New selective ERAP inhibitor canthin-6-one and uses thereof

Abstract

The present invention relates to canthin-6-one and compositions comprising it for the treatment and / or prevention of relapses of Hedgehog-dependent tumors (HH) tumors, or diseases characterized by aberrant expression of endoplasmic reticulum aminopeptidases (ERAP), the use of canthin-6-one as an ERAP inhibitor or marker for the detection of ERAP, and said therapeutic treatments comprising the administration of canthin-6-one in a therapeutically effective dosage to a patient in need thereof.

Description

[0001] NEW SELECTIVE ERAP INHIBITOR CANTHIN-6-ONE AND USES THEREOF

[0002] The present invention relates to canthin-6-one and compositions comprising it for the treatment and / or prevention of relapses of Hedgehog-dependent tumors (HH) tumors, or diseases characterized by aberrant expression of endoplasmic reticulum aminopeptidases (ERAP), the use of canthin-6-one as an ERAP inhibitor or marker for the detection of ERAP, and said therapeutic treatments comprising the administration of canthin-6-one in a therapeutically effective dosage to a patient in need thereof.

[0003] BACKGROUND

[0004] The Hedgehog (HH) signaling pathway plays a crucial role in embryogenesis, tissue development, and stem cell maintenance. Its aberrant activation, induced by alterations in the transcriptional activity of specific HH factors (such as Ptch, Smo, Sufu, Gli1 , and Gli2), promotes tumorigenesis mechanisms in various tumors, including basal cell carcinoma (BCC) and medulloblastoma (MB). Medulloblastoma (MB) is the most common malignant brain tumor in children. It is divided into four molecular subgroups: Wingless (WNT), Sonic-Hedgehog (SHH), Group 3 (G3), and Group 4 (G4). In the pathogenesis of the SHH subgroup, the SHH signaling pathway plays a key role. Conventional therapeutic approaches, such as surgery, radiation, and chemotherapy, often cause significant adverse effects. A thorough understanding of the molecular mechanisms underlying the development and progression of MB-SHH has led to the identification of new therapeutic strategies for the treatment of this tumor. To date, significant progress has been made in the development of SHH pathway inhibitors, especially for drugs that target the SMO activator receptor. Although three of these have entered clinical trials for the treatment of SHH-dependent tumors, the adverse side effects resulting from their administration and the emergence of drug resistance, mainly due to mutations in the SMO receptor, have greatly limited the safety and efficacy of these drugs. A further obstacle to the use of these inhibitors is the existence of SMO-independent Hedgehog pathway activation mechanisms, which highlight the need to identify new components of the pathway to use as alternative drug targets.

[0005] In this context, endoplasmic reticulum aminopeptidase 1 (ERAP1 in humans, ERAAP in mice; Saveanu L et al, Nat Immunol 2005; UniProt codes Q9NZ08 and Q9EQH2) has emerged as a valid drug target for combating HH-dependent tumors. This enzyme has recently been described as a new activator of the SHH pathway, promoting the stabilization of GLI transcription factors, the final effectors of the pathway. Specifically, it has been discovered that ERAP1 interacts with the deubiquitinase USP47 to promote the degradation of pTrCP, an F-box protein of the Skp1-Cul1-Fbox (SCF) E3 ligase complex normally stabilized by LISP47. Since the SCFpTrCPcomplex is responsible for regulating GLI activator proteins, the latter are no longer degraded and are able to promote the expression of SHH pathway target genes, promoting cell proliferation and tumorigenesis. In accordance with this molecular mechanism, it has been widely demonstrated that both genetic and pharmacological inhibition of ERAP1 are able to suppress SHH-MB growth in vitro and in vivo. It is also known that abnormalities in ERAP1 expression have been linked to autoimmune diseases such as ankylosing spondylitis and psoriasis. It is reported in the literature that inhibition of ERAP1 may help modulate the immune response and reduce inflammation in these diseases.

[0006] More generally, inhibitors of the enzymes ERAP 1 and 2 (endoplasmic reticulum aminopeptidases) are primarily being studied for their potential in the treatment of various autoimmune diseases and cancer, as they are involved in the processing of peptides for presentation by class I molecules of the major histocompatibility complex (MHC), which is crucial for the immune response. By inhibiting ERAP 1 and / or 2 enzymes, immune responses can be modulated, potentially reducing inflammation in autoimmune conditions or enhancing antitumor immunity in cancer therapy.

[0007] It is therefore of great interest to identify new ERAP inhibitors for use in the treatment of diseases wherein there is altered activity and / or expression of these enzymes, including HH-dependent tumors.

[0008] SUMMARY OF THE INVENTION

[0009] The authors of this invention have surprisingly discovered that Canthin-6-one (IUPAC name 1 ,6-diazatetracicloesadeca-3,5(16),6,8,10,12, 14-eptaen-2-one), an indole alkaloid already known in the literature for its anti-mycobacterial properties, is an effective inhibitor of ERAP. Surprisingly, the authors found and demonstrated that Canthin-6-one exhibits inhibitory activity on ERAP. This inhibitory activity, being exerted both on the enzymatic activity of ERAP and on its binding affinity to other proteins (Figures 1 and 3), interferes with the ability of ERAP to form complexes with other proteins, resulting in the inhibition of the functions performed by these complexes. Canthin-6-one is therefore an effective inhibitor of ERAP as it is able to counteract functions such as the enzymatic activity and binding affinity of this protein in an effective and targeted manner. The molecule in question has also been found to be particularly effective at a therapeutic level in in vitro and in vivo experiments, extremely specific and with low cytotoxicity. The invention, therefore, provides a molecule that allows for the targeted treatment of diseases related to an alteration in the expression and / or functions of the ERAP protein, in particular ERAP1 , such as HH-dependent tumors. In fact, the authors have shown that Canthin-6-one is surprisingly effective in counteracting the functions (both enzymatic and binding affinity to other proteins) of ERAP1 in SHH-dependent tumors and in reducing SHH-MB tumor growth. Furthermore, the authors surprisingly discovered that Canthin-6-one effectively crosses the blood-brain barrier and is also able to inhibit the proliferation and suppress the selfrenewal of MB cancer stem cells (MB-SLCs), a critical aspect in cancer therapy as it combats the formation of metastases and the occurrence of recurrences. Furthermore, the concentrations of Canthin-6-one that proved effective in experiments conducted by the authors of the invention on tumors associated with ERAP1 alterations (such as SHH-MB) are significantly lower than those reported in the literature on tumors not known to be associated with ERAP alterations, demonstrating a surprising and unexpected effect of Canthin -6-one when used in diseases associated with abnormal constitutive expression or abnormal overexpression of ERAP.

[0010] In the present invention, abnormal constitutive expression means constitutive expression of a particular protein, in this case ERAP1 , in tissues or stages of development wherein such constitutive expression is not present in healthy individuals; and abnormal overexpression means an expression of the molecule of interest (ERAP1) that is higher (in statistically significant amounts) than that measured in corresponding healthy control tissues.

[0011] The present invention therefore relates to:

[0012] -Canthin-6-one or a pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use in the treatment of Hedgehog (HH) dependent tumours or in the treatment and / or prevention of relapses of said tumours

[0013] - Canthin-6-one or the pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use in the treatment of conditions that exhibit aberrant positive expression of endoplasmic reticulum aminopeptidase (ERAP), wherein said aberrant positive expression is a constitutive expression of the ERAP protein in a tissue wherein it is normally absent, or an overexpression of ERAP compared to the expression thereof in a corresponding healthy control tissue

[0014] -A pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable carrier for use in the treatment of Hedgehog (HH)- dependent tumors or in the treatment and / or prevention of relapses of said tumors.

[0015] -A pharmaceutical composition comprising Canthin -6-one or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier for use in the treatment of conditions exhibiting aberrant positive expression of ERAP wherein said aberrant positive expression is a constitutive expression of the ERAP protein in a tissue wherein it is normally absent, or an overexpression of ERAP compared to the expression thereof in a corresponding healthy control tissue.

[0016] -The use in vitro or ex vivo of cathin-6-one as an inhibitor of ERAP.

[0017] -A method for treating HH-dependent tumors, for treating and / or preventing relapses of said tumors, or for treating diseases exhibiting aberrant positive expression of endoplasmic reticulum aminopeptidase (ERAP) wherein said aberrant positive expression is a constitutive expression of the ERAP protein in a tissue wherein it is normally absent, or an overexpression of ERAP compared to the expression thereof in a corresponding healthy control tissue, wherein said treatment comprises administering Canthin-6-one or a pharmaceutical composition comprising Canthin -6-one and at least one pharmaceutically acceptable carrier, in a therapeutically effective dosage, to a patient in need thereof.

[0018] The invention also covers the use of Canthin-6-one for the preparation of medicaments for the above-mentioned treatments.

[0019] GLOSSARY

[0020] The term ERAP refers to the endoplasmic reticulum aminopeptidase protein. Unless otherwise specified, the term generally refers to this protein and includes both human endoplasmic reticulum aminopeptidase 1 (ERAP1) and human endoplasmic reticulum aminopeptidase 2 (ERAP2). The term ERAP1 or ERAP 1 refers to the human Endoplasmic Reticulum Aminopeptidase 1 protein and ERAAP in mice (UniProt codes Q9NZ08 and Q9EQH2), while the term ERAP2 or ERAP 2 refers to the human endoplasmic reticulum aminopeptidase 2 protein (UniProt code D6RF46).

[0021] In this description, the terms “Hedgehog-dependent tumor” or “HH-dependent tumor” or “tumor dependent on the Hedgehog pathway” refer to tumors that have alterations of any type and at any level of the Hedgehog (HH) molecular pathway. In general, in the literature, an HH-dependent tumor is defined as a neoplasm whose onset is the result of genetic and / or cytogenetic mutations in the genes encoding the main components of the HH signal transduction pathway.

[0022] In this description, the term “aberrant positive expression of ERAP” indicates the presence of abnormal constitutive expression of this protein compared to control tissues or healthy individuals in whom the protein is not constitutively expressed, or the upregulation of ERAP protein expression compared to a healthy individual or healthy control tissue. In this description, diseases related to an alteration (positive) in ERAP expression refer to those diseases in which there is “aberrant positive expression” of ERAP, (1 and / or 2) as defined above.

[0023] In the present invention, the term “patient” refers to an individual affected by a disease or diseases, in which at least one of said diseases is related to or caused by an aberrant positive expression of the ERAP protein (constitutive expression of ERAP in a tissue in which it is not normally present or overexpression of the ERAP protein compared to its expression in a corresponding healthy control tissue).

[0024] In the present invention, the term “ERAP inhibitor” (meaning inhibitor of ERAP1, ERAP2, or both) refers to a compound capable of reducing the functions of the ERAP protein, such functions including enzymatic activity (aminopeptidase) and / or binding affinity of the ERAP protein with other proteins.

[0025] According to the present invention, the term “therapeutic treatment” refers to any treatment intended to cure, alleviate, eliminate, or attenuate symptoms, or to prevent or reduce the possibility of contracting disorders or malfunctions of the human or animal body, or even to limit the progression of a disease.

[0026] DETAILED DESCRIPTION OF THE FIGURES

[0027] FigurE 1. A. Relative expression of the H-2Kb / S8L complex in HeLa-Kb cells transfected with L13L and treated with increasing concentrations of compounds identified as potential ERAP1 inhibitors and analyzed by flow cytometry. Cells treated with Leu-SH were used as a positive control. Results from samples analysed in 3 independent tests are shown. Statistical significance was calculated using one-way ANOVA. *P <0.05, **P <0.01, ***P <0.001. B, D, F. Western blot representative of the thermostability of ERAP1 following heat shocks carried out at the temperatures indicated in the presence of Canthin-6-one (B), N3 (D), and N7 (F) or solvent alone. The result of Ponceau staining rendered in black and white is shown below as a sample loading control. C, E, G. Quantification of ERAP1 thermostability following treatment with Canthin-6-one (C), N3 (E), and N7 (G) in three independent experiments. The stabilization of ERAP1 is reported as ATm. (Mean ± SD). Statistical significance was calculated using the two-tailed Student's t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0028] Figura 2. A-B. Dose-response curve of a transcriptional activity assay of a Gli- responsive promoter in NIH 3T3 Shh Light II cells in which the SHH pathway was activated with SAG. Cells were treated for 48 hours with Canthin-6-one (A) or Leu-SH (B) at the indicated doses. C. WNT pathway activity was assessed in mouse embryonic fibroblasts (MEFs WT) transfected with the Top-flash luciferase reporter and p-catenin and treated with increasing doses of Canthin-6-one or solvent for 48 hours. Data show the results of three independent experiments (mean ± SD). Statistical significance was calculated using the two-tailed Student's t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

[0029] Figura 3. A-C. Protein (A, B) and mRNA (C) levels of SHH pathway target genes in WT MEFs treated for 48h with the agonist SAG and with increasing doses of Canthin- 6-one (C6O). C-G. Co-immunoprecipitation assays showing the effect of treatment with increasing doses of Canthin-6-one on ERAP1 / USP47 (D, E) and USP47-pTrCP (F, G) interactions in WT MEF cells. Figure 3E, in particular, shows the inhibition of ERAP1 / USP47 binding affinity exerted by canthin-6-one. The graph shows, in particular, how a minimum dose of canthin-6-one is able to inhibit the binding affinity of ERAP1 to other proteins. The graphs represent the averages of three independent experiments. Statistical significance was calculated using the two-tailed Student's t- test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

[0030] Figura 4. A,D. Effect of treatment with increasing doses of Canthin-6-one (C6O) (A) and LeuSh (D) on the proliferation of SHH-MB MED-1 murine cells assessed by Trypan Blue counting at the times indicated. B,E. Percentage of dead cells in the assay described in A and D. C,F Expression levels of SHH pathway target genes in MED-1 treated with increasing doses of Canthin-6-one (C) and LeuSh (F) for 48h. The graphs represent the means of three independent experiments. Statistical significance was calculated using the two-tailed Student's t-test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001.

[0031] Figura 5 A, D. Effect of treatment with increasing doses of Canthin-6-one (C6O) (A) and LeuSh (D) on the proliferation of murine BCC cells assessed by Trypan Blue counting at the times indicated. B, E. Percentage of dead cells in the assay described in A and D. C, F. Expression levels of SHH pathway target genes in BCCs treated with increasing doses of Canthin-6-one (C) and LeuSh (F) for 48h. The graphs represent the averages of three independent experiments. Statistical significance was calculated using the two-tailed Student's t-test. *p<0.05; **p<0.01; ***p<0.001 ; ****p<0.0001.

[0032] Figura 6 A. Primary SHH-MB cells derived from Math1-cre / PtcC / C mice that spontaneously develop tumors were cultured and treated with the indicated concentrations of Canthin-6-one. B. Percentage of cells dead following treatment in the proliferation assay described in A. C. Expression levels of SHH pathway target genes in primary cells assessed 48 hours after treatment with the indicated doses of Canthin- 6-one. The graphs represent the averages of three independent experiments. Statistical significance was calculated using the two-tailed Student's t-test. *p<0.05;

[0033] **p<0.01; ***p<0.001; ****p<0.0001.

[0034] Figura 7 Canthin-6-one has no effect in cells genetically silenced for ERAP1. A-B. Expression levels of SHH pathway target genes in Patched 1 KO murine fibroblasts (A) and primary mMB cells (B) assessed 48 hours after treatment with the indicated doses of Canthin-6-one. The graphs represent the means of three independent experiments. Statistical significance was calculated using the two-tailed Student's t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0035] Figura 8. Effect of treatment with different concentrations of Canthin-6-one (C6O) on the proliferation of MB PDX (A), D283 (B), and HDMB03 (C) cells belonging to group G3, and CHLA (D) cells belonging to group G4, evaluated by Trypan Blue counting at the indicated time points. The graphs represent the averages of three independent experiments. Statistical significance was calculated using the two-tailed Student's t- test. **p<0.01

[0036] Figura 9. A-B. Self-renewal capacity of SHH-MB cancer stem cells derived from Math1-cre / PtcC / C mice expressed as a percentage of neurosphere formation following 10 days of treatment with Canthin-6-one (C6O) at the concentrations indicated. C. qRT-PCR of some of the target genes of the SHH pathway and genes involved in sternness in MB cancer stem cells treated with increasing doses of Canthin-6-one for 48 hours. The graphs show the averages of three independent experiments. Statistical significance was calculated using the two-tailed Student's t-test. *p<0.05; **p<0.01 ;

[0037] ***p<0.001; ****p<0.0001.

[0038] Figura 10. A. Primary SHH-MB cells inoculated into the flanks of NSG mice. The resulting masses were treated with Canthin-6-one (C6O) or with solvent alone (n=6), and growth was monitored every three days using caliper measurements. B. Quantification of the post-excision size of treated masses compared to control masses. C-D. Evaluation of mRNA and protein expression levels of SHH pathway target genes in treated masses compared to controls. Mean ± SD. Statistical significance was calculated using the two-tailed Student's t-test. *p<0.05; **p<0.01 ; ***p<0.001 ;

[0039] ****p<0.0001.

[0040] Figura 11. Canthin-6-one is able to reduce SHH-MB tumor growth by crossing the blood-brain barrier. Primary SHH-MB cells were inoculated into the cerebellum of NSG mice. The mice were then treated with Canthin-6-one (C6O) or with the solvent alone (n=6). A. Representative H&E staining of the cerebellums of mice treated with solvent alone (CTR) and Canthin-6-one (C6O). Scale bar 1 mm. B. Quantification of H&E staining shown in A. Mean ± SD. Statistical significance was calculated using a two- tailed Student's t-test. **p<0.01.

[0041] DESCRIPTION OF THE SEQUENCES

[0042] SEQ ID NO:1 - mGlil For

[0043] GCC AAC TTT ATG TCA GGG TOO CAG

[0044] SEQ ID NO:2 - mGlil Rev

[0045] GGA GAG AGO COG CTT CTT TGT TAA

[0046] SEQ ID NO:3 - mGli2 For

[0047] CAG CCA CCC CAG CGT AGA CA

[0048] SEQ ID NO:4 - mGli2 Rev

[0049] GCC CCA GGT CGC ACT CTA G

[0050] SEQ ID NO:5 - mCycD2 For

[0051] AGA AGG ACA TCC AAC CGT ACA TG

[0052] SEQ ID NO:6 - mCycD2 Rev

[0053] CAT GGC CAG AGG AAA GAC CTC

[0054] SEQ ID NO:7 - mHPRT For

[0055] GCT TCC TCC TCA GAC CGC TT

[0056] SEQ ID NO:8 - mHPRT Rev

[0057] GGT CAT AAC CTG GTT CAT CAT GG

[0058] SEQ ID NO 9 Peptide S8L

[0059] SIINFEKL

[0060] SEQ ID NO 10 Peptide L13L

[0061] LEQLESIINFEKL

[0062] DETAILED DESCRIPTION

[0063] The authors of this invention have surprisingly discovered that Canthin-6-one is an effective inhibitor of ERAP. At the end of a selection process involving over 1,200 candidate products, the authors of the invention selected eleven compounds (N1-N11), characterized by high chemical diversity, as potential ERAP ligands, with particular interest in ERAP1 (Table 1) (see examples section).

[0064] The ability of the selected compounds to inhibit ERAP1 showed that four (N1 , N3, N7, and N10) of the tested compounds caused reduced enzymatic activity of the aminopeptidase ERAP1 (Figure 1A).

[0065] These four compounds were then further selected (compound N10 was subsequently discarded due to its high toxicity on cell viability even at the lowest concentrations) and their ERAP1 protein target was verified using a CETSA (CEIIular Thermal Shift Assay). Using this assay, the authors demonstrated that Canthin-6-one is surprisingly able to effectively bind ERAP1 and induce its thermostabilization by increasing its ATmby 10.8°C thanks to the formation of new bonds between the enzyme and Canthin-6-one itself (Figure 1 B, C); which does not occur between ERAP1 and the other molecules tested (Figure 1 F, G).

[0066] Therefore, from over 1200 compounds, the authors identified and selected Canthin-6- one as an effective inhibitor of ERAP1.

[0067] Docking studies (not reported here) show that this molecule may also be a possible inhibitor of ERAP2.

[0068] As mentioned above, a direct correlation between aberrant constitutive expression of ERAP1 (i.e. , the presence of constitutive expression in tissues where the protein is not constitutively expressed in healthy individuals) and SHH-MB has been reported in the literature. The experimental data provided by the inventors demonstrate the significant therapeutic efficacy of Canthin-6-one against SHH-MB. As is known, SHH-MB belongs to the class of SHH-dependent tumors. Therefore, one object of the present invention is Canthin-6-one for use in the treatment of SHH-dependent tumors or for the treatment and / or prevention of relapses of said tumors.

[0069] The authors of the present invention have, in fact, amply demonstrated that Canthin-6- one is capable of effectively inhibiting the SHH pathway by acting as an ERAP1 inhibitor.

[0070] In one embodiment, said HH-dependent tumor is a Sonic Hedgehog-dependent (SHH) tumor. Non-limiting examples of SHH-dependent tumors include Sonic Hedgehog subgroup medulloblastoma (SHH-MB), basal cell carcinoma, and rhabdomyosarcoma. Furthermore, altered dysregulation of the SHH pathway is involved in the neoplastic transformation of breast and ovarian cancer and in the maintenance of stem cell characteristics of epithelial cells in lung, liver, prostate, colon, bladder, and melanoma cancer. As can be seen from the examples, the authors demonstrated the ability of Canthin-6- one to specifically counteract the activity of the SHH pathway with a functional transcription assay in cells that constitutively express a synthetic SHH-responsive promoter (Gli-RE) upstream of the luciferase reporter gene (see examples section and Figure 2). Surprisingly, the test revealed that Canthin-6-one significantly reduces the activity of the SHH pathway in a dose-dependent manner.

[0071] In the examples section, it can be seen that the result was also confirmed in mouse embryonic fibroblasts (MEFs), in which treatment with increasing doses of Canthin-6- one led to a reduction in SHH pathway activity (Figure 3A-C); The observed effect is due to the ability of Canthin-6-one to counteract the binding between ERAP1 and LISP47 (Figure 3D, E). In fact, inhibition of the binding affinity of ERAP1 to LISP47 restores the function of LISP47 within the SHH pathway, leading to a decrease in the activity of this signaling pathway.

[0072] In one embodiment of the invention, Canthin-6-one can be advantageously used in the treatment of SHH-dependent medulloblastoma (SHH-MB). In fact, as evident in the examples section, Canthin-6-one is particularly suitable for the treatment of this tumor or for the treatment and / or prevention of recurrence of this tumor, as the authors have surprisingly discovered that Canthin -6-one has the ability to cross the blood-brain barrier (Figure 11) and inhibits the proliferation of MB stem cells (MB-SLCs) and suppresses their self-renewal (Figure 9).

[0073] The authors of the present invention have demonstrated, as evident from the experimental section and figures, the binding of Canthin-6-one to ERAP1 and the inhibition of the SHH signaling pathway by Canthin-6-one. As is known from the literature, the regulation of this pathway is closely related to the presence of ERAP1 and, therefore, the molecule is specific for this target. For example, the authors genetically silenced ERAP1 in Ptchi KO murine fibroblasts (Ptch' / _MEFs, Figure 7A), cells whose SHH signaling pathway is constitutively active, and in primary SHH-MB cells derived from Math1-cre / Ptcc / cmice (Figure 7B). The data obtained clearly show that the absence of ERAP1 renders these cells insensitive to the action of Canthin-6- one in promoting inhibition of the SHH pathway, demonstrating that the target of Canthin-6-one is indeed ERAP1.

[0074] The demonstration of Canthin-6-one's efficacy in inhibiting ERAP and its specificity make this molecule particularly suitable for the treatment of diseases that present altered expression of ERAP1 and / or ERAP2 (overexpression compared to a corresponding healthy control tissue). In fact, several tumorous and non-tumorous pathological forms caused by alterations in the expression of ERAP1 and / or ERAP2 proteins or by the presence of polymorphic forms of these proteins are reported in the literature, for which inhibition of ERAP and its functions, such as enzymatic activity and binding affinity, is therapeutically desirable (see, for example, Fruci et al. “Altered expression of endoplasmic reticulum aminopeptidases ERAP 1 and ERAP2 in transformed non-lymphoid human tissues” Journal of cellular physiology 2008 742-749, , Stoehr CG, Buettner-Herold M, Kamphausen E, Bertz S, Hartmann A, Seliger B. Comparative expression profiling for human endoplasmic reticulum-resident aminopeptidases 1 and 2 in normal kidney versus distinct renal cell carcinoma subtypes. Int J Clin Exp Pathol. 2013 May 15;6(6):998-1008. PMID: 23696916; PMCID: PMC3657351 , Ombrello MJ, Kastner DL, Remmers EF. Endoplasmic reticulum- associated amino-peptidase 1 and rheumatic disease: genetics. Curr Opin Rheumatol. 2015 Jul;27(4):349-56. doi: 10.1097 / BOR.0000000000000189. PMID: 26002026; PMCID: PMC4565054).

[0075] Examples of such diseases include autoimmune and autoinflammatory diseases (such as ankylosing spondylitis, Behget's disease, psoriasis, and Birdshot chorioretinopathy), hypertension, viral infections, and cancer (melanoma, leukemia, lymphoma, breast, colon, thyroid, lung, cervical, prostate, kidney, and bladder cancer).

[0076] According to the invention, the administration of Canthin-6-one induces its therapeutic effect by inhibiting ERAP, in particular ERAP1.

[0077] In one embodiment, the treatment comprises a step of determining whether a patient has aberrant positive expression of the ERAP protein and one or more steps of administering said Canthin-6-one to patients who have said aberrant expression.

[0078] As already mentioned, according to the invention, “having aberrant positive expression” means that the patient has either constitutive expression of ERAP (ERAP1 and / or ERAP2) in tissues where said protein is not constitutively expressed in the corresponding healthy control tissues, or overexpression of said protein in one or more tissues compared to the corresponding healthy control tissues. Clearly, an expert in the field is able to assess when the observed overexpression is statistically significant, for example by using multiple controls. The determination of ERAP expression can be carried out according to any method known to a technician in the field. If a tissue is analyzed in which this expression is normally silenced (in healthy individuals), it will be sufficient to detect the presence of the ERAP protein in that tissue. If, on the other hand, overexpression must be determined, all techniques commonly used in the field that allow the expression of a protein to be quantified more or less accurately may be used. Non-limiting examples of such techniques include Western blot, ELISA, mass spectrometry, flow cytometry, quantitative PCR, immunohistochemistry, Luminex xMAP technology, radioimmunoassays (RIA), etc.

[0079] The healthy control tissue may be tissue taken from healthy donors or a portion of tissue not affected by the disease taken from the patient themselves (for example, a portion of the same tissue adjacent to the portion affected by the disease).

[0080] The present invention also covers pharmaceutical compositions comprising Canthin-6- one and at least one pharmaceutically acceptable carrier for the same uses indicated above for Canthin-6-one, i.e., for use in the treatment of SHH-dependent tumors - dependent tumors or for the treatment and / or prevention of recurrence of said tumors, or also for use in the treatment of conditions that present an aberrant positive expression of ERAP1 , wherein said aberrant positive expression is a constitutive expression of the ERAP protein in a tissue wherein it is normally absent, or an overexpression of ERAP compared to the expression thereof in a corresponding healthy control tissue. Therefore, all the embodiments described above apply mutatis mutandis to the pharmaceutical compositions of the invention.

[0081] With regard to pharmaceutically acceptable excipients, a person skilled in the art will be able to select the most suitable one or ones based on the type of formulation to be produced, as well as any other additives, based on their knowledge of the pharmacopoeia without the need for inventive input.

[0082] It should be noted that the authors have surprisingly discovered the ability of Canthin-6- one to easily cross the blood-brain barrier. This allows for non-invasive forms of administration of the molecule even when the pathology affects the CNS.

[0083] The pharmaceutical composition according to the invention may be, for example, in liquid, solid, or semi-solid form, granular, etc.

[0084] In particular, the composition may be formulated, according to a non-limiting example, in the form of a tablet, solution, suspension, emulsion, syrup, soft or hard capsule, aerosol, injectable solution, injectable suspension, injectable emulsion, gel, ointment, cream, lotion, or spray.

[0085] According to a non-limiting example, the composition may be formulated for topical, enteral, oral, sublingual, nasal, oronasal-pharyngeal, systemic, intravenous, intraperitoneal, intrathecal, intramuscular, subcutaneous, intratumoral, or inhalation administration.

[0086] In jurisdictions where therapeutic treatments may be claimed, the invention relates to a method of treating SHH-dependent tumors or a method of treating and / or preventing said tumors comprising the administration of Canthin-6-one or the pharmaceutical composition comprising Canthin -6-one and at least one pharmaceutically acceptable carrier according to any of the embodiments of the invention.

[0087] In addition, the invention relates to a method for treating diseases exhibiting aberrant positive expression of the ERAP protein, wherein said aberrant positive expression is a constitutive expression of the ERAP protein in a tissue wherein it is normally absent, or an overexpression of ERAP compared to the expression thereof in a corresponding healthy control tissue, comprising administering Canthin-6-one -6-one or the pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable carrier according to any of the embodiments of the invention

[0088] The invention also relates to an in vitro or ex vivo use of cathin-6-one as an inhibitor or as a marker for the detection of ERAP.

[0089] In a further embodiment, canthin-6-one, conjugated to a suitable tag (e.g., fluorophore or other), may be used for the detection of ERAP, e.g., in diagnostics.

[0090] In summary, the invention relates to:

[0091] 1. Canthin-6-one for use in the treatment of Hedgehog (HH)-dependent tumors or in the treatment and / or prevention of recurrence of such tumors.

[0092] 2. Canthin-6-one for use according to point 1, wherein said tumors are Sonic Hedgehog (SHH)-dependent tumors.

[0093] 3. Canthin-6-one for use according to either point 1 or 2, wherein said tumors are selected from: Sonic Hedgehog (SHH-MB) medulloblastoma, basal cell carcinoma, rhabdomyosarcoma.

[0094] 4. Canthin-6-one for use according to point 3, wherein said tumor is SHH-MB.

[0095] 5. Canthin-6-one for use in the treatment of conditions that exhibit aberrant positive expression of endoplasmic reticulum aminopeptidase (ERAP), wherein said aberrant positive expression is a constitutive expression of the ERAP protein in a tissue wherein it is normally absent, or an overexpression of ERAP compared to the expression thereof in a corresponding healthy control tissue.

[0096] 6. Canthin-6-one for use according to point 5, wherein administration of said molecule causes a therapeutic effect by inhibiting ERAP.

[0097] 7. Canthin-6-one for use according to any of points 5 or 6, wherein the treatment comprises a step of determining whether a patient has aberrant positive expression of ERAP and one or more administration steps of said Canthin-6-one to patients that present said aberrant positive expression of ERAP. 8. A pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable carrier for use in the treatment of Hedgehog (HH)- dependent tumors or in the treatment and / or prevention of recurrence of said tumors.

[0098] 9. The pharmaceutical composition for use according to point 8, wherein said tumors are Sonic Hedgehog (SHH) tumors.

[0099] 10. The pharmaceutical composition for use according to any of points 8 or 9, wherein said tumors are selected from: medulloblastoma of the Sonic Hedgehog (SHH. MB) subgroup, basal cell carcinoma, rhabdomyosarcoma.

[0100] 11. The pharmaceutical composition for use according to point 10, wherein said tumor is SHH-MB.

[0101] 12. A pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable carrier for use in the treatment of diseases exhibiting aberrant positive expression of endoplasmic reticulum aminopeptidase (ERAP) wherein said aberrant positive expression is a constitutive expression of the ERAP protein in a tissue wherein it is normally absent, or an overexpression of ERAP compared to the expression thereof in a corresponding healthy control tissue.

[0102] 13. The pharmaceutical composition for use according to point 12, wherein administration of said composition causes a therapeutic effect by inhibiting ERAP.

[0103] 14. The pharmaceutical composition for use according to any of items 12 or 13, wherein the treatment comprises a step of determining whether a patient has aberrant positive expression of ERAP and one or more steps of administering said Canthin-6-one to patients who have said aberrant positive expression of ERAP.

[0104] 15. The pharmaceutical composition for use according to any of items 8 to 14, in liquid, solid, or semi-solid form.

[0105] 16. The pharmaceutical composition for use according to any of items 8 to 15 in the form of a composition for topical, enteral, oral, sublingual, nasal, oronasal-pharyngeal, systemic, intravenous, intraperitoneal, intrathecal, intramuscular, subcutaneous, intratumoral, or inhalation administration.

[0106] 17. The pharmaceutical composition for use according to any of points 8 to 16 in the form of a tablet, solution, suspension, emulsion, syrup, soft or hard capsule, aerosol, injectable solution, injectable suspension, injectable emulsion, gel, ointment, cream, lotion, spray.

[0107] 18. Canthin-6-one for use according to any of items 5 to 7 or composition for use according to any of items 12 to 14 wherein said ERAP protein is ERAP1 and / or ERAP2. 19. Canthin-6-one for use according to any one of items 5 to 7 or composition for use according to any one of items 12 to 14 wherein said pathology is an autoimmune and / or autoinflammatory disease, hypertension, a viral infection, or cancer.

[0108] 20. Canthin-6-one or composition for use according to point 19, wherein said 5 autoimmune and / or autoinflammatory diseases are ankylosing spondylitis, Behget's disease, psoriasis, Birdshot chorioretinopathy.

[0109] 21. Canthin-6-one or composition for use according to point 19, wherein said cancer is melanoma, leukemia, lymphoma, breast cancer, colon cancer, thyroid cancer, lung cancer, cervical cancer, prostate cancer, kidney cancer, bladder cancer.

[0110] 10 22. Use in vitro or ex vivo of cathin-6-one as an ERAP inhibitor.

[0111] At any point in this description and in the claims, the term “ERAP” may be replaced by: “ERAP1 and / or ERAP2”

[0112] “ERAP1”

[0113] 15 “ERAP2”.

[0114] At any point in the description and in the claims, the term ‘comprising’ may be replaced by the term “consisting of”.

[0115] In compliance with Art. 170bis, paragraph 2 of the Italian Industrial Property Code (C.P.I.) and in accordance with Art. 21, paragraph 2 of the C.P.I. Implementation 20 Regulations adopted by Ministerial Decree No. 33 of January 13, 2010, it is hereby declared that: the material of animal / vegetable origin underlying the invention covered by the above application indicated in the tables below has the origin indicated in the same tables.

[0116] 25

[0117] With regard to SHH-MB PDX cells, it is hereby declared that the aforementioned cells were obtained following the express, free and informed consent to their collection and use by the person from whom the material was collected, in accordance with current 5 legislation.

[0118] In compliance with Art. 170bis, paragraph 4 of the CPI, it is hereby declared that: with reference to the biological material containing genetically modified microorganisms or organisms, which is the subject of or used in this application, the obligations arising from national or Community legislation have been complied with, and in particular, the 10 provisions of paragraph 6 of Decree Laws No. 206 of 12 April 2001 and No. 224 of 8

[0119] July 2003 concerning such modifications.

[0120] EXAMPLES Canthin-6-one was identified following an in silica screening of an in-house library comprising more than 1,200 natural products and their derivatives against the crystallographic structure of the catalytic domain of ERAP1. The in silica study identified eleven compounds (N1-N11), characterised by high chemical diversity, as potential ERAP1 ligands (Table 1). Some of these compounds belong to the chemical class of phenolic compounds (N2-3, N5-9); in particular, two of them are phenolic acids (N2 and N9). Among the phenolic compounds of the flavonoid subgroup, characterised by a diphenylpropane skeleton, i.e. two benzene rings connected by a chain of three carbon atoms forming a pyran ring (heterocyclic ring containing oxygen) closed with the benzene ring A, which is called ring C, the following were selected: a flavanone aglycone (N5), characterised by the presence of a chiral centre at C-2 and a ketone function at position C-4 of ring C; a flavone (N7), characterised by a double bond between carbons C-2 and C-3 and a ketone function at position 4 of ring C; a flavanol (N8), with two asymmetric carbon atoms (C-2 and C-3). Among the phenolic compounds not belonging to the flavonoid subclass, two anthranoids characterised by an anthracene base skeleton were selected, an anthraquinone (N3) and a ferruginine (N6). Another compound identified is a pentacyclic triterpenoid (N4), belonging to a subclass of terpenes consisting of six isoprene units. Finally, three compounds belong to the chemical class of alkaloids, one of the largest families of natural products, characterised by vast structural diversity without a uniform classification: a p-carboline alkaloid (N1), an oxazole alkaloid (N10) and an imidazole-type alkaloid (N11).

[0121] The compounds selected in silica were subsequently tested as potential ERAP1 inhibitors using an antigen presentation assay based on the specific recognition of the SIINFEKL (S8L) epitope by antibody 25.D1.16. For this purpose, HeLa cells stably overexpressing the H-2Kb allele (HeLa-Kb) were transfected with a vector encoding the LEQLESIINFEKL (L13L) peptide, which requires the enzymatic activity of ERAP1 to generate the S8L epitope (York IA et al 2006; Towne CF et al, 2005). In the absence of an inhibitor, HeLa-Kb cells expressing L13L show high presentation of the H-2Kb / S8L complex on the cell surface, as determined by flow cytometry. Leucinthiol (LeuSH) was included as a positive control (James E et al, 2013). All compounds were tested except for N6, which was incompatible with the test because of its yellow colour. Four (N1 , N3, N7 and N10) of the 10 compounds tested showed a significant reduction in membrane antigen presentation, attributable to reduced enzymatic activity of the aminopeptidase ERAP1 (Figure 1A).

[0122] Therefore, the actual interaction between the selected compounds (N1 , N3 and N7; compound N10 was discarded due to high toxicity on cell viability even at the lowest concentrations) and their target protein ERAP1 was investigated using a CETSA (Cellular Thermal Shift Assay). This assay is one of the most widely used to evaluate and quantify the degree to which a compound binds to its target protein in a cellular context. It is based on the assumption that when a protein interacts with its specific ligand, it undergoes a change in thermostability, becoming more resistant to heat. This translates into a change in its denaturation profile (melting curve) and, consequently, a shift in its melting temperature (thermal shift) (Axelsson et al. 2004). Using this assay, it was possible to demonstrate that Canthin-6-one is able to bind and induce thermostabilisation of ERAP1 , increasing its ATmby 10.8°C thanks to the formation of new bonds between the enzyme and Canthin-6-one itself (Figure 1 B, C); which does not occur between ERAP1 and the other two molecules N3 (Figure 1D, E) and N7 (Figure 1 F, G). N10 is not represented because, in a toxicity assay, it was found to be extremely toxic at the same doses used for the other three compounds. Initially, the ability of Canthin-6-one to counteract the activity of the SHH pathway was evaluated with a functional transcription assay in cells that constitutively express a synthetic SH Fl- responsive promoter (Gli-RE) upstream of the luciferase reporter gene. In these cells, the pathway is induced following treatment with SAG, a known SMO agonist (Taipale et al, 2000; Chen et al, 2002). This in vitro test is based on the detection of flash-type luminescence emission in individual samples using a luminometer and revealed that Canthin-6-one significantly reduces SHH pathway activity in SAG-treated cells in a dose-dependent manner, with an IC50 of 44 nM (Figure 2A). L-Leucinthiol (Leu-SH), a known ERAP1 inhibitor, was used as a positive control, showing that the reduction in luciferase activity occurs at much higher doses than Canthin-6-one (Figure 2B).

[0123] It should be noted that Canthin-6-one is unable to modulate the activity of the WNT pathway, whose alteration is found in another molecular subgroup of MB (Figure 2C), indicating a specificity of action of this molecule for the SHH pathway.

[0124] This result was confirmed in murine embryonic fibroblasts (MEFs), in which the SHH pathway was activated with SAG. In this cell line, treatment with increasing doses of Canthin-6-one leads to a reduction in SHH pathway activity, attributable to an increase in the protein levels of PTrCP, F-box of the CRL1 E3-ubiquitin ligase complex responsible for the degradation of the activating factors Gli1 and Gli2, and of the repressive cleaved form Gli3R (Figure 3A-C). This effect is due to the ability of Canthin- 6-one to counteract the binding between ERAP1 and USP47 (Fig. 3D, E), thus promoting the interaction between USP47 and the E3 ligase PTrCP (Figure 3F, G). These events lead to an increase in PTrCP protein levels and the consequent degradation of the Gli activators (Gli1 and Gli2) and generation of the repressive form of the Gli3 factor (Gli3R) (Figure 3A and B). The target genes of the pathway are also reduced at the transcriptional level (Figure 30).

[0125] These data confirm the molecular mechanism previously identified by the research group of the Inventors (Bufalieri et al 2019) through which ERAP1 acts as an activator of the SHH signalling pathway. Subsequently, the anti-tumour properties of Canthin-6- one were evaluated in an immortalised SHH-MB MED-1 cell line (Figure 4A-C) by comparing its effect to that of Leu-SH (Figure 4D-F), a known ERAP1 inhibitor. Specifically, it was observed a dose-dependent decrease in cell proliferation (Figure 4A) with a consequent increase in cell mortality (Figure 4B). Again, treatment with Canthin-6-one causes a significant dose-dependent reduction in the expression of SHH pathway target genes (Figure 40). In contrast, treatment with LeuSh, used at the same doses as Canthin-6-one, had no effect on either proliferation or the target genes of the SHH pathway, confirming that Canthin-6-one is able to perform its activity as an inhibitor at much lower doses than the already known ERAP1 inhibitor LeuSh.

[0126] In addition, it was evaluated the anti-tumour capacity of Canthin-6-one in another type of cancer, basal cell carcinoma (BCC), which is also characterised by constitutive activation of the Hedgehog pathway and is considered one of the most relevant “HH- dependent” tumours. In this regard, it was considered the immortalised murine basal cell carcinoma line ASZ001, derived from a Ptch knockout mouse model that spontaneously develops this tumour. It was compared the ability of the two compounds on cell proliferation and relative cell mortality (Figures 5A-B and 5D-E). In particular, it was observed a dose-dependent reduction in proliferation and an increase in cell mortality in cells treated with Canthin-6-one (Figure 5A-B), but not in those treated with Leu-Sh at the same doses (Figure 5D, E). These data are associated with a reduction in SHH pathway target genes only in BCC cells treated with Canthin-6-one (Figure 5C, F). These data suggest that Canthin-6-one can be considered an ERAP1 inhibitor not only in SHH-MB but also in other SHH-dependent tumours in which ERAP1 is expressed.

[0127] It was then tested the inhibitory effect of Canthin-6-one on primary murine SHH-MB cells (Figure 6A-C) isolated from Math1-cre / Ptcc / cmice that spontaneously develop this type of tumour and treated with different concentrations of this compound (1-100 nM). In this tumour model, it was also observed a significant dose-dependent reduction in cell proliferation (20-70%) (Figure 6A) and an increase in cell mortality (Figure 6B). Consistent with the data shown above, this effect is associated with a reduction in the expression of SHH pathway target genes following treatment with increasing doses of Canthin-6-one (Figure 6C). To confirm that Canthin-6-one inhibition of the SHH signalling pathway requires the presence of ERAP1 and, therefore, that the compound is specific for this target, it was genetically silenced ERAP1 in Ptchi knockout murine fibroblasts (Ptch' / _MEFs, Figure 7A), cells whose SHH signalling pathway is constitutively active, and in primary SHH- MB cells derived from Math1-cre / Ptcc / cmice (Figure 7B). The absence of ERAP1 renders these cells insensitive to the action of Canthin-6-one in promoting SHH pathway inhibition, consistent with the inventors’ previous publication (Bufalieri, et al. 2019). These results demonstrate that ERAP1 is necessary for Canthin-6-one to act as an inhibitor of the SHH signalling pathway.

[0128] It is important to note that Canthin-6-one is also able to reduce the proliferation of human SHH-MB cells derived from tumour samples from patients transplanted into mice (PDX, Patient Derived Xenografts, SHH-MB PDX) at a dose of 1 pM (Figure 8A). In addition, to evaluate the specificity of Canthin-6-one's action on SHH-MB, treatments were carried out on different MB lines belonging to the G3 and G4 subgroups (D283, G3; HDMB03, G3, CHLA, G4), having previously excluded its efficacy in the WNT subgroup (Figure 20). Despite the use of Canthin-6-one concentrations equivalent to those effective for SHH-MB PDX, no reduction in cell proliferation was found in the different G3-MB or G4-MB cell lines (Figure 8B-D).

[0129] Given the important role that the SHH pathway plays in the processes of self-renewal and maintenance of stem cells, Canthin-6-one was also evaluated for its ability to modulate the behaviour of MB cancer stem cells (MB-SLCs), which represent the most difficult tumour cell component to eradicate and are primarily responsible for resistance to therapies and therefore for recurrences. Treatment with Canthin-6-one has a suppressive effect on the self-renewal capacity of MB-SLCs, particularly on their ability to form neurospheres (Figure 9A, B). Furthermore, Canthin-6-one has an inhibitory effect on the SHH pathway, assessed as a reduction in Gli1 transcript levels and in the expression levels of genes involved in sternness (Figure 9C).

[0130] The anti-tumour effect of Canthin-6-one was further validated in vivo using a heterotopic allograft model created by inoculating primary SHH-MB cells derived from Math1-cre / Ptcc / cmice into the flank of mice and treating the tumour masses with the molecule of interest for 18 days. Again, treatment with Canthin-6-one resulted in a reduction in tumour growth (Figure 10 A, B), with a consequent decrease in SHH pathway activity assessed both for the expression of signalling target genes at the mRNA and protein levels (Figure 10 C, D).

[0131] One of the major limitations in the study of new drugs concerns the passage of the blood-brain barrier, a physical barrier composed of astrocyte pedicels that is extremely selective only for small and selected molecules. Based on the data obtained in the above-described in vivo experiment, which confirmed the in vitro results, it was decided to conduct an orthotopic experiment to verify whether Canthin-6-one was actually able to overcome this major limitation and reach its target organ (the cerebellum) by crossing the blood-brain barrier. To this end, primary MB cells derived from Mathl- cre / Ptcc / cmice were inoculated directly into the cerebellum of immunocompromised NSG mice and subsequently treated intravenously with Canthin-6-one or vehicle alone as a control. Consistent with what has been shown so far, mice treated with Canthin-6- one show a clear and significant reduction in tumour mass compared to the control group (Figure 9), demonstrating that the compound is not only able to reduce the tumour growth of SHH-MB cells but also that it is able to cross the blood-brain barrier.

[0132] Materials and methods

[0133] Chemistry

[0134] All tested compounds (N1-N11) are known structures belonging to an in-house library of natural products available at the Organic Chemistry Laboratory of the Department of Chemistry and Drug Technologies of Sapienza University of Rome. The chemical identity of the compounds was confirmed by NMR and found to be consistent with the data reported in the literature.

[0135] The compound Canthin-6-one (1,6-diazatetraciclo[7.6.1.05’16.010’15]esadeca- 3, 5(16), 6, 8, 10, 12, 14-eptaen-2-one) showed NMR spectra identical to those reported in the literature (Choonong R et al. 2022).

[0136] Compound N2 (coumaric acid or (E)-3-(4-hydroxyphenyl)prop-2-enoic acid) showed NMR spectra identical to those reported in the literature (Govindan B et al. 2019).

[0137] Compound N3 (Reina or 4,5-dihydroxy-9,10-dioxoanthracene-2-carboxylic acid) was purchased from TCI (CAS: 478-43-3, Chuo-ku, Tokyo 103-0001 , Japan) and used without further purification.

[0138] Compound N4 (oleanolic acid or ((4aS,6aS,6bR,8aR,10S,12aR,12bR)-10-hydroxy- 2,2,6a,6b,9,9,12a-heptamethyl-1,3,4,5, 6, 6a, 6b, 7, 8, 8a, 9, 10,11,12, 12a, 12b, 13, 14b- octadecahydropecene-4a(2H)-carboxylic acid) was purchased from Sigma-Aldrich (CAS: 508-02-1, St. Louis, MO, USA) and used without further purification.

[0139] Compound N5 (Sakuranetin or (±)-5-hydroxy-2-(4-hydroxyphenyl)-7-2,3- dihydrochromen-4-one) showed NMR spectra identical to those reported in the literature (Park I et al. 2021). Compound N6 (Ferruginin B or 4,5,10-trihydroxy-7-methyl-1,1,3-tris(3-methylbut-2- enyl)anthracen-2-one) showed NMR spectra identical to those reported in the literature (Monache FD et al. 1979).

[0140] Compound N7 (Luteolin or 2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromen-4-one) showed NMR spectra identical to those reported in the literature

[0070] , Compound N8 (catechin hydrate or (2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7- triol hydrate) showed NMR spectra identical to those reported in the literature (Liu W et al. 2016).

[0141] Compound N9 (p-methoxydihydrocinnamic acid or 3-(4-methoxyphenyl)propanoic acid) showed NMR spectra identical to those reported in the literature (Yuan T et al. 2023). Compound N10 (halfordinol or 4-(2-pyridin-3-yl-1,3-oxazol-5-yl)phenol) showed NMR spectra identical to those reported in the literature (Mishra T et al. 2023).

[0142] Compound N11 (Pilocarpine nitrate or (3S,4R)-3-ethyl-4-[(3-methylimidazol-4- yl)methyl]oxolan-2-one nitrate) showed NMR spectra identical to those reported in the literature (Chiozzi RZ et al. 2018).

[0143] Chemicals and reagents

[0144] Optima mass spectrometry (MS) grade water, acetonitrile (ACN), methanol (MeOH) and isopropanol (i-PrOH) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All reagents and salts were purchased from Merck (Darmstadt, Germany).

[0145] Cell models

[0146] Cells used in the experiment and their relative origin

[0147] Animal models

[0148] Mouse models used in the experiment and their origin

[0149] Cell antigen processing assay

[0150] HeLa cells stably overexpressing H-2Kb were grown to 90% confluence in 12-well plates and transfected with the pTracer-CMV2-L13L construct having SEQ ID NO 10. Lipofectamine 2000 (Life Technologies) was used as the transfection reagent, according to the manufacturer's protocol. After 6 hours, the cells were treated with increasing concentrations of ERAP1 inhibitors (5-10-30 pM) or Leu-SH (30 pM) for 48 hours. The cells were then harvested and stained with monoclonal antibody 25-D1.16, which reacts with the complex formed by the S8L peptide, having SEQ ID NO 9, derived from ovalbumin bound to H-2Kb. The mean fluorescence intensity (MFI) of the S8L-H-2Kb complex was evaluated on the GFP-positive cell population by flow cytometry. Cell and Primary Cultures

[0151] NIH3T3 SHH-Light-ll, wild-type (WT) or Ptch' / _mouse MEFs, and Med1-MB cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Sigma Aldrich) plus 10% foetal bovine serum (FBS; Sigma Aldrich) or 0.5% FBS for NIH3T3 SHH-Light-ll cells. Murine ASZ001-BCC cells were cultured in 154-CF Cascade Biologies (Gibco) plus 0.05 mM CaCh and 2% chelated FBS. All media contained 1% penicillin-streptomycin and 1% L-glutamine (Sigma Aldrich). Primary MB cells were isolated from the tumour, which was mechanically destroyed with Pasteur pipettes drawn in HBSS with 1% penicillin-streptomycin and treated with DNase (10 pg / ml) for twenty minutes. The cells were centrifuged and resuspended in Neurobasal A (Sigma Aldrich) with B27 supplement without vitamin A (2%), penicillin-streptomycin (1%) and L-glutamine (1%). HH-dependent stable MB cells were cultured as neurospheres in DMEM / F12 (2% B27 without vitamin A; 3% 10* glucose; 0.2% insulin 10 mg / ml; 1% penicillin-streptomycin; 0.01% heparin 2 mg / ml; 0.06% N-Acetyl-L-Cysteine). When necessary, neurosphere cultures were pelleted and dissociated by incubation with Accutase to obtain a suspension of single cells. Mycoplasma contamination in cell cultures was routinely detected using a PCR detection kit (Applied Biological Materials, Richmond, BC, Canada).

[0152] Transfections and Lentiviral Infections

[0153] Transient transfections were performed using Dream FectTMGold reagent (Oz Biosciences SAS, Marseille, France) in accordance with the manufacturer's protocols. Lentiviral particles were generated in HEK293 cells by combining the packaging plasmids pCMV-dR8.74 and VSV-G / pMD2 with the pLKO.1 plasmid (shCTRL SHC002; shERAPI TRCN0000031119, Sigma-Aldrich), using the calcium phosphate transfection method. Ptch' / ' MEFs and primary MB cells were infected using the spin inoculation method.

[0154] Treatments

[0155] Where indicated, cells were treated with SAG (200 nM, Alexis Biochemicals Farmingdale, NY, USA) for 48 hours, and the compounds Canthin-6-one, N3, N7, N10 and Leu-SH for 48 hours at the concentrations indicated.

[0156] Plasmids, Antibodies, and Other Reagents pcDNA3.1-Flag-USP47 was generated in the inventors' laboratory using standard cloning techniques and verified by sequencing. shCTRL (SHC002) and shERAPI (TRCN0000031119) in pLKO.1 plasmids were purchased from Sigma-Aldrich. Anti-Gli1 mouse antibodies (L42B10, 1 :500), anti-pTrCP rabbit antibodies (D13F10, 1 :1000). Mouse anti-p-Actin C4 HRP (sc-47778, 1:2000), mouse anti-HA F-7 (sc-7392, 1:1000), and mouse anti-pTrCP C-6 (sc-390629, 2 pg) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-Gli3 (AF3690, 1:500), goat anti-Gli2 (AF3635, 1:1000), and mouse anti-ERAP1 6H9 (MAB2334, 1:500) antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Anti-Flag M2 (A8592, 1:2000) was purchased from Sigma Aldrich (St Louis, MO, USA). PE-Cyanine7-conjugated monoclonal antibody against the OVA257-264 (SIINFEKL) peptide bound to H-2Kb (25-D1.16) was purchased from Thermofisher Scientific (Waltham, MA, USA). HRP- conjugated goat anti-mouse IgG (1:5000), HRP-conjugated goat anti-rabbit IgG (1:5000) and HRP-conjugated donkey anti-goat IgG (1:5000) were purchased from Bethyl Laboratories (Montgomery, TX, USA).

[0157] Transcriptional activity assay

[0158] The SHH signalling pathway-dependent transcriptional activity assay was performed in NIH3T3 SHH-Light II cells, which stably express a Gli-responsive luciferase reporter and Renilla pRL-TK (normalisation control), treated for 48 hours with SAG (200 nM) and Canthin -6-one or L-Leu-Sh and / or DMSO as a control at the concentrations indicated. The WNT assay was performed in wt MEFs transfected with the Top Flashluciferase reporter and p-Catenin and treated with different concentrations of the compound Canthin-6-one or DMSO as a control for 48 hours. Luciferase and Renilla activities were measured using a luminometer according to the manufacturer's instructions (Biotium Inc., Hayward, CA, USA). Results were expressed as luciferase / renilla ratios and represent the mean ± S.D. of at least three experiments, each performed in triplicate.

[0159] Immunoblot analysis and immunoprecipitation

[0160] Cells were lysed using RIPA lysis buffer (50mM Tris-HCI at pH 7.6, 150mM NaCI, 0.5% sodium deoxycholate, 5 mM EDTA, 0.1% SDS, 100mM NaF, 2mM NaPPi, 1% NP-40) with added protease and phosphatase inhibitors. The lysates were centrifuged at 40,000 rpm for 20 min at 4°C and the resulting supernatants were resuspended in sample loading buffer, boiled for 5 min, separated by SDS-PAGE, transferred to nitrocellulose membranes (GVS North America, Sanford, ME, USA), blocked with 5% milk in TBS containing 0.1% Tween 20 (Sigma-Aldrich) and incubated with the indicated antibodies. Immunoprecipitation was performed using total cell extracts obtained by lysing cell pellets with Triton-containing lysis buffer (50 mM Tris-HCI pH 7.5, 250 mM sodium chloride, 50 mM sodium fluoride, 1 mM EDTA pH 8, 0.1% Triton), supplemented with protease and phosphatase inhibitors. Cell lysates were immunoprecipitated overnight at 4°C on a wheel with specific primary antibodies or IgG used as a control (2 pg / mg, Santa Cruz Biotechnology). The following day, the immunocomplexes were incubated with Protein G agarose beads (Santa Cruz Biotechnology) for 1 hour at 4°C on a wheel. The immunoprecipitates were then washed five times with the lysis buffer described above, resuspended in sample loading buffer, boiled for 5 min, separated by SDS-PAGE and then subjected to immunoblot analysis. Additional pull-down experiments were performed using Flag- LISP47 translated in vitro from the pcDNA3.1-Flag-USP47 plasmid and ERAP1 from the pCMV6-XL5-ERAP1 plasmid (SC31137, Origene, Rockville, MD, USA) using the TNT Quick Coupled Transcription / Translation system (Promega). Flag-USP47 and ERAP1 were placed in a 1:1 ratio in PC-100 buffer (HEPES pH 7.4 20 mM, MgCI22 mM, KCI 100 nM, glycerol 20%, EDTA 0.2 mM, Igepal 0.05%, BSA 100 mg / ml, DTT 1 mM) supplemented with protease and phosphatase inhibitors and with Canthin-6-one (1 pM) or solvent as a control. The samples were then immunoprecipitated overnight at 4°C on a wheel with anti-ERAP1 6H9 (2 pg / mg, MAB2334 from R&D Systems) or IgG used as a control (2 pg / mg, Santa Cruz Biotechnology). The following day, the immunocomplexes were analysed exactly as described above.

[0161] For all immunoblot analyses, reactive bands were visualised by chemiluminescence using WesternBright ECL HRP substrate (Advansta or Cyanagen).

[0162] Thermostabilisation assay (CETSA)

[0163] For the CETSA assay, protein extracts from MEFs were prepared in RIPA buffer (10 mM Tris HCI, pH 7.4, 150 mM NaCI, 1% Igepal, 1% sodium deoxycholate, 0.1% SDS, 0.1% glycerol, and 2 mM DTT), with the addition of protease and phosphatase inhibitors. Homogenisation was achieved by 3 cycles of freezing and thawing in dry ice. The soluble fraction was separated from the debris. The cell lysate was divided into two tubes: the first was treated with Canthin-6-one, N3 or N7 (1 pM), and the second was exposed only to the solvent (control). After 30 minutes of incubation at room temperature, the lysates were further divided into smaller aliquots (400 pg each) and heated individually at the temperatures listed for 3 minutes, followed by 3 minutes of cooling at room temperature. Subsequently, they were centrifuged to separate the soluble fractions from the precipitates. All supernatants were analysed by Western blot.

[0164] Cell Proliferation Assay

[0165] For MED1 , D283, HDMB03, and CHLA and primary MB cells, trypan blue counting was performed after a treatment period of 12, 24, 48, and 72 h with Canthin-6-one at the indicated doses. For the IncuCyte® S3 experiments, SHH-MB PDX were seeded in 96- well plates, 6 wells per condition, in complete medium and treated with Canthin-6-one or DMSO as a control. The plates were transferred to the IncuCyte® S3 incubator and incubated under physiological conditions (37 °C, 5% CO2) for 72 hours. Images were collected every 12 hours for SHH-MB PDX and proliferation was assessed as confluence. Experiments were performed in triplicate and data were analysed using the IncuCyte software package (Essen BioScience, Ann Arbor, Ml, USA). mRNA expression analysis

[0166] Total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) for mono-exonic genes, or with TRIzol reagent (Thermo Fisher Scientific) and reverse transcribed with the SensiFAST cDNA Synthesis Kit (Bioline Reagents Limited, London, UK). qRT-PCR analysis of Gli1, Gli2, CyclinD2, HHIP1, N-Myc, Patchedl, Nanog, and POU5F1 (OCT4) was performed using the ViiATM 7 Real-Time PCR System (Life Technologies). Standard parameters were used to amplify a reaction mixture containing the cDNA and the SensiFASTTM Probe Lo-ROX mixture (Bioline Reagents Limited) and Taqman Gene Expression Assays (Thermo Fisher Scientific). The average of two output cycles was used to calculate the amount of transcript in each sample amplified in triplicate (using SDS software version 2.3). mRNA quantification was calculated as the ratio of sample quantity to normaliser quantity expressed in arbitrary units. Data were normalised with the endogenous control (HPRT) and expressed as Fold Change relative to the control sample value.

[0167] The following primers were used to amplify the indicated genes:

[0168] . mGlil For 5' - GCC AAC TTT ATG TCA GGG TCC CAG - 3' (SEQ ID: NO 1) . mGlil Rev 5' - GGA GAG AGC CCG CTT CTT TGT TAA - 3' (SEQ ID: NO 2) . mGli2 For 5' - CAG CCA CCC CAG CGT AGA CA - 3' (SEQ I D: NO 3) . mGli2 Rev 5' - GCC CCA GGT CGC ACT CTA G - 3' (SEQ I D: NO 4)

[0169] . mCycD2 For 5' - AGA AGG ACA TCC AAC CGT ACA TG - 3' (SEQ I D: NO 5) . mCycD2 Rev 5' - CAT GGC CAG AGG AAA GAC CTC - 3' (SEQ I D: NO 6) . mHPRT For 5' - GCT TCC TCC TCA GAC CGC TT - 3' (SEQ ID: NO 7) . mHPRT Rev 5' - GGT CAT AAC CTG GTT CAT CAT GG - 3' (SEQ ID: NO 8) The following assays were used to amplify the genes listed below:

[0170] . mPtchl Mm00436026_m1

[0171] . mHHIPI Mm00469580_m1

[0172] • mNanog Mm02019550_s1

[0173] . mPou5f1 (OCT4) Mm03053917_g1

[0174] . mHPRT Mm03024075_m1

[0175] MB Neurosphere Formation Assay

[0176] MB-SLCs were seeded into 96-well plates in decreasing numbers (50, 25, 10, 5 cells; 12 wells per condition, 10 replicates per cell number / density) in neurosphere culture medium and treated with the indicated concentration of Canthin-6-one. The plates were placed in a humidified incubator at 37°C and 5% CO2. After ten days, all wells containing neurospheres were counted.

[0177] Animal Studies

[0178] Spontaneous tumours from Math1-cre / Ptcc / cmice were isolated, disrupted and pipetted to obtain a single-cell suspension. Equal amounts of cells (2 x 1OA6) were injected subcutaneously into the posterior flank of NSG mice (Charles River Laboratories, Lecco, Italy). The tumours were allowed to grow until they reached an average size of -200 mm3. The animals were randomly divided into two groups (n = 6) and injected intratumorally every two days with Canthin-6-one (1 mg / kg) and / or solvent for 17 days. Cells were resuspended in an equal volume of culture medium and Matrigel (BD Biosciences, Heidelberg, Germany) prior to s.c. injection. After injection, tumour growth was monitored and measured with a caliper. Changes in tumour volume were assessed using the formula (length x width) x 0.5 x (length + width).

[0179] For the orthotopic allograft model, adult NSG mice were anaesthetised by intraperitoneal (i.p.) injection of ketamine (10 mg / kg) and xylazine (100 mg / kg). The posterior cranial region was shaved and placed in a stereotaxic apparatus, and primary MB cells from Math1-cre / Ptcc / cmice were stereotaxically implanted into the cerebellum (2 x 10A5 / 3 pl) according to the coordinates of the Franklin and Paxinos atlas. After injection at a rate of 1 pl / min, the cannula was kept in place for 5 minutes and then the skin was closed with metal clips. Ten days after tumour implantation, the animals were randomly divided into two groups (n = 6) and treated i.v. every two days with Canthin- 6-one 1 mg / kg or solvent alone. After 25 days of treatment, the animals were sacrificed and their brains were fixed in 4% formaldehyde and embedded in paraffin. Tumour volume was calculated from 60 serial coronal sections of 5 pm each after H&E staining every 75 pm of brain slice. A microscope (Axio Imager M1 ; Leica Microsystems GmbH, Wetzlar, Germany) equipped with a motorised stage and Image Pro Plus 6.2 software was used to evaluate the tumour area of each slide, and tumour volume was calculated using the formula: tumour volume = sum of the area measured for each section x section thickness x sampling frequency.

[0180] All animal protocols were approved by the local ethical authorities (Ministry of Health) and conducted in accordance with Italian legislation (Legislative Decree 26 / 2014). European and national regulations for the care and use of animals for experimental and scientific purposes (Legislative Decree 26 / 2014) were followed.

[0181] Animal welfare

[0182] During the in vivo experiment described above (heterotopic or orthotopic implants of primary murine medulloblastoma cells, followed by intratumoral or intravenous injections of Canthin-6-one, respectively), the animals undergoing the procedure were kept under observation to check their health status on a daily basis with clinical condition assessment.

[0183] The table below was used as a reference to assess any signs of suffering in the animals during the experiment. If the score reaches 4, the designated veterinarian is immediately informed and monitors the animal as frequently as deemed appropriate for each individual case.

[0184] If the score reaches 6, indicating that the animal's health is severely compromised, the animal is euthanised.

[0185] In the case of the in vivo experimentation conducted for Canthin-6-one, no suffering was observed in the animals undergoing treatment, either in heterotransplantation or orthotransplantation experiments. The animals undergoing the procedure showed a score of zero, corresponding, as indicated in the table, to good health: normal coat, normal mucous membranes, normal physical activity, normal food and water consumption. All animals were sacrificed at the same time at the end of the treatment and not due to the animal's discomfort.

[0186] Statistical Analysis

[0187] For all experiments, the P-value was determined using a two-tailed Student's t-test and statistical significance was set at P < 0.05. Results are expressed as mean ± S.D. of an appropriate number of experiments (at least 3 biological replicates). For IC50 and IncuCyte experiments, statistical significance was determined using GraphPad Prism (version 8.0, San Diego, CA, USA). For IncuCyte® experiments, data were analysed using the IncuCyte® software package (Essen BioScience, UK). For animal studies, statistical significance was determined using ANOVA and reported as mean ± S.D.

Claims

CLAIMS1. Canthin-6-one or a pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use in the treatment of Hedgehog (HH) dependent tumours or in the treatment and / or prevention of relapses of said tumours2. Canthin-6-one or the pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use according to claim 1 wherein said tumours are Sonic Hedgehog (SHH) dependent tumours.

3. Canthin-6-one or the pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use according to any one of claims 1 to 2 wherein said tumours are selected within: Sonic Hedgehog subgroup medulloblastoma (SHH-MB), basal cell carcinoma, rhabdomyosarcoma, preferably wherein said tumour is SHH-MB.

4. Canthin-6-one or the pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use in the treatment of conditions that exhibit aberrant positive expression of endoplasmic reticulum aminopeptidase (ERAP), wherein said aberrant positive expression is a constitutive expression of the ERAP protein in a tissue wherein it is normally absent, or an overexpression of ERAP compared to the expression thereof in a corresponding healthy control tissue, preferably wherein said protein ERAP is ERAP1 and / or ERAP25. Canthin-6-one or the pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use according to claim 4 wherein the administration of said molecule causes a therapeutic effect through the inhibition of ERAP.

6. Canthin-6-one or the pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use according to any one of claims 4 or 5 wherein the treatment comprises a step for determining whether a patient presents an aberrant positive expression of ERAP and one or more administration steps of said Canthin-6-one to patients that present said aberrant positive expression of ERAP.

7. The pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use according to any one of claims from 1 to 6, in liquid, solid, or semi-solid form.

8. The pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use according to any one of claims from 1 to 7 in the form of a composition for topical, enteral, oral, sublingual, nasal, oro- nasopharyngeal, systemic, intravenous, intraperitoneal, intrathecal, intramuscular, subcutaneous, intratumoral, or inhalation administration, preferably in the form of a tablet, solution, suspension, emulsion, syrup, soft or hard capsule, aerosol, injectable solution, injectable suspension, injectable emulsion, gel, ointment, cream, lotion, or spray9. Canthin-6-one or the pharmaceutical composition comprising Canthin-6-one and at least one pharmaceutically acceptable vehicle for use according to any one of claims from 4 to 6 wherein said condition is an autoimmune and / or autoinflammatory disease, hypertension, a viral infection, or cancer, preferably wherein said autoimmune and / or autoinflammatory diseases are ankylosing spondylitis, Behget's disease, psoriasis, or Birdshot chorioretinopathy and preferably wherein said cancer is melanoma, leukemia, lymphoma, breast cancer, colon cancer, thyroid cancer, lung cancer, cervical cancer, prostate cancer, kidney cancer, bladder cancer.

10. In vitro or ex vivo use of cathin-6-one as an inhibitor of ERAP.

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