNFR1 after TNF treatment, suggesting that the formation of Complex I is not affected by phenuximide. Activation of downstream TNFR1 signaling also induces RIPK1-independent apoptosis using TNF / cycloheximide (CHX). Under these circumstances, phenuximide was unable to block caspase and PARP1 cleavage indicating apoptosis induction, and the number of apoptotic cells was measured by MTT and LDH assays (Figs. 5b and 5c). In addition, TRAIL-induced apoptosis was not affected by the presence of Phensuximide, which suggests that Phensuximide does not affect receptor-mediated complex formation that does not depend on RIPK1 kinase activity (Figs. 5d and 5e).

[0093]

[0094] [Example 5] Inhibition of RIPK1 kinase activity-dependent apoptosis and inflammatory response in macrophages by Phensuximide

[0095] It has been previously reported that necroptosis activates a transcriptional program that induces the expression of various inflammatory cytokines during the process of apoptosis. Since RIPK1 has become a promising target in various human inflammatory diseases, we investigated whether Phensuximide could suppress necroptosis-mediated inflammatory cytokine expression. Although TNF-induced necroptosis can increase IL-8 protein levels in a manner dependent on RIPK1 kinase activity, inhibition of RIPK1 activity by Phensuximide treatment suppressed IL-8 protein levels (Fig. 6a). Similar to Nec-1, the inhibitory effect of Phensuximide on inflammatory cytokine production was at the transcriptional level (Fig. 6b).

[0096] Necroptosis-mediated cell membrane rupture is the final step in releasing cellular components as damage-associated molecular patterns (DAMPs) that initiate an inflammatory response. HMGB1, one of the DAMPs, was detected in the supernatant under necroptosis conditions. However, when Phensuximide was administered, HMGB1 was no longer detected, suggesting the importance of inhibiting RIPK1 kinase activity in regulating the inflammatory response (Fig. 6c).

[0097] Macrophages play a pivotal role in both innate and adaptive immunity and can adopt a diverse spectrum of phenotypes, each possessing unique functions related to immune regulation, in response to changes in the inflammatory microenvironment. To understand the role of macrophages in the inflammatory response, bone marrow-derived macrophages (BMDMs) were isolated from bone marrow and necroptosis was induced. Phensuximide inhibited the RIPK1-mediated necroptosis pathway, thereby suppressing apoptosis through the inhibition of RIPK1 kinase activity (Figs. 6d and 6e). As expected, transcriptional activity of inflammatory cytokines via the necroptosis pathway was lost by Phensuximide (Fig. 6f).

[0098] Next, since the polarization state of macrophages plays a crucial role in regulating immune homeostasis, we investigated whether Phensuximide affects macrophage polarization in the context of inflammatory responses. The major phenotypes of macrophages are classified into pro-inflammatory macrophages (M1 macrophages) and anti-inflammatory macrophages (M2 macrophages). BMDM can differentiate into M1 macrophages upon exposure to lipopolysaccharide (LPS) / interferon (IFN)-γ and into M2 macrophages upon stimulation by interleukin (IL)-4. Phensuximide did not affect macrophage polarization (Figs. 6g and 6g). Inflammatory apoptosis, known as pyroptosis, is another method by which macrophages release pro-inflammatory cytokines and has been identified as a major cause of septic shock. Canonical pyroptotic apoptosis is orchestrated through the assembly of the inflammasome, a complex multimolecular complex activated in host defense against microbial infection, thereby promoting the development of adaptive immune responses. Data to date indicate that Phensuximide has inhibitory effects on necroptotic apoptosis and pro-inflammatory cytokine expression through the inhibition of RIPK1 kinase activity, confirming the effects of Phensuximide in relation to pyroptosis. Since the activation of Toll-like receptor 4 (TLR4) indirectly promotes inflammasome assembly and amplifies the inflammatory process, we first investigated whether Phensuximide affects TLR4 signaling. Phensuximide did not alter the downstream signaling of TLR4, including MAPK activation and NF-κB activation, which increase pro-inflammatory cytokines (Figs. 6i and 6j). Although there are fundamental differences among inflammasomes triggered by various stimuli, Canonical inflammasomes generally promote caspase-1 activation leading to the induction of pyroptosis, and Phensuximide did not affect inflammasome activation.It is noteworthy that pyroptosis is independent of RIPK1 kinase activity, suggesting that Phensuximide exerts specific effects on inflammatory diseases dependent on RIPK1 kinase activity.

[0099]

[0100] [Example 6] Phensuximide as a potential therapeutic candidate for necroptosis-related diseases

[0101] The data to date suggest that Phensuximide is an effective inhibitor that prevents RIPK1 kinase activity, and that this inhibitor could be applied as a potential therapeutic drug candidate for necroptosis-related diseases through drug repurposing. To demonstrate the potential of Phensuximide as a novel inhibitor for necroptosis-related diseases, it was next applied to a mouse model.

[0102] LPS-induced systemic inflammatory response syndrome (SIRS) is a clinically significant entity similar to septic shock. Importantly, it was confirmed that Phensuximide itself does not cause significant damage. The extent of damage was assessed by measuring tracheal and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. LPS was injected intraperitoneally, and Phensuximide or a vehicle was injected into mice starting one hour prior to LPS injection. Pharmacological inhibition of RIPK1 by Phensuximide significantly protected against LPS-induced death (Fig. 7a). Markers of cytotoxicity were analyzed by serum ALT and AST and blood urea nitrogen (BUN), which were significantly reduced in animals treated with Phensuximide. However, there was no difference in creatinine (Fig. 7b). Since death induced by SIRS is often accompanied by severe organ dysfunction, pathological changes occurring in the lungs of this mouse model were evaluated. Six hours after LPS stimulation, histological examination revealed severe edema and alveolar septal thickening in septic animals, whereas lung damage was reduced in Phensuximide-treated animals (Fig. 7c). Lung tissue correlated with these histological findings showed that while septic mice exhibited excessive inflammatory cytokine expression, Phensuximide-treated animals showed reduced inflammatory cytokine expression (Fig. 7d). Similar to the increased inflammatory cytokine expression in LPS-induced lung tissue, excessive inflammatory cytokine expression was also observed in the kidneys and liver (Fig. 7e). Although no significant damage was visualized during this time in hematoxylin and eosin-stained liver and kidney histological sections, these results suggest that Phen is promising as an effective therapeutic agent for resolving RIPK1-induced diseases.

[0103]

[0104] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0105] The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and the concept of equivalents thereof should be interpreted as being included within the scope of the present invention.

Claims

1. Containing phensuximide or a pharmaceutically acceptable salt thereof as an active ingredient, RIPK1 kinase inhibitor.

2. In Paragraph 1, The above phensuximide specifically inhibits the activity of RIPK1 kinase, RIPK1 kinase inhibitor.

3. In Paragraph 2, The above phensuximide does not affect NF-κB activation, RIPK1 kinase inhibitor.

4. In Paragraph 2, The above phensuximide does not affect MAPK activation, RIPK1 kinase inhibitor.

5. In Paragraph 1, The expression of inflammatory cytokines is suppressed by the above-mentioned phensuximide, RIPK1 kinase inhibitor.

6. In Paragraph 5, The above inflammatory cytokine is one that includes IL-8, RIPK1 kinase inhibitor.

7. In Paragraph 1, The inhibition of necroptosis by the above-mentioned phensuximide, RIPK1 kinase inhibitor.

8. A RIPK1 kinase inhibitor according to any one of claims 1 to 7 comprising as an active ingredient, Composition for the prevention or treatment of RIPK1-mediated inflammatory diseases.

9. In Paragraph 8, The above RIPK1-mediated inflammatory disease includes those selected from the group consisting of systemic inflammatory response syndrome (SIRS), sepsis, neurodegenerative disease, ischemic brain injury, liver disease, and combinations thereof. Composition for the prevention or treatment of RIPK1-mediated inflammatory diseases.

10. A RIPK1 kinase inhibitor according to any one of claims 1 to 7 comprising as an active ingredient, Health functional food for the prevention or improvement of RIPK1-mediated inflammatory diseases.

11. In Paragraph 10, The above RIPK1-mediated inflammatory disease includes those selected from the group consisting of systemic inflammatory response syndrome (SIRS), sepsis, neurodegenerative disease, ischemic brain injury, liver disease, and combinations thereof. Health functional food for the prevention or improvement of RIPK1-mediated inflammatory diseases.

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