TYROSINE-CONTAINING MOTIFS

Technical field of the invention

The present invention relates to covalent inhibitors that specifically bind and inhibit enzyme targets with pre-defined tyrosine-containing motifs, such as the enzyme sirtuin 5. In particular, the covalent inhibitors of the present invention may be used alone or in combination with other therapeutic agents for treatment of disease, such as cancer.

Background of the invention

Enzymes are a necessity for biochemical reactions at the cellular level to occur at rates sufficient to sustain life. Thus, control of the activity of enzymes is essential for homeostasis and incorrect regulation of enzymes may cause severe disease. Accordingly, there exist many diseases which is a direct consequence of improperly regulated or functioning enzymes. Such enzyme-related diseases may be treated e.g. by replacement of a deficient enzyme, activation of a dormant enzyme or inhibition of an enzymatic function, with the treatment depending on the mechanism of action of the target enzyme.

The NAD ⁺ -dependent sirtuin enzymes catalyze the cleavage of different e-N- acyllysine posttranslational modifications and therefore play key roles in regulating a range of biological processes, including gene transcription and metabolism. Seven different sirtuin isoforms (SIRT1-7) have been identified in mammals, which localize to separate cellular compartments and display preference for the recognition and cleavage of different lysine acyl groups. In particular, sirtuin 5 (SIRT5) primarily removes glutaryl, malonyl and succinyl posttranslational modifications to lysine residues in the mitochondria. Among other biological functions, SIRT5 has been shown to promote reactive oxygen species (ROS) and ammonia detoxification, inhibit inflammation, modulate mitophagy during starvation, maintain cardiac oxidative metabolism in response to cardiac stress, as well as regulate tumor growth and colorectal cancer metabolic reprogramming, highlighting the therapeutic potential associated with SIRT5 inhibition.

Despite the crucial physiological roles of the sirtuin 5 enzyme, the development of inhibitors with potent and selective cellular activity has been lacking behind.

When designing inhibitors for SIRT5, there are two main binding pockets to target; the NAD ⁺ binding site and the substrate binding site. Targeting of the NAD ⁺ binding site is disadvantaged due to the extended amounts of human enzymes that utilize NAD ⁺ as a co-substrate, which results in an increased risk of adverse effects. In contrast, the substrate binding site encompasses a relatively large and specific substrate binding pocket. The binding pocket comprises two specific non-hydrophobic residues, Argl05 and Tyrl02, which interact with the succinyl-lysine group of the substrate via hydrogen-bonding and electrostatic interactions. For this reason, many inhibitors have been designed with a carboxylate group, since this chemical moiety could be favoured to occupy the binding pocket of SIRT5.

An example of the development of SIRT5 inhibitors is presented by Kalbas et al. (2018) who developed peptide-based selective SIRT5 affinity probes by optimizing the acyl moiety in a systematic manner. The inhibitors presented therein all comprises a carboxylate moiety, yielding a reversible inhibitor. Other reversible peptide inhibitors of SIRT5 based on thiosuccinyl-lysine or Ne-carboxyethyl- thiourea-lysine residues have also been proposed.

Another example of a reversible SIRT5 inhibitor is presented by Rajabi et al.

(2017) who reported a mechanism-based SIRT5 inhibitor containing a thiourea functionality that takes advantage of the NAD ⁺ -mediated cleavage mechanism in sirtuins to form a stalled intermediate with ADP-ribose, therefore enhancing its residence time inside the SIRT5 substrate-binding pocket. In addition, the inhibitor of Rajabi et al. contains a terminal carboxylic acid that stabilizes the substrate inside the SIRT5 binding pocket with hydrogen bond interactions to the Tyrl02-Arg l05 motif. Similarly, WO 2014/197775 A2 discloses a selection of thiourea compounds for use as SIRT5 inhibitors. The thiourea group forms together with a carboxylate functional group a moiety with selectivity towards the active site of SIRT5. The inhibitors of WO 2014/197775 A2 are envisioned to be suitable for treatment of a range of different diseases.

Unfortunately, both thiourea and carboxylate functionalities have limited pharmacological relevance, due to lack of biostability and inferior cell membrane permeability.

Alternative strategies for development of potent and selective inhibitors of SIRT5 include also small molecule heterocyclic scaffolds. However, these constructs generally exhibit poor isozyme selectivity, which render them prone to causing adverse effects.

Thus, other designs of inhibitors are needed for identifying SIRT5 inhibitors with high therapeutic potential.

Hence, improved SIRT5 inhibitors which are highly potent and specific, would be advantageous. In particular, the provision of improved SIRT5 inhibitors, which furthermore possess enhanced biostability and cell membrane permeability, and may be used for treatment of disease, such as cancer, would be beneficial.

Summary of the invention

Herein is provided inhibitors of SIRT5 that are conceptually different from previously developed reversible SIRT5 inhibitors. The conventional functional groups of the inhibitor for targeting SIRT5 have been replaced with new functional moieties that enables highly selective covalent binding of the inhibitors to SIRT5, and ultimately overcomes the difficulties of other inhibitors of SIRT5 with lack of stability and poor cell membrane permeability.

Thus, an object the present invention relates to the provision of covalent SIRT5 inhibitors comprising functionalities that improves potency and specificity, while keeping the compounds stable and capable of permeating cell membranes. Another object of the present invention is to provide covalent SIRT5 inhibitors that may be effectively used in treatment of disease, either as single active component or in a combination therapy. Thus, one aspect of the invention relates to a covalent inhibitor of the sirtuin 5 enzyme according to formula (I) :

wherein :

Ri comprises an arylfluorosulfate group;

R2 is selected from amino acid side chains,

R3 is selected from H, COOR, CONH2, CONHR6 and triazolo, wherein R6 is selected from C3-C8 cycloalkanes, linear alkanes, aromatics and heteroaromatics,

R4 and Rs are independently selected from H, Cl, F, Br, I, CF3, NO2, C1-C10 alkyl esters, C1-C10 alkoxy, alcohols and CONH(CH2)R7, or R4 and Rs together form an aryl or heteroaryl ring system optionally substituted with one or more substituents selected from nitrogen, C1-C10 alkyl, alcohol, ether and halogen, wherein R7 IS selected from C2-C10 alkynes, azides, trans- cyclooctenes and cyclopropenes, and n is an integer selected from 2-6.

A preferred aspect of the invention relates to a covalent inhibitor of the sirtuin 5 enzyme according to formula (la) :

wherein :

Ri comprises an arylfluorosulfate group selected from 6- or 10-membered aromatic or heteroaromatic rings substituted with a fluorosulfate group;

R2 is selected from amino acid side chains,

R3 is selected from H, COOR, CONH2, CONHR6 and triazolo, wherein R6 is selected from C3-C8 cycloalkanes, linear alkanes, aromatics and heteroaromatics,

Rs is selected from formulas (Ila) or (lib);

R4 and Rs are independently selected from H, Cl, F, Br, I, CF3, NO2, C1-C10 alkyl esters, C1-C10 alkoxy, alcohols and CONH(CH2)R7, or R4 and Rs together form an aryl or heteroaryl ring system optionally substituted with one or more substituents selected from nitrogen, C1-C10 alkyl, alcohol, ether and halogen, wherein R7 IS selected from C2-C10 alkynes, azides, trans- cyclooctenes and cyclopropenes, n is an integer selected from 2-6, and

m is an integer selected from 2-6.

Another aspect of the present invention relates to a pharmaceutical composition comprising the covalent inhibitor of the sirtuin 5 enzyme as described herein or a pharmaceutically acceptable salt thereof.

Yet another aspect of the present invention relates to a covalent inhibitor of the sirtuin 5 enzyme or the pharmaceutical composition as described herein for use as a medicament. A further aspect of the present invention relates to a covalent inhibitor of the sirtuin 5 enzyme or the pharmaceutical composition as described herein for use in the amelioration or treatment of cancer. Still another aspect of the present invention is to provide a kit comprising :

i. the covalent inhibitor of the sirtuin 5 enzyme or the pharmaceutical composition as described herein,

ii. one or more additional therapeutic agents, and

iii. optionally, instructions for use.

A still further aspect of the present invention relates to a method for preparing a covalent inhibitor of the sirtuin 5 enzyme as described herein, said method comprising the following steps:

i. provision of a compound according to formula (Yl), and

ii. contacting the compound according to formula (Yl) with a compound according to formula (Y2), thereby obtaining a covalent inhibitor according to formula (I),

wherein R1-R7, n, and X are as defined herein.

Yet another aspect of the present invention relates to a method for preparing a covalent inhibitor of the sirtuin 5 enzyme as described herein, said method comprising the following steps: provision of a compound according to formula (Yla), and

ii. contacting the compound according to formula (Yla) with a compound according to formula (Y2), thereby obtaining a covalent inhibitor according to formula (la),

wherein Ri-Rs, n, m and X are as defined herein. Brief description of the figures

Figure 1 shows (A) Positive ion mode MALDI-TOF mass spectra of SIRT5 before and after incubation at room temperature with compounds (V) and (VI), respectively, at different time points. (B) Peptide mass fingerprinting experiment showing trypsinized fragment of SIRT5 conjugated to compound (VI). (C) Covalent labelling of SIRT5 by compounds (VI), (VI-c), (Vl-d) and (Vl-b).

Recombinant SIRT5 (2 mM) were incubated with any of the compounds (VI), (VI- c), (Vl-d) and (Vl-b) (20 mM) overnight at room temperature, followed by click chemistry with Fluor488-azide, SDS-PAGE gel electrophoresis, and in-gel fluorescence measurement. (D) Pull down of SIRT5 from enriched mitochondrial lysate using compound (X-6b). Lysed, enriched mitochondria from HEK293 cells (400 pg) were incubated with covalent inhibitor (10 or 100 mM), NAD ⁺ (0 or 500 mM), and protease inhibitor (IX) overnight at room temperature, followed by click chemistry with Biotin-PEG3-azide, pull-down using streptavidin beads, SDS- PAGE gel electrophoresis, and western blot using anti-SIRT5 primary antibody. (E) Pull-down of SIRT5 from enriched mitochondrial lysate using compounds (V-2c) and (VI). Lysed enriched mitochondria from HEK293 cells (400 pg) was incubated with covalent inhibitor (10 or 100 mM), NAD ⁺ (0 or 500 mM), and protease inhibitor (IX) overnight at room temperature, followed by click chemistry with Biotin-PEG3-azide, pull-down using streptavidin beads, SDS-PAGE gel

electrophoresis, and western blot using anti-SIRT5 primary antibody.

Figure 2 shows time-dependent inhibition of SIRT5 enzymatic activity by compounds (V) and (VI), respectively.

Figure 3 shows (A) LC-MS traces of compounds (V) and (VI) after incubation in buffer for 48 hours, showing exquisite chemical stability. (B) UPLC traces of compounds (V) and (VI) after incubation in serum for 15 min and 60 min, respectively. (C) Stability of compounds (V), (VI), and (V-2c) after incubation in human serum at 37 °C for 0-8 h.

Figure 4 shows covalent labelling selectivity among SIRT1-7 by compound (VI). Recombinant enzymes (2.5 mM) were incubated with compound (VI) (10 mM) overnight at room temperature, followed by click chemistry with Fluor488-azide, SDS-PAGE gel electrophoresis, and in-gel fluorescence measurement. Boxes represent the locations of the enzymes according to the corresponding Coomassie blue-stained gel.

Figure 5 shows that dose-dependent covalent labelling of SIRT5 (2.5 mM) by compound (VI) remains highly efficient in concentrated HEK293 lysate (total protein 8 mg/mL) and shows minimal labelling of other proteins. Enzyme denaturation by pre-boiling of SIRT5 for 5 minutes in Tris buffer (pH 8) completely abolishes covalent adduct formation. Figure 6 shows dose-response curves from MTT assays measuring toxicity against cancer cell lines HeLa, Jurkat, and MCF7 of a reversible inhibitor versus

compounds (V) and (VI).

Figure 7 shows that compound (VI) binds covalently to native SIRT5 isoforms inside HEK293 cells in culture. Biotin is then conjugated to the compound-proteins adduct, proteins are resolved by gel electrophoresis, transferred to a membrane, and visualized by streptavidin-conjugated HRP.

Figure 8 shows time-dependent inhibition of SIRT5 enzymatic activity by (A) compound (VI-c); (B) compound (Vl-d); (C) compound (Vl-b); (D) compound (X- 6b); (E) compound (V-2c); (F) compound (V-2b); (G) compound (X-6a), and (H) compound (VI-3a).

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

Detailed description of the invention

Definitions

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

Covalent inhibitor

In the present context, the term "covalent inhibitor" refers to an inhibitor that bind and bond to their target protein, e.g. a target enzyme, through a bond forming functional group. The covalent bond between the covalent inhibitor and the target protein involves sharing of electron pairs between atoms.

Covalent inhibitors bind irreversibly to a protein target and is to be distinguished from reversible inhibitors, which are in equilibrium with their target, /.e. binding and unbinding over time.

In the present invention, the bond-forming functional group is an arylfluorosulfate group. Sirtuin 5

In the present context, the term "sirtuin 5" refers to the protein known as silent mating type information regulation 2 homolog 5, which is in humans encoded by the SIRT5 gene. In the present context, the terms "sirtuin 5" and "SIRT5" are used interchangeably for the sirtuin 5 enzyme.

Proteins belonging to the sirtuin family comprises a sirtuin core domain and are dependent on NAD ⁺ as co-factor for their enzymatic activity. SIRT5 exhibits enzymatic activities as e.g. a deacetylase, deglutarylase desuccinylase, and demalonylase, capable of removing acetyl, glutaryl, succinyl, and malonyl groups from the lysine residues of proteins.

Sirtuin 5 corresponds to SEQ ID NO: l as recited in the Uniprot database under reference number Q9NXA8. It is to be noted that sirtuin 5 exist as different variants in different species, and the enzyme sirtuin 5 as used herein covers also variability across species. Thus, sirtuin 5 may e.g. be a protein comprising the amino acid sequence according to SEQ ID NO: l or to an amino acid sequence with at least 80% sequence identity to SEQ ID NO: l, such as 90% sequence identity, 95% sequence identity, or 99% sequence identity to SEQ ID NO: l.

It is to be understood that sequences with sequence identity to SEQ ID NO: l retain the enzymatic activity of sirtuin 5 comprising the amino acid sequence according to SEQ ID NO: l. Arylfluorosulfate

In the present context, the term "arylfluorosulfate" refers to a chemical group with the general formula Ar-O-SCh-F, wherein Ar corresponds to "aryl". Aryl is any functional group or substituent derived from an aromatic ring. The aromatic ring may e.g. be an aromatic hydrocarbon, such as phenyl, or a substituted aromatic hydrocarbon, such as pyridine. Further, the term "aryl" encompass aromatic or heteroaromatic ring systems comprising 6- or 10-membered rings, such as phenyl or naphthyl. Heteroaromatic rings are aromatic rings comprising one or more heteroatoms, such as N, O or S. The arylfluorosulfate group (Ar-O- SO2-F) is to be distinguished from the aryl sulfonyl fluoride group (Ar-SCh-F). The fluorosulfate group (-O-SO2-F) may be positioned in any position on the aromatic ring structure, i.e. in ortho, meta or para position.

The arylfluorosulfate groups may react chemoselectively in a sulfur (VI) fluoride exchange (SuFEx) reaction to covalent bind to a target protein. Specifically the arylfluorosulfate group may facilitate covalent binding to e.g. tyrosine (Tyr), lysine (Lys) or serine (Ser) amino acid residues.

Mitchondrial targeting motif

In the present context, the term "mitochondrial targeting motif" refers to a moiety that is capable of targeting the covalent inhibitor to the mitochondria organelle upon administration to a subject. Preferably, the mitochondrial targeting motif is triphenylphosphine, also abbreviated as PPh3 (IUPAC name: triphenylphosphane). Amino acid side chains

In the present context, the term "amino acid side chain" refers to the side chain of the 20 standard (or proteinogenic) amino acid encoded by codons of the genetic code. In the standard amino acids, the side chains are the carbon chains attached to the a-carbon, as depicted by the following formula:

Amino acid

Side chain

Each of the 20 standard amino acid contain a specific side chain. The amino acid may be grouped according to the chemical nature or properties of their side chain One such grouping is the aromatic side chains. Therefore, in the present context, the term "aromatic amino acid side chains" refers to the side chains of histidine, phenylalanine, tryptophan and tyrosine. Another grouping is the basic side chains Therefore, in the present context, the term "basic amino acid side chains" refers to the side chains of arginine, histidine and lysine. Halogen

In the present context, the term "halogen" refers to the elements contained within group 17 of the periodic table. Thus, halogens may be fluorine (F), chlorine (Cl), bromine (Br), iodine (I), astatine (At), and tennessine (Ts). Preferably, the halogens are selected from fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

Therapeutic agent

In the present context, the term "therapeutic agent" refers to any molecule or substance that have a beneficial effect on a disease or condition when

administered to a subject. Examples of therapeutic agents include, but are not limited to, proteins, peptides, small molecule drugs, anti-cancer agents and pharmaceutically acceptable salts thereof.

Anti-cancer agent

In the present context, the term "anti-cancer agent" refers to any molecule or substance that have a beneficial effect on a cancer when administered to a subject. Examples of classes of anti-cancer agents include, but are not limited to, alkylating agents, antimetabolites, natural products, hormones and small molecule drugs.

Herein, anti-cancer agents may be any of these molecules or substances, which may be administered together with the covalent inhibitors as part of a therapeutic regimen for treatment of a cancer. Administration of the anti-cancer agent and covalent inhibitor may be sequentially, separately or simultaneously.

Combination therapy

In the present context, the term "combination therapy" refers to a therapy in which the covalent inhibitor is used together with one or more therapeutic agents as part of a single treatment. The covalent inhibitor and one or more therapeutic agents may be co-formulated or given as separate compounds. Administration of the covalent inhibitor and one or more therapeutic agents may be sequentially, separately or simultaneously. Pharmaceutical composition

In the present context, the term "pharmaceutical composition" refers to a composition suspended in a suitable amount of a pharmaceutical acceptable diluent or excipient.

In one embodiment, the term "composition" refers to any such composition suitable for administration to a subject, and such compositions may comprise a pharmaceutically acceptable carrier or diluent, for any of the indications or modes of administration as described. The active materials in the compositions of this invention can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.

Such composition is usually known as a pharmaceutical composition. Preferably the composition is a pharmaceutically acceptable composition. Thus, in the present context, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. Pharmaceutically acceptable salt

In the present context, the term "pharmaceutically acceptable salt" refers to a salt that can be formulated into a composition for pharmaceutical use including, but not limited to, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

Carrier

In the present context, the term "carrier" refers to any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Excipient

In the present context, the term "excipient" refers to a diluent, adjuvant, carrier, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in

"Remington's Pharmaceutical Sciences" by E. W. Martin.

Adjuvant

In the present context, the term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and as a lymphoid system activator, which non-specifically enhances the immune response. Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response.

Sequence identity

In the present context, the term "sequence identity" refers to the sequence identity between genes or proteins at the nucleotide, base or amino acid level, respectively. Specifically, a DNA and a RNA sequence are considered identical if the transcript of the DNA sequence can be transcribed to the identical RNA sequence.

Thus, in the present context "sequence identity" is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (/. e. , % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are the same length.

In another embodiment, the two sequences are of different length and gaps are seen as different positions. One may manually align the sequences and count the number of identical amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990). BLAST nucleotide searches may be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength =

3 to obtain amino acid sequences homologous to a protein molecule of the invention.

To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized. Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilizing the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).

Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment. In the context of the present invention, the BLASTN and PSI BLAST default settings may be advantageous.

The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted. An embodiment of the present invention thus relates to sequences of the present invention that has some degree of sequence variation.

The terms "selected from" and "selected from the group consisting of" are used interchangeably herein.

Covalent inhibitors of enzyme targets

Sirtuins (SIRTs) are a family of nicotinamide adenine dinucleotide (NAD+)- dependent lysine deacetylases, which amongst other regulate important biological pathways and processes ranging from apoptosis, age-associated

pathophysiologies, adipocyte and muscle differentiation, and energy expenditure to gluconeogenesis. Sirtuin 5 (SIRT5) is one of seven sirtuin proteins and has received significant attention as SIRT5 has been found to have weak deacetylase activity but strong desuccinylase, demalonylase and deglutarylase activities, while also being associated with several human diseases such as cancer, Alzheimer's disease, and Parkinson's disease.

Currently, the biological functions and therapeutic possibilities of SIRT5 have been well documented. However, inhibitors of this important biological target that are also suitable for pharmaceutical use remain scarce. Several peptide or small molecule inhibitors have been developed for either academic or therapeutic use. Unfortunately, as described above, many of the designs utilized in the known inhibitors of SIRT5 have limited pharmacological relevance due to drawbacks such as lack of biostability, poor cell membrane permeability or inadequate selectivity. Common for many of the known SIRT5 inhibitors are that they are reversible inhibitors, which relies on a carboxylate functionality stabilizing the inhibitor in the binding pocket of SIRT5. Herein are provided inhibitors of SIRT5 that are conceptually different from previously developed reversible SIRT5 inhibitors. Thus, the inhibitors of the present invention are not reversible inhibitors, but instead developed for covalent binding to the target protein.

Selective covalent modification of a targeted protein is a powerful tool in chemical biology and drug discovery, with applications ranging from identification and characterization of proteins and their functions to the development of targeted covalent inhibitors. Some covalent ligands contain an "affinity motif" and an electrophilic functional moiety that reacts with a nucleophilic residue of the targeted protein. Ideally, this facilitates formation of a covalent, irreversible bond with just the intended functional group among thousands of

nucleophiles in the proteome.

Covalent inhibitors are typically met with scepticism in the medical field because of the risk of irreversible adverse effects and has been abolished from drug discovery programs for a long time, mainly due to concerns regarding their idiosyncratic toxicity. Consequently, any covalent inhibitor is required to be highly specific for the target protein to minimize adverse effects.

Herein, are provided covalent inhibitors that may be used for selective covalent binding of tyrosine, lysine or serine residues. Preferably, the covalent inhibitors described herein is for covalent binding of tyrosine residues. The covalent inhibitors of the present invention are based on an arylfluorosulfate functional moiety (Ar-0-S02-F), which under appropriate conditions undergo a

sulfur(VI)-fluoride exchange (SuFEx) reaction to specifically bind to a tyrosine, lysine or serine residue. The covalent inhibitors react exclusively upon activation within the binding site of the target protein. Thus, without being bound by theory, the surrounding protein environment requires basic residues (Arg, Lys, or His) that decrease the pK _a of the targeted nucleophilic residue and/or that facilitate fluoride ion departure (e.g. by hydrogen bonding interactions) to achieve the SuFEx reaction. Consequently, the covalent inhibitors of the present invention are basically unreactive in the presence of amino acids or natural products that does not contain this specific protein microenvironment.

The covalent inhibitors of the present invention are in some variants developed for inhibition of the SIRT5 enzyme. Through structure-activity relationship (SAR) studies, covalent inhibitors with high potency and selectivity have been

developed. The low and narrow intrinsic reactivity of the arylfluorosulfate-based functional moiety assures minimal covalent reactivity with off-target nucleophiles in a cellular context, making these compounds largely unreactive towards the human proteome. These traits makes the covalent inhibitors of the present invention good pharmaceutical candidates for use in the treatment of disease, such as cancer.

Thus, an aspect (termed "aspect I") of the present invention relates to a covalent inhibitor of the sirtuin 5 enzyme according to formula (I) :

wherein :

Ri comprises an arylfluorosulfate group;

R2 is selected from amino acid side chains,

R3 is selected from H, COOR, CONH2, CONHR6 and triazolo, wherein R6 is selected from C3-C8 cycloalkanes, linear alkanes, aromatics and heteroaromatics,

R4 and Rs are independently selected from H, Cl, F, Br, I, CF3, NO2, C1-C10 alkyl esters, C1-C10 alkoxy, alcohols and CONH(CH2)R7, or R4 and Rs together form an aryl or heteroaryl ring system optionally substituted with one or more substituents selected from nitrogen, C1-C10 alkyl, alcohol, ether and halogen, wherein R7 IS selected from C2-C10 alkynes, azides, trans- cyclooctenes and cyclopropenes, and n is an integer selected from 2-6. Alternatively, the covalent inhibitor of the present invention may also be a compound in which the benzene ring has only a single substituent, i.e. the substituent Rs is absent (or is H). Thus, an embodiment of the present invention relates to a covalent inhibitor of the sirtuin 5 enzyme according to formula (1-2) :

(1-2) wherein R1-R4, R6-R7 and n are as defined in "aspect l" or "aspect la". Preferably, the sirtuin 5 (SIRT5) enzyme is human SIRT5 as defined in the Uniprot database (reference no. Q9NXA8) by the amino acid sequence according to SEQ ID NO: l. Thus, an embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the sirtuin 5 enzyme is defined by the amino acid according to SEQ ID NO: l or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: l, such as 90% sequence identity, 95% sequence identity, or 99% sequence identity to SEQ ID NO: l .

Another aspect of the present invention relates to an inhibitor of the sirtuin 5 enzyme according to formula (I) :

wherein :

Ri comprises an arylfluorosulfate group;

R2 is selected from amino acid side chains, R3 is selected from H, COOR, CONH2, CONHR6 and triazolo, wherein R6 is selected from C3-C8 cycloalkanes, linear alkanes, aromatics and heteroaromatics,

R4 and Rs are independently selected from H, Cl, F, Br, I, CF3, NO2, C1-C10 alkyl esters, C1-C10 alkoxy, alcohols and CONH(CH2)R7, or R4 and Rs together form an aryl or heteroaryl ring system optionally substituted with one or more substituents selected from nitrogen, C1-C10 alkyl, alcohol, ether and halogen, wherein R7 IS selected from C2-C10 alkynes, azides, trans- cyclooctenes and cyclopropenes, and n is an integer selected from 2-6. One of the main functions of SIRT5, i.e. removal of glutaryl, malonyl and succinyl posttranslational modifications to lysine residues, takes place in the mitochondria. Thus, variants of the covalent inhibitors may advantageously encompass a mitochondrial targeting motif to enhance the delivery of the covalent inhibitors to the specific organelle. An example of a mitochondrial targeting motif is

triphenylphosphine (PPI73), which is an organophosphorous chemical moiety with the formula P(C6Hs)3. PPhi3 is stable in biological systems and display low reactivity towards non-relevant cellular components.

Thus, another aspect (termed "aspect la") of the present invention relates to a covalent inhibitor or inhibitor of the sirtuin 5 enzyme according to formula (la) :

wherein :

Ri comprises an arylfluorosulfate group selected from 6- or 10-membered aromatic or heteroaromatic rings substituted with a fluorosulfate group;

R2 is selected from amino acid side chains,

R3 is selected from H, COOR, CONH2, CONHR6 and triazolo, wherein R6 is selected from C3-C8 cycloalkanes, linear alkanes, aromatics and heteroaromatics,

Rs is selected from formulas (Ila) or (lib); R ₄ and R5 are independently selected from H, Cl, F, Br, I, CF ₃ , NO ₂ , C ₁ -C ₁₀ alkyl esters, C ₁ -C ₁₀ alkoxy, alcohols and CONH(CH2)R7, or R ₄ and Rs together form an aryl or heteroaryl ring system optionally substituted with one or more substituents selected from nitrogen, C ₁ -C ₁₀ alkyl, alcohol, ether and halogen, wherein R _{7 IS} selected from C ₂ -C ₁₀ alkynes, azides, trans- cyclooctenes and cyclopropenes, n is an integer selected from 2-6, and

m is an integer selected from 2-6. As described herein, the arylfluorosulfate moiety is the functional group, which under appropriate conditions covalently binds to the target protein. The

arylfluorosulfate moiety may be attached to the covalent inhibitor in a variety of configurations that enables the SuFEx reaction to occur. The arylfluorosulfate group may be a phenyl or naphthyl group substituted with a fluorosulfate group, with the phenyl or naphthyl group optionally comprising one or more heteroatoms. The heteroatoms may be N, O or S. Thus, an embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the arylfluorosulfate moiety comprises or consists of a phenyl group substituted with a fluorosulfate group, said phenyl group optionally comprising one or more heteroatoms independently selected from N, O and S. Another embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the arylfluorosulfate moiety comprises or consists of a naphtyl group substituted with a fluorosulfate group, said naphtyl group optionally comprising one or more heteroatoms independently selected from N, O and S.

In variants of the covalent inhibitors, the arylfluorosulfate group may be a phenyl or naphthyl group substituted with a fluorosulfate group and one or more substituents selected from the group of carboxylic acids, amines, alkyl or hydroxyl groups. Thus, an embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the arylfluorosulfate moiety comprises or consists of a phenyl group substituted with a fluorosulfate group and one or more substituents selected from the group of carboxylic acids, amines, alkyl or hydroxyl groups, preferably a carboxylic acid.

In some variants of the covalent inhibitors, the arylfluorosulfate moiety is attached directly to the amide functional group next to Ri according to formula

(I)· Thus, a preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein Ri is defined by the formula (II):

wherein X is selected from CH and N, preferably X is N.

The exact positioning of the electrophilic fluorosulfate group (-O-SO2-F) may be varied to obtain the most efficient fluorosulfate substitution. Thus, the covalent inhibitors of the present invention are not limited to a single regioisomeric variant. An embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the fluorosulfate group (-O-SO2-F) is positioned in the meta position on the aromatic ring.

Thus, a preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (III): wherein X is selected from CH and N, preferably X is N. In an alternative variant, the covalent inhibitor of the present invention may also be a compound in which the benzene ring has only a single substituent, i.e. the substituent Rs is absent (or is H). Thus, an embodiment of the present invention relates to a covalent inhibitor of the sirtuin 5 enzyme according to formula (III-2):

wherein R2-R4, R6-R7 and n are as defined in "aspect I".

Another embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (Ilia) : wherein X is selected from CH and N, preferably X is N.

The binding pocket of SIRT5 comprises several non-hydrophobic residues that are predicted to be involved in the fluorosulfate substitution. Without being bound by theory, two specific non-hydrophobic residues, Argl05 and Tyrl02, are proposed to be involved in the fluorosulfate substitution. The covalent inhibitors of the present invention may be substituted on the aryl group to further enhance positioning of the electrophilic sulfur(VI) towards Tyrl02 and/or achieve a better stabilization of the leaving fluoride anion by Argl05 in SIRT5. One such

substitution may be featuring a fluorosulfate-substituted pyridine. Thus, a preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IV) :

R2 are selected from amino acid side chains of the 20 standard amino acids. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R2 is a side chain of an amino acid selected from the group consisiting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, tryptophan, tyrosine and valine.

R2 are preferentially selected from amino acid side chains with aromaticity. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R2 is selected from aromatic amino acid side chains. Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R2 is selected from the side chain of tryptophan and tyrosine.

A preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IVa) :

Also preferred are amino acid residues with basic side chains. Thus, an

embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R2 is selected from basic amino acid side chains. Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R2 is selected from group consisiting of the side chain of arginine, histidine and lysine.

A further embodiment of the present invention relates to a covalent inhibitor as described herein, wherein R2 is selected from the group consisting of the side chain of arginine, tryptophan and tyrosine, preferably the side chain of arginine.

Thus, a preferred embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula

wherein X is selected from CH and N, preferably X is N.

Variants of the covalent inhibitor includes the combination of an arginine moiety with a benzenesulfonamide. Therefore, an embodiment of the present invention relates to a covalent inhibitor as described herein, wherein Rs is defined by formula (Ila).

Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IV-2a) : wherein X is selected from CH and N, preferably X is N, and wherein R3, R6 and n are as defined in "aspect la". Another embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (V-2) :

(V-2)

wherein X is selected from CH and N, preferably X is N. Another embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (V-2a): (V-2a)

wherein X is selected from CH and N, preferably X is N.

A further embodiment relates to of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula

Yet another embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (V-2c):

(V-2c) Other variants of the covalent inhibitor includes the combination of an arginine moiety with the mitochondrial targeting motif, triphenylphosphine (PPhi3). Thus, an embodiment of the present invention relates to a covalent inhibitor as described herein, wherein Rs is defined by formula (lib). PPhi3 may be linked to the amide through alkyl chains of varying length. As an example, the

mitochondrial targeting motif may be incorporated in the covalent inhibitor as triphenyl(propyl)phosphine. Thus, an embodiment of the present invention relates to a covalent inhibitor as descreibed herein, wherein m is 3. Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IV-2b) :

wherein X is selected from CH and N, preferably X is N, and

wherein R3, R6 and n are as defined in "aspect la".

A further and preferred embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula

(V-3) : Yet another embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (V-3a):

wherein X is selected from CH and N, preferably X is N.

Variants of the covalent inhibitor includes combination of a tryptophan moiety with a fluor-substituted benzene ring. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IV-h) or (IV-i) : wherein R3, R6 and n are as defined in "aspect I". Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R3 is CONHR6. These variations of the covalent inhibitors may be with either one or two substituents on the aryl-SCh benzene ring, with the single substituent preferably being fluorine in the meta position. Therefore, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula

wherein R4-R7 and n are as defined in "aspect I". Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IV-d) or (IV-e) :

wherein R6 and n are as defined in "aspect I". For the R ₆ group, it is preferred that the substituent is bulky. Thus, an

embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R6 is selected from C3-C8 cycloalkanes. Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R3 is selected from H, CONH2, and CONH(CmH2m-i), with m being an integer between 3-8.

A preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R6 is cyclobutane. Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R3 is CONH(C4H7). Thus, a preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IV-c) : (IV-c)

An alternative variant of the covalent inhibitor of the present invention may be a compound in which the benzene ring has only a single substituent, i.e. the substituent Rs is absent (or is H). Thus, an embodiment of the present invention relates to a covalent inhibitor of the sirtuin 5 enzyme according to formula (IV-

wherein R4, R7 and n are as defined in "aspect I". In general, it may be preferred that the aryl-SCh benzene ring comprises only a single substituent. Thus, a preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein Rs is H.

In variants of the covalent inhibitors the aryl-SCh benzene ring is substituted with a halogen, preferably fluorine. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R4 is a halogen. A preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein R4 is F. Preferred variations of the covalent inhibitors may comprise the combinations of moieties including tryptophan, an amide attached cyclobutane and an aryl-SCh benzene ring substituted with a halogen, preferably with fluorine positioned in the meta position. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IV-f):

wherein n is as defined in "aspect I".

A preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IV-g): Yet another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein n is 4.

Other variants of the covalent inhibitors are featuring a fluorosulfate-substituted pyridine in combination with CONHR ₆ . Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by a formula selected from the group consisting of formulas (IV-j) to (IV-I):

wherein R2, R4-R7 and n are as defined in "aspect I". Other variants of the covalent inhibitors are featuring a fluorosulfate-substituted pyridine in combination with an amide attached cyclobutane. Thus, an

embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by a formula selected from the group consisting of formulas (IV-m) to (IV-o):

wherein R2, R4-R5, R7 and n are as defined in "aspect I". Other variants of the covalent inhibitors are featuring a fluorosulfate-substituted pyridine in combination with an aryl-S0 ₂ benzene ring substituted with fluorine, preferably with fluorine positioned in the meta position. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IV-p) or (IV-q) :

wherein R2-R3, R ₆ and n are as defined in "aspect I". A preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (V) : The covalent inhibitor may be further defined by the stereochemistry. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (V-a) :

For some applications it may be considered beneficial to label the covalent inhibitor. To this end, a click chemistry label may be comprised in the covalent inhibitor. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein F is CONH(CH2)R7 and R7 is selected from C2-C10 alkynes, azides, trans- cyclooctenes and cyclopropenes. A preferred embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (VI) :

Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (Vl-a) :

(VI-a)

Yet another embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (Vl-b): (Vl-b)

A further embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (VI-c) :

(VI-c)

Another embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (Vl-d):

(Vl-d)

An even further embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula

wherein X is selected from CH and N, preferably X is N.

Yet another embodiment of the present invention relates to a covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (VI-3) :

(VI-3) wherein X is selected from CH and N, preferably X is N.

Thus, a covalent inhibitor of the present invention is defined by formula (VI-3), wherein X is N. This covalent inhibitor is termed VI-3a.

Labelling of the covalent inhibitors may therefore be performed by well-known click chemistry reactions, such as copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), strain-promoted alkyne-nitrone cycloaddition (SPANC) or reactions of strained alkenes.

The covalent inhibitors described herein includes also variants encompassing arylfluorosulfates for targeting SIRT5 in combination with different other functional moieties. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (VII) or (VII-2):

wherein R2- R7 and n are as defined in "aspect I", and X is selected from CH and N, preferably X is N.

The arylfluorosulfate moiety may in some variants of the covalent inhibitors be combined with the aromatic side chain of the amino acid tryptophan. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by a formula selected from the group consisting of formulas (Vll-a) to (Vll-i):

wherein R3- R7 and n are as defined in "aspect I", and X is selected from CH and N, preferably X is N.

The arylfluorosulfate moiety may in some variants of the covalent inhibitors be combined with an amide, preferably further comprising a cyclobutane moiety and/or a fluorine atom. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by a formula selected from the group consisting of formulas (Vll-j) to (VII-o):

wherein R2, R4-R7, and n are as defined in "aspect I", and X is selected from CH and N, preferably X is N.

The arylfluorosulfate moiety may in some variants of the covalent inhibitors be combined with an aryl-SCh benzene ring substituted with a fluorine atom, preferably with fluorine positioned in the meta position. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (VII-p) or (Vll-q) :

wherein R2-R3, R6, and n are as defined in "aspect I", and X is selected from CH and N, preferably X is N.

The covalent inhibitors described herein includes also variants featuring a fluorosulfate-substituted pyridine for targeting SIRT5 in combination with different other functional moieties. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (VIII) or (VIII-2): wherein R2-R7 and n are as defined in "aspect I". In other variants of the covalent inhibitors, the fluorosulfate-substituted pyridine is combined with the aromatic side chain of the amino acid tryptophan. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by a formula selected from the group consisting of formulas (Vlll-a) to (Vlll-i):

 wherein R3-R7 and n are as defined in "aspect I".

In further variants of the covalent inhibitors, the fluorosulfate-substituted pyridine is combined with an amide, preferably further comprising a cyclobutane moiety and/or an aryl-SCh benzene ring substituted with a fluorine atom, preferably with fluorine positioned in the meta position. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by a formula selected from the group consisting of formulas (Vlll-j) to (VIII-o):

wherein R2, R4-R7, and n are as defined in "aspect I". In even further variants of the covalent inhibitor, the fluorosulfate-substituted pyridine is combined with an aryl-SCh benzene ring substituted with a fluorine atom, preferably with fluorine positioned in the meta position. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (VIII-p) or (Vlll-q) :

wherein R2-R3, R ₆ , and n are as defined in "aspect I".

The covalent inhibitors described herein include variants in which the

arylfluorosulfate moiety is combined with different other functional moieties that supports efficient inhibition of SIRT5. Some variants are based on the aromatic side chain of the amino acid tryptophan in combination with other functional moieties. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by a formula selected from the group consisting of formulas (IX-a) to (IX-i) :

wherein Ri, R3-R7 and n are as defined in "aspect l" or "aspect la".

Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (X-l) or (X-

wherein Ri, R3, R ₆ and n are as defined in "aspect la". Other variants of the covalent inhibitor are defined by the inclusion of amide, preferably further comprising a cyclobutane moiety and/or an aryl-S0 ₂ benzene ring substituted with a fluorine atom, preferably with fluorine positioned in the meta position. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by a formula selected from the group consisting of formulas (IX-j) to (IX-o):

wherein Ri, R2, R4-R7, and n are as defined in "aspect l" or "aspect la".

Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by a formula selected from the group consisting of formulas (X-3) to (X-6):

wherein Ri, R2, R6, and n are as defined in "aspect la". A further embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (X-6a) or

Yet other variants of the covalent inhibitor is based on an aryl-SCh benzene ring substituted with a fluorine atom, preferably with fluorine positioned in the meta position. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (IX-p) or (IX-q):

wherein Ri, R2-R3, R ₆ , and n are as defined in "aspect l" or "aspect la". Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is defined by formula (X-7) :

(X-7)

wherein Ri, R2-R3, R6, and n are as defined in "aspect la".

The covalent inhibitors described herein all comprise the a arylfluorosulfate moiety enabling the inhibitors to participate in a SuFFex reaction to covalent bind to SIRT5. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is capable of binding covalently to the sirtuin 5 enzyme.

The covalent inhibitors bind covalent to Tyrl02 of SIRT5 to effective block the active site of the SIRT5 and ensure efficient inhibition. Therefore, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is capable of binding covalently to a tyrosine residue of the sirtuin 5 enzyme. Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is capable of binding covalently to Tyrl02 of the sirtuin 5 enzyme. The capability of the covalent inhibitor to bind covalently to a tyrosine residue of the sirtuin 5 enzyme may be measured according to the methods described in Examples 1 and 2.

The covalent inhibitors described herein are based on the specific covalent binding of the arylfluorosulfate moiety to a tyrosine, lysine or serine residue. Thus, an aspect of the present invention relates to a covalent inhibitor of a target enzyme according to formula (I) : wherein :

Ri comprises an arylfluorosulfate group;

R2 is selected from amino acid side chains,

R3 is selected from H, COOR, CONH2, CONHR6 and triazolo, wherein R6 is selected from C3-C8 cycloalkanes, linear alkanes, aromatics and heteroaromatics,

R4 and Rs are independently selected from H, Cl, F, Br, I, CF3, NO2, C1-C10 alkyl esters, C1-C10 alkoxy, alcohols and CONH(CH2)R7, or R4 and Rs together form an aryl or heteroaryl ring system optionally substituted with one or more substituents selected from nitrogen, C1-C10 alkyl, alcohol, ether and halogen, wherein R7 IS selected from C2-C10 alkynes, azides, trans- cyclooctenes and cyclopropenes, and n is an integer selected from 2-6.

The target enzyme may be any enzyme which is susceptible to a sulfur(VI)- fluoride exchange (SuFEx) reaction. Thus, an embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the target enzyme comprises a tyrosine, lysine and/or serine residue. Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the covalent inhibitor is capable of binding covalently to a tyrosine residue of the target enzyme.

The covalent inhibitors react exclusively upon activation within the binding site of the target enzyme, i.e. the SuFEx reaction is only effected if the surrounding protein environment favours the reaction to occur. Thus, the surrounding protein environment requires basic residues, e.g. arginine (Arg), lysine (Lys) or histidine (His), that decreases the pK _a of the targeted nucleophilic residue and/or that facilitate fluoride ion departure (e.g. by hydrogen bonding interactions) to achieve the SuFEx reaction. Thus, an embodiment of the present invention relates to the inhibitor of the present invention, wherein one or more basic amino acid residues are located in close proximity to the tyrosine residue within the binding pocket of the target enzyme. Another embodiment of the present invention relates to the covalent inhibitor as described herein, wherein one or more basic amino acid residues are located less than 10 A, such that less than 8 A, such that less than 5 A, apart from the tyrosine residue within the binding pocket of the target enzyme. A further embodiment of the present invention relates to the covalent inhibitor as described herein, wherein the at least one or more basic amino acid residue is located within 10 amino acid residues of the tyrosine residue, such as within 8 amino acid residues, such as within 6 amino acid residues, such as within 5 amino acid residues, such as within 4 amino acid residues, such as within 3 amino acid residues, such as within 2 amino acid residues, such as within 1 amino acid residues of the tyrosine residue. A preferred embodiment relates to the covalent inhibitor as described herein, wherein the one or more basic amino acid residues are arginine and/or lysine residues.

The covalent inhibitors as described herein potently and selectively inhibits target enzymes which are susceptible to a sulfur(VI)-fluoride exchange (SuFEx) reaction. Specifically, the covalent inhibitors inhibit efficiently and selectively SIRT5.

Therefore, the covalent inhibitors as described herein are suitable for treatment of diseases which are associated with SIRT5 functioning. Diseases to which the enzymatic activity of SIRT5 are considered to be associated with include, but are not limited to, cancer, Alzheimer's disease, and Parkinson's disease.

Thus, an aspect of the present invention relates to a pharmaceutical composition comprising the covalent inhibitor of the sirtuin 5 enzyme as described herein or a pharmaceutically acceptable salt thereof.

An embodiment of the present invention relates to the pharmaceutical

composition as described herein, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant. Another embodiment of the present invention relates to the pharmaceutical composition as described herein, wherein the pharmaceutical composition further comprises one or more additional therapeutic agents.

Cancers represent a major health risk and many forms are still very

poorly managed clinically due to resistance development or metastases.

Therefore, the need for novel therapeutics with alternative mechanisms of action are needed. To this end, sirtuins, and specifically SIRT5, constitutes important regulatory enzymes with potential as drug targets. As the human sirtuin 5 isozyme has been implicated in tumorigenesis, the SIRT5 enzyme may be targeted with covalent inhibitors for the treatment of cancers.

Thus, an embodiment of the present invention relates to the pharmaceutical composition as described herein, wherein the one or more therapeutic agents are anti-cancer agents.

The covalent inhibitors as described herein may in principle be beneficial in combination with any type of anti-cancer agent. An embodiment of the present invention relates to the pharmaceutical composition as described herein, wherein the anti-cancer agents are selected from the group consisting of Doxorubicin, Paclitaxel, Docetaxel, Cisplatin, Oxaliplatin, Cetuximab, FOLFOX, 5-FU, Vorinostat, Romidepsin, Trastuzumab, Pertuzumab and Panitumumab.

5-FU is an abbreviation for the anti-cancer agent fluorouracil. FOLFOX is a combination drug, mainly for use in the treatment of colorectal cancer. FOLFOX consists of folinic acid, fluorouracil and oxaliplatin.

A preferred embodiment of the present invention relates to the pharmaceutical composition as described herein, wherein the anti-cancer agents are FOLFOX and/or Trastuzumab. Another embodiment of the present invention relates to the pharmaceutical composition as described herein, wherein the anti-cancer agents are Cetuximab and/or Panitumumab. An aspect of the present invention relates to a covalent inhibitor of the sirtuin 5 enzyme as described herein or the pharmaceutical composition as described herein for use as a medicament.

Another aspect of the present invention relates to a covalent inhibitor of the sirtuin 5 enzyme as described herein or the pharmaceutical composition as described herein for use in the amelioration or treatment of a disease selected from the group consisting of cancer, Alzheimer's disease, and Parkinson's disease. The covalent inhibitors are considered particularly relevant for treatment of cancer.

Thus, a further aspect of the present invention relates to a covalent inhibitor of the sirtuin 5 enzyme as described herein or the pharmaceutical as described herein for use in the amelioration or treatment of cancer.

The cancer may be, but is not limited to, acute lymphocytic leukaemia, acute myeloid leukaemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukaemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, soft tissue cancer, testicular cancer, thyroid cancer, ureter cancer, urinary bladder cancer, and digestive tract cancer such as, e.g., oesophageal cancer, gastric cancer, pancreatic cancer, stomach cancer, small intestine cancer, gastrointestinal carcinoid tumor, cancer of the oral cavity, colorectal cancer, and hepatobiliary cancer.

An embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the cancer is selected from the group consisting of glioblastoma, leukaemia, lymphoma, myeloma, ovarian cancer, breast cancer, colorectal cancer, lung cancer, skin cancer and pancreatic cancer.

A preferred embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the cancer is breast cancer or colorectal cancer.

Combination therapies are widely used to treat diseases such as cancer, tuberculosis, leprosy, malaria, and HIV/AIDS. Many combination therapies benefit from increased efficiency as compared to monotherapies and additional major benefit of combination therapies is that they reduce development of drug resistance since a pathogen or tumor is less likely to have resistance to multiple drugs simultaneously. Within oncology, it has furthermore been demonstrated that simultaneous administration of multiple targeted drugs minimizes the risk of relapse when no single mutation confers cross-resistance to both drugs. Thus, the covalent inhibitors as described herein may be especially effective and beneficial when used as part of a combination therapy.

Therefore, an embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the covalent inhibitor or pharmaceutical composition is administered as a combination therapy.

Another embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the combination therapy comprises one or more anti-cancer agents selected from the group consisting of Doxorubicin, Paclitaxel, Docetaxel, Cisplatin, Oxaliplatin, Cetuximab, FOLFOX, 5-FU, Vorinostat, Romidepsin, Trastuzumab, Pertuzumab and Panitumumab.

A further embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the cancer is breast cancer or colorectal cancer and the anti-cancer agents are FOLFOX and/or Trastuzumab. A preferred embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the cancer is breast cancer and the anti-cancer agent is Trastuzumab.

Another embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the cancer is colorectal cancer and the anti-cancer agent is FOLFOX.

Other treatments include combination of the covalent inhibitors of SIRT5 with EGF receptor inhibitors, such as Cetuximab and Panitumumab. Therefore, an embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, and head and neck cancer and the anti-cancer agent is Cetuximab and/or Panitumumab.

The covalent inhibitors and the pharmaceutical compositions described herein may be administered by any conventional method. Thus, an embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the covalent inhibitor or pharmaceutical composition and additional therapeutic agents are administered sequentially, separately or simultaneously.

In this regard it is noted that combination therapy as used herein refers to both co-formulations of the covalent inhibitor and additional therapeutic agents, as well as administration of the covalent inhibitor and additional therapeutic agents as separate entities. The route of administration may be any conventional route of administration, wherein the route of administration is chosen to fit the specific treatment. Therefore, an embodiment of the present invention relates to the covalent inhibitor or the pharmaceutical composition for use as described herein, wherein the covalent inhibitor or pharmaceutical composition is administered by a route selected from the group consisting of orally, parenterally, intravenously, intradermally, subcutaneously, and topically, in liquid or solid form. The components for the treatment described herein may be provided as a kit for easy application to a subject in need of the treatment. Thus, an aspect of the present invention relates to a kit comprising:

i. the covalent inhibitor of the sirtuin 5 enzyme as described herein or the pharmaceutical composition as described herein,

ii. one or more additional therapeutic agents, and

iii. optionally, instructions for use.

Instructions may include guidance with regard to route of administration, dosage regimen, etc. The therapeutic agents comprised in the kit may be similar to those described herein for the treatment of disease, such as cancer. Therefore, an embodiment of the present invention relates to the kit as described herein, wherein the one or more therapeutic agents are anti-cancer agents. Another embodiment of the present invention relates to the kit as described herein, wherein the anti-cancer agents are selected from the group consisting of Doxorubicin, Paclitaxel, Docetaxel, Cisplatin, Oxaliplatin, Cetuximab, FOLFOX, 5- FU, Vorinostat, Romidepsin, Trastuzumab, Pertuzumab and Panitumumab. A further embodiment of the present invention relates to the kit as described herein, wherein the covalent inhibitor or pharmaceutical composition of (i) and additional therapeutic agents of (ii) are for sequential, separate or simultaneous administration. The covalent inhibitors may be prepared by conventional synthetic steps. Thus, an aspect of the present invention relates to a method for preparing a covalent inhibitor of the sirtuin 5 enzyme as described herein, said method comprising the following steps:

i. provision of a compound according to formula (Yl), and

ii. contacting the compound according to formula (Yl) with a compound according to formula (Y2),

thereby obtaining a covalent inhibitor according to formula (I),

wherein R1-R7, n, and X are as defined herein.

Another aspect of the present invention relates to a method for preparing a covalent inhibitor of the sirtuin 5 enzyme as described herein, said method comprising the following steps:

i. provision of a compound according to formula (Yla), and

ii. contacting the compound according to formula (Yla) with a compound according to formula (Y2), thereby obtaining a covalent inhibitor according to formula (la), wherein Ri-Rs, n, m and X are as defined herein. An embodiment of the present invention relates to the method as described herein, wherein the free amino group of compound (Yl) or (Yla) is protected by a protection group. Another embodiment of the present invention relates to the method as described herein, wherein the protected amino group of compound (Yl) or (Yla) is deprotected prior to step (ii). Yet another embodiment of the present invention relates to the method as described herein, wherein the protection group is a NHBoc group. A further embodiment of the present invention relates to the method as described herein, wherein deprotection is performed by mixing with DCM/TFA/TIPS. In another embodiment of the present invention, the Cl atom of compound (Y2) may be substituted with any group capable of reacting with the free amino acid of compound (Yl) or (Yla), such as a halogen.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

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

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

Example 1 : Binding of covalent inhibitor to sirtuin 5 enzyme

Synthesis of covalent inhibitors

General procedure for amide bond formation 1 :

A solution of acyl chloride (2 eq) in CH2CI2 (20 mL/mmol) is treated with the amine (1 eq) in CH2CI2 (20 mL/mmol) at 0°C, followed by slow addition of N,N- Diisopropylethylamine (DIPEA) (3 eq) in dichloromethane (DCM). The reaction mixture is stirred at room temperature overnight, monitored by thin-layer chromatography (TLC). When the reaction finish, it is washed twice with 1M HCI, and extracted with CH2CI2. The combined organic layers are dried over Na2S04 and concentrated. The crude final product is purified by preparative high- performance liquid chromatography (HPLC) (10% B to 100% B) and the solvent is freeze dried to afford the pure product as a white solid. It is noted that A represents a heteroatom, such as N in the case of pyridine. R represents a fluorosulfate group.

Carboxylic acid (1.5 eq), 1-hydroxybenzotriazole (HOBt) (1.5 eq), iPrNH2 (3 eq) and the amine (1 eq) are dissolved in anhydrous CH2CI2 (10 mL/mmol) and cooled to 0 °C. l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.5 eq) is added and the reaction mixture is stirred at 0°C for 5 minutes and is then stirred overnight at ambient temperature. The reaction mixture is concentrated under reduced pressure and the crude residue is dissolved in ethyl acetate (EtOAc) and washed with aqueous KHSO4 (5%), saturated aqueous NaHC03, and brine. The organic phase is dried over Na2S04 and concentrated under reduced pressure. The crude final product is purified by preparative HPLC (10% B to 100% B) and the solvent is freeze dried to afford the pure product as a white solid. It is noted that R represents a fluorosulfate group.

3-fffS)-6-fffS)-l-fcvciobutviamino)-3-f lH-indoi-3-vi)-l-oxopropan-2-vi)amino)- 5-((3-fiuorophenvi)suifonamido)-6-oxohexyi)carbamovi)-5-meth viphenvi sulfurofluoridate - compound (V)

Compound (V) is synthesized according to the general procedure for amide bond formation 1 using 3-(chlorocarbonyl)-5-methylphenyl sulfurofluoridate (0.2 mmol), DIPEA (0.3 mmol) and (S)-6-amino-N-((S)-l-(cyclobutylamino)-3-(lH- indol-3-yl)-l-oxopropan-2-yl)-2-((3-fluorophenyl)sulfonamido )hexanamide (0.1 mmol) in 70% yield.

^! H NMR (600 MHz, DMSO-c/e) d 10.80 (d, J = 2.4 Hz, 1H, NH), 9.13 (d, J = 1.7 Hz, 1H, CH, Ar), 9.05 (d, J = 2.7 Hz, 1H, CH, Ar), 8.80 (t, J = 5.5 Hz, 1H, NH), 8.44 (dd, J = 2.7, 1.7 Hz, 1H, CH, Ar), 8.11 (d, J = 8.5 Hz, 1H, NH), 8.04 (d, J = 7.9 Hz, 1H, NH), 8.00 (d, J = 7.8 Hz, 1H, NH), 7.53 (dt, J = 8.5, 2.1 Hz, 1H, CH, Ar), 7.51 - 7.46 (m, 2H, 2 CH, Ar), 7.38 - 7.33 (m, 2H, 2 CH, Ar), 7.31 (dd, J = 8.1, 1.0 Hz, 1H, CH, Ar), 7.05 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H, CH, Ar), 7.01 (d, J = 2.3 Hz, 1H, CH, Ar), 6.96 (ddd, J = 7.9, 6.9, 1.0 Hz, 1H, CH, Ar), 4.22 (q, J = 7.0 Hz, 1H, CH), 4.07 (h, J = 8.1 Hz, 1H, CH), 3.78 (td, J = 8.5, 5.5 Hz, 1H, CH), 3.17 (dtd, J = 13.4, 8.5, 7.7, 4.3 Hz, 2H), 2.90 (dd, J = 14.4, 6.8 Hz, 1H), 2.74 (dd, J = 14.5, 6.9 Hz, 1H), 2.07 (dtt, J = 10.9, 7.6, 3.8 Hz, 1H), 1.98 (dtt, J = 13.8, 6.2, 3.9 Hz, 1H), 1.83 - 1.75 (m, 1H), 1.69 - 1.61 (m, 1H), 1.56 - 1.50 (m, 2H), 1.49 - 1.45 (m, 1H), 1.42 (p, J = 7.3 Hz, 3H), 1.24 (d, J = 5.9 Hz, 1H), 1.12 (ddd, J = 13.8, 9.8, 6.4 Hz, 1H). ¹³ C NMR (151 MHz, DMSO) d 170.2 (CO), 169.6 (CO), 162.6 (CO), 161.50 (d, J = 247.7 Hz, C, Ar), 148.7 (CH, Ar), 146.6 (C, Ar), 144.8 (CH, Ar), 143.0 (d, J = 7.2 Hz, C, Ar), 136.0 (C, Ar), 131.9 (C, Ar), 131.1 (d, J = 7.7 Hz, CH, Ar), 128.0 (CH, Ar), 127.4 (C, Ar), 123.4 (CH, Ar), 122.6 (d, J = 2.7 Hz, CH, Ar), 120.8 (CH, Ar), 119.3 (d, J = 21.4 Hz, CH, Ar), 118.4 (CH, Ar), 118.1 (CH, Ar), 113.6 (d, J = 24.2 Hz, CH, Ar), 111.2 (CH, Ar), 109.6 (C, Ar), 56.2 (CH), 53.2 (CH), 43.8 (CH), 40.1 (CH2), 32.5 (CH2), 30.0 (CH2), 29.9 (CH2), 28.2 (CH2), 27.8 (CH2), 22.4 (CH2), 14.6 (CH2). ¹⁹ F NMR (376 MHz, DMSO) d 40.2 (OS02F), -73.5 (CF3, TFA), -111.0 (F, Ar). HRMS calc for [M + H] ⁺ : C33H37F2N608S2 ⁺ : 747.2004, found : 747.2066. General scheme for the synthesis of alkyne containinq compounds:

fS 6-fffS l-fcvclobutylamino 3-f lH-indol-3-vn-l-oxopropan-2-vnamino 6- oxo-5-ff4-(prop-2-vn-l-ylcarbamovnphenvnsulfonamido ^’ )hexan-l-aminium - compound (XX!

A mixture of tert-butyl ((S)-5-amino-6-(((S)-l-(cyclobutylamino)-3-(lH-indol-3- yl)-l-oxopropan-2-yl)amino)-6-oxohexyl)carbamate (1 mmol), 4-(prop-2-yn-l- ylcarbamoyl)benzenesulfonyl chloride (2 mmol), and DIPEA (2.5 mmol) are dissolved in anhydrous DCM (25 ml.) and stirred overnight at ambient

temperature. The reaction mixture is concentrated under reduced pressure and the crude residue is purified by chromatography column (0®4% CH3OH in

CH2CI2) . N-boc deprotection is carried out by dissolving the compound in a mixture of DCM/TFA/TIPS (5: 3:0.5) and stirring at room temperature for 1 h. Volatiles are removed in vacuo to afford the sulfonamide XX (614 mg, 87%), as a white solid. It is noted that R represents a fluorosulfate group.

^! H NMR (600 MHz, DMSO-c/e) d 10.81 (d, J = 2.4 Hz, 1H, NH), 9.06 (t, J = 5.5 Hz, 1H, NH), 8.13 (d, J = 8.3 Hz, 1H, NH), 8.03 (dd, J = 9.8, 7.9 Hz, 2H, 2 NH), 7.94 - 7.88 (m, 2H, 2 CH, Ar), 7.82 - 7.76 (m, 2H, 2 CH, Ar), 7.70 - 7.61 (m,

3H, NH3), 7.48 (d, J = 8.0 Hz, 1H, CH, Ar), 7.33 - 7.29 (m, 1H, CH, Ar), 7.05

(ddd, J = 8.1, 6.9, 1.1 Hz, 1H, CH, Ar), 7.01 (d, J = 2.3 Hz, 1H, CH, Ar), 6.95

(ddd, J = 7.9, 6.9, 1.0 Hz, 1H, CH, Ar), 4.23 (q, J = 7.3 Hz, 1H, CH), 4.12 - 4.06

(m, 1H, CH), 4.06 - 4.03 (m, 2H, CH2), 3.72 (td, J = 8.4, 5.6 Hz, 1H, CH), 3.15 (t, J = 2.5 Hz, 1H, CH, alkyne), 2.94 (dd, J = 14.5, 6.6 Hz, 1H), 2.80 (dd, J = 14.5, 7.3 Hz, 1H), 2.63 (dq, J = 12.4, 5.9 Hz, 2H), 2.08 (dtd, J = 11.5, 7.4, 3.2 Hz, 1H), 1.97 (qt, J = 7.6, 3.8 Hz, 1H), 1.83 - 1.75 (m, 1H), 1.64 (tt, J = 10.5, 8.7 Hz, 1H), 1.54 (ddt, J = 13.7, 5.9, 3.6 Hz, 2H), 1.46 - 1.32 (m, 4H), 1.16 (tdd, J = 14.9, 10.4, 7.0 Hz, 1H), 1.10 - 0.99 (m, 1H). ¹³ C NMR (151 MHz,

DMSO) d 170.0 (CO), 169.7 (CO), 164.8 (CO), 143.3 (C, Ar), 136.9 (C, Ar), 135.9 (C, Ar), 127.9 (2 CH, Ar), 127.3 (C, Ar), 126.6 (2 CH, Ar), 123.5 (CH, Ar), 120.8 (CH, Ar), 118.4 (CH, Ar), 118.1 (CH, Ar), 111.1 (CH, Ar), 109.6 (C, Ar), 80.9 (C, alkyne), 73.1 (CH, alkyne), 56.2 (CH), 53.2 (CH), 43.8 (CH), 38.6 (CH2), 32.1 (CH2), 30.0 (CH2), 29.9 (CH2), 28.6 (CH2), 27.8 (CH2), 26.4 (CH2), 21.8 (CH2), 14.6 (CH2). HRMS calc for [M + H] ⁺ : C36H47N607S ⁺ : 707.3149, found :

707.3214. fcvclobutylaminoV3-f lH-indol-3-vn-l-oxoDroDan-2-vnaminoV

6-oxo-5-ff4-(prop-2-vn-l-ylcarbamovnphenvnsulfonamidoIhexync arbamovn- pyridin-3-yl sulfurofluoridate - compound (VI)

tert-Butyl ((S)-6-(((S)-l-(cyclobutylamino)-3-(lH-indol-3-yl)-l-oxoprop an-2- yl)amino)-6-oxo-5-((4-(prop-2-yn-l-yl-carbamoyl)phenyl)sulfo namido)hexyl)- carbamate (0.1 mmol) is dissolved in a mixture of DCM/TFA/TIPS (5: 3:0.5) and stirred at room temperature for 1 h. The volatiles are removed under vacuum to afford 4-(N-((S)-6-amino-l-(((S)-l-(cyclobutylamino)-3-(lH-indol-3- yl)-l- oxopropan-2-yl)amino)-l-oxohexan-2-yl)sulfamoyl)-N-(prop-2-y n-l-yl)benzamide

(TFA salt). Compound VI is then synthesized according to the general procedure for amide bond formation 2 using 5-((fluorosulfonyl)oxy)nicotinic acid (2 eq), HOBt (1.5 eq), / PrNHz (3 eq), EDC (1.5 eq) and 4-(N-((S)-6-amino-l-(((S)-l- (cyclobutylamino)-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)- l-oxohexan-2- yl)sulfamoyl)-N-(prop-2-yn-l-yl)benzamide (0.1 mmol, 1 eq) in 83% yield.

^! H NMR (600 MHz, DMSO-c/e) d 10.79 (d, J = 2.4 Hz, 1H, NH), 9.12 (d, J = 1.7 Hz, 1H, CH, Ar), 9.06 - 9.00 (m, 2H, NH and CH, Ar), 8.79 (t, J = 5.5 Hz, 1H,

NH), 8.44 (dd, J = 2.7, 1.7 Hz, 1H, CH, Ar), 8.12 (d, J = 8.2 Hz, 1H, NH), 8.02 (d, J = 8.0 Hz, 1H, NH), 7.98 (d, J = 7.8 Hz, 1H, NH), 7.92 - 7.87 (m, 2H, 2 CH, Ar), 7.82 - 7.74 (m, 2H 2 CH, Ar), 7.48 (dd, J = 7.8, 1.1 Hz, 1H, CH, Ar), 7.30 (dd, J = 8.1, 1.0 Hz, 1H, CH, Ar), 7.04 (ddd, J = 14.8, 6.8, 1.8 Hz, 2H, 2 CH, Ar), 6.95 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H, CH, Ar), 4.25 (q, J = 7.2 Hz, 1H, CH), 4.11 - 4.05 (m, 1H, CH), 4.05 - 4.02 (m, 2H, CH2), 3.73 (td, J = 8.3, 5.7 Hz, 1H, CH), 3.19 - 3.08 (m, 3H, CH2 and alkyne), 2.94 (dd, J = 14.5, 6.5 Hz, 1H), 2.80 (dd, J =

14.5, 7.2 Hz, 1H), 2.07 (dtt, J = 10.8, 7.6, 3.8 Hz, 1H), 1.97 (dtt, J = 10.9, 7.8,

3.9 Hz, 1H), 1.83 - 1.73 (m, 1H), 1.65 (tt, J = 10.6, 8.7 Hz, 1H), 1.56 - 1.50 (m, 2H), 1.48 - 1.44 (m, 1H), 1.38 (p, J = 7.3, 6.7 Hz, 3H), 1.23 - 1.15 (m, 1H),

1.12 - 1.03 (m, 1H). ¹³ C NMR (151 MHz, DMSO) d 170.2 (CO), 169.6 (CO), 164.8 (CO), 162.6 (CO), 148.7 (CH, Ar), 146.6 (C, Ar), 144.7 (CH, Ar), 143.3 (C, Ar),

136.9 (C, Ar), 135.9 (C, Ar), 131.9 (C, Ar), 128.0 (CH, Ar), 127.8 (2 CH, Ar), 127.3 (C, Ar), 126.6 (2 CH, Ar), 123.5 (CH, Ar), 120.8 (CH, Ar), 118.4 (CH, Ar), 118.1 (CH, Ar), 111.1 (CH, Ar), 109.6 (C, Ar), 80.9 (C, alkyne), 73.1 (CH, alkyne), 56.3 (CH), 53.1 (CH), 43.8 (CH), 40.1 (CH2), 32.4 (CH2), 30.0 (CH2),

29.9 (CH2), 28.6 (CH2), 28.3 (CH2), 27.7 (CH2), 22.4 (CH2), 14.6 (CH2). ¹⁹ F NMR (376 MHz, DMSO) d 40.2 (OS02F), -74.5 (CF3, TFA). HRMS calc for

[M + H] ⁺ : C37H41FN709S2 ⁺ : 810.2313, found : 810.2373.

Mass Spectrometric Determination of Covalent Labelling (MALDI-TOF)

His-SIRT5 (9 mM) is incubated with compounds (V) or (VI) (90 mM, 10 eq) in 50 mM Tris, pH 8, 150 mM NaCI in the presence of NAD+ (200 mM) at room

temperature. At different time points, adduct formation is stopped by mixing 2 pl _¬ ot the corresponding sample with 2 mI_ of matrix, and 1 pL of this mixture is placed on an 384 ground steel target plate and dried in air. A saturated solution of sinapic acid in H2O (0.1% TFA)/MeCN (1 : 1) is used as matrix. Spectra are obtained over the m/z range of 27.000-35.000, using a MALDI-TOF mass spectrometer. Peptide mass fingerprinting compound (V) and SIRT5

His-SIRT5 (15.48 mM) is incubated with compound (V) (154.8 mM, 10 eq) in 50 mM Tris, pH 8, 150 mM NaCI at room temperature overnight. Adduct formation is stopped by mixing 10 mI_ of the corresponding sample with 2 mI_ 0.5 mg/ml_ trypsin and 40 pL 0.1 M (NH4)2C03. The sample incubated at 37 °C for 6 h. The tryptic peptides are desalted using C18 ZipTips according to the manufacturer's protocol and analyzed by MALDI-TOF using aCHCA as matrix.

Peptide mass fingerprinting compound (VI) and SIRT5

His-SIRT5 (7.74 mM) is incubated with compound (VI) (77.4 mM, 10 eq) in 50 mM Tris, pH 8, 150 mM NaCI at room temperature for 5 h. Adduct formation is stopped by mixing 10 mI_ of the corresponding sample with 2 mI_ 0.5 mg/ml_ trypsin and 40 mI_ 0.1 M (NH4)2CC>3. The sample incubated at 37 °C overnight. The tryptic peptides are desalted using C18 ZipTips according to the manufacturer's protocol and analyzed by MALDI-FTICR with aCHCA as matrix.

Results

Positive ion mode MALDI-TOF mass spectra of SIRT5 before and after incubation at room temperature with the compounds (V) for 12 hours and alkyne-tagged compound (VI) at different time points show efficient time-dependent covalent labelling of recombinant SIRT5 (Figure 1A).

Peptide mass fingerprinting confirms that Tyrl02 in the SIRT5 active site is the targeted residue by identification of the peptide fragment 98-VWEFYHYRREVMGSK- 112 (according to SEQ ID NO: l) linked to the residual part of compound (VI) after fluoride displacement (Figure IB).

Conclusion

The example demonstrates that the compounds as described herein bind covalently to the target enzyme. Furthermore, it is demonstrated that the inhibitors bind to a residue in the enzyme active site of SIRT5, which establishes proof of the fundamental compound design. Example la : Binding of covalent inhibitor with different arylfluorosulfates to sirtuin 5 enzyme

Recombinant SIRT5 (2 mM) is incubated with covalent inhibitor (20 mM) and NAD ⁺ (200 mM) overnight at room temperature, followed by click chemistry with

Fluor488-N3, SDS-PAGE gel electrophoresis, and in-gel fluorescence

measurement.

Copper(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) "click" reaction

After incubation of covalent inhibitor, NAD ⁺ and enzyme in Tris buffer (pH = 8), Azide-AlexaFluor-488 (50 mM), CuSC>4 (100 mM), 2-(4-((bis((l-(tert-butyl)-lH- l,2,3-triazol-4-yl)methyl)amino) methyl)-lH-l,2,3-triazol-l-yl)acetic acid

(BTTAA) (100 mM), and sodium ascorbate (1 mM) are added. The click reactions are incubated at room temperature for two hours.

In-gel fluorescence SDS-PAGE

After the "click" reaction, 4X loading buffer (5 mI_) and 10X reducing agent (2 mI_) are added and the samples are heated to 85 °C for 10 minutes. The samples are then resolved by Sodium Dodecyl Sulfate - Poly Acrylamide Gel Electrophoresis (SDS-PAGE). The gel is then visualized on an image analysis system (excitation 473 nm, emission filter 558 nm).

Results

In-gel fluorescence showed covalent adduct formation between recombinant SIRT5 and compound (VI), compound (VI-c), compound (Vl-d) and compound (Vl-b). (Figure 1C).

Conclusion

This example demonstrates that different types of arylfluorosulfates are able to covalently bind SIRT5 as compound (VI), compound (VI-c), compound (Vl-d) and compound (Vl-b) are based on 4 different types of arylfluorosulfates. Example lb: Pull-down of endogenous sirtuin 5 enzyme from mitochondrial Ivsate from mammalian cells using covalent inhibitors with arylfluorosulfate

Mitochondrial lysate from HEK cells (400 pg) is incubated with covalent inhibitor (10 or 100 mM), NAD ⁺ (0 or 500 mM), and protease inhibitor (IX) overnight at room temperature, followed by click chemistry with Biotin-PEG3-N3, pull-down using streptavidin beads, SDS-PAGE gel electrophoresis, and western blot.

Copper(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) "click" reaction

After incubation of covalent inhibitor, NAD ⁺ , and protease inhibitor overnight Biotin-PEG3-N ₃ (500 mM), CuS0 ₄ (1 mM), BTTAA (2 mM), sodium ascorbate (20 mM), and SDS (0.1 %) are added. The click reactions are incubated at room temperature for two hours.

Pull-down procedure

The click reaction is quenched by addition of EDTA (5 mM). The protein content is then precipitated in a mixture of Me0H : H20:CHCl3 (volume ratio 2: 1 :0.5 compared to the sample volume). The mixture is pelleted by centrifugation (5 min, 14000 G, 4 °C) and the pellet is washed with ice cold MeOH (5 volumes compared to the sample volume after the click reaction), followed by

centrifugation (5 min, 14000 G, 4 °C) and removal of the MeOH. The pellet is dissolved in phosphate buffered saline (PBS) containing 2% SDS and sonicated until dissolved. The sample is diluted with PBS to contain 0.2% SDS and a protein concentration of 1 g/L. A sample of 13 pg of lysate is removed for blotting the sample before pull-down. The rest of the sample is incubated with streptavidin magnetic beads for 2 hours at room temperature under agitation. The beads are then washed five times with 1 ml. PBS containing 0.2% SDS. The enriched proteins are then eluted by boiling the beads for 10 minutes in 4X loading buffer (10 pl_) and 10X reducing agent (4 pL). The samples are then resolved by SDS- PAGE.

Western blot procedure

After elution from the beads in the pull-down step, the enriched proteins are resolved by SDS-PAGE followed by transfer to a PVDF membrane. The membrane is blocked with 5% skimmed milk in TBST at room temperature for 1 hour. Subsequently the membrane is washed 3 times with TBST for 5 min followed by incubation with anti-SIRT5 antibody in 5% bovine serum albumin in TBST

(1 : 1000) overnight at 4 °C. The membrane is washed 3 times with TBST for 5 min. Then the membrane is incubated with HRP conjugated secondary antibody in 2% skim milk in TBST (1 : 10,000) for 1 hour. After washing with TBST 3 times for 5 min and with TBS 1 time for 5 min the membrane is visualized using enhanced chemiluminescent reagents on an image analysis system. L = lysate in figures ID and E. Results

The western blot shows that the compounds (X6-b), (V2-c) or (VI) in

concentrations 10 mM or 100 pM pull down SIRT5 from the mitochondrial fraction of lysates from HEK293 cells both with and witout NAD ⁺ present (Figure 1D-E). Conclusion

This example demonstrates that the covalent inhibitors are able to covalently label endogenous SIRT5 in enriched mitochondrial fractions of cell lysate without spiking the lysate with recombinant SIRT5. Example 2: Inhibition of sirtuin 5 enzyme bv covalent inhibitor

SIRT5 pre-incubation assays

Sirtuin 5, inhibitor, and NAD ⁺ are preincubated for 5 min, 5h or 21 h at rt in 50 mM Tris, pH 8, 150 mM NaCI in a total volume of 253 pL. Preincubated sample (20 pL) is added to each well in a black low binding 96-well microtiter plate and mixed with substrate for a final volume of 25 pL. The following concentrations are used : SIRT5 (187.5 nM during pre-incubation, giving 150 nM after substrate addition), inhibitor (250/200 pM, 166.7/133.3 pM, 125/100 pM, 111.1/88.9 pM, 74.1/59.3 pM, 69.4/55.6 pM, 49.4/39.5 pM, 38.6/30.9 pM, 32.9/26.3 pM, 21.9/17.6 pM, 21.4/17.1 pM, 14.6/11.7 pM, 11.9/9.5 pM, 9.8/7.8 pM, 6.6/5.3 pM, 6.5/5.2 pM, 4.3/3.5 pM, 3.7/2.9 pM, 2.8/2.3 pM, 2.0/1.6 pM, 1.1/0.91 pM, 0.63/0.50 pM, 0.35/0.28 pM or 0.19/0.16 pM), NAD ⁺ (625/500 pM), and substrate (0/50 pM). The plate is incubated at 37 °C for 30 min. Then, trypsin and nicotinamide (25 pL, 0.4 mg/ml_ and 8 mM, respectively; final concentrations 0.2 mg/ml_ and 4 mM, respectively) are added and the assay development is allowed to proceed for 15 min at room temperature. DMSO concentration in the final assay solution does not exceed 2% (v/v) and control wells without either enzyme (negative control) or inhibitor (positive control) are included in each plate. The plates are analyzed using a plate reader with excitation at 360 nm and detecting emission at 460 nm. Fluorescence measurements (RFU) are converted to [AMC] concentrations based on a [AMC]-fluorescence standard curve.

Results

Compound (V) and alkyne-tagged compound (VI) show time-dependent enzymatic inhibition of SIRT5, consistent with time-dependent covalent labelling of recombinant SIRT5 and thus covalent inhibition (Figure 2).

Conclusion

The example demonstrates that the compounds not only bind to the target enzyme, but also inhibits its enzymatic activity, which is essential for biological activity.

Example 2a : Inhibition of sirtuin 5 enzyme bv a selection of different covalent inhibitors

SIRT5 pre-incubation assays

Sirtuin 5, covalent inhibitor, and NAD ⁺ are preincubated for 5 min, 4 h, 5 h, 16 h, 21 h or 24 h at room temperature in 50 mM Tris, pH 8, 150 mM NaCI. The preincubated sample is added to a well in a black low binding 96-well microtiter plate and mixed with substrate for a final volume of 25 pL. The following concentrations are used in the assay: 150 nM SIRT5, 500 pM NAD ⁺ , 50 pM substrate and varying concentrations of covalent inhibitor. The plate is incubated at 37 °C for 30 min. Then, 25 pL developer mix containing 0.4 mg/ml_ trypsin and 8 mM nicotinamide are added and the assay development is allowed to proceed for 15 min at room temperature. DMSO concentration in the final assay solution does not exceed 2% (v/v) and control wells without either enzyme (negative control) or covalent inhibitor (positive control) are included in each plate. The plates are analyzed using a plate reader with excitation at 360 nm and detecting emission at 460 nm. Results

Compound (VI-c) (Figure 8A), compound (Vl-d) (Figure 8B), compound Vl-b (Figure 8C), compound X-6b (Figure 8D), compound V-2c (Figure 8E), compound V-2b (Figure 8F), compound X-6a (Figure 8G) and compound VI-3a (Figure 8H) showed time-dependent enzymatic inhibition of SIRT5.

Conclusion

The example demonstrates that the compounds not only bind to the target enzyme, but also inhibits its enzymatic activity, which will be essential for biological activity.

Example 3: Stability of covalent inhibitor

Chemical Stability Assays

A 10 mM DMSO stock solution of compounds (V) or (VI) is diluted to a final concentration of 50 mM in aqueous Tris buffer (pH=8) at 37 degrees. Chemical stability of the compound is measured after incubation for 48 hours. All time points are measured simultaneously by liquid chromatography mass spectrometry (LC-MS) and the chemical stability is evaluated relative to the time=0 (Figure 3A). Stability in serum

A 10 mM DMSO stock solution of compounds (V or VI) is diluted to a final concentration of 100 pM in human serum and incubated at 37 °C. Then, 45 pL of sample is quenched with urea (50 pL, 6 M) at different time points (t=0, 15 min, and 1 h) and incubated at 4 °C for 10 min. Acetonitrile (100 uL) is added to the sample, which is incubated for another 10 min at 4 °C. The samples are

centrifuged for 30 min at 14000 g. The supernatant is mixed with 50 uL Milli-Q water and filtered in a 0.2 pm filter before analysis by UPLC (Figure 3B).

Similarly, the reversible inhibitor of Rajabi et al. (2017) is compared to

compounds (V) and (V-2c) (Figure 3C).

Results

After 48 hours, no sign of degradation in buffer is observed by LC-MS, showing excellent chemical stability of the arylfluorosulfate-containing compounds (Figure 3A). Stability of peptide-based compounds can be low in serum; however, both compounds exhibited intense peaks in the UPLC analysis after incubation for 1 hour (Figure 3B). The half life of compounds (V2-c) and (V) had a half-life 3.3 h and 1.3 h respectively, which is more the half time of the reversible inhibitor (half-life = 0.9 h) (Figure 3C).

Conclusion

It can be concluded that the arylfluorosulfate moiety and remaining scaffolds of compounds (V) and (VI) are highly chemically stable upon incubation in buffer (Figure 3A). Stability in serum is also demonstrated (Figure 3B-C).

Example 4: Specificity of covalent inhibitor against sirtuin 5 versus other sirtuin enzymes

Recombinant SIRT1-7 (2.5 mM) are incubated with compound (VI) (10 pM) overnight at room temperature, followed by click chemistry with Fluor488-N3, SDS-PAGE gel electrophoresis, and in-gel fluorescence measurement (Figure 4). Boxes represent the locations of the enzymes according to the corresponding Coomassie blue-stained gel.

Copper(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) "click" reaction

After incubation of compound and enzyme (12 h) or mitochondrial lysate (8 h) in Tris buffer (pH = 8), rhodamine 110-azide (for in-gel fluorescence) or diazo biotin azide (for streptavidin enrichment) is added (100 eq. to probe). Then, CuS04 (200 eq.), BTTAA (400 eq.), and sodium ascorbate (1000 eq.) are added. The click reactions are incubated at 25 °C for two hours.

In-gel fluorescence SDS-PAGE

After the "click" reaction, 4X loading buffer (5 pL) and 10X reducing agent (2 pL) are added and the samples are heated to 85 °C for 10 minutes. The samples are then resolved by Sodium Dodecyl Sulfate - Poly Acrylamide Gel Electrophoresis (SDS-PAGE). The gel is then visualized on an image analysis system (excitation 473 nm, emission filter 558 nm). Results

Incubation of recombinant SIRT1-7 with compound (VI) showed selectivity for covalent binding of SIRT5 over other SIRT isoforms. The in-gel fluorescence analysis demonstrates that only SIRT5, containing the Tyrl02-Argl05 motif in the appropriate location of the sirtuin pocket, forms the corresponding covalent adduct with the electrophilic warhead of compound (VI) (Figure 4).

Conclusion

This example establishes that the compounds as described herein covalently bind SIRT5 in a highly selective manner over the other six enzymes in the sirtuin class.

Example 5: Specificity of covalent inhibitor against sirtuin 5 in cell Ivsate

Recombinant SIRT5 (2.5 mM) is incubated with compound (VI) (0, 5, 10 or 20 mM), HEK293 whole cell extract (WCE) (80 pg), protease inhibitor (lx), and NAD+ (200 pM) in 50 mM Tris, pH 8, 150 mM NaCI overnight at room temperature, followed by click chemistry with Fluor488-N3, SDS-PAGE gel electrophoresis, and in-gel fluorescence measurement. Coomassie-stained gel is shown as control for enzyme loading. Results

Dose-dependent covalent labelling of SIRT5 (2.5 pM) by compound (VI) remains highly efficient in concentrated HEK293 lysate (total protein 8 mg/ml_) and shows minimal labelling of other proteins (Figure 5). Enzyme denaturation by pre-boiling SIRT5 for 5 minutes in Tris buffer (pH 8) completely abolishes covalent adduct formation (Figure 5).

Conclusion

This example demonstrates (i) that the inhibitor selectively forms a conjugate with SIRT5 when challenged with potential interference from a complex proteome environment, and (ii) that the active site composition is required for covalent labelling, which shows the low extent of off-target binding. Example 6: Cytotoxicity of covalent inhibitor against cancer cells

Cells are seeded into 96-well plates at 5,000-10,000 cells per well. After 24 h, test compounds are added to final concentrations ranging from 0.10 to 100 mM. Cells are incubated for 72 h and cell viability is measured (MTT assay kit) following the manufacturers protocol. Relative cell viability in presence of test compounds is normalized to the DMSO-treated controls after background subtractions. All viability assays are performed as biological duplicates in technical triplicate.

Results

Incubation of three different immortalized cancer cell lines with compounds (V), (VI) or a previously reported non-covalent, reversible inhibitor (Rajabi et al. 2017) reveal higher potencies of covalent, arylfluorosulfate-based inhibitors in general, as the non-covalent, reversible inhibitor does not exhibit cytotoxicity at the highest concentrations (Figure 6).

Conclusion

Covalent inhibitors of SIRT5 demonstrate the ability to kill cancer cells in culture. The previously reported, reversible inhibitor (Rajabi et al. 2017) was less efficient, suggesting poor cell permeability.

Example 7: Cell permeability of covalent inhibitor

1,800,000 cells are seeded into 10 cm plates. After 24 h, test compound (VI) is added to final concentrations ranging from 1 to 20 pM. Cells are incubated for additional 48 h and harvested, fractionated, and lysed to provide mitochondrial extracts. This is followed by click chemistry with biotin-N3, SDS-PAGE gel electrophoresis, and in-gel measurement of intracellular SIRT5 labelling by incubation with streptavidin conjugated to horseradish peroxidase (HRP).

Results

The mitochondrial extracts from cells incubated without compound or varying concentrations of compound (VI), resolved by gel electrophoresis and visualized by biotin-streptavidin-HRP show that several isoforms of SIRT5 are labeled with compound (VI) inside the mitochondria (Figure 7). Conclusion

It can be concluded that compound (VI) is able to cross the cell membrane of living cancer cells in culture and covalently bind to native SIRT5 inside the mitochondria.

Example 8: Efficiency of covalent inhibitor in mouse model

Nude mice (nu/nu, male, 5 weeks old) are injected subcutaneously with HCT116 or MCF7 cells (5 x 1,000,000 cells) stably expressing SIRT5 WT. Mice are then treated with compounds (V) or (VI) in varying dosing regiments using subcutaneous injection. The diameters of tumors are measured using calipers every 2 or 3 days. At the end point, the tumors are dissected and analyzed.

Results

It is expected that the results demonstrate a significant reduction in tumor size compared to vehicle-treated control animals.

Conclusion

It can be concluded from the expected results that SIRT5 inhibition by the compounds (V) and (VI) has the potential to kill cancer cells in vivo.'

References

Kalbas et al. (2018), J. Med. Chem., 61, 2460-2471

Rajabi et al. (2017), Angew. Chem. Int. Ed., 56, 14836-14842

WO 2014/197775 A2