Use of immunoproteasome subunit inhibitors for treatment or prevention of mitochondriopathies

Abstract

The present invention relates to use of immunoproteasome subunit inhibitors for treatment or prevention of mitochondriopathies resulting from a decreased level of mitochondrial proteins associated with mutations in nuclear-encoded genes causing defects in respiratory chain.

Description

[0001] USE OF IMMUNOPROTEAS OME SUBUNIT INHIBITORS FOR TREATMENT OR PREVENTION OF MITOCHONDRIOPATHIES

[0002] TECHNICAL FIELD

[0003] The present invention relates to the use of immunoproteasome subunit inhibitors for treatment or prevention of mitochondriopathies resulting from a decreased level of mitochondrial proteins associated with mutations in nuclear-encoded genes causing defects in respiratory chain.

[0004] BACKGROUND ART

[0005] Although mitochondrial diseases are considered to be rare (e.g. single families described with a particular type of a mutation of a particular gene), conservative estimates of the prevalence nuclear DNA mutations in affected adults are 2.9 per 100,000 (Gorman et al., 2016). It is now recognized in the field that it may not be feasible to differentiate between specific syndromes, as mitochondriopathies may show a very diverse phenotype, with a dysfunction of almost any organ system. In addition genotype-phenotype correlations in mitochondrial diseases are often poor (Thompson et al., 2020). Mutations in different genes can cause the same phenotype and the same pathogenic mutation can cause a range of different phenotypes (Schon et al., 2020). Currently, the recommended approach tends to be not to diagnose a particular syndrome but to evaluate the genetic background of a disorder associated with a mitochondrial dysfunction. When a mitochondrial disease is suspected clinically, the strategy for reaching a final diagnosis may rely on excluding other common or metabolic disorders, and on subsequent biochemical and histochemical analysis of affected tissue, with a growing importance of whole genome sequencing (Thompson et al., 2020).

[0006] In an important conclusion of the above, due to numerous challenges in diagnosis and possible variability in symptoms between individuals, a specific type of mitochondriopathy is defined more clearly not by reference to a particular syndrome(s) but by defining the cause at the molecular level. Some mitochondrial diseases are caused by insufficient import of mutated protein to mitochondria, which is degraded in the cytosol (Mohanraj et al., 2019). Indeed, one of the features of many mitochondrial diseases is an apparent absence or a low level of a mutated protein, suggesting a common mechanism.

[0007] In recent years a variety of different mitochondriopathies treatments have been investigated. One of the approaches, which was presented in WO2019166588, is based on a discovery that mutations in nuclear encoded mitochondrial proteins, such as RES Al, result in reduced levels of said proteins in mitochondria. Moreover, these reduced levels of said mitochondrial proteins have been attributed to an increased cytosolic degradation of the mutant proteins due to a slower import into the mitochondria. Furthermore, it was demonstrated that the degradation of newly synthesized mutated mitochondrial proteins can be counteracted by a process of proteasome inhibition. Consequently, WO2019166588 discloses a treatment of mitochondriopathies with proteasome inhibitors, wherein said treatment overcomes the cellular mechanism defect underlying said mitochondriopathies.

[0008] Proteasomes, present in many species from an archaebacterium to human, are large (2.5 MDa), a barrel- shaped, multi-subunit proteolytic complexes located in a cytoplasm and nuclei of a cell, which are important for cellular protein homeostasis. Proteasomes are responsible for ATP dependent, highly specific degradation of normal, damaged, mutated and misfolded proteins, which are recognized by the proteasome by their ubiquitin tags, a small (8.6 kDa) proteins consisting of 76 amino acids. The term proteasome usually refers to the 26S proteasome and includes a 20S core catalytic complex with 19S regulatory subunits. The catalytic core consists of two a- and two P-rings and each ring contains seven subunits. The outer a-rings are responsible for regulation of substrate access into the inner catalytic part formed by P-subunits. Three of the seven P-type subunits are proteolytically active in the catalytic core of the proteasome, PSMB6 (P 1), PSMB7 (P2) and PSMB5 (P5), showing caspase -like, trypsin-like and chymotrypsin-like activity, respectively. These subunits cleave proteins after acidic, basic and hydrophobic residues, respectively (Rousseau and Bertolotti, 2018).

[0009] Moreover, it has been demonstrated that ubiquitin-proteasome pathway (UPP) disturbances are involved in a variety of diseases, including cancer, neurodegenerative and autoimmune disorders. Interference of proteasome inhibitors with the ubiquitin proteasome pathway involved in protein turnover in the cell leads to the accumulation of proteins engaged in cell cycle progression, which ultimately put a halt to cancer cell division and induce apoptosis. Upregulation of many tumor suppressor proteins involved in cell cycle arrest are known to play a role in PI (proteasome inhibitors) induced cell cycle arrest in a variety of cancer cells. Pls has been reported to significantly increase the expression of p21 and p27 proteins in many cancers thereby causing cell cycle arrest (Rastogi & Mishra, 2012; Bonvini et al., 2007). Accumulation of non-degraded proteins increase unfolded protein response (UPR) leading to induction of endoplasmic reticulum stress, which again causes cell cycle arrest to release cells from this cellular stress. Pls induced ROS generation in many cancers is responsible for DNA damage mediated cell cycle arrest (Rastogi & Mishra, 2012).

[0010] Therefore, in recent years a variety of the ubiquitin system inhibitors, especially proteasome inhibitors, were designed and evaluated. Examples of authorized proteasome inhibitors include bortezomib, carfilzomib and ixazomib. However, the FDA-approved proteasome inhibitors inhibit the constitutive proteasomes. Thus, their clinical use is limited by the toxicities they induce. The most common side effect of proteasome inhibitors include, for example, peripheral neuropathy (Arastu-Kapur et al., 2011), thrombocytopenia (Richardson et al, 2005), as well as gastrointestinal (Stansborough and Gibson, 2017), renal (Wanchoo et al., 2017), and cardiac toxicities (Gavazzoni et al., 2018).

[0011] Hence, there exist a need for therapeutic agents, which would display a fewer number of adverse effect and still prevent degradation of mitochondrial proteins necessary in the treatment of mitochondriopathies.

[0012] The immunoproteasome is a proinflammatory cytokine-induced form of proteasome characterized by a different composition of catalytic subunits. In the immunoproteasome these catalytic subunits from constitutive proteasome are substituted by inducible - counterparts: PSMB9 ( li), PSMB10 ( 2i) and PSMB8 ( 5i), which have similar activities with exception to PSMB9 that displays chymotrypsin-like and not caspase-like activity. Additional proteasome subtypes expressing a mix of constitutive and inducible subunits are also found in normal tissues and in some human cancer cell lines and are called “intermediate proteasomes”. The immunoproteasome core particle can bind either PA700 (19S) regulatory particle or IFN-y induced PA28aP (IIS) regulatory particle, and is involved in ATP and ubiquitin-dependent or -independent protein degradation, respectively (Motosugi and Murata, 2019; Murata et al., 2018). Differences between proteasome and immunoproteasome are summarized in Table 1 below.

[0013] Table 1

[0014] The immunoproteasome is expressed in a broad range of immune cells, such as T- cells, B-cells and antigen presenting cells. Since the immunoproteasome is highly expressed in immune cells, immunoproteasome- specific inhibitors selectively affect the function of activated immune cells while sparing other cell types that would be damaged by treatment with bortezomib, an anticancer drug approved by the US Food and Drug Administration (FDA) that inhibits the P5 subunit of both the constitutive CP and the immunoproteasome (Murata et al., 2018).

[0015] Current consensus states, however, that this type of inducible proteasome is also expressed in nonimmune cells in response to proinflammatory cytokines and other stress cues such as nitric oxide (NO), heat shock, H2O2, and hyperglycemia (Angeles et al., 2012). Assembly of the immunoproteasome is approximately four times faster than that of the 26S proteasome (Murata et al., 2018). The immunoproteasome has also a shorter half-life of 27 h compared to the 26S proteasome (133 h), which serves its specific upregulation in stress and under cytokine exposure, after which it is not needed in large quantities (Morozov and Karpov, 2019). It has the ability to hydrolyze proteins at greater rates compared to the standard proteasome, and therefore may contribute to maintenance of cellular homeostasis by rapid removal of potentially harmful proteins under pathological conditions.

[0016] The immunoproteasome plays a critical role in the immune system because it degrades intracellular proteins, for example, those of viral origin, into small proteins. Contrary to the proteasome the expression of immunoproteasome subunits is not essential as demonstrated by experiments in which mice devoid of all three immunoproteasome subunits were fully viable (Kincaid et al, 2012). Moreover, peptide library-based screening revealed that immunoproteasome and proteasome have overlapping but distinct substrate specificities (Winter et al 2017). This suggests a distinct physiological role of the immunoproteasome that may be exploited to minimize the collateral damage caused by inhibition of proteasome.

[0017] The expression of immunoproteasome catalytic subunit PSMB9 in cells with defective mitochondria has been recently described in an article (Kim et al., 2023). As it was observed in cells with different mitochondrial defects, PSMB9 protein was assembled into the mature proteasomal complex, which was followed by an increase of immunoproteasome- specific catalytic activity. However, no link between the increased immunoproteasome activity in mitochondria-defective cells and a use of immunoproteasome subunit inhibitors for treatment or prevention of mitochondriopathies was previously presented.

[0018] The immunoproteasome and proteasome have similar structures of their catalytic core, which is composed of a ring of P subunits. Substrates interact with domains exposed to a substrate binding channel, composed of primed and non-primed specificity (S) pockets that bind target polypeptides in the C- to N-terminal direction. The peptide localized between primed and non-primed is cleaved by the active site, which is constituted by Thrl residue. While the active site and two proximal S and S’ pockets are localized in the active subunit, the distal specificity pockets are localized to adjacent -subunits (Huber and Groll, 2021).

[0019] The immuno- and constitutive proteasome crystal structures revealed differences in inhibitor specificity for different subunits (Huber et al., 2012; Zhang et al., 2020). The structures of the PSMB7 (P2c) and PSMB10 (P2i) subunits are essentially identical in terms of their substrate binding pockets, except for the D53E substitution in PSMB 10 (Huber et al., 2012). The PSMB9 (Pli) subunit showed a structure distinct from its constitutive analog PSMB6 (pic). Key polar residues in the substrate binding channel of subunit PSMB6 are replaced by hydrophobic ones in PSMB9 (Huber and Groll, 2021). Substitutions T20V, T3 IF, R45L, and T52A in PSMB9 increase the hydrophobicity of the S 1 pocket and reduce it in size leading to change in preference of hydrolysis preferentially after small, hydrophobic, and branched residues. This specificity agrees with specific cleavage of Anorogenic peptide substrate Ac-PAL-AMC (Acetate-Pro-Ala-Leu-7- aminomethylcoumari) designed to analyze PSMB9-specific proteasome activity (Huber et al., 2012; Xi et al., 2019). As with p2-subunits, the PSMB5 (P5c) and PSMB8 (P5i) substrate binding channels only slightly differ in sequence and structure. The most pronounced distinction is the size of their SI -specificity pockets, originating from a conformational change of M45 and mutation of V31 of constitutive proteasome PSMB5 into M31 resulting in a larger SI pocket. Thus, while inhibitors specific for PSMB5 require small hydrophobic residues like Ala, Vai and Leu in SI -pocket, the SI -pocket of PSMB9 is enlarged and promotes cleavage after large hydrophobic groups such as Phe, Tyr or Trp (Huber and Groll, 2021; Xi et al., 2019; Zhang et al., 2020).

[0020] The above structural differences allowed to design subunit-specific inhibitors. Current challenge in medicine is to develop inhibitors selective to immunoproteasome subunits with less toxicity for the treatment of hematologic malignancies, nervous system diseases and immune system diseases. However, prior art is silent about use of subunitspecific immunoproteasome inhibitors in treatment of mitochondriopathies.

[0021] DISCLOSURE OF INVENTION

[0022] The inventors of the present invention have shown that immunoproteasome subunit inhibitors can be used for the treatment or prevention of mitochondriopathies and, at the same time, display decreased toxicity when compared to proteasome inhibitors, also referred to in the prior art as pan-proteasome or constitutive proteasome inhibitors. A common phenomenon of many mitochondrial diseases is an apparent absence or a low level of a mutated protein. The mutated proteins that are not efficiently imported to mitochondria are degraded in the cytosol by proteasome (Friederich et al., 2017; Mohanraj et al., 2019). As shown by the present inventors in the cells derived from patients with mitochondriopathies an increased immunoproteasome activity is observed. Thus, immunoproteasome subunit inhibitors will have more specific and less toxic effect in treatment of mitochondriopathies.

[0023] The present inventors have observed increased expression of immunoproteasome subunits in fibroblasts derived from patients suffering from mitochondrial diseases and demonstrated a rescue of mutated mitochondrial protein by inhibitors of immunoproteasome subunits. Thus, a therapeutic effect of immunoproteasome subunit inhibitors with respect to mitochondriopathies has been demonstrated. Moreover, a treatment with immunoproteasome subunit inhibitors is less toxic than previously proposed treatment with proteasome inhibitors.

[0024] In a first aspect of the present invention, therefore, there is provided an immunoproteasome subunit inhibitor for use in the treatment or prevention of a mitochondrial disease, wherein the immunoproteasome subunit inhibitor is a selective immunoproteasome subunit inhibitor, which is administered in the amount not sufficient to inhibit a proteasome activity. A term “mitochondrial disease” or “mitochondriopathy”, as used interchangeably in the present description, is a collective term for clinical disorders united by the common feature of failure of mitochondrial function and aberrant energy metabolism (Gorman et al., 2016; Schon et al., 2020; Thompson et al., 2020). Predominantly, mitochondriopathies are caused by defects in respiratory chain. In many cases, such defects are associated with mutations in nuclear-encoded genes. Consequently, according to the present invention the immunoproteasome subunit inhibitors are used to treat mitochondrial diseases, which are characterized by a decreased level of mitochondrial proteins.

[0025] The mitochondriopathy according to the present invention is preferably selected from a group comprising mitochondrial myopathy, preferably progressive mitochondrial myopathy, cardiomyopathy, preferably dilated 1GG; mitochondrial myopathy with congenital cataract, hearing loss, and developmental delay, isolated autosomal dominant mitochondrial myopathy, mitochondrial myopathy with extrapyramidal signs, cardioencephalomyopathy, preferably fatal infantile cardioencephalomyopathy, frontotemporal dementia and / or amyotrophic lateral sclerosis 2, spinal muscular atrophy, preferably Jokela type spinal muscular atrophy, myopathy with extrapyramidal signs, mitochondrial complex I deficiency, mitochondrial complex IV deficiency, Mohr- Tranebjaerg syndrome, reticular dysgenesis, Leigh syndrome associated to cytochrome c oxidase deficiency, mitochondrial hepatoencephalopathy, Cowden syndrome 3, somatic merkel cell carcinoma, mitochondrial complex II deficiency, pagangliomas 1, preferably with or without deafness; paragangliomas 5, sulfate oxidase deficiency, mitochondrial complex III deficiency, preferably nuclear type 2, combined oxidative phosphorylation deficiency 22, Mitochondrial complex V deficiency, preferably nuclear type 1, 3 or 4; mitochondrial complex I deficiency, Leigh syndrome associated to mitochondrial complex I deficiency, Leigh syndrome associated to COX IV deficiency, and mitochondrial complex III deficiency, preferably nuclear type 2, 3, 4, 5, 7, 8, or 9.

[0026] In the preferred embodiment, the mitochondrial diseases that can be treated using immunoproteasome subunit inhibitors according to the present invention comprise diseases with decreased levels of complex IV caused by mutations in genes encoding for complex IV subunits or assembly factors. Gene mutations as used herein refer to one or more mutation in one gene, as well as more than one mutations in more than one gene.

[0027] Examples of such mitochondriopathies are set out in Table 2 below.

[0028] Table 2

[0029] More preferably, the mitochondrial diseases that can be treated using immunoproteasome subunit inhibitors according to the present invention is selected from a group of diseases associated to mutation in genes C0X6B1 and COA7. According to the present invention, an immunoproteasome subunit inhibitor is used, more preferably, in the treatment of the mitochondrial complex IV deficiency, nuclear type 7 or spinocerebellar ataxia, autosomal recessive, with axonal neuropathy 3, and most preferably in the treatment of the mitochondrial complex IV deficiency, nuclear type 7.

[0030] Therefore, in the most narrow interpretation the data presented hereinbelow in the Examples concern mitochondrial complex IV deficiency, nuclear type 7 (MIM: 619051) as sociated with homogenou s mutation c .221 G> A in exon 2 of C OX6B1. Mutation c .221 G> A in exon 2 of COX6B1 gene was associated with cases of severe infantile encephalomyopathy (Massa et al., 2008). COX6B1 is a subunit of respiratory complex IV, also known of cytochrome c oxidase, which participates in ATP production via oxidative phosphorylation in mitochondria. The resulting substitution R19H in COX6B1 protein reduced expression of this protein and led to drastic decrease respiratory complex IV activity. COX6B is a mitochondrial IMS protein, which is imported into mitochondria via MIA pathway. The protein connects the two monomers of cytochrome c oxidase into the physiological dimeric form and is also believed to interact with cytochrome c. Similar substitution (R20C) in COX6B1 protein was reporter by Abdulhag and colleagues and associated with decreased levels of COX6B1 protein and complex IV deficiency (Abdulhag et al., 2015).

[0031] In an alternative embodiment, the mitochondrial diseases that can be treated using immunoproteasome subunit inhibitors according to the present invention comprise diseases with decreased levels of complex IV caused by mutations in genes other than those encoding for complex IV subunits or assembly factors. Examples of such mitochondriopathies are presented in Table 3 below.

[0032] Table 3

[0033] Examples of genetic mutations causing mitochondriopathy and protein / gene to disease relation between mitochondrial protein / gene and mitochondriopathies that can be treated using an immunoproteasome subunit inhibitor are also presented in WO2019166588, table 1 on pages 15 - 24. As used herein, the term “treatment” or “treating” of a disease includes inhibiting the disease state, i.e., hindering the development of the disease state or its clinical symptoms, or alleviating the disease state, i.e., causing temporary or permanent regression of the disease state or its clinical symptoms. The term “prevention” or “preventing” of a disease means causing the clinical symptoms of the disease state not to develop in a patient that can be predisposed to the disease, but does not yet experience or display symptoms of said disease.

[0034] The term “immunoproteasome subunit inhibitor” as used herein should be understood as an entity decreasing or blocking activity of the immunoproteasome- specific subunits, thus resulting in an increase of the mutated mitochondrial protein. In the present description the term “immunoproteasome subunit inhibitor” is used interchangeably with a term “immunoproteasome inhibitor”. Examples of the immunoproteasome subunit inhibitors are known from the prior art and include those presented in Xi et al., 2019 and Huber & Groll, 2021: ONX-0914, PR-924, KZR-616, KZR-504, KZR-109, UK-101, LU-015i, LU- 005i, LU-045i, LU-025i, LU-035i, LU-002i, YU-102, LU-OOli, ML604440, IPSI-OO1, 2- CA, 4-CA, PKS2251, PKS2252, PKS2279, PKS21221, PKS21293, Argyrin B, Rol9, HT1171, HT2004, HT1213, HT2111, DB-310, DPLG3, PRN1126, HT2004, Piperlongumine, M3258, Compound 1, Compound la, Compound lb, Compound 3, Compound 6, Compound 7, Compound 8, Compound 16, and Compound 22. Among the above-identified immunoproteasome subunit inhibitors, the immunoproteasome inhibitors selective for immunoproteasome subunit pii (PSMB9) (Xi et al., 2019; Huber & Groll, 2021) are of particular interest. These include the following compounds: KZR-504, KZR- 109, Compound 7 (name according to Huber and Groll, 2021), Compound 4 (name according to Xi, 2019), DB-310, LU-001, ML60444, UK-101 and YU-102.

[0035] Any of the above-identified immunoproteasome subunit inhibitors alone or in combination can be used according to the present invention for the treatment of mitochondriopathies. In a more preferred embodiment the immunoproteasome subunit inhibitor is an epoxyketone peptide-based immunoproteasome subunit inhibitor. In the most preferred immunoproteasome subunit inhibitors for use of the invention include KZR-504, KZR-616 and ONX-0914 or a combination thereof.

[0036] KZR-504 is a dipeptidyl epoxyketone derivative developed by Kezar Life Sciences and is the most immunoproteasome- selective subunit inhibitors reported up to now. KZR- 504, displays approximately 100-fold stronger inhibitory activity toward PSMB9 (pii) activity compared against PSMB6 (P 1 c) with IC50 values of 0.05 and 46.27 pM, respectively, determined in MOLT-4 (human T cell leukemia) cell lysates. KZR-504 is of interest for the treatment of autoimmune disease (Huber and Groll, 2021; Zhang et al., 2020). Evaluating the inhibition of PSMB9 (pii) in mouse tissues reveals that KZR-504 is both selective and potent in vivo with >50% target inhibition achieved at >1 mg / kg in all tissues tested except brain [Johnson et al., 2017]. Most pii-selective compounds share a cyclic residue at P3 site (amino acid side chains of a peptide interacting with S3 pocket). KZR-504 feature a 2- pyridone at P3 site (Huber and Groll, 2021). The chemical structure of KZR-504 is presented below.

[0037] ONX-0914 (PR-957), on the other hand, is a tripeptide epoxyketone and the first PSMB8 (P5i) selective inhibitor reported in literature developed by Onyx Pharmaceuticals. ONX-0914 was recently classified as pan-selective inhibitor targeting both PSMB8 and PSMB9 immunoproteasome subunits with 80-fold higher selectivity towards PSMB8 than PSMB9 (Huber and Groll, 2021). Literature data indicate its higher selectivity to immunoproteasome analogs compared to constitutive subunits (IC50 values of 0,0057 pM for PSMB8 compared to 0,054 pM for PSMB5 and IC50 values of 0,46 pM for PSMB9 compared to over 10 pM for PSMB6) (Huber and Groll, 2021). The phenyl side chain of ONX-0914 perfectly fits into the spacious SI pocket of PSMB8 but not into the smaller one of PSMB5, which explains the subunit specificity of this inhibitor. The forces necessary to enlarge the SI pocket of PSMB5 by pushing Met45 aside explain around 10 times higher IC50 value of ONX-0914 for PSMB8 (Huber et al., 2012; Huber and Groll, 2021). The chemical structure of ONX-0914 is presented below.

[0038] ONX0914 has previously been reported as useful for treatment of autoimmune diseases including experimental rheumatoid arthritis, inflammatory bowel disease (IBD), Hashimoto’s thyroiditis, Graves’ disease, systemic lupus erythematosus -like disease (SLE), experimental autoimmune encephalomyelitis (EAE), and multiple sclerosis (MS) (Basler et al., 2015). KZR-616, a pan-selective immunoproteasome inhibitor targeting both PSMB8 and PSMB9 ( 5i and 01i) immunoproteasome subunits developed by Kezar Life Sciences with an activity profile similar to ONX-0914. KZR-616 is a tripeptide ketoepoxide and analog of ONX0914. KZR616 was developed based on the optimization of peptidyl epoxyketone derivatives ONX-0914 and PR-924 (Zhang et al., 2020). KZR-616 indicates higher selectivity to immunoproteasome analogs compared to constitutive subunits (IC50 values of 0,039 pM for PSMB8 compared to 0,668 pM for PSMB5 and IC50 values of 0,131 pM for PSMB9 compared to over 10,6 pM for PSMB6) (Huber and Groll, 2021). KZR-616 has been approved for phase II clinical trials for treatment of several autoimmune diseases, such as systemic lupus erythematosus with and without nephritis, autoimmune hepatitis, active polymyositis or dermatomyositis and active autoimmune hemolytic anemia or immune thrombocytopenia (Huber and Groll, 2021). The chemical structure of KZR-616 is presented below.

[0039] In another aspect of the present invention a method of treatment or prevention of treatment or prevention of a mitochondrial disease in a patient is provided, which comprises administration of one or more immunoproteasome subunit inhibitors. The immunoproteasome subunit inhibitors are administered in the amount sufficient to obtain a therapeutic or prophylactic effect, respectively.

[0040] The term “a selective immunoproteasome subunit inhibitor” as used herein refers to the immunoproteasome subunit inhibitor having at least 5-fold, preferably at least 6-fold, more preferably at least 7-fold, even more preferably at least 8-fold, also more preferably at least 9-fold, most preferably at least 10-fold stronger inhibitory activity toward an immunoproteasome subunit than toward a proteasome subunit. In the preferred embodiment, the selective immunoproteasome subunit inhibitors can exhibit even higher inhibitory activity toward an immunoproteasome subunit than toward a proteasome subunit, such as at least 15, 17, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 times higher inhibitory activity. Alternatively, the immunoproteasome subunit inhibitor can have at least 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900- or even 1000-fold stronger inhibitory activity toward an immunoproteasome subunit than toward a proteasome subunit.

[0041] In terms of IC50 values, “a selective immunoproteasome subunit inhibitor” refers to the immunoproteasome subunit inhibitor having at least 5 times lower IC50 value for an immunoproteasome subunit compared to the IC50 value for a proteasome subunit. In the preferred embodiment the immunoproteasome subunit inhibitor has at least one order of magnitude, more preferably at least two orders of magnitude, most preferably at least three orders of magnitude lower IC50 value for an immunoproteasome subunit compared to the IC50 value for a proteasome subunit. In a specific embodiment of the present invention the IC50 values with respect to proteasome and immunoproteasome subunits correspond to those exhibited by KZR-504, ONX0914 and KZR-616.

[0042] According to the present invention a selective immunoproteasome subunit inhibitor is administered in the amount not sufficient to inhibit a proteasome activity. It means that the amount of a selective immunoproteasome subunit inhibitor used according to the present invention decreases or blocks activity of at least one immunoproteasome- specific subunit without affecting the activity of any of the proteasome subunits. This selective blocking of immunoproteasome- specific subunits without blocking any of the proteasome activity is important, as it ensures a decreased level of toxicity in the treatment of mitochondriopathies. Therefore, according to the present invention the therapeutic effect results from blocking or decreasing of immunoproteasome subunit activity and not from blocking or decreasing of proteasome subunit activity.

[0043] Based on their discovery of immunoproteasome subunit PSMB9 increase in cells with defective mitochondria, the inventors searched for the same phenomenon in fibroblasts derived from patients suffering from mitochondrial diseases. The inventors found that expression of catalytic subunits of immunoproteasome is increased in fibroblasts derived from patients with mutations in nuclear genes that encode subunits of the mitochondrial respiratory chain and its assembly factors. Specifically, fibroblasts with mutation in COX6B1 gene display higher level of subunit PSMB9 that has a chymotrypsin-like activity. Similarly, fibroblasts with mutations in C0A7 gene display an increased level of PSMB9. In addition, C0A7 mutated fibroblasts present an increased level of another immunoproteasome subunit PSMB8, which also has a chymotrypsin-like catalytic activity. Notably, these mutations in C0X6B and C0A7 genes correspond to the nuclear mutations of a patient with severe infantile encephalomyopathy (Massa et ah, 2008), mitochondrial leukoencephalopathy and cytochrome c oxidase deficiency (Martinez Lyons et ah, 2016) respectively. In both cases original reports noticed a radically decreased level of respective protein in samples form patients. This demonstrates that instances of mitochondriopathy, in which level of mutated protein is decreased, are correlated with an increased level of immunoproteasome subunits.

[0044] It was further shown by the inventors that COX6B mutated fibroblasts display an increased catalytic activity specific to PSMB9 subunit, as well as general chymotrypsin-like activity. Thus the PSMB9 subunit expressed in these cells was active and most probably effectively folded and assembled into functional units of immunoproteasome or intermediate immunoproteasome .

[0045] It was previously shown that patients with this particular mutation of COX6B gene display a decreased expression of this protein. The inventors have concluded that preventing the excessive proteolytic activity in the cytosol by chemical inhibition of PSMB9 may rescue level of mutated COX6B protein. The inventors tested the effects of inhibitors of immunoproteasome subunits on fibroblasts with mutated COX6B. Initially they compared protein ubiquitination level in cells treated with KZR-504, an inhibitor of PSMB9, KZR-616 and ONX-0914, which also to some extent inhibits proteasome. All the inhibitors did not trigger an increase in protein ubiquitination at the concentrations tested compared to proteasome inhibitor bortezomib. It was further confirmed that ONX-0914 and KZR-616 are effective against global chymotrypsin-like catalytic activity.

[0046] The inventors further analyzed the effects of ONX-0914, KZR-504 and KZR-616 on expression of mutant version of COX6B protein in patient fibroblasts. Notably they have found that all tested compounds increased the level of mutated COX6B protein. Thus, the rescue of COX6B protein level can be achieved by inhibiting solely the activity of immunoproteasome even if the inhibition is specifically targeted to subunit PSMB9 of immunoproteasome . In previous reports proteasome inhibitor bortezomib was used to rescue mitochondrial function by inhibition of excessive degradation of mutated mitochondrial proteins (Mohanraj et al., 2019). Bortezomib is known, however, for its toxic influence on cells caused by global change in protein homeostasis. In order to verify if inhibitors of immunoproteasome subunits are less toxic than bortezomib the inventors tested the effects of KZR-504, ONX-0914 and bortezomib on cell proliferation. In contrast to bortezomib which caused a significant decrease of proliferation, cells treated with KZR-504 and ONX-0914 displayed a similar proliferation dynamics to untreated fibroblasts. Moreover, ONX-0914, KZR-504 and KZR-616 did not or to much lesser extent than bortezomib increased the level of stress-induced HSP70 protein, corroborating lower cellular stress caused by the inhibitors of immunoproteasome subunits.

[0047] In conclusion ONX-0914, KZR-504 and KZR-616 effectively increased level of mutated COX6B protein, which depletion is associated with cases of severe infantile encephalomyopathy. At the same time ONX-0914 and KZR-504 did not affect either protein ubiquitination pattern nor the proliferation of fibroblasts and all three tested inhibitors did not induce cellular stress. Thus, inhibition of immunoproteasome subunits as an effective way to prevent excessive degradation of mitochondrial proteins in mitochondrial diseases.

[0048] BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 presents SDS-PAGE and western blot analysis result, which shows that immunoproteasome catalytic subunit PSMB9 is upregulated in patient fibroblasts with COX6B-R19H mutation.

[0050] Figure 2 presents SDS-PAGE and western blot analysis result, which shows that immunoproteasome catalytic subunits PSMB8 and PSMB9 are upregulated in patient fibroblasts with COA7 mutations (Y 137 Cl exon2A).

[0051] Figure 3 presents a diagram of PSMB9 (A) and chymotrypsin-like (B) proteolytic activities in fibroblast homogenates, which show that these activities are increased in patient fibroblasts with COX6B R19H mutation. Figure 4 presents SDS-PAGE and western blot analysis result, which shows that protein ubiquitination is not affected in fibroblasts treated with immunoproteasome subunit inhibitors ONX-0914, KZR-504 and KZR-616.

[0052] Figure 5 presents a diagram of chymotrypsin-like activity in fibroblast homogenate, which shows global chymotrypsin-like activity inhibition by immunoproteasome subunit inhibitors ONX-0914, KZR-504 and KZR-616.

[0053] Figure 6 presents a diagram of chymotrypsin-like activity specific to PSMB9, which shows proteolytic activity of immunoproteasome subunit PSMB9 inhibition by immunoproteasome subunit inhibitors ONX-0914, KZR-504.

[0054] Figure 7 presents SDS-PAGE and western blot analysis result, which shows that level of COX6B-R19H protein is increased in patient fibroblasts after treatment with immunoproteasome subunit inhibitors ONX-0914, KZR-504, KZR-616.

[0055] Figure 8 presents growth curve of fibroblasts treated with ONX-0914, KZR-504 and bortezomib, respectively, over time, which shows that inhibitors of immunoproteasome subunits ONX-0914 and KZR-504 have no impact on fibroblast proliferation in contrast to proteasome inhibitor bortezomib.

[0056] EXAMPLES

[0057] Materials and Methods

[0058] Growth condition of fibroblasts cells

[0059] Fibroblast cells were cultured at 37°C with 5% CO2 in standard DMEM containing high glucose (4.5g / L), supplemented with 10% (v / v) heat inactivated fetal bovine serum, 2 mM glutamine, 100 U / ml penicillin and 10 pg / ml streptomycin sulfate, 1 mM sodium pyruvate and 50 pg / ml uridine. For experiments, cells were grown in Dulbecco’s modified Eagle medium (DMEM) containing low glucose (1.1 g / L) or galactose (1.8 g / L) as and when described.

[0060] Immortalized skin fibroblast from a patient and a healthy donor

[0061] Mt4229i immortalized skin fibroblast from a patient (19-year-old woman) harboring a COA7 mutations were used. Mt8987i immortalized skin fibroblast from a patient (10-year-old man) harboring a

[0062] C0X6B mutation were used.

[0063] Immortalized skin fibroblast from a healthy donor were received from Prof. Massimo Zeviani laboratory, Mitochondrial Biology Unit, MRC, Cambridge.

[0064] Protein cellular extract

[0065] Fibroblasts were grown in DMEM-low glucose medium for 24 h, and then cells were transferred to galactose medium for 48h. Cells were harvested by cell scrapper and washed twice with IX PBS (without Ca2+and Mg2+) by centrifugation (1000 x g, 5 min, 4 °C). The cell pellet was solubilized in RIPA buffer containing PMSF (2mM) for 30 min at 4 °C. The cell lysate was clarified by centrifugation (20,000 x g, 20min, 4 °C) and supernatant was collected. A small fraction was used to determine the protein concentration by RotiQuant protein assay. The remaining fraction was solubilized in Laemmli sample buffer containing 50 mM DTT and analyzed by reducing SDS-PAGE and western blotting.

[0066] RIPA buffer: 65 mM Tris base, 150 mM NaCl, 1% v / v NP-40, 0.25 % sodium deoxycholate, 1 mM EDTA and 2mM PMSF, pH 7.4

[0067] In solution assay of chymotrypsin-like activity of immunoproteasome subunits

[0068] Cells were seeded in concentration of 27xl03 / cm on 150mm cell culture dish in low glucose medium for 24h. The next day medium was changed for galactose for 48h. Next cells were harvested and transferred to epi-tubes and centrifuged 1000g / 4°C / 5min. Cells pellets were lysed in 200pl cold proteasome lysis buffer (50mM Tris-HCl, pH 7.4, lOMgCh, 250mM sucrose, 2mM ATP, 2mM PMSF, ImM DTT) and homogenized in Dounce homogenizer (Sartorius, cat bo. BBI-85407050). The homogenate was clarified by centrifugation at 10000g / 4°C / 15min and protein concentration was measured using Bradford method with BSA as a standard. Ipg protein were incubated with 50 pM Suc-Leu-Leu- Val-Tyr-AMC (chymotrypsin-like activity; Bachem, cat. no. 1-1395) and 4pg protein were incubated with 50 pM Ac-Pro-Ala-Leu-AMC (Cayman Chemical, cat. no. 26592) to measure the activity of PSMB9 in a final volume of 200pl of lysis buffer in a 96-well plate. Fluorescence (excitation wavelength 380 nm, emission wavelength 460 nm) was measured every 5 min for 2 h at 25°C using Synergy Hl Hybrid Multi-Mode Microplate Reader (BioTek, 548 cat. no. H1MFDG). The rate of kinetic reaction of proteasome activity was calculated and data are represented in a fold change compared to wild-type cells.

[0069] Proliferation assay

[0070] Fibroblasts from a healthy donor were seeded at concentration of lxl04 / cm in 6-well plates in low glucose culture medium. After 24h cells from 3 wells were counted to obtain the initial cell number (Oh). Cells were washed IX PBS (without Ca2+and Mg2+) and incubated with trypsin (0.5 g / L) for 5 minutes. Next trypsin was blocked by an equal volume of culture medium and cells were counted in Countess II Automated Cell Counter. The culture medium on other cells was changed to galactose culture medium containing either DMSO (a vehicle) or ONX0914 (100 nM), KZR504 (10 nM) or bortezomib (20 nM). Every 24 h for 3 days cells from separate wells were counted as described above.

[0071] Inhibitors of immunoproteasome subunits

[0072] ONX-0914 - CAS Number: 960374-59-8, Cayman, Cat. No. 16271

[0073] KZR-504 - CAS Number: 1629052-78-3, MedChemExpress, Cat. No. HY-101786

[0074] KZR-616 - CAS Number: 1629677-75-3, MedChemExpress, Cat. No. HY-114419 Proteasome inhibitor

[0075] Bortezomib - CAS Number: 179324-69-7, Velcade®, Millennium Pharmaceuticals

[0076] EXAMPLE 1. Characterization of immunoproteasome subunit expression and activity in fibroblasts derived from patients carrying mutations in COA7 and COX6B

[0077] In order to verify expression of immunoproteasome subunits in cells carrying mutations in mitochondrial proteins fibroblasts derived from patients with mitochondriopathies were analyzed. Mt8987i cells carrying mutation in COX6B gene (R19H) (Massa et al., 2008) and control fibroblast from a healthy donor were seeded in culture medium containing low glucose. After 24h culture medium was exchanged to galactose containing medium for 48h. Cells were harvested and protein cellular extract was prepared. Protein levels were analyzed by SDS-PAGE and western blotting with specific antibodies. The results are shown in Figure 1. In a further experiment mt4221i fibroblasts carrying mutations in COA7 gene (Y137C / exon2A) (Martinez Lyons et al., 2016) and control fibroblasts from a healthy donor were seeded in culture medium containing low glucose. After 24h cell medium was exchanged for galactose containing medium and cell were allowed to grow for 12h. Cells were harvested and protein cellular extract was prepared. Protein levels were analyzed by SDS-PAGE and western blotting with specific antibodies. The results are shown in Figure 2.

[0078] Significantly higher levels of immunoproteasome subunits were detected in both patient fibroblasts. In COX6B-R19H carrying cells expression of immunoproteasome subunit PSMB9 was increased while in COA7-Y137C / exon2Acells PSMB9 and PSMB8 were increased in comparison to the fibroblasts from a healthy donor (Figs 1 and 2).

[0079] In a further experiment proteolytic activity of immunoproteasome subunit PSMB9 was assessed in patient fibroblasts by an in-solution assay. Cellular homogenates were prepared from both patient fibroblasts and healthy fibroblasts grown as described above. Homogenates were incubated with Anorogenic peptides responding to general chymotrypsin-like activity and chymotrypsin-like activity specific to PSMB9 (Fig. 3).

[0080] In comparison to healthy fibroblasts, COX6B-R19H patient fibroblasts displayed an increased chymotrypsin-like activity measured by the peptide specific to PSMB9 subunit, as well as by the peptide detecting general chymotrypsin-like proteolytic activity (Fig. 3). These results demonstrate that an increased expression of immunoproteasome subunits in cells with defective mitochondria is accompanied by increased proteolytic activity.

[0081] EXAMPLE 2. Effects of immunoproteasome subunit inhibitors on human fibroblasts carrying mutation in COX6B

[0082] In order to investigate the inAuence of immunoproteasome subunit inhibitors on patient fibroblasts several concentrations of ONX-0914, KZR-504 and KZR-616 were tested for the induction of protein ubiquitination. COX6B-R19H carrying cells were seeded and cultivated in low glucose culture medium for 24h with or without indicated immunoproteasome subunit inhibitors. Next cells were transferred to galactose culture medium with or without indicated immunoproteasome subunit inhibitors and cultivated another 24h. Protein cellular extract was prepared and analyzed by SDS-PAGE and western blotting with antibody against ubiquitin. The results are shown in Fig. 4

[0083] In comparison with cells treated with vehicle only the cells treated with immunoproteasome subunit inhibitors did not display an increased level of ubiquitination (Fig. 4). This result indicates that the selected concentrations of immunoproteasome subunit inhibitors do not affect global level of ubiquitination and thus are safer to use than inhibitors of proteasome.

[0084] In a further experiment an inhibition level of immunoproteasome subunits by selected inhibitors was tested. The results are presented in Figs 5 and 6. Cellular homogenates were prepared from COX6B-R19H fibroblasts grown 24h in low glucose and later next 24h in galactose medium in both conditions with the presence of DMSO (vehicle), KZR-504 (30 nM) or ONX-0914 (100 nM) or KZR-616 (50nM). Homogenates were incubated with Anorogenic peptides responding to global chymotrypsin-like activity and chymotrypsin-like activity specific to PSMB9. KZR-504 did not inAuence global chymotrypsin-like activity, while ONX-0914 and KZR-616 effectively decreased it by 70% and 60%, respectively (Fig. 5). On the other hand, KZR-504 and ONX-0914 were equally effective against chymotrypsin-like activity specific to PSMB9 and decreased it by 20% (Fig-6).

[0085] In order to explore the effects of immunoproteasome subunit inhibition on the level of mutant proteins, COX6B-R19H fibroblasts were cultivated with selected concentrations of ONX-0914 (30, 50 and 100 nM) or KZR-504 (5, 10 and 30 nM) or KZR-616 (10, 30 and 50 nM) as described above. After treatment protein cellular extract was prepared and analyzed with SDS-PAGE and western blotting. The results are shown in Fig. 7

[0086] Immunodecoration with antibody against COX6B protein revealed an increased presence of the mutant protein in fibroblasts treated with all three inhibitors ONX-0914, KZR-504 and KZR-616 (Fig. 7). Furthermore, immunoproteasome subunit inhibitors did not increase the level of HSP70 stress responsive protein, thus are safer to use than inhibitors of proteasome, like bortezomib (Fig. 7). EXAMPLE 3. Effects of immunoproteasome subunit inhibitors on cell proliferation

[0087] The effect of immunoproteasome subunit inhibitors and proteasome inhibitor on cell proliferation was tested. To this end fibroblasts from a healthy control were seeded in low glucose culture medium. After 24h cells were transferred to galactose culture medium containing 100 nM ONX-0914, 10 nM KZR-504 or 20 nM bortezomib. Cells were counted every 24 h for 3 days, starting from the addition of immunoproteasome subunit inhibitors. The results are shown in Fig. 8.

[0088] This experiment revealed that proliferation of fibroblasts was not affected by the indicated concentrations of ONX-0914 and KZR-504 in contrast to proteasome inhibitor bortezomib, which decreased fibroblast proliferation significantly. The result clearly demonstrates that inhibitors of immunoproteasome subunits exhibit lower toxicity than bortezomib.

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Claims

PATENT CLAIMS1. An immunoproteasome subunit inhibitor for use in the treatment or prevention of a mitochondrial disease in a patient, wherein the immunoproteasome subunit inhibitor is a selective immunoproteasome subunit inhibitor, which is administered in the amount not sufficient to inhibit a proteasome activity.

2. The immunoproteasome subunit inhibitor of proteolytic activity for use according to claim 1, wherein the mitochondrial disease results from a decreased level of mitochondrial proteins associated with mutations in nuclear-encoded genes causing defects in respiratory chain.

3. The immunoproteasome subunit inhibitor of proteolytic activity for use according to claim 1 or 2, wherein the mitochondrial disease is selected from a group consisting of mitochondrial myopathy, preferably progressive mitochondrial myopathy, cardiomyopathy, preferably dilated 1GG; mitochondrial myopathy with congenital cataract, hearing loss, and developmental delay, isolated autosomal dominant mitochondrial myopathy, mitochondrial myopathy with extrapyramidal signs, cardioencephalomyopathy, preferably fatal infantile cardioencephalomyopathy, frontotemporal dementia and / or amyotrophic lateral sclerosis 2, spinal muscular atrophy, preferably Jokela type spinal muscular atrophy, myopathy with extrapyramidal signs, mitochondrial complex I deficiency, mitochondrial complex IV deficiency, preferably mitochondrial complex IV deficiency nuclear type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22, Mohr-Tranebjaerg syndrome, reticular dysgenesis, Leigh syndrome associated to cytochrome c oxidase deficiency, mitochondrial hepatoencephalopathy, Cowden syndrome 3, somatic merkel cell carcinoma, mitochondrial complex II deficiency, pagangliomas 1, preferably with or without deafness; paragangliomas 5, sulfate oxidase deficiency, mitochondrial complex III deficiency, preferably mitochondrial complex III deficiency nuclear type 2, combined oxidative phosphorylation deficiency 22, mitochondrial complex V deficiency, preferably mitochondrial complex V deficiency nuclear type 1, 3 or 4; mitochondrial complex I deficiency, Leigh syndrome associated to mitochondrial complex I deficiency, Leighsyndrome associated to COX IV deficiency, and mitochondrial complex III deficiency, preferably mitochondrial complex III deficiency nuclear type 2, 3, 4, 5, 7, 8, or 9.

4. The immunoproteasome subunit inhibitor of proteolytic activity for use according to claim 3, wherein the mitochondrial disease is a disease characterized by decreased levels of complex IV caused by one or more mutations in genes selected from a group comprising SURF1, SCO2, COXIO, SCO1, LRPPRC, C0X15, C0X6B1, TACO1, C0A5, C0X14, COX20, PET100, C0A6, C0A3, C0X8A, COX4I1, APOPT1, COX6A2, PET117, C0X5A, COXFA4, C0X16, COA7, GFM1, MRPS16, TSFM, MRPS22, AIFM1, MTRFR, AARS2, MRPL3, MTFMT, ATP5F1A, MARS2, TRMT5, MRPS7, TRIT1, MRPS2, MIC0S13, GATC, MRPS23, MRPS28, PTCD3, PRORP, POLRMT, TTC19, UQCC2, GFER, CHCHD10, YARS2 and MICU1.

5. The immunoproteasome subunit inhibitor of proteolytic activity for use according to claim 4, wherein the mitochondrial disease is a disease characterized by decreased levels of complex IV caused by mutations in genes encoding for complex IV subunits or assembly factors caused by one or more mutations in genes selected from a group comprising SURF1, SCO2, COXIO, SCO1, LRPPRC, C0X15, C0X6B1, TACO1, C0A5, C0X14, COX20, PET100, C0A6, C0A3, C0X8A, C0X4I1, APOPT1, COX6A2, PET117, C0X5A, C0XFA4, C0X16 and COA7.

6. The immunoproteasome subunit inhibitor of proteolytic activity for use according to claim 5, wherein the mitochondrial disease is a disease caused by one or more mutations in gene C0X6B 1 and / or C0A7.

7. The immunoproteasome subunit inhibitor of proteolytic activity for use according to claim 6, wherein the mitochondrial disease is a disease caused by one or more mutations in gene C0X6B1.

8. The immunoproteasome subunit inhibitor of proteolytic activity for use according to any of claims 1 to 7, wherein the immunoproteasome subunit inhibitor is selected from a group comprising KZR504, ONX0914 and KZR-616 or a combination thereof.

9. The immunoproteasome subunit inhibitor of proteolytic activity for use according to any of claims 1 to 8, wherein one or more mutations in genes selected from a group comprising SURF1, SCO2, COXIO, SCO1, LRPPRC, C0X15, C0X6B1, TAC01, C0A5,C0X14, COX20, PET100, COA6, COA3, COX8A, COX4I1, APOPT1, COX6A2, PET117, COX5A, COXFA4, COX16, COA7, GFM1, MRPS16, TSFM, MRPS22, AIFM1, MTRFR, AARS2, MRPL3, MTFMT, ATP5F1A, MARS2, TRMT5, MRPS7, TRET1, MRPS2, MICOS13, GATC, MRPS23, MRPS28, PTCD3, PRORP, POLRMT, TTC19, UQCC2, GFER, CHCHD10, YARS2 and MICU 1 are detectable in a biological sample obtained from the patient.

10. The immunoproteasome subunit inhibitor of proteolytic activity for use according to 9, wherein one or more mutations are detectable in genes selected from a group comprising SURF1, SCO2, COXIO, SCO1, LRPPRC, COX15, COX6B1, TACO1, COA5, COX14, COX20, PET100, COA6, COA3, COX8A, COX4I1, APOPT1, COX6A2, PET117, COX5A, COXFA4, COX 16 and COA7.

11. A method for treating or preventing a mitochondrial disease in a patient by administrating one or more immunoproteasome subunit inhibitors, wherein the immunoproteasome subunit inhibitor is a selective immunoproteasome subunit inhibitor, which is administered in the amount not sufficient to inhibit a proteasome activity.

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