An inhibitor of SUMO e1-enzyme for use in tissue regeneration

Abstract

The present invention relates to the use of inhibitors or modulators of the SUMOylation pathway for use in a method of increasing or facilitating tissue regeneration in a mammal subject, such as a human subject in need thereof. In particular, the present invention relates to modulation of SUMOylation levels for use in the treatment of conditions associated to damaged intestinal epithelium, such as Crohn's disease and ulcerative colitis, in a mammal subject, such as a human subject in need thereof.

Description

[0001] AN INHIBITOR OF SUMO E1 -ENZYME FOR USE IN TISSUE REGENERATION

[0002] Technical field of the invention

[0003] The present invention relates to the use of inhibitors or modulators of the SUMOylation pathway for use in a method of increasing or facilitating tissue regeneration. In particular, the present invention relates to modulation of SUMOylation levels for use in the treatment of conditions associated to damaged intestinal epithelium, such as Crohn's disease and ulcerative colitis.

[0004] Background of the invention

[0005] Many debilitating and incurable diseases are characterized by damage or loss of a specific tissue in the body, leading to an abrupt or progressive functional failure. Although the human body possesses a 'regenerative potential', the endogenous healing process is not always capable of restoring normal tissue homeostasis, especially when damage is too extensive. Thus, strategies to enhance the body's natural ability to repair itself hold great promise to treat, and potentially cure, a wide range of conditions, including trauma, genetic diseases, and infections. The development of such therapeutic strategies can significantly reduce hospitalization times, improve the general well-being of affected individuals and, ultimately, dramatically reduce the enormous societal costs associated to caring of patients.

[0006] Among these currently incurable conditions, inflammatory bowel diseases (IBDs), including ulcerative colitis and Crohn's disease, are a growing problem in the Western world. Currently, around 0.4% and 1.0% of the population is affected IBDs in Europe and the US, respectively, and incidence is on the rise worldwide. Although the genetic and immune components of IBDs are complex, these diseases invariably involve damage to the gut epithelium, which forms a protective barrier against the trillions of microbes residing in the gut. Exposure to the luminal content causes inflammation, which can lead to further damage to the epithelium and adjacent tissues. As the lining of the intestines becomes ulcerated and functional cells are lost, patients become unable to adequately process food and absorb water, and experience severe pain, diarrhoea, weight loss and other highly debilitating symptoms. Currently, IBD patients are treated with immune modulatory drugs to reduce inflammation levels and often remain on this therapy for a lifetime. Despite the introduction of several new drugs into clinical management in the past few decades, curative options are still lacking. While aiming at reducing the inflammation burden, current treatment strategies fail to promote restoration of the intestinal epithelium and, therefore, have palliative effects only. The development of therapeutic regimens that enhance regeneration of the intestinal lining is, therefore, of high clinical relevance for IBD patients.

[0007] In Europe, an estimated 2.5-3 million people are affected by IBDs, with a direct healthcare cost of approximately €5bn per year. Similarly, in the US, a study found that the annual direct cost of health care for a person living with inflammatory bowel disease was more than three times higher than it is for those who do not have IBD (Park K. T. et al., 2020), with the total annual financial burden being estimated between approx. $14 to $30bn in 2014 (Mehta F., 2016).

[0008] Mustafa S. A. et al., 2017, discloses e.g. that SUMOylation pathway alteration coupled with downregulation of SUMO E2 enzyme at mucosal epithelium modulates inflammation in inflammatory bowel disease and that a decrease of the SUMO E2 conjugating enzyme Ubc9 is associated with inflammation in intestinal epithelium.

[0009] Karhausen J. et al., 2018, discloses e.g. a critical role of the SUMO2 / 3 isoforms in modulating responses to intestinal ischemia reperfusion damage and provide evidence that SUMO1 deletion activates a compensatory process of overexpression of SUMO2 / 3 that blunts intestinal inflammatory responses after intestinal ischemia reperfusion damage.

[0010] Suhail A. et al., 2019, discloses that inflammatory bowel disease is associated with SUMOylation. Suhail A. et al., further discloses that the DeSUMOylase SENP7 was upregulated specifically in intestinal epithelial cells in both human inflammatory bowel disease and in a mouse model and concluded that epithelial SENP7 is necessary and sufficient for controlling gut inflammation. In view of the challenges in the art, enhancing epithelial tissue regeneration following damage could significantly reduce the burden on individual patients and society at large, potentially revolutionizing how patients with inflammatory bowel disease (IBD) and other ulcerative conditions disorders are treated clinically. Of note, gastrointestinal tract ulcerations can also affect pets and farm animals (Barrett, MacDonalds, 2023; Daure E. et al., 2017). Therefore, the development of methods to enhance tissue regeneration is also of relevance for veterinary medicine purposes.

[0011] Hence, an improved method or pharmaceutical approach that enhances epithelial tissue repair would be advantageous.

[0012] Summary of the invention

[0013] Intestinal organoids are complex three-dimensional cellular aggregates that mimic the cellular and architectural organization of the intestinal epithelium and represent a tractable in vitro system to investigate tissue physiology. Interestingly, the process of organoid formation from single epithelial cells recapitulates cellular and transcriptional aspects of epithelial regeneration in vivo (Yui S. et al., 2018; Serra D. et al., 2019; Lukonin I. et al., 2020). This in vitro regenerative state is characterized by increased expression of known regeneration-associated markers, such as Seal and Trop2, and activation of pro-regenerative pathways, such as YAP / TAZ.

[0014] To identify pathways regulating intestinal epithelial regeneration, the inventors set up an unbiased small-molecule compound screen and an independent CRISPR / Cas9 genetic screen in murine intestinal organoids. Using this in vitro system, the inventors serendipitously found that: i) pharmacological inhibition of SUMO El ligase and ii) ablation of the gene Sumo2 lead to enhancement of the pro- regenerative state. These findings indicate that inhibition of the SUMOylation pathway can be used to enhance epithelial healing. This pathway has not been associated with tissue regeneration previously. In the context of tissue regeneration, inhibition of SUMOylation can be used to support epithelial healing following damage and, thereby, complement existing treatment strategies that target inflammation in e.g. patients suffering from inflammatory bowel diseases. Treatment with inhibitors of the SUMOylation pathways can also be used to treat other conditions that involve ulceration of the intestinal epithelium, such as viral, bacterial, or parasitic infections, or recovery from chemo- or radiotherapy, as well as ulceration in other tissues.

[0015] One aspect of the invention relates to an inhibitor or modulator of the SUMOylation pathway for use in a method of increasing or facilitating regeneration of a tissue in a mammal, such as intestinal epithelial tissue, in a human subject in need thereof.

[0016] Another aspect of the present invention relates to an inhibitor or modulator of the SUMOylation pathway for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, in a mammal subject, such as a human subject in need thereof.

[0017] Yet another aspect of the present invention is to provide a method of increasing tissue regeneration in a mammal subject, such as a human subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor or modulator of the SUMOylation pathway.

[0018] Still another aspect of the present invention is to provide a method of treating a mammal subject, such as a human subject in need thereof, suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to a subject an inhibitor or modulator of the SUMOylation pathway.

[0019] Brief description of the figures

[0020] Figure 1 shows illustrative representation of SUMOylation conjugation pathway. The covalent attachment of SUMO to its substrates is processed by the stepwise action of an enzymatic cascade involving El, E2 and E3 enzymes. First, SUMO (S) is matured by SUMO specific proteases (Prot), revealing a carboxy-terminal Gly-Gly motif. This enables SUMO to become adenylated by the SUMO-activating enzyme subunit 1 (SAEl)-ubiquitin-like activating enzyme subunit 2 (UBA2) El complex in an ATP-dependent reaction and transferred to the catalytic site of the UBA2 subunit. SUMO is then transferred to the E2 (Ubc9, ubiquitin-like conjugating enzyme 9) Finally, SUMO is conjugated directly or with the help of an E3 ligase to its substrate, forming an isopeptide bond (solid line). Protein substrates can be modified with a single SUMO (monosumoylation), with multiple SUMOs (multisumoylation) or with a SUMO chain (polysumoylation). SUMOylation is reversed by SUMO specific proteases that cleave SUMO from the substrate. The known number of SUMO enzymes, given in brackets, is limited, in contrast to the thousands of known substrates. Figure 1 is modified from Pichler A. et al., 2017.

[0021] Figure 2 shows the results from the CRISPR / Cas9 loss-of-function screen in intestinal organoids for three independent sgRNAs targeting the gene Sumo2. The normalized read counts for each sgRNA in the entire cell population compared to the regenerative-like cell population are plotted.

[0022] Figure 3 shows a bar plot summarizing the results of the compound screening in mouse intestinal organoids. Organoid circularity was used as proxy readout of the regenerative state. Bars show mean ± standard deviation of circularity values extracted from images of organoids treated with the indicated compounds.

[0023] Figure 4 shows representative phase contrast microscopy images of organoids treated with DMSO (vehicle) or the SUMOylation inhibitors ML-792 or TAK-981 as indicated.

[0024] Figure 5 shows bar plots indicating the expression levels of genes Ly6a (Seal) and Tacsdt2 (Trop2) in organoid cultures treated with DMSO or the SUMOylation inhibitors ML-792 or TAK-981. Bars represent mean ± standard deviation. Dots represent the exact values from individual experimental replicates. TPM - transcripts per million.

[0025] Figure 6 shows bar plots indicating percentage of the cells expressing the markers Seal and Trop2 in organoid cultures treated with DMSO or the SUMOylation inhibitors ML-792 or TAK-981. Bars represent mean ± standard deviation. Dots represent the exact value from individual experimental replicates.

[0026] Figure 7 shows representative immunofluorescent images showing subcellular localization of YAP1 protein in organoids treated with DMSO (vehicle) or the SUMOylation inhibitors ML-792 or TAK-981. The arrows indicate cells with nuclear YAP1 signal. Bar plots on the right depicts percentage of cells per organoid with nuclear YAP1 upon the indicated treatments. Bars represent mean ± standard deviation. Dots represent the exact value from individual organoids.

[0027] Figure 8 shows summary of gene set enrichment analyses (GSEA) indicating enrichment of the colitis-associated gene signatures (Yui S. et al., 2018) in the transcriptome of organoids treated with the SUMOylation inhibitor ML-792

[0028] Figure 9 shows summary of gene set enrichment analyses (GSEA) indicating enrichment of the fetal intestinal (Pikkupeura L. M. et al., 2023) in the transcriptome of organoids treated with the SUMOylation inhibitor ML-792.

[0029] Figure 10 shows summary of gene set enrichment analyses (GSEA) indicating enrichment of the colitis-associated gene signatures (Yui S. et al., 2018) in the transcriptome of organoids treated with the SUMOylation inhibitor TAK-981.

[0030] Figure 11 shows summary of gene set enrichment analyses (GSEA) indicating enrichment of the fetal intestinal (Pikkupeura L. M. et al., 2023) in the transcriptome of organoids treated with the SUMOylation inhibitor TAK-981.

[0031] Figure 12 shows body weight curves of animals administered with DSS and subsequently treated with either vehicle or the SUMOylation inhibitor TAK-981 during tissue repair. Blue arrows indicate the days when animals were injected with either vehicle or TAK-981. The lines represent the average weight relative to the starting point ± SEM in vehicle or TAK-981 treated groups (n = 12 animals per group). Figure 13 shows plot relative body weight of animals in each treatment group at day 11. Bars represent mean ± standard deviation. Significance was assessed using unpaired Student t -test.

[0032] The present invention will now be described in more detail in the following.

[0033] Detailed description of the invention

[0034] Definitions

[0035] Prior to discussing the present invention in further details, the following terms and conventions will first be defined :

[0036] CRISPR-Cas9: abbreviation of "clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9"

[0037] ML-792: commercially available SUMO El ligase inhibitor.

[0038] SUMO: abbreviation of "Small ubiquitin-like modifiers"

[0039] SUMOylation pathway (SUMO conjugation cascade): when used herein, the SUMOylation pathway refers to a cascade of energy-dependent enzymes and their reactions. Three enzymatic steps (El: activating ligase enzyme; E2: conjugating enzyme and E3: ligases) are involved in the pathway. The SUMO enzymatic cascade catalyzes the dynamic posttranslational modification process of SUMOylation (i.e. covalent attachment of SUMO moieties to target proteins).

[0040] SUMO El ligase: also known as SUMO activating enzyme (SAE). A modular, multidomain 110 kDa heterodimer comprised of SUMO-activating enzyme 1 (Sael) and 2 (Sae2 or Uba2) subunits that harbor two catalytic activities required for activation of SUMO: adenylation and thioester bond formation. This comprises the first critical step of the SUMO conjugation cascade. In humans, SUMO El ligase complex is encoded by the two genes SAE1 (SUMO-activating enzyme subunit 1) and UBA2 (SUMO-activating enzyme subunit 2). SUMOylome: when used herein, SUMOylome refers to the compendium of proteins modified by the SUMOylation pathway.

[0041] TAK-981 : commercially available SUMO El ligase inhibitor

[0042] YAP1: abbreviation for Yes-associated protein 1. A protein in humans encoded by the YAP1 gene.

[0043] Embodiments of the invention - medical uses

[0044] In one embodiment of the present invention there is provided an inhibitor or modulator of the SUMOylation pathway for use in a method of increasing or facilitating regeneration of a mammal tissue, preferably human intestinal epithelial tissue, in a human subject in need thereof.

[0045] In another embodiment of the present invention there is provided an inhibitor or modulator of the SUMOylation pathway for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy in a mammal subject, such as a human subject in need thereof.

[0046] In still another embodiment of the present invention there is provided an inhibitor chosen from ubiquitin-like 1-activating enzyme of the SUMO pathway (SUMO El inhibitor) or a ubiquitin-like 2-activating enzyme of the SUMO pathway (SUMO E2 inhibitor) or a combination of a SUMO El inhibitor and a SUMO E2 inhibitor or a modulator of the SUMOylation pathway for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy in a mammal subject, such as a human subject in need thereof.

[0047] In still another embodiment of the present invention there is provided an inhibitor has the structure represented by the formula (ML-792):

[0048] for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy in a mammal subject, such as a human subject in need thereof.

[0049] In still another embodiment of the present invention there is provided an inhibitor that has the structure represented by the formula (TAK-981): for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy in a mammal subject, such as a human subject in need thereof.

[0050] In still another embodiment of the present invention there is provided an inhibitor or modulator of the SUMOylation pathway for use in a method of increasing or facilitating regeneration of a mammal tissue, preferably human intestinal epithelial tissue, in a human subject in need thereof, wherein the inhibitor or modulator is used in combination with immunomodulatory treatment.

[0051] In still another embodiment of the present invention there is provided an inhibitor or modulator of the SUMOylation pathway for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy in a mammal subject, such as a human subject in need thereof, wherein the inhibitor or modulator is used in combination with immunomodulatory treatment.

[0052] In still another embodiment of the present invention there is provided an inhibitor chosen from ubiquitin-like 1-activating enzyme of the SUMO pathway (SUMO El inhibitor) or a ubiquitin-like 2-activating enzyme of the SUMO pathway (SUMO E2 inhibitor) or a combination of a SUMO El inhibitor and a SUMO E2 inhibitor or a modulator of the SUMOylation pathway for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy in a mammal subject, such as a human subject in need thereof, wherein the inhibitor or modulator is used in combination with immunomodulatory treatment.

[0053] In still another embodiment of the present invention there is provided an inhibitor that has the structure represented by the formula (ML-792): for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy in a mammal subject, such as a human subject in need thereof, wherein the inhibitor is used in combination with immunomodulatory treatment.

[0054] In still another embodiment of the present invention there is provided an inhibitor that has the structure represented by the formula (TAK-981): for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy in a mammal subject, such as a human subject in need thereof, wherein the inhibitor is used in combination with immunomodulatory treatment.

[0055] Embodiments of the invention - method of treatment (tissue regeneration)

[0056] In still another embodiment of the present invention there is provided a method of increasing tissue regeneration in a mammal subject, such as a human subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor or modulator of the SUMOylation pathway.

[0057] In still another embodiment of the present invention there is provided a method of increasing tissue regeneration in a mammal subject, such as a human subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor or modulator of the SUMOylation pathway, wherein the inhibitor or modulator of the SUMOylation pathway is used in combination with immunomodulatory treatment.

[0058] In still another embodiment of the present invention there is provided a method of increasing tissue regeneration in a mammal subject, such as a human subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor or modulator of the SUMOylation pathway, wherein the inhibitor is an inhibitor of ubiquitin-like 1-activating enzyme of the SUMO pathway (SUMO El inhibitor) or a ubiquitin-like 2-activating enzyme of the SUMO pathway (SUMO E2 inhibitor) or a combination of a SUMO El inhibitor and a SUMO E2 inhibitor.

[0059] In still another embodiment of the present invention there is provided a method of increasing tissue regeneration in a mammal subject, such as a human subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor or modulator of the SUMOylation pathway, wherein the inhibitor is an inhibitor of ubiquitin-like 1-activating enzyme of the SUMO pathway (SUMO El inhibitor) or a ubiquitin-like 2-activating enzyme of the SUMO pathway (SUMO E2 inhibitor) or a combination of a SUMO El inhibitor and a SUMO E2 inhibitor and wherein the inhibitor or modulator of the SUMOylation pathway is used in combination with immunomodulatory treatment.

[0060] In still another embodiment of the present invention there is provided a method of increasing tissue regeneration in a mammal subject, such as a human subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor or modulator of the SUMOylation pathway, wherein the inhibitor has the structure represented by the formula (ML- 792):

[0061] In still another embodiment of the present invention there is provided a method of increasing tissue regeneration in a mammal subject, such as a human subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor or modulator of the SUMOylation pathway, wherein the inhibitor has the structure represented by the formula (TAK- 981): In still another embodiment of the present invention there is provided a method of increasing tissue regeneration in a mammal subject, such as a human subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor or modulator of the SUMOylation pathway, wherein the inhibitor has the structure represented by the formula (ML- 792):

[0062] and wherein the inhibitor is used in combination with immunomodulatory treatment.

[0063] In still another embodiment of the present invention there is provided a method of increasing tissue regeneration in a mammal subject, such as a human subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor or modulator of the SUMOylation pathway, wherein the inhibitor has the structure represented by the formula (TAK- 981): and wherein the inhibitor is used in combination with immunomodulatory treatment.

[0064] Embodiments of the invention - method of treatment (medical treatment)

[0065] In still another embodiment of the present invention there is provided a method of treating a mammal subject, such as a human subject in need thereof, suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to the subject an inhibitor or modulator of the SUMOylation pathway.

[0066] In still another embodiment of the present invention there is provided a method of treating a mammal subject, such as a human subject in need thereof, suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to the subject an inhibitor or modulator of the SUMOylation pathway and wherein the inhibitor or modulator of the SUMOylation pathway is used in combination with immunomodulatory treatment.

[0067] In still another embodiment of the present invention there is provided a method of treating a mammal subject, such as a human subject in need thereof, suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to the subject an inhibitor or modulator of the SUMOylation pathway, wherein the inhibitor is an inhibitor of ubiquitin-like

[0068] 1-activating enzyme of the SUMO pathway (SUMO El inhibitor) or a ubiquitin-like

[0069] 2-activating enzyme of the SUMO pathway (SUMO E2 inhibitor) or a combination of a SUMO El inhibitor and a SUMO E2 inhibitor.

[0070] In still another embodiment of the present invention there is provided a method of treating a mammal subject, such as a human subject in need thereof, suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to the subject an inhibitor or modulator of the SUMOylation pathway, wherein the inhibitor is an inhibitor of ubiquitin-like

[0071] 1-activating enzyme of the SUMO pathway (SUMO El inhibitor) or a ubiquitin-like

[0072] 2-activating enzyme of the SUMO pathway (SUMO E2 inhibitor) or a combination of a SUMO El inhibitor and a SUMO E2 inhibitor and wherein the inhibitor or modulator of the SUMOylation pathway is used in combination with immunomodulatory treatment. In still another embodiment of the present invention there is provided a method of treating a mammal subject, such as a human subject in need thereof, suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to the subject an inhibitor having the structure represented by the formula (ML-792): In still another embodiment of the present invention there is provided a method of treating a mammal subject, such as a human subject in need thereof, suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to the subject an inhibitor having the structure represented by the formula (TAK-981): In still another embodiment of the present invention there is provided a method of treating a mammal subject, such as a human subject in need thereof, suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to the subject an inhibitor having the structure represented by the formula (ML-792): and wherein the inhibitor is used in combination with immunomodulatory treatment.

[0073] In still another embodiment of the present invention there is provided a method of treating a mammal subject, such as a human subject in need thereof, suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to the subject an inhibitor having the structure represented by the formula (TAK-981): and wherein the inhibitor is used in combination with immunomodulatory treatment. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0074] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0075] The invention will now be described in further details in the following non-limiting examples.

[0076] Examples

[0077] Example 1 (Genetic screen in intestinal organoids)

[0078] To identify genes and pathways regulating intestinal epithelial regeneration, the inventors devised an unbiased CRISPR / Cas9 loss-of-function screen in murine intestinal organoids. Organoids were engineered to express a doxycycline-inducible Cas9 transgene, and then transduced with the genome-wide Brie sgRNA library targeting 19,674 protein-coding genes (Doench et al., 2016). Regenerative-like cells were sorted based on high expression Seal and Trop2. sgRNA sequences, which integrated into the genomic DNA were amplified and sequenced by next generation sequencing. Comparison of sgRNA frequency in the regenerative-like (Scal / Trop2-postive) versus the non-regenerative cell population (Scal / Trop- negative) revealed a significant enrichment of 3 independent sgRNAs targeting Sumo2. The gene Sumo2 encodes the protein SUMO2 (Small Ubiquitin-like modifier 2), which is the most abundant isoform of the three SUMO family members (SUMO1, SUMO2 and SUMO3) in vertebrates. This finding supports that reduction in SUMOylation levels has pro-regenerative effects.

[0079] Table 1. Normalized read counts for three independent sgRNAs targeting Sumo2 in the non-regenerative (Scal-Trop2 negative) and the regenerative-like cell populations (Scal-Trop positive) isolated from mouse organoids transduced with genome-wide CRISPR-Cas9 library.

[0080] Example 2 (SUMO El inhibitors trigger regenerative-like responses in organoid model)

[0081] Using an independent experimental set-up, the inventors observed that mechanical disruption of organoid structures during conventional culture maintenance triggers a regenerative-like response that is independent of single cell dissociation. Using this novel approach, the inventors carried out a small-molecule compound screening and identified ML-792 as a potent enhancer of the regenerative response. ML-792 is a selective inhibitor of the SUMO El-ligase, which blocks the entire SUMOylation conjugation pathway. Further confirming these findings, the inventors observed that treatment with TAK-981 - another selective SUMO El-ligase inhibitor - enhances the regenerative response in intestinal organoids. Both compounds lead to increased expression of regeneration-associated markers (Seal and Trop2), and activation of the pro-regenerative transcriptional factor protein YAP1.

[0082] Table 2. Gene expression levels (average transcripts per million ± standard deviation) of Scal / Ly6a and Trop2 / Tacstc / 2 determined by RNA-sequencing in organoids treated with DMSO or SUMOylation inhibitors.

[0083] Table 3. Protein expression levels (percentage ± standard deviation of positive cells) of Seal and Trop2 determined by flow cytometry analysis in organoids treated with DMSO or SUMOylation inhibitors.

[0084] Table 4. Quantification of activated (nuclear) YAP protein (percentage ± standard deviation of positive cells) determined by immunofluorescent imaging analysis in organoids treated with DMSO or SUMOylation inhibitors.

[0085] Example 3 (evidence of regenerative properties of epithelial cells at gene expression level)

[0086] The inventors observed that at the gene expression level, organoids treated with selective SUMOylation inhibitors (ML-792 and TAK-981) display a fetal-like regenerative transcriptional signature. This transcriptional state characterises epithelial cells with increased regenerative properties. (Ayyaz A. et al., 2019; Yui S. et al., 2018).

[0087] Table 5. Quantification of regeneration-associated signatures (normalized enrichment score) determined by gene set enrichment analysis from transcriptomic (RNA-seq) profiling of organoids treated with DMSO or SUMOylation inhibitors.

[0088] Example 4 (proteomics analysis)

[0089] The inventors deployed a proteomics approach to define proteins modified by the SUMOylation pathway (SUMOylome) in intestinal epithelial cells. A diverse range of nuclear proteins directly SUMOylated was identified. These included many nuclear regulators with known pro-regenerative functions.

[0090]

[0091]

[0092]

[0093] Table 6. List of top 50 mostly SUMOylated proteins specifically identified during the regenerative state through mass spectrometry-based profiling of organoid cultures.

[0094] Example 5 (intestinal epithelial regeneration in vivo)

[0095] To validate the regeneration-promoting effect of treatment with SUMOylation inhibitors, the inventors employed an in vivo study using a chemical-induced colitis murine model - a well-established experimental model of intestinal epithelial regeneration. The mice were wild-type C57BI6 strain, females, 12 week-old (Provided by Taconic Biosciences). Mice were administered dextran sulfate sodium (DSS) for 5 days in drinking water to induce formation of ulcers in the epithelium of large intestine that leads to severe body weight loss. Subsequently, mice were left to recover while treated with the SUMOylation inhibitor TAK-981, or vehicle solution, for 4 consecutive days (1 daily intra-peritoneal injections from day 8 to 11). Mice were monitored for 16 days after the onset of DSS administration and body weight was used to quantitatively describe the changes in the functional recovery process. Treatment with the SUMOylation inhibitor led to a faster recovery in body weight compared to vehicle-treated controls. The inventors, thus, found that transient inhibition of SUMOylation pathway can be used to improve regeneration in vivo. Table 7 - Quantification of body weight changes (percentage relative to day 1 ± standard deviation) in non-injured (control) and DSS-injured animals injected with vehicle solution or TAK-981

[0096] References

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[0107] Suhail A. et al., 2019, "DeSUMOylase SENP7-Mediated Epithelial

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Claims

1. An inhibitor of SUMO El-ligase for use in a method of increasing or facilitating regeneration of an intestinal epithelial tissue in a mammal subject.

2. An inhibitor of SUMO El-ligase for use in the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, in a mammalian subject in need thereof.

3. The inhibitor of SUMO El-ligase for use according to any of claims 1-2, wherein the inhibitor has the structure represented by the formula (ML-792):

4. The inhibitor of SUMO El-ligase for use according to any of claims 1-2, wherein the inhibitor has the structure represented by the formula (TAK-981):

5. The inhibitor of SUMO El-ligase for use according to any of claims 1-4, wherein the inhibitor is used in combination with immunomodulatory treatment.

6. The inhibitor of SUMO El-ligase for use according to any of claims 1-5, wherein the mammal subject is a human subject.

7. A method of increasing intestinal epithelial tissue regeneration in a mammal subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of an inhibitor of SUMO El-ligase.

8. A method of treating a mammal subject suffering from inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative conditions, epithelial damage, conditions involving ulceration of the intestinal epithelium caused by viral, bacterial, or parasitic infections, or ulceration caused by chemo- or radiotherapy, by administering to the subject an inhibitor of SUMO El-ligase.

9. The method according to any of claims 7-8, wherein the inhibitor of SUMO El- ligase has the structure represented by the formula (ML-792):

10. The method according to any of claims 7-8, wherein the inhibitor of SUMO El- ligase has the structure represented by the formula (TAK-981):

11. The method according to any of claims 7-10, wherein the inhibitor of SUMO El-ligase is used in combination with immunomodulatory treatment.

12. The method according to any of claims 7-11, wherein the mammal subject is a human subject.

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