EP3652542A1

EP3652542A1 - Methods and means for diagnosing and/or treating a fatiguing illness

Info

Publication number: EP3652542A1
Application number: EP18737937.5A
Authority: EP
Inventors: Christiaan Hubert Simon Roelant; Kenny Leo De Meirleir
Original assignee: Protea Biopharma NV
Current assignee: Protea Biopharma NV
Priority date: 2017-07-14
Filing date: 2018-07-16
Publication date: 2020-05-20
Also published as: WO2019012159A1

Abstract

The invention relates to methods of typing a human comprising determining a level of activity of a 2-oxoglutarate-dependent dioxygenase, preferably an asparaginyl β-hydroxylase, in a cell of said human. Said typing preferably provides an indication that the human is suffering from a fatiguing illness. The invention relates to methods of treating a human that is typed as suffering from a fatiguing illness.

Description

Title: Methods and means for diagnosing and/or treating a fatiguing illness

1. FIELD: The invention relates to methods and means for diagnosing and/or treating a human suffering from a fatiguing illness. Said methods and means are directed to a newly discovered link between altered mitochondrial functioning and said fatiguing illness.

2. BACKGROUND OF THE INVENTION

Chronic Fatigue Syndrome (CFS), also termed Myalgic Encephalomyelitis

(ME), Systemic Exertion Intolerance Disease (SEID) or Chronic Fatigue Immune Deficiency Syndrome (CFIDS), is an unexplained syndrome only defined by consensus criteria formulated by a panel of clinical experts (Carruthers et al., 2011. J Intern Med 270: 327-338). These criteria are extremely diverse, comprising unresolved fatigue not related to any known medical condition and affecting considerably day to day activities due to discomfort after exercise, unrefreshing sleep, short term memory or concentration, pain in muscles, sore throat, joint pain without swelling and redness, headaches and soft lymph nodes, orthostatic intolerance, gastro-intestinal problems, extreme nociceptive abnormalities afflicting 0,7% of the northern hemispheric western population in a 3 to 1 female/male ratio (Holmes et al., 1988. Ann Intern Med 108: 387-389; Working Group of the Royal Australasian College of Physicians. Chronic fatigue syndrome. Clinical practice guidelines 2002. Med J Aust 76: S23-56). Many potential triggers and causes have been suggested and described making hard to believe CFS is in fact a "real disease" and therefore after many decades the condition still remains a subject of biased discussions (White et al., 2011. Lancet 377: 823-836; Ward (Ed), "Meanings of ME: Interpersonal and Social. Dimensions of Chronic Fatigue".

Springer, 2015). Although mitochondrial involvement in the etiology of CFS has been suggested and variations in ATP content have been observed with white blood cells, no disease mechanism has been delineated thus far (Campling and Sharpe (Eds), "Chronic Fatigue Syndrome (CFS/ME)". Oxford University press, 2nd edition, 2008; Lawson et al., 2016. J Nat Sci 2: 1-14). Several other fatiguing illnesses, including Gulf War Illness (GWI), have a significant overlap in symptoms with CFS, including constant fatigue, joint aches and muscle weakness.

Up to 95% of oxygen delivered to the body is used by mitochondrial respiration, a process providing up to 80%-90% of ATP to cells of mammalian tissues. Therefore, a link between fatiguing illnesses such as CFS and altered mitochondrial functioning would be attractive and it has been proposed that an aberrant "ATP profile" is indicative of a person suffering of CFS (Myhill et al., 2009. Int J Clin Exp Med 2: 1-16). However, published studies in general do not always take into account the fact that CFS might relate to altered mitochondrial functioning.

There is thus a need to establish a causative link between altered

mitochondrial functioning and fatiguing illnesses such as CFS, in order to provide methods and means of diagnosing and treating of a person suffering from such fatiguing illnesses.

3. SUMMARY OF THE INVENTION

The present invention has established a relation between hypoxia, NADH metabolism and mitochondrial activity that may be causative to fatiguing illnesses such as CFS. According to this model, a central role is provided by a 2-oxoglutarate (20G)-dependent dioxygenase, in particular "Factor Inhibiting Hypoxia inducible factor" (FIH) and its interaction with Ankyrin Repeat Domain-containing proteins.

Starting from observations made on urine redox capacity and

chemiluminometric analysis of NADH-induced reactive oxygen species generation by peripheral white blood cells it was postulated that hypoxia- mediated shifts of dioxygenase activities between indoleamine 2,3-dioxygenase and 2-oxoglutarate- dependent dioxygenases are key to the full understanding of the unexplained symptoms of Chronic Fatigue Syndrome. We propose that altered mitochondrial SOD2 activity and reduced hydrogen peroxide efflux result in increases of inhibitor of the hypoxia-inducible factor (FIH) -mediated interactions and hydroxylation of arginyl/asparaginyl positions in the Ankyrin Repeat Domains of substrates such as, MYPT1, RNase-L, IkBa, Notch, TRPV, Ankyrinl, Tankyrase cause the complex symptomatology of Chronic Fatigue Syndrome. The invention therefore provides a method of typing a human comprising determining a level of activity of a 2-oxoglutarate- dependent dioxygenase, preferably an asparaginyl β-hydroxylase such as FIH, in a cell of said human. Said level of activity may be determined directly or indirectly, as is known to a skilled person and is detailed herein below.

Said typing in a preferred method according to the invention provides an indication that the human is suffering from a fatiguing illness, preferably from chronic fatigue syndrome, fibromyalgia, multiple sclerosis, gulf war / veterans syndrome, amyotrophic lateral sclerosis, post-Lyme disease or related disorders.

A level of activity of 2-oxoglutarate-dependent dioxygenase, preferably an asparaginyl β-hydroxylase such as FIH, may be determined by determining a ratio of intracellular nicotinamide adenine dinucleotide NADH/NAD⁺, preferably by imaging NADH auto-fluorescence, preferably by fluorescence lifetime imaging.

A level of activity of 2-oxoglutarate-dependent dioxygenase, preferably an asparaginyl β-hydroxylase such as FIH, may be determined by determining a level of asparaginyl and/or histidinyl hydroxylation of at least one ankyrin repeat domain. Said at least one ankyrin repeat domain may be present in a protein that is exogenous provided to a relevant cell of a human that is typed with a method according to the invention, preferably an isolated cell. In a preferred method according to the invention, said ankyrin repeat domain is present in an endogenous protein, preferably in ankyrin.

A level of activity of 2-oxoglutarate-dependent dioxygenase, preferably an asparaginyl β-hydroxylase such as FIH, may be determined by determining a level of activity of plasma membrane associated NADH oxidase. A preferred method comprises isolating intact cells from the human; incubating the isolated cells with a chemiluminescent reagent and NADH; determining a level of chemiluminescence; comparing said determined level of chemiluminescence to a control level; and typing said human on the basis of the comparison.

The invention further provides a method of treating a human suffering from a fatiguing illness, comprising administering a therapeutically effective amount of an inhibitor of an asparaginyl β-hydroxylase to said human.

Said inhibitor preferably is a nucleic acid molecule, preferably a siRNA molecule or miRNA molecule, or a low molecular weight (< 1 kdalton) molecule. Said inhibitor preferably is or comprises a chromene derivative and/or a hydroxypyridone compound, or is or comprises a manganese superoxide dismutase, or a mimic thereof, preferably manganese(III) tetrakis(l-methyl-4- pyridyl)porphyrin, and/or a peroxide donor, preferably an alkali percarbonate such as sodium percarbonate and/or a peroxide such as magnesium peroxide or carbamide peroxide.

The invention further provides a pharmaceutical composition comprising an inhibitor of an asparaginyl β-hydroxylase, preferably of FIH, and a

pharmaceutically acceptable excipient. Said pharmaceutical composition preferably is for use in a method of treating a human suffering from a fatiguing illness.

The invention further provides a use of an inhibitor of an asparaginyl β- hydroxylase, preferably of FIH, in a method for the preparation of a medicament for the treatment of a human suffering from a fatiguing illness

4. FIGURE LEGENDS

Fig. 1: Overall scheme of non-heme iron and heme iron-dependent

dioxygenase activities linked by mitochondrial interaction in response to a diversity of triggers and consequences relevant to the onset of Chronic Fatigue Syndrome.

Fig. 2: Urine redox potential measured as tetrazolium-reduction after 10 minutes at pH 9.0. Range of O.D. values for controls and CFS samples are compared without and with ascorbate oxidase (100 U ).

Fig. 3: Ranges of chemiluminescence kinetics observed as a function of time with a group of diagnosed CFS patients (n= 163) versus controls (n=82) and maximal light output (E-max). Light units are expressed as counts per minute (CPM). PBMC's obtained from controls show a mean energetic demand of 13 + /- 3 % versus 40+/- 18% observed with PBMC's obtained from CFS patients and measured as NADH induced chemiluminescence. Max. E-demand is induced with ΙΟΟμΜ Verapamil (2-(3,4-dimethoxyphenyl)-5-[2-(3,4-dimethoxyphenyl)ethyl- methylamino]-2-propan-2-ylpentanenitrile), a first generation calcium channel blocker. Fig. 4: Extracellular addition of NADH to mammalian cells ( e.g. PBMC ) activates NADH-oxidase at the plasma membrane.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1 Definitions

The term "typing", as is used herein, refers to assessing presence or absence of a fatiguing illness, preferably to discriminating between a human that is likely to suffer from a fatiguing illness and a human that is not likely to suffer from a fatiguing illness. Said typing provides an additional or alternative treatment decision-making factor. The methods of the invention find particular use in choosing appropriate treatment for humans that are likely to suffer from a fatiguing illness.

The term "2-oxoglutarate-dependent dioxygenase", as is used herein, refers to a protein that catalyzes hydroxylation of proteins by incorporating a dioxygen molecule, using ferrous iron as cofactor and 2-oxoglutarate as a co-substrate which is decarboxylated to succinate and CO2. 2-Oxoglutarate-dependent dioxygenases are sensors of energy metabolism. A preferred 2-oxoglutarate-dependent

dioxygenase is an asparaginyl β-hydroxylase, most preferably Factor Inhibiting Hypoxia-inducible factor (FIH), also termed Hypoxia-inducible factor 1-alpha inhibitor (HIFAN) or Hypoxia-inducible factor asparagine hydroxylase.

The term "Hypoxia-Inducible transcription Factor (HIF)", as is used herein, refers to an αβ heterodimer that has an essential role in adaptation to low oxygen availability. HIF is regulated by two oxygen-dependent hydroxylation events.

Hydroxylation of specific proline residues by HIF prolyl 4-hydroxylases targets the HIF-a subunit for proteasomal degradation. Hydroxylation of an asparagine in the C-terminal transactivation domain prevents its interaction with the transcriptional coactivators CBP/p300, thereby inhibiting transcriptional activation of downstream genes by HIF.

The term "Factor Inhibiting Hypoxia-inducible factor (FIH)", as is used herein, refers to an Fe(II)/2-oxoglutarate-dependent dioxygenase that acts as a negative regulator of the hypoxia-inducible factor (HIF) by catalysing 6- hydroxylation of an asparaginyl residue in the C-terminal transcriptional activation domain of HIF. FIH also catalyzes asparaginyl hydroxylation of ankyrin repeat domain-containing proteins, including Nuclear Factor κΒΙ (NFKB1), NFKBIA, NOTCH 1, NOTCH2, NOTCH3, Tankyrase 2 (TNKS2) and Protein Phosphatase 1 Regulatory Subunit 12A, and hydroxylates histidinyl residues within the ankyrin repeat domain of, for example, Tankyrase-2 (Yang et al., 2011. FEBS J 278 1086-97).

The term "fatiguing illnesses", as is used herein, refers to diseases or disorders that are characterized by their complex, multi-organ chronic signs and symptoms, including muscle pain, chronic fatigue, headaches, memory loss, nausea, gastrointestinal problems, joint pain, lymph node pain, which last for at least 6 months. Examples of such diseases or disorders are chronic fatigue syndrome, fibromyalgia, multiple sclerosis, Parkinson's disease, gulf war / veterans syndrome, amyotrophic lateral sclerosis, cancer, post-Lyme disease and related disorders such as sick building syndrome and mercury/amalgam disease.

The term "inhibitor of an asparaginyl β-hydroxylase" as used herein, refers to a therapeutic compound of any type, including small molecule-, peptide-, protein, antibody-, antisense-, small interfering RNA-, or microRNA-based compound, that inhibits, blocks or counteracts hydroxylation of an asparaginyl residue in at least one ankyrin-repeat domain-containing protein by an asparaginyl β-hydroxylase. Preferably, such an inhibitor is an inhibitor of Factor Inhibiting Hypoxia-inducible factor (FIH).

The term "ankyrin repeat domain", as is used herein, refers to a domain of about 33 amino acid residues in proteins that mediates protein— protein

interactions. An ankyrin repeat domain is defined by its structure rather than its function. A consensus ankyrin repeat domain comprises the amino acid sequence:

N-terminus -L23-X24-X25-X26-X27-X28-D/E29-V30-N31-, in which the numbering indicates the position of the amino acid residue within the 33 amino acid domain. A preferred consensus sequence is:

D1X2X3G4X5T6P7L8H9L10 A11A12X13X14G15H16L17E18I19V20 E21V22L23L24K25

Z26G27A28D29V30N31A32X33

(Kohl et al., 2003. PNAS 100: 1700-1705).

As used herein, the term "treatment" or "treating" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed during the course of clinical pathology. Desirable effects of the treatment include preventing occurrence or recurrence of the illness, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the illness, and/or amelioration or palliation of the state of the illness. In the present invention, "treatment" or "treating" is preferably understood to mean improving at least the physical capacity of a human suffering from a fatiguing illness by administering a pharmaceutical composition comprising an inhibitor of an asparaginyl β-hydroxylase.

The term "therapeutically effective amount" refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Ideally, a therapeutically effective amount of an agent is an amount sufficient to inhibit or treat the disease or condition without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic agent. Preferably, the term refers to (i) an amount sufficient to inhibit, block or counteract hydroxylating activity of an asparaginyl β-hydroxylase, preferably of Factor Inhibiting Hypoxia-inducible factor (FIH), and/or (ii) an amount sufficient to treat a human suffering from a fatiguing illness. It is within the knowledge and capabilities of the skilled practitioner to determine therapeutically effective dosage regimens.

The term "administering", as used herein, refers to the physical introduction of an agent or therapeutic compound that inhibits, blocks or counteracts

hydroxylating activity of an asparaginyl β-hydroxylase, preferably of Factor Inhibiting Hypoxia-inducible factor (FIH) to a human suffering from a fatiguing illness, using any of the various methods and delivery systems known to those skilled in the art. The skilled person is aware of suitable methods for

administration and dosage forms. Administration of small molecules described herein can be performed by non-parenteral administration such as by oral and enteral administration. Preferred routes of administration for protein-based agents such as antibodies is by parenteral administration, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, executed inter alia by injection or infusion in the form of a solution. Administering can be performed, for example, once, a plurality of times, and/or over one or more extended periods of time. 5.2 Methods of typing

Key to the present invention is that mitochondrial disfunctioning results in activation of "hypoxia inducing factor inhibitor" (FIH), leading to aberrant hydroxylation of ankyrin repeat domains (ARD) in intracellular structural and metabolic proteins. This aberrant hydroxylation is an important factor in fatiguing illnesses such as chronic fatigue syndrome, fibromyalgia, multiple sclerosis, gulf war / veterans syndrome, amyotrophic lateral sclerosis, post-Lyme disease and related disorders.

Without being bound by theory, said mitochondrial disfunctioning might be triggered by diverse factors such as, e.g. hypoxia, bacterial-, viral- or mycoplasma infections, or interactions with metals, which cause changes in intracellular calcium-fluxes, thereby influencing mitochondrial functioning. Continued triggering might result, in some individuals, to mitochondrial disfunctioning, resulting in decreased efflux of hydrogen peroxide from the mitochondria and activation of FIH. The efflux of hydrogen peroxide that is produced by superoxide dismutase 2 (SOD2)-catalyzed dismutation of O^2" is proportional to the

NADH/NAD+ ratio in the mitochondrial matrix.

Dismutation of superoxide by intramitochondrial SOD2 normally leads to sufficiently high concentrations of hydrogen peroxide (Afanas'ev, 2001. Circulation Res 89: e46). However, PBMC obtained from CFS patients presumably show a lower efflux of hydrogen peroxide, as compared to controls (Marcu et al., 2014. Mol Cell Biol 34: 2890-2902). Less hydrogen peroxide efflux implies suboptimal SOD2 activity and/or aquaporin mediated hydrogen peroxide transport creating a higher risk of mitochondrial damage. In addition, FIH is highly sensitive to an inhibitory action of hydrogen peroxide. When hydrogen peroxide efflux drops, the activity of FIH increases (Tarhonskaya et al., 2015. J Biol Chem 290: 19726-19742; Murphy, 2009. Biochem J 417: 1-13).

FIH is a dioxygenase, which is a group of enzymes requiring oxygen as a substrate. As such, they are oxygen sensors and play a key role in health and disease. 2-Oxoglutarate (20G)-dependent dioxygenases (20G) such as FIH have been found to play a central role in processes by which cells sense and respond to hypoxia by hydroxylation of hypoxia inducible factor HIFl-oc (Franke et al., 2013. Blood 122: 1122-1128; Tian et al., 2011. J Biol Chem 286: 13041-13051). FIH controls hypoxia inducible factor (HIFl-oc) association with the key activators p300 and CREB Binding Protein (CBP) and regulates the transcriptional activity of HIF1. FIH targets a conserved asparaginyl residue in the C-terminal

transactivation domain of HIFl-oc, which suppresses its transcriptional activity (Tarhonskaya et al., 2015. J Biol Chem 290: 19726-197).

Ankyrin repeat Domains (ARD) are common interaction motifs of proteins that have been shown to be efficiently hydroxylated by FIH. An ankyrin repeat is a structural motif in proteins forming an elongated stacked structure build of 4-6 copies with a continuous hydrophobic core and a large solvent-accessible surface (Wilkins et al., 2016. Chem Med Chem 11: 773-786). It is a protein-protein interaction unit involved in a diverse set of cellular activities such as cell-cycle regulation, cytoskeletal integrity, ion transport through ion channels, signal transduction and other. The first reported ARD substrates for FIH were pl05 and IkBa, components of the NF-kB signaling system which palsy a key role in immune responses. Because hydroxylated ARDs bind less tight to FIH, when compared to unhydroxylated ARDs, ARD hydroxylation has the potential to enable a "memory" effect of hypoxic events (Masson et al., 2012. EMBO Rep 13: 251-257). It has been demonstrated that FIH may also hydroxylate histidinyl and aspartyl residues making FIH a highly promiscuous oxygenase, a property it shares with other 2 OG- oxygenases acting on proteins including JmjC histone demethylases (Markolovic et al., 2015. J Biol Chem 290: 20712-20722). The interaction of all these factors are summarized in Fig. 1.

Modulation of FIH activity, especially activation of FIH activity, is thus a key factor in fatiguing illnesses such as chronic fatigue syndrome. A method of typing a human according to the invention comprises determining a level of activity of a 2- oxoglutarate-dependent dioxygenase, preferably an asparaginyl β-hydroxylase such as FIH, in a cell of said human.

A level of activity of a 2-oxoglutarate-dependent dioxygenase, as is used herein, preferably a level of activity of an asparaginyl β-hydroxylase such as FIH, may be determined directly, by determining a catalytic activity of 2-oxoglutarate- dependent dioxygenase and/or indirectly by determining a ratio of intracellular nicotinamide adenine dinucleotide NADH/NAD+, by determining a level of asparaginyl and/or histidinyl hydroxylation of at least one ankyrin repeat domain, and or by determining a level of activity of plasma membrane associated NADH oxidase.

Methods for determining directly a level of activity of a 2-oxoglutarate- dependent dioxygenase, as is used herein, preferably a level of activity of an asparaginyl β-hydroxylase such as FIH, are known in the art. For example, Kim and Yang, 2015 (Kim and Yang, 2015. Molecules 20: 20551-20568) describe several assays, including radioactive isotope-based assays, fluorescence-based assays and mass spectrometry-based assays that can be used for directly measuring activity of a 2-oxoglutarate-dependent dioxygenase, as is used herein, preferably a level of activity of an asparaginyl β-hydroxylase such as FIH.

A level of activity of 2-oxoglutarate-dependent dioxygenase, preferably of preferably an asparaginyl β-hydroxylase such as FIH, may be indirectly

determined by determining a ratio of intracellular nicotinamide adenine

dinucleotide NADH/NAD+.

A ratio of intracellular nicotinamide adenine dinucleotide NADH/NAD+ may be determined by imaging NADH auto-fluorescence. It is known to a person skilled in the art that live tissues illuminated with ultraviolet light emit blue fluorescence, arising primarily from mitochondrial NADH. The nicotinamide moiety of NADH absorbs light of wavelength 340±30 nm and emits fluorescence at 460±50 nm

(Chance et al., 1979. J Biol Chem 254, 4764-4771). An increase in precision may be obtained by applying low temperature measurements of NAD(P)H fluorescence (Chance et al., 1979. J Biol Chem 254, 4764-4771). Changes in autofluorescence intensity may reflect changes in either [NADH] or [NADPH], often denoted as NAD(P)H to indicate the uncertain origin of the signal. However, NADH

fluorescence predominates over that of NADPH ( Avi-Dor et al., 1962. J Biol Chem 237: 2377-2383), rendering this method suitable for determining a ratio of intracellular nicotinamide adenine dinucleotide NADH/NAD+.

A ratio of intracellular nicotinamide adenine dinucleotide NADH/NAD+ preferably is determined by fluorescence lifetime imaging, allowing to differentiate between NADH and NADPH at the level of a single cell (Blacker et al., 2014.

Nature Communications 5,: 3936 (2014). With excitation at about 700 nm and a 435-485 nm detection window, the intracellular fluorescence lifetimes of NADH and NADPH were found to vary to be 1.5±0.2 and 4.4±0.2 ns respectively.

A level of activity of 2-oxoglutarate-dependent dioxygenase, preferably of an asparaginyl β-hydroxylase such as FIH, may further be determined by determining a level of asparaginyl and/or histidinyl hydroxylation of at least one ankyrin repeat domain. Any method known in the art to determine a level of asparaginyl and/or histidinyl hydroxylation of at least one ankyrin repeat domain may be used. For example, a hydroxylation status of an ankyrin repeat domain (ARD) may be analyzed using, for example, mass spectrometry (MS) or, preferably, tandem MS (MS/MS). A preferred method for determining a hydroxylation status of an ARD comprises high performance liquid chromatography (HPLC), preferably coupled to tandem mass spectrometry (LC-MS/MS). The LC-MS/MS analysis may be performed, for example by using a HPLC chromatographic system coupled to a triple- quadrupole mass-spectrometer.

Said ankyrin repeat domain may be exogenously present in a cell of a human.

For example, a peptide or protein comprising said ankyrin repeat domain may be introduced into said cell. For example, said ARD may be introduced by infection or transfection of a vector encoding the peptide or protein comprising said ankyrin repeat domain. Alternatively, said peptide or protein comprising said ankyrin repeat domain may be introduced, for example by providing said peptide of protein with a cell-penetrating domain. A cell-penetrating domain, as is used herein, refers to peptides that facilitate cellular intake/uptake of various molecules. Examples of suitable cell-penetrating domains are penetratin or Antenapedia (N- terminus RQIKWFQNRRMKWKK), TAT (N-terminus YGRKKRRQRRR), SynBl (N- terminus RGGRLSYSRRRFSTSTGR), SynB3 (N-terminus RRLSYSRRRF), PTD-4 (N-terminus PIRRRKKLRRLK), and PTD-5 (N-terminus RRQRRTSKLMKR).

A level of asparaginyl and/or histidinyl hydroxylation of at least one ankyrin repeat domain may further be determined by analysis of an ankyrin repeat domain that is present in an endogenous protein, preferably in ankyrin. Suitable downstream targets of 2-oxoglutarate-dependent dioxygenase, preferably of an asparaginyl β-hydroxylase such as FIH, include the myosin binding subunit of Myosin Phosphatase D, termed MYPT1, RNase L, IkB-a, Notch, TRPV, Ankyrin, Ankyrin G and Tankyrase 2, all of which can be implicated in clinical symptoms as being described in humans suffering from a fatiguing illness such as chronic fatigue syndrome.

MYPT1 comprises an ARD that is a target of FIH hydroxylation (Webb et al., 2009. Biochem J 420: 327-333). Altered hydroxylation of MYPT1 may compromise relaxation of smooth muscles. Smooth muscles are involved in blood circulation in particular "arterioles". Altered contraction and relaxation of smooth muscles surrounding arterioles may result in "painful" ischemic conditions (Selvaraju et al., 2014. Antioxid Redox Signal 20: 2631-2665), as frequently experienced by CFS patients and a majority of women suffering from fibromyalgia. In particular, women are more sensitive because of the fact that muscle contraction is under control of oestradiol. Another impact of altered smooth muscle activity relates to altered gut motility and defective valve function (valve of Bauhin, separating aerobic intestine from the anaerobic colon ) in the gut resulting in alterations of microbiome composition and equilibrium. Heart function may be affected by altered Ankyrin repeat domain hydroxylation of smooth muscle types involved in pumping blood "into the heart" (diastolic). It is known but remains unexplained that CFS patients have a lower throughput of blood in their heart, described as CFS associated cardiomyopathy, displaying a pattern of blood flow through the heart called "pseudonormalization" which is indicative of an intermediate case of diastolic dysfunction. This would indicate that the left ventricle has stiffened sufficiently to prevent it from filling during the second (a) phase of the diastolic pulse which is also merely a problem observed with female patients.

RNase L is a mediator of type 1 interferon-induced anti-viral activity playing diverse and critical cellular roles including the regulation of cell proliferation, differentiation, senescence, apoptosis, tumorigenesis and the control of the immune response (De Meirleir et al., 2000. Am J Med 108: 99-105). RNase L monomers binding to 2'-5' linked oligoadenylates (2-5A) induces dimerization and activation of RNase L. 2-5A binding occurs in the ARD domains 2-4 that are located in the N- terminal portion of RNase L. RNase L is inhibited by the RNase L inhibitor (Englebienne and De Meirleir (EDs). 2002. "Chronic Fatigue Syndrome: A

Biological Approach". CRC Press, ISBN:0849 31046 6). RNase L activity may become activated because excess hydroxylation by FIH of its ARD prevents RNase L to dimerize optimally because of steric hindrance and therefore may escape full control of the RNase L Inhibitor (RLI). Uncontrolled RNase L activity may result in mRNA destruction and cell apoptosis (Nys and De Meirleir, 2005. In Vivo 19: 1013- 1021); Brennan-Laun et al., 2014. J Interferon & Cytokine Res 34: 275-288).

IkB-a, an inhibitor of Nuclear Factor- κΒ (NF-kB), also contains ARDs that are hydroxylated by FIH. Hydroxylated ARDs render IkB-a unstable and prone to degradation, resulting in translocation of NF-kB to the nucleus where it associates with various activators and initiates the transcription of many genes involved in the inflammatory response (Brennan-Laun et al., 2014. J Interferon & Cytokine Res 34: 275-288). Since the NF-kB pathway is known to be important in the inflammatory response, modulation of the activity of the Ankyrin proteins in the pathway by inhibition of 2 OG oxygenases that modify Ankyrin proteins, so that their stability is altered, is useful for treatments of diseases associated with the inflammatory response. Desirable medicinal effects include the regulation of inflammation and immunity such as is achieved by reducing or increasing the interaction between NF-kB proteins such as pl05 and IkB-a and the p50/p65 transcripitional process ( D'Ignazio and Rocha, 2016. Cells 5: 10; Wilkins et al., 2016. ChemMedChem 11: 773-786).

Notch is another substrate for FIH hydroxylation of its ARD (Coleman et al., 2007. J Biol Chem 282: 24027-24038; Myllymaki et al., 2017. Mol Cell Biol 37: e00529-16; Zheng et al.. 2008. Proc Natl Acad Sci USA 105: 3368-3373). Altered

Notch activity has serious consequences for cell signaling, cytoskeleton movement, control through tyrosine kinase, apoptosis, actin control, bone generation and osteoporosis, gut ligands, pancreatic differentiation, cardiac valve homeostasis and neuronal functions, of which deregulations are frequently observed in fatiguing illnesses such as CFS (Englebienne and De Meirleir (EDs), 2002. "Chronic Fatigue Syndrome: A Biological Approach. CRC Press, ISBN:0849 31046 6).

Ionic channel activity, in particular Vanilloid (TRPV) activity, may be altered in CFS through altered hydroxylation of ARD in the channel. This alteration may explain nociceptive defects in CFS. TRPV4, a calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation and bone homeostasis. Recent studies reported that mutations in TRPV4, including mutation in some in its ankyrin repeat domain, are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. Ion channelopathy is a presumed dysfunction in CFS (Englebienne and De Meirleir (EDs), 2002. "Chronic Fatigue Syndrome: A Biological Approach. CRC Press, ISBN:0849 31046 6).

Altered hydroxylation of ARD may result in impaired interaction of ankyrin 1 protein with spectrin in reticulocytes before mitochondrial extrusion, creating symptoms of hereditary spherocytosis (Zhang et al., 2009. Blood 114: 157-164; White et al., 1990. Proc Natl Acad USA 87: 3117-3121). This may explain the altered deformability of red blood cells and abnormal sedimentation rates observed with CFS.

Altered hydroxylation of Ankyrin G (AnkG) also plays an important role in the course of a fatiguing illness such as CFS. AnkG regulates neuronal plasticity, maintenance of axons, possibly through interaction with NF-kB (Konig et al., 2017. Sci Rep 7: 42006; doi: 10.1038/srep42006), and interacts with membrane-bound Na+ ion channel Epithelial Sodium Channel (ENaC) (Christine A. Klemens, 2017. PhD thesis. University of Pittsburgh) . The interaction with ENaC is likely to modulate numerous hormones, including aldosterone. Altered AnkG hydroxylation may explain a chronic failure of the aldosterone-renin- angiotensin system ( the so called aldosterone-renin paradox in CFS/ME), altered Na+/K+ fluxes and Mg2+ reuptake leading to disturbed electrolyte balances and fatigue, as is observed in patients suffering from a fatiguing illness such as CFS and in patients with Adison's disease. A poly-ADP-ribosyltransferase, termed tankyrase-2, uses NAD+ as a cosubstrate to link ADP-ribose polymers to target proteins, resulting in a post- translational modification referred to as PARsylation (Hsiao and Smith, 2008. Biochimie 90: 83-92). Tankyrase-2 is also known to be involved in vesicle trafficking and in telomere length regulation (Hsiao and Smith, 2008. Biochimie 90: 83- 92). Multiple FIH-dependent Asn hydroxylation sites have been identified in tankyrase-2 ARD (Cockman et al., 2009. Mol Cell Proteomics 8:535-546) and two histidinyl residues in the tankyrase-2 ARD are also substrates for FIH- catalysed 6-hydroxylation (Yang et al., 2011. FEBS J 278: 1086-1097).

Interestingly, hypoxia has been implicated in telomere regulation and

hydroxylation of Tankyrase 2 could contribute to this phenomenon. Very recently, telomere shortening has been observed in persons suffering from a fatiguing illness such as chronic fatigue syndrome (Unger et al., 2016. FASEB J 30: Supplement Ib459).

A level of activity of 2-oxoglutarate-dependent dioxygenase, preferably of an asparaginyl β-hydroxylase such as FIH, may further be determined by determining a level of activity of plasma membrane associated NADH oxidase.

The activity of a NADH oxidase at the cell surface is regulated by a coenzyme Q/QH2 oxidoreductase-mediated plasma membrane electron transport accepting electrons from cytosolic [NADH/NAD+]_cytosoi controlled by the aspartate/malate shuttle-mediated electron flux which itself is proportional to the mitochondrial matrix [NAD H/NAD +] mito .

A method of typing a human comprising determining a level of activity of a 2- oxoglutarate-dependent dioxygenase, preferably an asparaginyl β-hydroxylase, in a cell of said human by determining an activity of a plasma membrane associated NADH oxidase is preferably performed by a method comprising isolating intact cells from the human; incubating the isolated cells with a chemiluminescent reagent and NADH; determining a level of chemiluminescence; comparing said determined level of chemiluminescence to a control level; and typing said human on the basis of the comparison. Cationic chemiluminescent reagent, including but not limited to chemiluminogenic probe (CLP²⁺) will bind to the negatively charged cell surface and becomes univalently reduced. The resulting CLP⁺ reacts with oxygen to produce superoxide anion radicals that may react back with CLP+ to produce excited methylacrida (MA) and long lived luminescence (hv). Free unbound CLP remains in the CLP2+ state and does not participate in the reaction process further. Further preferred chemiluminescent reagents are imidazopyrazinone derivatives, lophine derivatives and acridinium esters such as phenoxy-substituted acridinium ester (di-ortho-bromophenyl- acridinium ester), oxalate esters, as is known to a skilled person.

Said cells preferably are isolated from a liquid sample, such as blood, urine, stool, or bodily fluids. Preferably, said cells are or comprise mononuclear peripheral blood cells. Methods to isolate mononuclear peripheral blood cells from blood are known to a skilled person, and include ficoll isopaque density gradient

centrifugation (GE Healthcare Bio-Sciences AB). As is known to a person skilled in the art, typing of a sample can be performed in various ways. In one method, a coefficient or stimulation index is determined that is a measure of a similarity or dissimilarity of a sample with a previously established reference level that is specific to a certain cell type, tissue, disease state or any other interesting biological or clinical interesting sample group. Typing of a sample can be based on its (dis) similarity to a reference, or based on multiple references such as, for example, a reference from at least one person suffering from a fatiguing illness such as chronic fatigue syndrome, and a reference from at least one person not suffering from a fatiguing illness. According to the invention, the references are representative of samples from humans that are known to suffer from a fatiguing illness, and/or that are known not to suffer from a fatiguing illness. Said reference preferable refers to the average level obtained in a mixture of samples from humans that are known to suffer from a fatiguing illness, and/or that are known not to suffer from a fatiguing illness. A threshold may be provided that demarcates a sample of a person suffering from a fatiguing illness such as chronic fatigue syndrome, and a sample a person not suffering from a fatiguing illness. Said threshold may be a value of a level of activity of a 2-oxoglutarate-dependent dioxygenase, as is used herein, preferably a level of activity of an asparaginyl β-hydroxylase such as FIH, or a value of a coefficient or stimulation index, as is known to a person skilled in the art.

5.3 Methods of treatment

The invention further provides a method of treating a human suffering from a fatiguing illness, comprising administering a therapeutically effective amount of an inhibitor of an asparaginyl β-hydroxylase, preferably of FIH, to said human. Said human preferably is typed as suffering from a fatiguing illness by a method of the invention.

Said inhibitor preferably is a nucleic acid molecule, preferably a siRNA molecule or miRNA molecule, or a low molecular weight (< 1 kdalton) molecule.

A preferred inhibitor of an asparaginyl β-hydroxylase, preferably of FIH, is by employing RNA interference (RNAi) by administering one or more small interfering RNA (siRNA) molecules. RNAi is based on the generation of short, double-stranded RNA (dsRNA) which activates a normal cellular process resulting in highly specific RNA degradation (Zamore et al., 2000. Cell 101: 25-33) and/or suppression of translation. Recent studies have demonstrated that RNA

interference is mediated by the generation of 18-to 23-nucleotide dsRNA molecules with 2 nucleotide-long 3' overhangs termed small interfering RNA (siRNA) duplexes. RNAi allows silencing of a gene on the basis of its sequence. For expression of dsRNA molecules in a cell or cell line, a heterologous expression cassettes encoding the two strands of the dsRNA duplex molecule may be constructed. Said heterologous expression cassettes preferably comprise a polymerase III enhancer/promoter. A preferred polymerase III enhancer/promoter is selected from the U6 and HI promoter. A polymerase III enhancer/promoter preferably drives expression of small interfering RNA (siRNA) strands that, upon duplex formation by base-pairing, comprise 18-23 (typically 19) nucleotide-long double-stranded siRNA molecules with 2 nucleotide-long 3' overhangs with one of the strands exhibiting extensive homology to a part of a mRNA transcript from a gene encoding an asparaginyl β-hydroxylase, preferably FIH. Said siRNA activates the RNA interference (RNAi) pathway and interferes with the expression of said gene.

A further preferred heterologous expression cassette preferably comprises a polymerase II or polymerase III promoter that drives expression of a short hairpin RNA (shRNA) or a miRNA. A short hairpin RNA (shRNA) comprises a 50-100 nucleotide long RNA molecule comprising two stretches of 20-25 nucleotides that are complementary and can base-pair, whereby the two stretches are

interconnected through a hairpin turn. The shRNA hairpin structure is cleaved by the cellular machinery into double stranded siRNA, which silences gene expression via RNA interference. miRNA molecules are transcribed by polymerase II as pri- miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. These pre-miRNAs are then processed to mature double stranded miRNAs of about 18-23 nucleotides in the cytoplasm which silence gene expression via RNA interference, primarily by suppressing translation.

Said heterologous expression cassette is provided to a relevant cell with the aid of a vector comprising said expression cassette. Said vector preferably is a viral vector. A variety of viral vector systems may be utilized to express a nucleic acid molecule in cells according to the methods of the invention. Suitable vectors include vectors based on gammaretrovirus, lentivirus, adenovirus (AdV), adeno-associated virus (AAV) and herpes simplex virus (HSV). Methods for targeting cells are known in the art, including pseudotyping of retroviral vectors with a viral glycoprotein that binds to a specific membrane receptor of a specific cell type. Furthermore, a viral glycoprotein can be fused with a ligand protein or antibody that recognizes cell type-specific surface molecules, providing a versatile way of cell type-specific gene delivery (Vannucci et al., 2013. New Microbiol 36, 1-22).

A low molecular weight (< 1 kdalton) molecule for use in methods of the invention preferably is a low molecular weight molecule of 500 Dalton or less. Said molecule preferably shows good absorption and permeation in biological systems and is consequently more likely to be a successful drug candidate than a molecule with a molecular weight above 1 kdalton or even above 500 Dalton (Lipinski et al., 1997. Advanced Drug Delivery Reviews 23: 3-25). Synthetic compound libraries (e.g. LOP AC™, Sigma Aldrich) or natural compound libraries (Specs, TimTec) may be screened to identify said molecules.

A preferred inhibitor is or comprises a chromene derivative and/or a hydroxypyridone compound. Suitable chromene derivatives have been described in US20100331400, which is hereby incorporated by reference. Said chromene derivatives inhibit hydroxylation by FIH, and are thereby suitable for use in the methods of the present invention. Suitable hydroxypyridone compounds have been described in US Appl. No.2 08/991,913, which is hereby incorporated by reference.

A further preferred inhibitor is or comprises a manganese superoxide dismutase, or a mimic thereof, preferably manganese(III) tetrakis(l-methyl-4- pyridyl)porphyrin, and/or a peroxide donor, preferably an alkali percarbonate such as sodium percarbonate and/or a peroxide such as magnesium peroxide or carbamide peroxide.

Manganese(III) tetrakis(l-methyl-4-pyridyl)porphyrin is a manganese- porphyrin which acts as a superoxide dismutase (SOD) mimetic with increased stability to pH and hydrogen peroxide. The rate constant for superoxide

dismutation is 3.9 x 107 M-ls-1.

The invention further provides a method of treating a human suffering from a fatiguing illness, comprising administering a therapeutically effective amount of a compound that reduces or increases interaction between, for example, pl05 and IkB-a and the p50/p65 transcriptional complex (Rius et al., 2008. Nature 453: 807- 11; Cockman et al., 2006. Proc Natl Acad Sci USA 103: 14767-14772) to said human. Since the NF-kB pathway is known to be important in the inflammatory response, desirable medicinal effects can be obtained by administering a

dithiocarbamate as described in Schreck et al., 1992. J Exp Med 175: 1181-1194), a monoterpene or triterpene composition as described in US20060148732, or a thiophene derivative as described in US20040192943, which citations are hereby incorporated by reference.

The invention further provides a method of treating a human suffering from a fatiguing illness, comprising administering a therapeutically effective amount of a compound that stimulates hydrogen peroxide emission of mitochondria by interference with oxidation of free fatty acids, preferably of palmitoyl-carnitine and mildronate. Examples of compounds that stimulate hydrogen peroxide emission of mitochondria include carnitine, malate, palmitoyl carnitine, and combinations thereof such as malate and carnitine, palmitoyl carnitine and carnitine, and malate and palmitoyl carnitine.

The invention further provides a method of treating a human suffering from a fatiguing illness, comprising administering a therapeutically effective amount of a compound that regulates peroxisomal catalase activity. Examples of compounds that regulate peroxisomal catalase activity include flavonoids such as genistein, luteolin, kaempferol, and quercetin, and a phytoestrogen such as bioclanin A which also acts as a peroxisome proliferator- activated receptor gamma agonist.

pharmaceutically acceptable excipient.

Said inhibitor of an asparaginyl β-hydroxylase, preferably of FIH, preferably is selected from baicalein (5,6,7-trihydroxyflavone), 3,4- dihydroxybenzoate, N-oxalylglycine, oxaloacetate, citrate, and combinations thereof.

Said pharmaceutically acceptable excipient preferably is selected from diluents, binders or granulating ingredients, a carbohydrate such as starch, a starch derivative such as starch acetate and/or maltodextrin, a polyol such as xylitol, sorbitol and/or mannitol, lactose such as a-lactose monohydrate, anhydrous a-lactose, anhydrous 6-lactose, spray-dried lactose, and/or agglomerated lactose, sugars such as dextrose, maltose, dextrate and/or inulin, glidants (flow aids) and lubricants, and combinations thereof.

A pharmaceutical composition according to the invention preferably is for use in a method of treating a human suffering from a fatiguing illness.

The invention further provides a use of an inhibitor of an asparaginyl β- hydroxylase, preferably of FIH, as described herein above in a method for the preparation of a medicament for the treatment of a human suffering from a fatiguing illness.

The content of publications mentioned herein are incorporated herein by reference.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be

appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

6. Examples

Example 1

Materials and Methods

All materials used were of analytical grade. MTT (3-(4,5'-dimethylthiazol-2- yl)-2,5-dipehenyl tetrazolium), Lucigenin (9,9'-bis(N-methylacridinium) di-nitrate), NADH ( beta- nicotinamide adenine dinucleotide, reduced form), ascorbic acid, ascorbate oxidase (from Cucurbita sp.) and Verapamil ((RS)-2-(3,4- dimethoxyphenyl) - 5- [2- (3, 4- dimethoxyphenyl) ethyl- methyl- amino] - 2- ( 1 - methylethyl)pentanenitrile) were obtained from Sigma-Aldrich. PBS/Tris buffer, pH 9.00 was prepared by mixing of equal volumes of PBS ( phosphate buffered saline solution ) and 100 mM Tris buffer, pH 9. PBMC isolation tubes (50 ml) were purchased from Greiner Bio One. O.D. readings were performed using a BioRad (i- Mark) plate reader and chemiluminescence was observed using a Beckman LSC6500 scintillation counter operated in in-coincidence Tritium window setting.

Patients

Patients: 163 patients from Himmunitas Clinic gave informed consent to participate in the study. They all fulfilled the ICC criteria for ME/CFS (Carruthers et al., 2011. J. Intern Med 270: 327-338).

Controls: 82 healthy people referred to the laboratory for general check-up were tested. They had a normal white blood cell count, no inflammation, CRPC<1 mg/1, no clinical history of immune disease nor diabetes mellitus. Overnight fasting first morning urine samples and venous blood for isolation of PBMC were used in our study.

Urine redox potential

Briefly, to 700 μΐ of first morning urine samples, 100 μΐ of a methyl- tetrazolium salt (MTT) dissolved at 3 mg/ml in 100 mM Tris Buffer, pH 9 was added. 200 μΐ samples were taken, transferred to a 96-well microplate and color produced after 3 minutes was read at 495 nm and compared to samples containing 100 U/ml ascorbate oxidase (ascorbate oxidase catalyzes the reaction of ascorbate to dehydro-ascorbate and water).

Luminometric observation of peripheral blood mononuclear cells (PBMC) PBMC were isolated from heparinized whole blood (drawn one day before further manipulation) by centrifugation (450g, 15 min.) using 50 ml Greiner PBMC isolation tubes. After collection and washing, PBMC were suspended in PBS atlO⁶ PBMC/ml. Next, 100 μΐ PBMC suspension were transferred to transparant

Eppendorf tubes to which 100 μΐ PBS and 100 μΐ lucigenin- solution (lucigenin 10 ³ M dissolved in water) were added. The chemiluminescence reaction was started by the addition of 700 μΐ NADH solution (5.10 ⁴ M). Finally, the chemiluminescent Eppendorf vials were closed and placed in a scintillation vial and counted in a Beckman 6500 scintillation counter using the Tritium window and in-coincidence setting. Counts observed over time are expressed as counts per minute (CPM). Results

Under the conditions described, urine of CFS patients tends to produce more formazan (reduced tetrazolium) compared to healthy controls. Furthermore, formazan production in urine of CFS patients was significantly inhibited after the addition of 100 U of ascorbate oxidase. Urine samples collected from CFS patients scored significantly higher (mean O.D. 1.3+/- 0.62) than control samples (mean O.D. 0.32+/- 0.12). In the presence of ascorbate oxidase, mean scores were comparable (mean OD. 0.15+/- 0.07 versus mean O.D. 0.18+/- 0.09), meaning that differences in urine redox potential are largely determined by ascorbate content as summarized in Fig.2.

When 9,9'-N,N-biacridinium dinitrate (CLP2+) and NADH were admixed to a somatic mammalian cell suspension of which cells were resting and were not energy-depleted, almost no chemiluminescence was produced. However, with cells that were energy- demanding, a glow type chemiluminescence response (CL) was observed of which the intensity was proportional to the degree of energy depletion. The CL-reaction we have developed is a chemiluminometric variant of the ENMA method described by Nadlinger et al. (Nadlinger et al, 2002. BBA 1573: 177-182; Roelant , US patent 5,306,624), and is largely applicable to any mammalian cell type with an intact plasma membrane and containing mitochondria (Afanas'ev, 2001. Circulation Res 89: e46). Disrupted cells or cell membrane preparations to which CLP²⁺ and NADH were admixed, did not produce any chemiluminescent response, indicating that this type of chemiluminescence is based on the capacity of intact cells to reduce CLP²⁺ at their cellular membrane and the production of ROS interacting with CLP²⁺ to produce an excited state (CLP*) and light (hv): CELLS + CLP²⁺ +NADH +0₂ CLP⁺ + 0₂-— > CLP* + hv

Of all ROS known, univalent reduced CLP2+ preferentially reacts with superoxide anion radicals (O2-) to produce an excited dioxetane (CLP*) and light (hv)(Afanas'ev, 2001. Circulation Res 89: e46). In agreement with the ENMA method we observed CLP²⁺-dependent chemiluminescence based on NADH-induced O2-, reflecting intra-cellular energy requirements. Univalently reduced CLP (CLP+) is produced by plasma membrane-associated NADH-oxidase:

CLP2++ NADH + 0₂ ► CLP+- + NAD + + 0₂°- + H +

CLP+° + 0₂°- CLP-0₂ ► Methyl -Acridon ^* + hv

Under the conditions described, the chemiluminescence observed is a measure of cellular energetic demand. Freshly isolated PBMC do hardly produce any chemiluminescence, whereas cells that are depleted of ATP produce significant luminescence upon addition of NADH. The NADH consumption by the plasma- membrane quantified by the chemiluminescence (hv) response is proportional to the proton motive force or the redox state of the NADH pool of the mitochondria and their efflux of hydrogen peroxide acting as a retrograde signal to the cell to enable the activity, transcription, translation, import or degradation of

mitochondrial components to be adjusted accordingly. This type of

chemiluminescence was also observed with mammalian cells after storage in energy depleting conditions (overnight storage in PBS, results not shown). These results show that PBMC isolated from heparinized whole blood 24 hours after drawing of the blood and obtained from CFS patients produced altered

chemiluminescence kinetics compared to controls (Fig.3). A schematic of the observed chemiluminescence mechanism is shown in Fig. 4.

Claims

1. A method of typing a human comprising determining a level of activity of a 2-oxoglutarate-dependent dioxygenase, preferably an asparaginyl β-hydroxylase, in a cell of said human.

2. The method according to claim 1, wherein said typing provides an indication that the human is suffering from a fatiguing illness, preferably from chronic fatigue syndrome, fibromyalgia, multiple sclerosis, gulf war / veterans syndrome, amyotrophic lateral sclerosis, post-Lyme disease or related disorders.

3. The method according to claim 1 or claim 2, wherein the level of activity of 2-oxoglutarate-dependent dioxygenase is determined by determining a ratio of intracellular nicotinamide adenine dinucleotide NADH/NAD+.

4. The method according to any one of claims 1-3, wherein the level of activity of 2-oxoglutarate-dependent dioxygenase is determined by imaging NADH auto-fluorescence, preferably by fluorescence lifetime imaging.

5. The method according to claim 1 or claim 2, wherein the level of activity of 2-oxoglutarate-dependent dioxygenase is determined by determining a level of asparaginyl and/or histidinyl hydroxylation of at least one ankyrin repeat domain.

6. The method according to claim 5, wherein said ankyrin repeat domain is present in an endogenous protein, preferably in ankyrin.

7. The method according to claim 1 or claim 2, wherein the level of activity of 2-oxoglutarate-dependent dioxygenase is determined by determining a level of activity of plasma membrane associated NADH oxidase.

8. The method according to claim 7, comprising

- isolating intact cells from the human;

- incubating the isolated cells with a chemiluminescent reagent and NADH;

- determining a level of chemiluminescence;

- comparing said determined level of chemiluminescence to a control level; and

- typing said human on the basis of the comparison.

9. A method of treating a human suffering from a fatiguing illness, comprising administering a therapeutically effective amount of an inhibitor of an asparaginyl β-hydroxylase to said human.

10. The method of claim 9, wherein said inhibitor is a nucleic acid molecule, preferably a siRNA molecule or miRNA molecule, or a low molecular weight (< 1 kdalton) molecule.

11. The method of claim 9 or claim 10, wherein said inhibitor is or comprises a chromene derivative and/or a hydroxypyridone compound.

12. The method of claim 9 or claim 10, wherein said inhibitor is or comprises a manganese superoxide dismutase, or a mimic thereof, preferably

manganese(III) tetrakis(l-methyl-4-pyridyl)porphyrin, and/or a peroxide donor, preferably an alkali percarbonate such as sodium percarbonate and/or a peroxide such as magnesium peroxide or carbamide peroxide.

13. A pharmaceutical composition comprising an inhibitor of an asparaginyl β-hydroxylase, preferably of FIH, and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition of claim 13, for use in a method of treating a human suffering from a fatiguing illness.

15. A method of treating a human suffering from a fatiguing illness, comprising administering a therapeutically effective amount of a compound that stimulates hydrogen peroxide emission of mitochondria by interference with oxidation of free fatty acids.

16. A method of treating a human suffering from a fatiguing illness, comprising administering a therapeutically effective amount of a compound that regulates peroxisomal catalase activity.

17. A use of an inhibitor of an asparaginyl β-hydroxylase, preferably of FIH, in a method for the preparation of a medicament for the treatment of a human suffering from a fatiguing illness.