Patent Application
20240391887
The present invention relates to new VASH inhibitors, particularly useful in the prevention and/or the treatment of VASH-peptidase associated disorders and/or as research tools. The present invention also relates to a conjugate of said VASH inhibitors with a biomolecule and its use as drug or as research tool.
Microtubules (MTs) are the major types of cytoskeletal elements, and are particularly abundant in neurons. They are formed by polymerization of alpha- and beta-tubulin heterodimers. Within a cell, MTs have the ability to grow and shrink, a phenomenon that has been termed dynamic instability. The dynamic behavior of MTs allows them to support numerous cellular processes including intracellular transport, cell motility, cell division and cell morphogenesis. The functional diversity of microtubules is due to the various and numerous post-translational modifications (PTM) that affect tubulins. Known PTMs include acetylation, polyglutamylation, polyglycylation, phosphorylation, and tyrosination/detyrosination cycles. These modifications have an impact on microtubule dynamics, their organization, and their interactions with other cellular compounds and compartments.
Dysfunctions of MTs are associated with neurodevelopmental disorders as well as neurodegeneration. Well-described dysfunctions have been linked to mutations either in tubulins, the building blocks of MTs, or MT-associated proteins (MAPs) that regulate MT functions. The binding of many MAPs is mediated by the C-terminal tails of α- and β-tubulin that protrude at the surface of MTs known to be heavily subjected to various posttranslational modifications. The most abundant tubulin modifications in neurons is polyglutamylation, which generates multiple glutamate sidechains of variable length on both α- and β-tubulin C-termini. The negatively charged glutamates are added to the C-terminal tails that provide the binding sites for various MT-associated proteins (MAPs) and molecular motors. Accordingly, polyglutamylation has been shown to regulate the activity or binding of a number of MT-interacting proteins. Early studies have suggested that the binding of neuronal MAPs such as tau, a key player in Alzheimer's disease, is regulated by MT polyglutamylation. More recent reports have demonstrated that the activity of MT severing enzymes such as spastin and katanin, which play important roles in axonal growth and plasticity, is also controlled by this modification. Furthermore, polyglutamylation appears to regulate the velocity and processivity of KIF1A kinesin motor protein, which is involved in the delivery of synaptic vesicles and as such regulates synaptic transmission. Taken together tubulin polyglutamylation controls various important neuronal processes. Polyglutamylation is reversible and catalyzed by the enzymes belonging to the Tubulin Tyrosine Ligase Like (TTLL) family. In contrast, the reverse enzymes are members of the Cytosolic Carboxypeptidase (CCP) family. As such, the overall level of tubulin polyglutamylation in a particular cell is established as a result of the competition between both activities.
The first tubulin modification to be discovered was detyrosination, which is specific to alpha-tubulin and consists of the removal of the very C-terminal tyrosine residue of the MT. The tyrosination/detyrosination cycle is particularly involved with microtubule regulation in the neuronal cells, muscle cells, and dividing cells. Altered microtubule detyrosination is thus involved in disorders such as neurodegenerative diseases, neuronal regeneration disorders, cancers, muscular dystrophies, heart diseases, vascular disorders, retinal degeneration, infertility or ciliopathies.
Although detyrosination has been discovered almost half a century ago, the enzymes involved in its generation, which possess tubulin carboxypeptidase (TCP) activity, have been identified only very recently (Nieuwenhuis J. et al. Science, 2017, 358 (6369): 1453-1456; Aillaud C. et al. Science, 2017, 358 (6369:1448-1453). The members of the vasohibin family VASH1 and VASH2 are the enzymes that catalyze this modification. Recent research notably led to the identification of reversible and irreversible inhibitors for this protein enzymatic activity which are of particular interest for the treatment of neurodegenerative disorders including but not limited to tauopathies (e.g. Alzheimer's disease, frontotemporal dementia, corticobasal dementia and others) as well as the above recited disorders (see WO 2019/016259).
The identification of VASHs as tubulin detyrosinases has been made possible by employing a high affinity purification step using custom-designed inhibitors compatible with click chemistry (Aillaud, C. et al., Science. 2017, 358 (6369): 1448-1453). The most efficient among said inhibitors, referred as compound EPO-Y (Aillaud et al. 2017), is composed of an active group called transepoxysuccinate (TES) ethyl ester linked to a tyrosine residue, represented as follows:
EPO-Y (also termed Epo-Y) is an irreversible VASHs inhibitor initially considered as potential candidate for the treatment of disorders involving altered microtubule detyrosination. However, this compound shows a moderate IC50 of about of 15 μM in cellulo. In light of potential biomedical applications, high specificity and potency for VASHs are required for an ideal inhibitor. A small and highly electrophile epoxide molecule such as EPO-Y might interact with free thiols present on other proteins and in cysteine protease active sites especially at high μM range concentrations. Additionally, it could react with nucleophilic lysine residues in the tyrosine kinases' active sites, resulting in a loss of specificity. Strikingly, the only other reported inhibitor of detyrosination, called parthenolide, which was identified in the past via a cell-based screen (Fonrose et al. Cancer Res 2007; 67 (7): 3371-8), does not inhibit VASHs in vitro even at highly elevated concentrations (Hotta T. et al., Curr. Biol., 2021, 31 (4): 900-907). This suggests that the observed in cellulo effect of parthenolide is not mediated by a direct inhibition of VASHs.
Thus, there is a need for improving the existing VASHs inhibitor specificity, potency and bioavailability in order to provide a suitable candidate useful in the treatment of disorders involving altered microtubule detyrosination. In this invention, we describe highly efficient low nanomolar to picomolar, in cellulo VASHs inhibitors as potential drugs for the treatment of neurodegenerative diseases.
Using medicinal chemistry, libraries of compounds were generated, measured the effect in an ideally predictive in vitro system and used bioinformatics to model and understand the structural properties responsible for the variations in biological activities of the compounds. Hence, the inventors have developed new VASHs inhibitors that present highly enhanced in vitro and/or in cellulo activity as compared to EPO-Y.
The present invention thus relates to a compound of the following formula (I):
and R3 is OH, R1 is not O—C1-C6 alkyl.
According to another aspect, the present invention relates to a pharmaceutical composition comprising at least one compound of formula (I) as defined above and at least one pharmaceutically acceptable excipient.
According to another aspect, the present invention also relates to the compound of formula (I) or the pharmaceutical composition as defined above for use as a drug.
The present invention also relates to the compound of formula (I) for use as a research tool.
According to another aspect, the present invention also relates to a conjugate comprising a compound of formula (I) linked to a biomolecule.
According to another aspect, the present invention relates to the conjugate as defined above for use as drug or as research tool.
The term “stereoisomers” used in this invention refers to configurational stereoisomers and more particularly to optical isomers.
In the present invention, the optical isomers result in particular from the different position in space of the substituents attached to X. The carbon atoms or the nitrogen atoms of the X group to which the substituents are attached thus represent chiral or asymmetric centres. Optical isomers that are not mirror images of one another are thus designated as “diastereoisomers”, and optical isomers, which are non-superimposable mirror images are designated as “enantiomers”.
An equimolar mixture of two enantiomers of a chiral compound is designated as a racemic mixture or racemate.
In the context of the present invention, depending of the position of the substituents attached to the X group, the compound of the present invention may be a diastereoisomer of configuration (S,S), of configuration (R,R), of configuration (S,R) or of configuration (R,S) as illustrated hereafter:
When the position of the substituents is not specified in the compound, said compound corresponds to any one of the above diastereoisomers or to a mixture of said diastereoisomers.
For the purpose of the invention, the term “pharmaceutically acceptable” is intended to mean what is useful to the preparation of a pharmaceutical composition, and what is generally safe and non-toxic, for a pharmaceutical use.
The term “pharmaceutically acceptable salt and/or solvate” is intended to mean, in the framework of the present invention, a salt and/or solvate of a compound which is pharmaceutically acceptable, as defined above, and which possesses the pharmacological activity of the corresponding compound.
The pharmaceutically acceptable salts comprise:
Acceptable solvates for the therapeutic use of the compounds of the present invention include conventional solvates such as those formed during the last step of the preparation of the compounds of the invention due to the presence of solvents. As an example, mention may be made of solvates due to the presence of water (these solvates are also called hydrates) or ethanol.
The term “halogen”, as used in the present invention, refers to a fluorine, bromine, chlorine or iodine atom.
The term “Cx-Cy aliphatic chain” designates a linear or branched hydrocarbon chain, completely saturated or containing one or more unsaturations, but not aromatic, comprising from x to y carbon atoms, notably from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms. According to the present invention, the term “aliphatic chain” includes substituted or unsubstituted, linear or branched, alkyl, alkenyl or alkynyl groups.
The term “C1-C6 alkyl”, as used in the present invention, refers to a straight or branched monovalent saturated hydrocarbon chain containing from 1 to 6 carbon atoms including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, and the like.
The term “C2-C6 alkenyl”, as used in the present invention, refers to a straight or branched monovalent unsaturated hydrocarbon chain containing from 2 to 6 carbon atoms and comprising at least one double bond including, but not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like.
The term “C2-C6 alkynyl”, as used in the present invention, refers to a straight or branched monovalent unsaturated hydrocarbon chain containing from 2 to 6 carbon atoms and comprising at least one triple bond including, but not limited to, ethynyl, propynyl, propynyl, butynyl, pentynyl, hexynyl and the like.
The term “aryl”, as used in the present invention, refers to an aromatic hydrocarbon group preferably comprising from 6 to 12 carbon atoms and comprising one or more fused rings, such as, for example but not limited to, a phenyl or naphthyl group. Advantageously, it is a phenyl group.
The term “heteroaryl”, as used in the present invention, refers to an aromatic group comprising one or several, notably one or two, fused hydrocarbon cycles in which one or several, notably one to four, advantageously one or two, carbon atoms each have been replaced with a heteroatom selected from a sulfur atom, an oxygen atom and a nitrogen atom, preferably selected from an oxygen atom and a nitrogen atom. It can be a furyl, thienyl, pyrrolyl, pyridyl, oxazolyl, isoxazolyl, thiazolyle, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolyl, isoquinolyl, quinoxalyl or indyl.
The term “C1-C6-alkyl aryl” or “C1-C6-alkyl heteroaryl” as used in the present invention, refers to an alkyl group as defined above substituted respectively by an aryl or a heteroaryl group as defined above. Advantageously, a “C1-C6-alkyl aryl” is a benzyl group.
In the context of the present invention, “optionally substituted” means that the group in question is optionally substituted with one or more substituents which may be selected in particular from halogen, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkene, C2-C6 alkyne, aryl, N3, oxo, NRaRb, CORc, CO2Rd, CONReRf, ORg, N+RhRiRj, CN and NO2 wherein Ra to Rj are, independently of one another, H, C1-C6 alkyl or aryl, preferably H or C1-C6 alkyl.
The term “C1-C6 haloalkyl” refers to a C1-C6 alkyl chain as defined above wherein one or more hydrogen atoms are replaced by a halogen atom selected from fluorine, chlorine, bromine or iodine, preferably a fluorine atom. For example, it is a CF3 group.
In the context of the present invention, “unsaturated” means that the hydrocarbon chain may contain one or more unsaturation(s), i.e. a double bond C═C or a triple bond C≡C, advantageously one.
The term “pharmaceutical composition” is meant in the framework of the present invention a composition having preventive and curative properties.
The term “peptide coupling” refers to a chemical reaction between an amine function and a carboxylic acid function. The peptide coupling will be advantageously carried out in the presence of a coupling agent, such as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), carbonyldiimidazole (CDI), hexafluorophosphate 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetraméthyluronium (HBTU), tetrafluoroborate 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium (TBTU), hexafluorophosphate O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium (HATU), (benzotriazol-1-yloxy)tripyrrolodinophosphonium hexafluorophosphate (PyBOP), 7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) or propylphosphonic anhydride; optionally associated with an additive or a base, such as N-hydroxy-succinimide (NHS), N-hydroxy-benzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), I-hydroxy-7-azabenzotriazole (HAt), N-hydroxysylfosuccinimide (sulfo NHS), dimethylaminopyridine (DMAP), diisopropylethylamine (DIEA) or N-methylmorpholine (NMM).
The term “VASH (enzyme)”, as used in the present invention, refers to a tubulin carboxypeptidase enzyme (TCPase) involved in microtubules detyrosination mechanisms associated with neurodegenerative disorders, mental disorders, neurological disorders, ciliopathies, cancers and muscular dystrophies.
The term “biomolecule” refers to a molecule having biological properties. In the context of the present invention, it refers to a protein, a peptide, a biomarker, such as a photolabeling agent, or a E3 ubiquitin ligase recruiter including but not limited to Thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, PROTAC6, ZNL-02-096 or d9A-2.
The term “PROTAC”, as used in the present invention, is shortened form of PROteolysis Targeting Chimeras. A “PROTAC E3 ligase recruiter” is a conjugate comprising two ligands connected by a linker useful as tool for selective and complete protein degradation. One of the ligands is intended to bind the protein targeted for degradation, while the other should target an ubiquitin ligase. As a consequence, PROTAC E3 ligase recruiter can bring into proximity the protein targeted for degradation and the ligase which leads to the (poly) ubiquitination, i.e. the addition of a single or multiple ubiquitins, which are small (8.6 kDa) regulatory proteins, of the target protein to a substrate protein. The (poly) ubiquitin tag, attached onto the protein targeted for degradation, sends it for degradation by the proteasome. Contrary to ‘conventional’ inhibitors, PROTAC E3 ligase recruiter induces selective proteolysis, thereby suppressing all its biological functions. In the context of the present invention, the protein targeted for degradation is the VASH enzyme. Thus, the ligand of the PROTAC E3 ligase recruiter intended to bind the VASH enzyme is a fragment of a compound of formula (I) and the ligand intended to target the ubiquitin ligase is typically the Thalidomide.
The term “prodrug” relates to a typically pharmacologically inactive or less active derivative of an active drug that undergoes in cellulo or in vivo biotransformation to release the active drug by chemical or enzymatic cleavages. By “pharmacologically inactive or less active derivative”, it is understood, in the context of the invention, that the prodrug does not have relevant activity with respect to the inhibition of the VASH active site in an in vitro setting. However, they are active in in cellulo and in vivo assays because such tests allow the transformations required to provide the active drug. Prodrugs can offer many advantages over parent drugs such as increased cellular penetration, solubility, enhanced stability, improved bioavailability, reduced side effects, and better selectivity. Activation of prodrugs can involve many enzymes including, but not limited to, oxidoreductases like CYP450 and DT-diaphorase as well as hydrolytic enzymes like carboxylesterase and β-glucuronidase.
In the context of the invention, the prodrug compounds as defined in the present disclosure may be inactive in vitro. However, the prodrug compounds possess increased cell penetration. After cell penetration, the prodrug compounds are hydrolyzed to provide the corresponding active drugs, i.e. the potent VASH inhibitors.
The compound according to the present invention can be in the form of a stereoisomer or a mixture of stereoisomers, such as a mixture of enantiomers or diastereoisomers, notably a racemic mixture. According to a specific embodiment, the compound of formula (I) is in the form of one diastereoisomer of configuration (S,S), of configuration (R,R), of configuration (S,R) or of configuration (R,S) as defined above.
The formula (I) as defined herein encompasses both active compounds, i.e drugs, and prodrugs thereof.
In particular, the term “prodrug” according to the present invention refers to compounds of formula (I) in which R3 is not OH.
Preferably, the prodrugs according to the present invention are compounds of formula (I) in which R3 is O—C1-C6 aliphatic chain, O-aryl, O—C1-C6 alkyl-aryl, O-heteroaryl, O—C1-C6 alkyl-heteroaryl or NHOH, wherein up to 4 methylene units of said aliphatic chain are optionally replaced by O, C(O), NH or N—C1-C6alkyl, said aliphatic chain, aryl, heteroaryl, alkyl-heteroaryl or alkyl-aryl being optionally substituted. In prodrugs, R3 is notably O—C1-C6 aliphatic chain, such as O—C1-C6 alkyl, in particular O-ethyl, or O—C1-C6 alkyl-aryl, such as O-benzyl.
In the context of the present invention, the term “drug” refers to compounds of formula (I) in which R3 is OH.
Typically, R3 together with the adjacent carboxyl group forms an ester group. When being converted from the prodrug to the drug, this ester formed by R3 together with the adjacent carboxyl group is typically hydrolyzed in the corresponding carboxylic acid.
In the context of the present invention, the drug differs from its corresponding prodrug in that the ester groups presents in the prodrug are converted to corresponding carboxylic acids in the drug. Preferably, the prodrug comprises only one ester group being formed by R3 together with the adjacent carboxyl group. Therefore, the drug corresponding to a given prodrug has typically the same formula as said prodrug except the R3 group. In other words, in a given prodrug and the drug thereof, substituents R1, X and R are typically respectively identical. Alternatively, the prodrug may comprise two or more ester groups. In such case, all the ester groups are converted to carboxylic acids in the corresponding drug.
Preferably, the compound of formula (I) is in the form of a diastereoisomer of configuration (S,S) and responds to the following formula (I-A):
In a more preferred embodiment, the compound of formula (I-A) corresponds to the following enantiomer (I-A′):
In a preferred embodiment, X is
preferably
and R1, R2 and R3 are as defined in the present disclosure.
In another particular embodiment, X is
preferably
and R1, R2 and R3 are as defined in the present disclosure.
According to this embodiment, R4 is notably H or a C1-C12 aliphatic chain wherein up to 4 methylene units are optionally replaced by O, C(O), NH or N—C1-C6alkyl, said C1-C12 aliphatic chain being optionally substituted with one or more OH groups. In particular, R4 is selected in the group consisting of H, COOH, C(O)—C1-C6alkyl, (CH2)p—C(O)—C1-C6alkyl, C(O)—(CH2)p—C(O)—C1-C6alkyl, C(O)NHOH, (CH2)p—C(O)—NHOH and C(O)—(CH2)p—C(O)—NHOH, p being an integer from 1 to 4, said C1-C6 alkyl being notably methyl or ethyl, preferably ethyl.
In another particular embodiment, X is
preferably
and R1, R2 and R3 are as defined in the present disclosure.
According to a preferred embodiment, R is OR2.
According to some embodiments, R2 is H, a C1-C6 aliphatic chain, such as C1-C6 alkyl, or C1-C6 alkyl-aryl, said aliphatic chain or alkyl-aryl being optionally substituted. Preferably, R2 is H, optionally substituted C1-C6 alkyl, or an optionally substituted C1-C6 alkyl-aryl, such as benzyl. When R2 is a C1-C6 alkyl, such as methyl or ethyl, it is notably unsubstituted or substituted with one or more halogen, OH, NH2, N(C1-C6alkyl)2 or N+(C1-C6alkyl)3, in particular N+(CH3)3. More preferably, R2 is H, C1-C6 alkyl or benzyl. Even more preferably, R2 is C1-C6 alkyl, such as ethyl, or benzyl.
According to some embodiments, R2 is preferably not H. In such embodiments, R2 is thus preferably a C1-C6 aliphatic chain, such as C1-C6 alkyl, or C1-C6 alkyl-aryl, said aliphatic chain or alkyl-aryl being optionally substituted, and more preferably R2 is C1-C6 alkyl, such as ethyl or benzyl.
According to another embodiments, R is NH—S(O)2—R9, with R9 being C1-C6 aliphatic chain or an aryl, said aliphatic chain or aryl being optionally substituted. Preferably, R9 is a C1-C6 alkyl, such as methyl or ethyl, or an optionally substituted aryl. When R9 is an aryl, notably a phenyl, it is preferably unsubstituted or substituted with OH or a C1-C6 alkyl, such as a methyl or an ethyl.
According to some other embodiments, R3 is OH, O—C1-C6 aliphatic chain, O—C1-C6 alkyl-aryl, or NHOH, said aliphatic chain being optionally substituted. Preferably, R3 is OH, optionally substituted O—C1-C6 alkyl, O—C1-C6 alkenyl, such as O-allyl, O—C1-C6 alkyl-aryl, such as O-benzyl, or NHOH. When R3 is a O—C1-C6 alkyl, such as O-methyl or O-ethyl, said alkyl is notably unsubstituted or substituted with one or more halogen, OH, NH2, NH—C1-C6alkyl, N(C1-C6alkyl)2 or N+(C1-C6alkyl)3, in particular N+(CH3)3. More preferably, R3 is OH or O—C1-C6 alkyl, in particular O-ethyl.
According to some embodiments, R1 is O—C1-C6 alkyl or O—C2-C6 alkenyl, provided that when R3 is OH, R1 is not O—C1-C6 alkyl. Preferably, R1 is O-methyl, O-ethyl or O-allyl, provided that when R3 is OH, R1 is not O—C1-C6 alkyl. When R1 is O—C1-C6 alkyl, R3 is preferably O—C1-C6 alkyl such as O-methyl or O-ethyl, unsubstituted or substituted with one or more halogen, OH, NH2, NH—C1-C6alkyl, N(C1-C6alkyl)2 or N+(C1-C6alkyl)3, in particular N+(CH3)3. More preferably, when R1 is O—C1-C6 alkyl, notably O-ethyl, R3 is O-ethyl. When R1 is O—C1-C6 alkyl, R2 is preferably not H.
According to other embodiments, R1 is NR1aR1b, with R1a being H or a C1-C6 alkyl, such as methyl or ethyl, and R1b being OH or C1-C6 alkyl, said alkyl being optionally substituted with C(O)OH, C(O)O—C1-C6 alkyl or an aryl. In such embodiments, R1 may notably be NHOH, NH—C1-C6 alkyl or N(C1-C6 alkyl)2, said alkyl, such as a methyl, an ethyl, a n-propyl or a n-butyl, being unsubstituted or substituted with C(O)OH, C(O)—C1-C6 alkyl, notably with C(O)-methyl or C(O)-ethyl, or with an aryl, such as a phenyl.
According to a preferred embodiment, R1 is
preferably
The compound of formula (I) may be a compound of the following formula:
in which R, R3, R5, R6, X and Y are as defined in the present disclosure.
Preferably, the compound of formula (I) is a compound of formula (I-Aa) having the following configuration:
in which R, R3, R5, R6, X and Y are as defined in the present disclosure.
More preferably, the compound of formula (I) is a compound of formula (I-A′a) having the following configuration:
in which R, R3, R5, R6, X and Y are as defined in the present disclosure.
Y may notably be
In such an embodiment, R5 is preferably OH, O—C1-C6 alkyl, O-aryl, O-heteroaryl, O—C1-C6 alkyl-aryl, O—C1-C6 alkyl-heteroaryl, C(O)OH, C(O)O—C1-C6 alkyl, C(O)NHOH, C(O)NH2, C(O)NH—C1-C6 alkyl, C(O)NH—O—C1-C6 alkyl, NH—C1-C6 alkyl, N(C1-C6 alkyl)2, NH—C(O)—C1-C6 alkyl or NH—S(O)2—R9, said alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkyl-aryl or alkyl-heteroaryl being optionally substituted and R9 being as defined above. More preferably, R5 is OH, O—C1-C6 alkyl, such as O-methyl or O-ethyl, O—C1-C6 alkyl-aryl, such as O-benzyl, C(O)NH—O—C1-C6 alkyl, such as C(O)—NH—O-tert-butyl, or NH(S(O)2—R9, with R9 being preferably a phenyl unsubstituted or substituted with OH or a C1-C6 alkyl, such as a methyl or an ethyl. In particular, R5 is O—C1-C6 alkyl and notably O-ethyl.
Alternatively, Y may be —(CH2)m—, with m being an integer from 0 to 6, preferably from 0 to 4. More preferably, m is 1 or 2, even more preferably m is 2. In the embodiment wherein Y is —(CH2)m, R5 is preferably C(O)OH, C(O)O—C1-C6 alkyl or C(O)NHOH, said alkyl being optionally substituted. More preferably, R5 is C(O)OH, C(O)OEt or C(O)NHOH, in particular COOH.
According to some embodiments, R6 is OH or O—C1-C6 aliphatic chain, such as O-methyl, O-ethyl or O-allyl, notably O-ethyl, in particular when Y is
According to other specific embodiments, R6 is NH—CH(R7)—(CH2)n—R8, in particular when Y is —(CH2)m—, notably is —(CH2)2—. In such embodiments, R7 is preferably H, C1-C6 alkyl, C1-C6 alkyl-aryl, such as benzyl or C1-C6 alkyl-heteroaryl, such as CH2-indole group. R8 is preferably C(O)NH2, aryl, such as phenyl, heteroaryl, such as indole, SH, or S—C1-C6alkyl, such as S—C(CH3)3 and n is preferably 0, 1, 2 or 3. In some embodiments, R7 is C1-C6 alkyl, R8 is C(O)NH2 and n is 0. In some other preferred embodiments, R7 is H, R8 is phenyl and n is 1, 2 or 3, preferably 1.
According to a preferred embodiment, when R3 is OH, R1 is
preferably
In a preferred embodiment, the compound of formula (I) is a compound of formula (I-A′a) as defined above, in which X, R and R3 are as defined above, R1 is
preferably
with:
R5 being OH, O—C1-C6 alkyl, such as O-methyl or O-ethyl, O—C1-C6 alkyl-aryl, such as O-benzyl, and R6 being OH or O—C1-C6 aliphatic chain, such as O-methyl, O-ethyl or O-allyl.
According to another preferred embodiment, the compound of formula (I) is of formula (I-A′) in which X is
R1 is O—C1-C6 alkyl, in particular O-ethyl, R is OR2 with R2 being preferably H or C1-C6 alkyl, in particular ethyl, and R3 is OH or O—C1-C6 alkyl, in particular O-ethyl.
In a more preferred embodiment, the compound of formula (I) is of formula (I-A′a) in which X is
R is OR2 with R2 being preferably H, C1-C6 alkyl, in particular ethyl, or benzyl, R3 is OH or O—C1-C6 alkyl, in particular O-ethyl, and R1 is
preferably
in which:
R5 and R6 are independently OH or O—C1-C6 aliphatic chain, such as O-methyl, O-ethyl or O-allyl, in particular O-ethyl.
In another more preferred embodiment, the compound of formula (I) is of formula (I-A′) in which X is
R1 is NR1aR1b, with R1a being preferably H and R1b being preferably C1-C6 alkyl, such as a methyl, an ethyl, a n-propyl or a n-butyl, said alkyl being substituted with C(O)-methyl or C(O)-ethyl, R is OR2 with R2 being preferably C1-C6 alkyl, in particular ethyl, and R3 is OH or O—C1-C6 alkyl, in particular O-ethyl.
According to a particular embodiment, the compound of formula (I) is of the following formula (I-bis):
and R3 is OH, R1 is not O—C1-C6 alkyl.
In particular, the compound of formula (I) is not:
Preferably, the compound of formula (I) is not
which is described in William R. Roush et al. Synthesis, 1999, p. 1500-1504.
Preferably, the compound of formula (I) is not
which is described as compound SD-139 in Aillaud, C. et al. Science, 2017, 358, p. 1448-1453, Supplementary material.
According to a particular embodiment, the compound of formula (I) is an amino-acid or peptide based-VASH inhibitor. By ‘amino-acid or peptide based-VASH inhibitor’ according to the invention, it means a VASH inhibitor containing an amino-acid or a peptidic moiety constituted of 1 to 20 amino acids, wherein the most C-terminal amino acid is selected from Y or F, and able to target and inhibit, at least partially, the activity of proteins having a tubulin carboxypeptidase activity, and thereby inhibit microtubule detyrosination.
The term “amino-acid moiety” refers to 1 amino acid and the term “peptidic moiety” refers to a moiety containing at least two amino acids and at most 20 amino acids. When the peptidic moiety comprises two or more amino acids, said amino acids are linked together by peptide bonds and chemically modified or not.
According to a preferred embodiment, the compound of formula (I) is selected among:
and the pharmaceutically acceptable salts and/or solvates thereof.
In a particular embodiment, the compound of formula (I) according to the invention is a prodrug as defined above, notably selected in the group consisting of:
In a particular embodiment, the compound of formula (I) according to the invention is a drug as defined above, notably selected in the group consisting of:
In a particular and preferred embodiment, the compound of formula (I) according to the invention is selected in the group consisting of compounds LV-80 to LV-124 or conjugates thereof.
In a particular embodiment, the compound of formula (I) according to the invention is the compound LV-80 or a conjugate thereof.
In a particular embodiment, the compound of formula (I) according to the invention is the compound LV-91 or a conjugate thereof.
In a particular embodiment, the compound of formula (I) according to the invention is the compound LV-104 or a conjugate thereof. LV-104 may be a prodrug of the corresponding active drug IBMT11.
In a particular embodiment, the compound of formula (I) according to the invention is the compound LV-111 or a conjugate thereof. LV-111 may be a prodrug of the corresponding active drug.
In a particular embodiment, the compound of formula (I) according to the invention is the compound IBMT23 or a conjugate thereof. IBMT23 may be a prodrug of the corresponding active drug.
The compound of formula (I) as described above, or a pharmaceutically acceptable salt and/or solvate thereof, may be obtained by a method comprising the following steps:
with a compound of formula (III):
wherein RZ is R1 as defined above or OH,
The compound of formula (III) may be obtained according to methods well-known from the skilled person in the art.
The reaction between compound of formula (II) and formula (III) is notably a peptide coupling as defined above.
The compound of formula (II) is notably
Optionally, additional steps of protection/deprotection and/or of functionalization well-known from the skilled person in the art may occur before or after the reaction between compounds of formula (II) and (III) to afford compound of formula (I) with the suitable substituents as described above.
In particular, when RZ is OH, the compound resulting from step (a) undergoes a peptide coupling with
to provide a compound of formula (I) in which R1 is
The one or more peptide coupling carried out in the method of preparation of compound of formula (I) are notably achieved in presence of PyAOP as coupling agent. Preferably, the base DIEA is also used.
The peptide coupling may be carried out on a solid support, notably by using a resin to which one of the reagents, the amine part or the acid part, is attached. Such methods are well-known from the skilled person in the art.
The present invention also relates to a conjugate comprising a fragment of a compound of formula (I) as described above linked to a biomolecule such as a peptide, a protein, a biomarker, as for example a photolabelling agent, such as Rhodamine, cyanines derivatives or fluoresceine, an affinity probe such as biotin, or a E3 ubiquitin ligase recruiter including but not limited to Thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, PROTAC6, ZNL-02-096 or d9A-2.
The term “fragment of a compound of formula (I)” refers to the compound of formula (I) in which one extremity is modified due to the bonding to a biomolecule, for example via a linker. Typically, the R1 group is thus modified for allowing said bonding. For instance, in a conjugate according to the invention, the fragment of a compound of formula (I) refers to the following moiety:
According to a particular embodiment, the conjugate according to the present invention has the following formula (I′):
According to a preferred embodiment, the conjugate of formula (I′) is a conjugate of formula (I′-A) having the following configuration:
In particular, the conjugate of formula (I′) is a conjugate of formula (I′-Aa) having the following configuration:
In the conjugate of formula (I′), in particular of formula (I′-Aa), X is preferably
When Y is —(CH2)m—, in particular —(CH2)2—, R5 is preferably C(O)NHOH, R6 is preferably NH—CH2—(CH2)n—R8, with R8 being advantageously aryl, such as phenyl, and n being advantageously 1, 2 or 3, notably 1. When Y is
R5 is preferably OH or O—C1-C6 alkyl, such as O-methyl or O-ethyl, in particular O-ethyl.
According to some embodiments, the linker L corresponds to a divalent radical derived from a C1-C12 aliphatic chain, wherein one or more methylene unit(s) are replaced by structural linkers selected from arylene or fragments —O—, —S—, —C(═O)—, —SO2— or —N(C1-C6 alkyl)-, wherein said aliphatic chain is unsubstituted or is substituted with one or more radicals selected from halogen, OH, a C1-C6 alkyl and/or a C1-C6 alkyl aryl group, such as benzyl group.
Preferably, the biomolecule is E3 ligase recruiter, notably Thalidomide, and the conjugate is thus a PROTAC E3 ligase recruiter.
In a preferred embodiment, the conjugate is thus a PROTAC E3 ligase recruiter of the following formula (II′):
in which X, Y, L, R2, R3 and R5 are as defined above.
For example, the PROTAC E3 ligase recruiter is
in which X is as defined above, in particular X is
In another embodiment, the biomolecule is an affinity probe and the conjugate of formula (I′) may be:
In another embodiment, the biomolecule is a photolabeling agent and the conjugate of formula (I′) may be:
in which a compound of formula (I) is linked to Rhodamine.
The present invention also relates to a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and at least one compound of formula (I) as described above or a pharmaceutically acceptable salt and/or solvate thereof.
The present invention also relates to a pharmaceutical composition comprising at least one conjugate as described above, such as a conjugate of formula (I′), or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.
The pharmaceutical compositions of the invention can be intended to oral or parenteral (including but not limited to subcutaneous, intramuscular, intravenous, ocular, intravitreal, topical, sublingual administration, preferably oral or intravenous administration. The active ingredient can be administered in unit forms for administration, mixed with conventional pharmaceutical carriers, to animals, preferably mammals including humans.
For oral administration, the pharmaceutical composition can be in a solid or liquid (solution or suspension) form.
A solid composition can be in the form of tablets, gelatin capsules, powders, granules and the like. In tablets, the active ingredient can be mixed with pharmaceutical vehicle(s) such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic and the like before being compressed. The tablets may be further coated, notably with sucrose or with other suitable materials, or they may be treated in such a way that they have a prolonged or delayed activity. In powders or granules, the active ingredient can be mixed or granulated with dispersing agents, wetting agents or suspending agents and with flavor correctors or sweeteners. In gelatin capsules, the active ingredient can be introduced into soft or hard gelatin capsules in the form of a powder or granules such as mentioned previously or in the form of a liquid composition such as mentioned below.
A liquid composition can contain the active ingredient together with a sweetener, a taste enhancer or a suitable coloring agent in a solvent such as water. The liquid composition can also be obtained by suspending or dissolving a powder or granules, as mentioned above, in a liquid such as water, juice, milk, etc. It can be for example a syrup or an elixir.
For parenteral administration, the composition can be in the form of an aqueous suspension or solution which may contain suspending agents and/or wetting agents. The composition is advantageously sterile. It can be in the form of an isotonic solution (in particular in comparison to blood).
The compound of formula (I), the pharmaceutically acceptable salt and/or solvate thereof, or the pharmaceutical composition according to the invention act as VASH inhibitors, meaning that it is able to inhibit the peptidase activity of VASH that catalyzes microtubules detyrosination. When this VASH peptidase activity is deregulated, and notably abnormally increased, it induces severe disorders as defined below.
The compounds of formula (I) in which X is
are notably irreversible VASH inhibitors, which covalently bind to the VASH enzymes.
The compounds of formula (I) in which X is
are notably reversible VASH inhibitors, meaning that VASH peptidase enzymatic activity may be recovered.
The present invention relates to a compound of formula (I) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, for use as a drug, in particular in the prevention and/or the treatment of a VASH peptidase activity associated disorder.
In other terms, the present invention relates to the use of a compound of formula (I) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, for the manufacture of a drug, notably intended in the prevention and/or the treatment of a VASH peptidase activity associated disorder.
In other terms, the present invention relates to the use of a compound of formula (I) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof for the prevention and/or the treatment of a VASH peptidase activity associated disorder.
In other terms, the present invention relates to a method for the prevention and/or the treatment of a VASH peptidase activity associated disorder comprising the administration to a person in need thereof of an effective dose of a compound of formula (I) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof.
According to another aspect, the present invention relates to a pharmaceutical composition according to the invention for use as a drug, in particular in the prevention and/or the treatment of a VASH peptidase activity associated disorder.
In other terms, the present invention relates to the use of a pharmaceutical composition according to the invention for the manufacture of a drug, notably intended in the prevention and/or the treatment of a VASH peptidase activity associated disorder.
In other terms, the present invention relates to the use of a pharmaceutical composition according to the invention for the prevention and/or the treatment of a VASH peptidase activity associated disorder.
In other terms, the present invention relates to a method for the prevention and/or the treatment of a VASH peptidase activity associated disorder comprising the administration to a person in need thereof of an effective dose of a pharmaceutical composition according to the invention.
According to another aspect, the present invention relates to a conjugate according to the invention for use as a drug, in particular in the prevention and/or the treatment of a VASH peptidase activity associated disorder.
In other terms, the present invention relates to the use of a conjugate according to the invention for the manufacture of a drug, notably intended in the prevention and/or the treatment of a VASH peptidase activity associated disorder.
In other terms, the present invention relates to the use of a conjugate according to the invention for the prevention and/or the treatment of a VASH peptidase activity associated disorder.
In other terms, the present invention relates to a method for the prevention and/or the treatment of a VASH peptidase activity associated disorder comprising the administration to a person in need thereof of an effective dose of a conjugate according to the invention.
A preferred embodiment relates to a PROTAC E3 ligase recruiter according to the present invention, notably of formula (I″) as defined above, for use as a drug, in particular in the prevention and/or the treatment of a VASH peptidase activity associated disorder. Such a PROTAC E3 ligase recruiter is able to selectively target VASH enzyme and to induce its total degradation by proteasome.
The conjugate of formula (I″) in which X is
is notably an irreversible PROTAC E3 ligase recruiter.
The conjugate of formula (I″) in which X is
is notably reversible PROTAC E3 ligase recruiter.
According to the present invention, a VASH peptidase activity-associated disorder is preferably selected from fibrosis, cancer and a tubulin carboxypeptidase associated disease.
In particular, the VASH peptidase activity associated disorder is a disorder involving altered microtubule detyrosination and/or polyglutamylation, notably selected from neurodegenerative diseases, such as Alzheimer disease or Parkinson disease, glaucoma, psychiatric disorders, neuronal disorders, cancers, such as colon cancer and neuroblastoma, muscular dystrophies, infertility, retinal degeneration, Purkinje cell sicknesses, infantile onset degeneration, male infertilities and ciliopathies, in particular neurodegenerative diseases, cancers and muscular dystrophies.
In particular, a VASH peptidase activity-associated disorder is a tubulin carboxypeptidase associated disease including, without being limited to, disorder involving altered microtubule detyrosination.
According to a particular embodiment, a disorder involving altered microtubule detyrosination may be selected from neurodegenerative diseases, such as Alzheimer disease or Parkinson disease, glaucoma, psychiatric disorders, and neuronal disorders, cancers, such as colon cancer and neuroblastoma, muscular dystrophies, infertility, retinal degeneration and ciliopathies, in particular neurodegenerative diseases, cancers and muscular dystrophies.
In another aspect, a VASH peptidase activity-associated disorder is a tubulin carboxypeptidase associated disease including, disorder involving polyglutamination.
According to a particular embodiment, a disorder involving polyglutamination may be selected from neurodegeneration, neurodevelopmental disorders, Purkinje cell sicknesses, infantile onset degeneration, ciliopathies, male infertilities, cancer, respiratory disorder, retinal degeneration, bleeding disorders, non-Mendelian inheretence disorders.
According to another aspect, the present invention relates to a compound of formula (I) according to the invention or a conjugate thereof for use as research tool for research and development activities, in particular selected in the group consisting of:
These R&D activities as examples of illustrative but not limitative uses of said compounds for formula (I) or conjugate thereof.
The said compounds for formula (I) or conjugate thereof is used in particular as positive control in said in vitro or in cellulo screening assays or methods.
The said compounds for formula (I) may be coupled for example but not limited to fluorescent dyes, UV-sensitive dyes, HRP, alkaline phosphatase, biotin.
In a particular embodiment, a compound of formula (I) or a conjugate thereof according to the invention is used in an in vitro screening assay of VASH inhibitors (primary screening assay), wherein the compound of formula (I) is used as positive control.
As an example, the screening assay is an immuno-assay wherein the compound of formula (I) or a conjugate thereof is coated on plates as positive control.
The invention also concerns an in vitro kit assay for a primary screening assay of VASH inhibitors comprising a compound of formula (I) or a conjugate thereof as positive control.
In another particular embodiment, a compound of formula (I) or a conjugate thereof according to the invention is used in an in cellulo screening assay of VASH inhibitors (secondary screening assay), wherein the compound of formula (I) or a conjugate thereof is used as positive control.
The invention also concerns an in cellulo kit assay for a secondary screening assay of VASH inhibitors comprising a compound of formula (I) or a conjugate thereof as positive control.
In a preferred embodiment, the in cellulo kit assay comprises CHL cell lines and a compound of formula (I) as positive control.
In another particular embodiment, a compound of formula (I) or a conjugate thereof according to the invention is used as tool to better understand the role of tubulin detyrosination, in particular in tubulin detyrosination associated disorder, such as its occurrence, aggressiveness and progression (for example cancer).
So the invention also concerns an in vitro screening assay for identification of VASH inhibitors comprising the steps of:
In a particular embodiment, the substrate of VASH1 or VASH2 enzyme is selected in the group consisting of purified and recombinant alpha-tubulin and recombinant engineered telokin as disclosed in the application WO2020/012002.
In a particular embodiment, the step (ii) of detection of the C-terminal free tyrosine uses a colorimetric assay using a tyrosinase.
In another particular embodiment, the in vitro screening assay is an immuno-assay, in which the step (ii) comprises:
In particular, the antibody is labelled with a marker selected in the group consisting of an enzyme, a fluorescent compound or fluorophore, a (chemo) luminescent compound or a radioactive element, preferably an enzyme and more preferably a peroxidase.
In particular, the immunoassay is an enzyme immunoassay, a fluoroimmunoassay, a luminescent immunoassay or a radioimmunoassay, preferably a dot-blot or an enzyme immunoassay (ELISA).
In a particular embodiment, the in vitro screening immunoassay for identification of VASH inhibitors occurs in solid phase, wherein:
The present invention also concerns a kit for performing the in vitro screening immuno-assay for identification of VASH inhibitors as disclosed above, comprising:
In another particular embodiment, a compound of formula (I) or a conjugate thereof according to the invention is used in an in cellulo method for quantifying VASH peptidase activity.
The present invention also concerns an in cellulo screening assay for identification of VASH inhibitors comprising:
The present invention also concerns a kit for performing the in cellulo screening assay for identification of VASH inhibitors comprising:
In another particular embodiment, a compound of formula (I) or a conjugate thereof according to the invention is used in an in vitro diagnostic method for detecting a VASH peptidase activity associated disorder.
So the invention also concerns a kit assay for in vitro diagnostic method, wherein the compound of formula (I) or a conjugate thereof is used as positive control.
All of the Fluorenylmethyloxycarbonyl (Fmoc) protected amino acids and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU) were provided by Iris Biotech GmbH. Piperidine, N,N-Diisopropylethylamine (DIEA), TFA, Triisopropylsilane (TIS), dichloromethane (DCM), 1,2-Dichloroethane (DCE), N,N-Dimethylformamide (DMF), N-methylpyrrolidinone (NMP) and ethyl ether (Et2O) were provided by Sigma Aldrich. AmphiSpheres 40 RAM 0.38 mmol/g 75-150 μm resin was purchased from Agilent Technologies. Solvents used for HPLC and LC/MS were of HPLC grade.
Peptide synthesis was performed using a standard SPPS protocol. Each synthesis was performed using Fmoc-Rink amide AmphiSpheres 40 resin (0.37 mmol/g). The Fmoc protected amino acids (4 eq), HATU (4 eq) and DIEA (6 eq) were added to the syringe reactor and the mixture was stirred for 1 hour at room temperature. After each coupling reaction, the peptide-resin was submitted to two 5 min deprotection cycles with DMF/piperidine 80/20 v/v solution.
All crude compounds were purified by preparative HPLC (Waters 4000 apparatus) on a C18 reversed-phase column (C18 Deltapak column, 100 mm×40 mm, 15 μm, 100 Å) at a flow rate of 50 mL/min of a H2O+0.1% TFA and CH3CN+0.1% TFA mixture in gradient mode with UV detection at 214 nm. Fractions containing the pure product were collected and lyophilized.
Samples were prepared in an acetonitrile/water (50/50 v/v) mixture containing 0.1% TFA. The LC/MS system consisted of a Waters Alliance 2690 HPLC coupled to a Micromass (Manchester, UK) ZQ spectrometer (electrospray ionization mode, ESI+). All analyses were carried out using a C18 Chromolith Flash 25×4.6 mm column. A flow rate of 3 mL/min and a gradient of 0-100% Acetonitrile over 5 min of a H2O+0.1% HCOOH and CH3CN+0.1% HCOOH mixture in gradient mode with UV detection at 214 nm. Positive-ion electrospray mass spectra were acquired at a solvent flow rate of 100-200 μl/min. Nitrogen was used for both the nebulizing and drying gas. The data were obtained in a scan mode ranging from 200 to 1700 m/z in 0.1 s intervals; ten scans were summed up to obtain the final spectrum. Retention times are given in minutes. Solvents used for HPLC and LC/MS were of HPLC grade.
1.2.1) Synthesis of compounds of formula (I) according to the present invention
Boc-Tyr(Bn)-OH (2 g, 5.4 mmol, FLUKA AG) was dissolved in 3 ml of anhydrous DMF followed by Cs2CO3 (1.8 g, 5.4 mmol). The RM was stirred at RT during 5 min before allyl bromide (696.6 μl, 8.1 mmol) was added. After a 2-hour-long reaction at RT, RM was diluted in brine and EtOAc; then extracted 2 times. The regrouped organic phases were then washed 2 times with 1M KHSO4 solution, 2 times with NaHCO3 saturated solution and 2 times with brine; then organic phase was dried on MgSO4, filtered, and concentrated to afford Boc-Tyr(Bn)-OAll (2.45 g, yield: 110%, tR=2.19, MS (ESI+): m/z=412.3 [M+H]+). The product was used as it is without further purification for the next step.
Boc-Tyr(Bn)-OAll (2.22 g, 5.4 mmol) was dissolved in 15 ml of DCM, followed by 15 ml of TFA/TIS/H2O 95/2.5/2.5 v/v/v. The RM was then stirred for 1 h10 at RT. The RM was concentrated dry, and the remaining oil was precipitated with cold ether. The precipitate was filtered and dried over reduced pressure to afford compound 1 as a white solid. The product was used as it is without further purification.
(2,3S)-Oxirane-2,3-dicarboxylic acid (66 mg, 0.5 mmol) was dissolved in 3 ml of NMP, followed by compound 1 (212 mg, 0.5 mmol) and DIEA (374 μl, 2 mmol). The RM was stirred and 260 mg (0.5 mmol) of PyAOP were added. The RM was stirred at room temperature for 20 min, and then poured in 50 ml of cold water and kept in ice until precipitation appeared. The precipitate was removed by filtration and the filtrate pH was adjusted to 3 with 1M HCl solution. The filtrate was then extracted 3 times with EtOAc and the organic phase was washed 3 times with 1M HCl solution, one time with brine, dried over MgSO4 and evaporated dry to afford 100 mg of compound 2 as transparent oil. The product was used as it is without further purification.
HPLC purity=95%, tR=3.33, MS(ESI+): m/z=426.2 [M+H]+, (expected m/z=426.16)
Fmoc-Aad(OtBu)-OH (1 g, 2.28 mmol) was dissolved in 5 ml of NMP followed by the 2-phenylethan-1-amine (316 μl, 2.5 mmol) and the DIEA (1.175 ml, 6.83 mmol). The RM was stirred and the PyAOP was added (1.18 g, 2.28 mmol). The RM was stirred for 15 min at RT. It was then poured in 50 ml of EtOAc, washed 1 time with water/brine 50/50 v/v mixture, 3 times NaHCO3 saturated solution, 3 times 1M HCl solution, 1 time with brine, dried over MgSO4 and concentrated dry to afford 1.24 g of compound 3 as transparent oil. The compound was used without further purification.
Compound 3 (542 mg, 1 mmol) was dissolved in 5 ml of NMP followed by the addition of the diethylamine (208 μl, 2 mmol). The RM was then stirred at RT for one hour. Next, the RM was poured in 50 ml of water and the pH was adjusted to 11 by 1M NaOH solution. The aqueous phase was then extracted 3 times with EtOAc, the regrouped organic phases were washed with brine, dried over Na2SO4 and concentrated dry to afford 294 mg of compound 4 as transparent oil. The product was used without any further purification.
Compound 4 tert-butyl(S)-5-amino-6-oxo-6-(phenethylamino) hexanoate (131 mg, 0.41 mmol) and compound 2 HO-TES-Tyr(OBn)-OAll (174 mg, 0.41 mmol) were solubilized in DMF followed by the addition of DIEA (139 μl, 0.82 mmol) and BOP (173 mg, 0.41 mmol). After 2 hours, the product was isolated by extraction. The obtained white powder was then dried over night at 0.2 mbar to afford 260 mg of intermediate A as white solid.
(+,−)-trans-Oxirane-2,3-dicarboxylic acid (200 mg, 1.52 mmol, TCI chemicals) was dissolved in 5 ml of NMP, followed by the HCl·H-Tyr (OtBu)-OtBu (500 mg, 1.52 mmol, Iris Biotech GmbH) and the DIEA (1.340 ml, 8 mmol). The PyAOP was then added (792 mg, 1.52 mmol) and the reaction mixture (RM) was stirred for 80 min at room temperature (RT). The RM was then poured in 180 ml of cold water. The obtained precipitate was removed by filtration. The pH of the filtrate was then adjusted to 3 with 1M KHSO4 solution and extracted 3 times with EtOAc. The organic phases were regrouped, washed with brine, dried over Na2SO4 and concentrated dry to afford 433 mg of compound 5 as transparent oil.
Intermediate A (100 mg, 0.137 mmol) was dissolved in 15 ml of DCM, followed 15 ml of TFA/TIS/H2O. The RM was then stirred for 30 min at RT. The RM was concentrated dry and the remaining oil was precipitated with cold EtOEt. The precipitate was filtered and dried over reduced pressure. Next, the precipitate was dissolved in 1 ml of dry DMF and the allyl ester was removed by treatment with Pd0 Tetrakis (5.15 mg, 0.0045 mmol) and PhSiH3 (37 μl, 0.3 mmol). After 20 min the RM was dissolved in 20 ml EtOAc that was washed 1 time with 1M HCl solution, 1 time with brine and concentrated dry. The obtained precipitate was then purified by preparative HPLC to obtain 41 mg of LV-80 as white solid.
Intermediate A (100 mg, 0.137 mmol) was dissolved in 15 ml of DCM, followed 15 ml of TFA/TiS/H2O. The RM was then stirred for 30 min at RT. The RM was concentrated dry and the remaining oil was precipitated with cold Et2O. The precipitate was filtered and dried over reduced pressure to obtain Intermediate B.
Intermediate B (15 mg, 0.022 mmol) was dissolved in NMP, followed by the addition of cesium carbonate (7.15 mg, 0.022 mmol) and iodoethane (2.34 μl, 0.029 mmol). The RM was then stirred at RT for 1 hour. Next, the RM was poured in 30 ml of water. The aqueous phase was extracted 3 times with EtOAc. The regrouped organic phases were washed 3 times with saturated NaHCO3, 3 times with KHSO4 1M then washed with brine, dried over Na2SO4 and evaporated dry to afford 17 mg white solid. Next, the white solid was dissolved in 1 ml of dry DMF and the allyl ester was removed by treatment with Pd0 Tetrakis (1 mg, 0.00087 mmol), PhSiH3 (3 μl, 0.024 mmol). After 20 min the RM was dissolved in 20 ml EtOAc that was washed 1 time with 1M KHSO4 solution, 1 time with brine and concentrated dry. The obtained precipitate was then purified by preparative HPLC to obtain 8.1 mg of LV-86 as white solid.
1H NMR (500 MHZ, DMSO) δ 8.70 (s, 1H), 8.60 (s, 1H), 8.18 (t, J=5.5, 1H), 7.44 (d, J=7.5, 2H), 7.38 (t, J=7.4, 2H), 7.32 (t, J=7.3, 1H), 7.27 (t, J=7.5, 2H), 7.21-7.16 (m, 2H), 7.14 (d, J=8.3, 2H), 6.93 (d, J=8.2, 2H), 5.05 (s, 2H), 4.38 (d, J=4.6, 1H), 4.23 (dd, J=13.6, 7.6, 1H), 4.04 (q, J=7.1, 2H), 3.57 (d, J=5.1, 2H), 3.22 (td, J=13.0, 6.9, 2H), 3.02 (dd, J=13.8, 4.4, 1H), 2.84 (dd, J=13.8, 9.4, 1H), 2.69 (t, J=7.2, 2H), 2.24 (t, J=7.2, 2H), 1.57 (m, J=6.7, 1H), 1.50 (m, 1H), 1.45 (m, J=16.4, 8.0, 3H), 1.17 (t, J=7.1, 3H) 13C NMR (126 MHz, DMSO) δ 172.63, 172.50, 170.67, 165.65, 165.49, 157.11, 139.33, 137.20, 130.23, 129.57, 128.73, 128.46, 128.32, 127.85, 127.79, 126.15, 114.52, 69.15, 59.80, 53.95, 52.58, 52.46, 52.35, 50.63, 35.77, 35.04, 33.09, 31.52, 20.80, 14.17.
Intermediate B (10 mg, 0.015 mmol) was dissolved in anhydrous THF. N-Methylmorpholine (3.82 μL, 0.030 mmol) was added to the solution. The reaction mixture was then cooled down to −15° C. and stirred for 5 min. Isobutylchloroformate (1.94 μL, 0.015 mmol) was added, and the mixture was stirred for 10 min at −15° C. O-Tritylhydroxylamine (8.25 mg, 0.030 mmol) was added and the stirring continued for 15 min at −15° C. and 30 min at room temperature. Afterward, the mixture was poured into EtOAc (20 mL) washed 1M KSHO4, saturated solution of NaHCO3, dried over MgSO4 and concentrated dry to afford 14 mg of intermediate C as a white powder
Intermediate C (13.92 mg, 0.015 mmol) was dissolved in 10 ml of DCM, followed 10 ml of TFA/TiS/H2O. The RM was then stirred for 30 min at RT. The RM was concentrated dry and the remaining oil was precipitated with cold EtOEt. The precipitate was filtered and dried over reduced pressure. Next, the precipitate was dissolved in 0.5 ml of dry DMF and the allyl ester was removed by treatment with Pd0 Tetrakis (1 mg, 0.00087 mmol), PhSiH3 (2.2 μl, 0.0175 mmol). After 20 min the RM was dissolved in 20 ml EtOAc that was washed 1 time with 1M HCl solution, 1 time with brine and concentrated dry. The obtained precipitate was then purified by preparative HPLC to obtain 2 mg of LV-87 as white solid.
1H NMR (500 MHZ, DMSO) δ 8.80 (d, J=8.0, 1H), 8.70 (s, 1H), 8.61 (d, J=8.1, 1H), 8.17 (t, J=5.6, 1H), 7.44 (d, J=7.2, 2), 7.38 (d, J=14.8, 2H), 7.32 (t, J=7.3, 1H), 7.29-7.26 (m, 2H), 7.20-7.17 (m, 3H), 7.15 (d, J=8.6, 2H), 6.94 (d, J=8.7, 2H), 5.06 (s, 2H), 4.44-4.38 (m, 1H), 4.26-4.19 (m, 1H), 3.58 (s, 2H), 3.26-3.20 (m, 2H), 3.02 (dd, J=13.8, 4.6, 1H), 2.84 (dd, J=13.9, 9.4, 1H), 2.72-2.67 (m, 2H), 1.90 (dd, J=13.0, 6.6, 2H), 1.60-1.53 (m, 1H), 1.49-1.43 (m, 2H), 1.42-1.37 (m, 1H).
13C NMR (500 MHZ, DMSO) δ 172.50, 168.75, 165.75, 165.45, 157.14, 139.32, 137.18, 130.20, 128.71, 128.45, 128.35, 127.84, 127.78, 126.15, 114.43, 69.14, 53.82, 52.53, 52.37, 48.50, 35.07, 31.98, 31.79, 30.14, 21.63.
The H-Aad(tBu)Phe-sequence was synthetized via SPPS on cystamine-Trt resin (CA) charged 0.5 mmol/g. Next, the solid supported sequence was attached via an amide bond to compound HO-TES-Tyr(OBn)-OAll (95 mg, 0.225 mmol) via PyAOP (95 mg, 0.225 mmol) and DIEA (77 μl, 0.450 mmol) activation, overnight at RT. After 2 hours, Pd Tetrakis (8.66 mg, 0.008 mmol) and PhSiH3 were added, leading to the deprotection of the OAll ester. The thiolated version of LV-80 was cleaved from the resin by a 2 hours long treatment with TFA/DCM/H2O/TiS 50/50/2.5/2.5. The filtrate was then concentrated dry to provide a brown oil that was precipitated in cold Et2O to obtain a white solid that was isolated by filtration. The compound was then purified by preparative HPLC to obtain 32 mg of LV-81 as white solid.
HCl·H-Tyr-OEt (100 mg, 0.407 mmol) and (2S,3S)-trans-Oxirane-2,3-dicarboxylic acid (80.8 mg, 0.611 mmol) were dissolved in 1.5 mL of NMP, followed by the addition of DIEA (1,628 mmol, 374 μL). The mixture was stirred and HATU (309.5 mg, 0,814 mmol) was added. The mixture was stirred at room temperature for 20 min. The reaction mixture was poured in a Na2CO3 1 M solution and extracted 3 times with 30 ml of diethyl ether. The aqueous phase was acidified with KHSO4 1M and the resulting solution was extracted 3 times with 30 mL of ethyl acetate. The organic layers were combined, washed with brine then dried over MgSO4, filtered and concentrated under reduced pressure to afford intermediate D as a yellow oil. The product was used as it is without further purification.
Cs2CO3 (314 mg, 0.966 mmol) was added to Intermediate D (104 mg, 0.321 mmol) dissolved in anhydrous DMF and the reaction was stirred for 15 min. Iodoethane (77.6 μL, 0.966 mmol) was added to the solution, and the reaction was stirred at room temperature overnight and monitored by HPLC. The solution was poured in 30 ml of water and extracted 3 times with 30 ml of ethyl acetate then washed 3 times with a solution of NaHCO3 1 M and 3 times with a solution of KHSO4 1 M. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to afford the desired product as white crystals. The product was then purified by preparative chromatography to obtain 51.5 mg of LV-111 as white solid.
1H NMR (500 MHZ, DMSO) δ 8.78 (d, J=7.9, 1H), 7.07 (d, J=8.3, 2H), 6.78 (d, J=8.2, 2H), 4.42 (dd, J=14.2, 8.4, 1H), 4.17-3.89 (m, 6H), 3.61 (s, 1H), 3.40 (s, 1H), 2.93 (dd, J=13.8, 5.7, 1H), 2.83 (dd, J=13.7, 9.2, 1H), 1.29-1.05 (m, 9H).
13C NMR (126 MHz, DMSO) δ 171.40, 167.43, 165.49, 157.81, 130.66, 128.90, 114.60, 63.32, 62.06, 61.24, 54.20, 53.06, 51.69, 36.17, 15.14, 14.34.
HRMS (micrOTOF-Q): calculated for C19H25NO7 [M+H]+: 380.1631, found: 380.1704.
HCl·H-Tyr-OEt (100 mg, 0.407 mmol) and (2S,3S)-trans-Oxirane-2,3-dicarboxylic acid (27 mg, 0.204 mmol) were dissolved in 1.5 ml of NMP, followed by DIEA (1,628 mmol, 374 μL). The mixture was stirred for 5 min and HATU (154.7 mg, 0,407 mmol) was added. The mixture was stirred at room temperature for 20 min. The solution was extracted 3 times with ethyl acetate. The organic layers were washed 3 times with KHSO4 1M and NaHCO3 1 M, then one time with brine and dried over MgSO4, filtered and concentrated under reduced pressure to afford the desired product as a white powder.
Cs2CO3 (126.8 mg, 0.389 mmol) was added to LV-101 (100 mg, 0.195 mmol) dissolved in anhydrous DMF and the reaction was stirred for 15 min. Iodoethane (47.0 μL, 0.585 mmol) was added to the solution, and the reaction was stirred at room temperature overnight and monitored by HPLC. The solution was poured in 30 ml of water and extracted 3 times with 30 mL of ethyl acetate then washed 3 times with a solution of NaHCO3 1 M and 3 times with a solution of KHSO4 1 M. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to afford the desired product as a white powder. The product was then purified by preparative chromatography to obtain 75.6 mg of LV-104 as white powder.
1H NMR (500 MHZ, DMSO) δ 8.87 (d, J=7.7, 2H), 7.07 (d, J=8.2, 4H), 6.78 (d, J=8.1, 4H), 4.38 (dd, J=14.2, 8.3, 2H), 4.02 (q, J=7.1, 4H), 3.93 (q, J=6.9, 4H), 3.45 (s, 2H), 2.92 (dd, J=13.8, 5.7, 2H), 2.82 (dd, J=13.8, 9.2, 2H), 1.25 (t, J=6.9, 6H), 1.08 (t, J=7.1, 6H).
13C NMR (126 MHz, DMSO) δ 170.97, 165.50, 157.29, 130.25, 128.36, 114.14, 62.70, 60.62, 53.79, 52.17, 35.51, 14.62, 13.91.
HRMS (micrOTOF-Q): calculated for C30H38N2O9 [M+H]+: 571.6390, found: 571.2650
Carbonyldiimidazole CDl (113.6 mg; 1.2 eq) was dissolved in a minimum of DMF and the RM was placed at −10° C. Boc-NH—NH2 (92.5 mg; 1.2 eq) was also dissolved in a minimum of DMF and added to the RM which was then stirred during 30 min at −10° C. After this, TFA·HTyr (OEt) OEt (203.6 mg; 1 eq) was also dissolved in a minimum of DMF and was added to the RM followed by Et3N (168 μL). The RM was then stirred at room temperature during 2 h50. For the work up step, distilled water and AcOEt were added in the RM and placed in a separation funnel. The organic phase was then washed with HCl 1M (×2), saturated NaHCO3 (×2) and brine (×2). The solution obtained was then dried with MgSO4, filtered and concentrated to give an oil (154.3 mg; Yield: 56%). This obtained oil was solubilized in a TFA/TIS/H2O (95/2.5/2.5) solution and stirred at room temperature during 30 min and then concentrated dry to give Intermediate E as crude oil. The compound was used without any further purification.
CDl (63.3 mg; 1.2 eq) was dissolved in a minimum of DMF and the RM was placed at −10° C. Intermediate E (159.7 mg; 1.2 eq) was also dissolved in a minimum of DMF and added to the RM which was then stirred during 30 min at −10° C. The pH was adjusted to 8 with few drops of Et3N. After this, TFA·HTyr (OEt) OEt (115.9 mg; 1 eq) was also dissolved in a minimum of DMF and was added to the RM followed by Et3N (102.2 μL; 2.2 eq). The RM was then stirred at room temperature during 3 h. For the work up step, distilled water and AcOEt were added in the RM and placed in a separation funnel. The organic phase was then washed with HCl 1M (×2), saturated NaHCO3 (×2) and brine (×2). The solution obtained was then dried with MgSO4, filtered and concentrated to give an oil.
The raw product was then purified on HPLC and lyophilized to give very light and white crystals (43.9 mg; Yield: 24%; Purity: >96%).
1H NMR (500 MHz, CDCl3) δ 6.99 (d, J=8.5, 4H), 6.75 (d, J=8.6, 4H), 6.50 (s, 2H), 6.05 (d, J=8.1, 2H), 4.60 (dd, J=14.3, 6.6, 2H), 4.16-4.04 (m, 4H), 3.92 (q, J=7.0, 4H), 3.03-2.89 (m, 4H), 1.38-1.28 (m, 6H), 1.18 (t, J=7.1, 6H).
Boc-5-Ava-OH (1 g, 4.6 mmol) was dissolved in anhydrous DMF (30 ml) and EtOH (1.3 ml, 23 mmol) was added. Then was added in this order: DMAP (56.4 mg, 0.46 mmol), NMM (605 μl, 5.5 mmol), OxymaPure (653.7 mg, 4.6 mmol) and EDCi (1.1 g, 5.98 mmol). The RM was stirred at RT during 2h05. Three AcOEt extractions were proceeded. The organic layer was then washed with NAHCO3 (sat.), KHSO4 (1M), and brine three times each. Organic layer was then dried over MgSO4, filtered, and concentrated dry.
Intermediate F (100 mg, 0.41 mmol) was dissolved in DCM (700 μl), followed by TFA/TIS/H2O 95/2.5/2.5 v/v/v (663 μl). The RM was then stirred for 1 h at RT and concentrated dry.
Intermediate G (106.2 mg, 0.41 mmol) and IBMT7 (49.2 mg, 0.14 mmol) were dissolved in anhydrous DMF (911 μl). Then was added in this order: DMAP (1.2 mg, 0.01 mmol), NMM (127.6 μl, 1.16 mmol), OxymaPure (19.9 mg, 0.14 mmol) and EDCi (34.5 mg, 0.18 mmol). The RM was stirred at RT overnight. Three AcOEt extractions were proceeded. The organic layer was then washed with NAHCO3 (sat.), KHSO4 (1M), and brine three times each. Organic layer was then dried over MgSO4, filtered, and concentrated dry to afford 47.03 mg of desired product. The crude product was purified on inverse phase preparative HPLC.
Boc-Phe(4-NH2)—OH (100 mg, 0.36 mmol) was dissolved in NMP (691 μl) and DBU (54 μl, 0.36 mmol) was added. Iodoethane (29 μl, 0.36 mmol) was previously put at 0° C. and then added to the reaction mixture dropwise. After a 30 min stirring at RT, H2O and AcOEt were added and then poured in a separation funnel. Three AcOEt extractions were proceeded, and the organic layer was then washed with KHSO4 (1M) and brine three times each. Organic layer was then dried over MgSO4, filtered, and concentrated dry.
Intermediate H (47.9 mg, 0.16 mmol) was dissolved in anhydrous DMF (139 μl) and Pyridine (78 μl, 0.96 mmol) was added. Ms-Cl (37 μl, 0.48 mmol) was then added and the RM was stirred during 1 h at RT. H2O and AcOEt were added and then poured in a separation funnel. Three AcOEt extractions were proceeded. The organic layer was then washed with NAHCO3 (sat.), KHSO4 (1M), and brine three times each. Organic layer was then dried over MgSO4, filtered, and concentrated dry.
Intermediate I (19.8 mg, 0.05 mmol) was dissolved in DCM (200 μl), followed by TFA/TIS/H2O 95/2.5/2.5 v/v/v (122 μl). The RM was then stirred for 1 h at RT and concentrated dry.
Intermediate J (20 mg, 0.05 mmol) was dissolved in NMP (240 μl), followed by (2S,3S)-trans-Oxirane-2,3-dicarboxylic acid (6.6 mg, 0.05 mmol) and DIEA (61.2 μl, 0.35 mmol). The RM was stirred and HATU (19.1 mg, 0.05 mmol) was added. The RM was stirred at room temperature for 3 h. The RM was then extracted 3 times with EtOAc and the organic phase was washed 3 times with HCl (1M), one time with brine, dried over MgSO4, filtered, and evaporated dry to obtain the 18 mg mixture of 1 and 2. The crude product was purified on inverse phase preparative HPLC.
(IBMT35) Expected mass: 16.7 mg; Obtained mass: 3.2 mg; Yield: 19%; Purity: 98% (MS (ESI+): m/z=669.0 [M+H]+; time=2.64 min) (expected m/z=668.7)
Boc-Phe(4-NH2)—OH (50 mg, 0.18 mmol) was suspended in a solution of NaOH (1M) and the RM was placed at 0° C. Then was added Ts-Cl and the RM was stirred during 2h at 0° C. Acidification was proceed with HCl (1M) until pH 2-3 and the RM was extracted three times with AcOEt.
Then organic layer was washed three times with KHSO4 (1M) and two times with brine, dried over MgSO4, filtered, and concentrated dry.
Intermediate K (78.1 mg, 0.18 mmol) was dissolved in anhydrous DMF (1.2 ml) and EtOH (62.6 μl, 1.08 mmol) was added. Then was added in this order: DMAP (2.5 mg, 0.02 mmol), NMM (24.2 μl, 0.22 mmol), OxymaPure (25.6 mg, 0.18 mmol) and EDCi (44 mg, 0.23 mmol). The RM was stirred at RT during 40 min. Three AcOEt extractions were proceeded. The organic layer was then washed with NAHCO3 (sat.), KHSO4 (1M), and brine three times each. Organic layer was then dried over MgSO4, filtered, and concentrated dry.
Intermediate L (69.4 mg, 0.15 mmol) was dissolved in DCM (400 μl), followed by TFA/TIS/H2O 95/2.5/2.5 v/v/v (365 μl). The RM was then stirred for 1 h at RT, concentrated dry, and precipitated in diethyl ether.
Intermediate M (69.8 mg, 0.15 mmol) was dissolved in anhydrous DMF (385 μl), followed by (2S,3S)-trans-Oxirane-2,3-dicarboxylic acid (13.2 mg, 0.1 mmol) and DIEA (63.8 μl, 0.38 mmol).
The RM was stirred and HATU (57 mg, 0.15 mmol) was added. The RM was stirred at room temperature for 2h20. The RM was then directly purified on inverse phase preparative HPLC.
Compound 81 (7.35 mg, 0.01 mmol) was dissolved in 800 μl of DMF followed by the addition of biotin-maleimide (4.5 mg, 0.01 mmol) and 400 μl PBS solution. The mixture was lightly heated in order to solubilize both reagents and then stirred at RT for 1 h. The RM was then directly purified by preparative HPLC to afford 7 mg of LV-82 as white solid.
Fmoc-Aad(OtBu)-OH (88 mg, 0.216 mmol) was attached via a standard SPPS HATU (82 mg, 0.216 mmol) DIEA (50 μl, 0.288 mmol) activation. The RM was stirred with 200 mg of rink amide (RA) resin charged at 0.37 mmol/g in solid phase synthesis reactor for 2 h. After standard washings, the Fmoc protection was removed by two times 10 min DMF/Pip 80/20 v/v treatment. As a next step compound 5 (88 mg, 0.216 mmol) was attached to the liberated amine via a PyAOP (113 mg, 0.216 mmol), DIEA (545 μl, 0.63 mmol) activation. After overnight reaction, the resin was washed 3 times DMF, 3 times DCM and the inhibitor was cleaved by a 20 ml TFA treatment for 50 min. The product containing TFA filtrate was then evaporated dry, and the peptide was precipitated in EtOEt, filtered, purified by preparative HPLC and lyophilized to obtain 10.7 mg of LV-43 as a white solid.
Human CHL-1 cells (melanoma derived cell line, Nieuwenhuis J. et al. Science, 2017, 358 (6369): 1453-1456) were routinely cultured in DMEM containing 10% (v/v) fetal bovine serum (FBS), 50 U/ml penicillin and 50 μg/ml streptomycin (pen/strep) and incubated at 37° C. Prior testing, the CHL-1 cells were seeded in adequate plates (96, 48, 24, 12 or 6 wells) at 30% confluency. Next day cells were treated and collected for further analysis (immunoblotting, qPCR, immunofluorescence . . . ).
Cortical cell cultures were seeded either on glass coverslips or plastic plates coated with 0.1 mg/ml poly-L-lysine (Sigma) at ˜105 cells per cm2 or 2×104 cells per cm2 for low-density cultures. Neurons were plated in DMEM containing 10% (v/v) fetal bovine serum (FBS), 50 U/ml penicillin and 50 μg/ml streptomycin (pen/strep), and 1-2 h later the medium was replaced with Neurobasal medium supplemented with 2% B27, pen/strep, 0.6% glucose and 1% Glutamax (all reagents from Life Technologies).
Drosophila were raised at 25° C. yw flies were used as control for yw; αTub84BΔ3 mutant. Total males were disrupted in laemli loading buffer, boiled, sonicated and loaded for immunoblotting.
We used the methods as disclosed in the application WO2020/012002.
Human VASH1 (hVASH1) was cloned in the expression vector with a poly-histidine tag. Bacteria were transformed and induced with Isopropyl β-d-1-thiogalactopyranoside (IPTG) overnight or for 4 hours. Bacteria were collected and disrupted using a HTU-DIGI-F press (Heinemann). Recombinant proteins were purified using nickel-based affinity chromatography (IMAC) according to the manufacturer's protocol (GE Healthcare).
Spodoptera frugiperda Sf9 cells were grown, lysed and used for tubulin purification by affinity chromatography. Microtubules were obtained with taxol and stored until use. In vitro analysis of detyrosination activities was performed using the recombinant hVASH1. Detyrosination assays were performed in the presence of 0.5 μM microtubules. Reactions were stopped by addition of the denaturing loading buffer followed by 5 min incubation at 95° C., and samples were loaded on SDS PAGE for immunoblot analysis.
Standardized primary ELISA-based in vitro detyrosination assay in a 96-wells format has been developed according to the method disclosed in WO2020/012002. Briefly, the substrate of the VASH enzyme has been produced and 100 μL of the enzymatic mix (enzyme and inhibitor) is added to each well, then the plate is incubated 5 minutes at 37° C. Primary (rabbit anti-detyrosinated tubulin) is incubated for 1 hour and after washes, secondary (anti-rabbit) antibody is added to each well. The plate is incubated 1 h at room temperature. Development consists of addition of TMB (3,3′,5,5′-Tetramethylbenzidine) to each well, incubation 30 minute at room temperature. The colour development is stopped by addition of 0.5 M sulfuric acid. OD is measured at 450 nm.
Deglutamylation assays have been performed as previously described (Rogowski et al., Cell, 2010, 143 (4): 564-78).
Plasmid transfections were performed and cells were collected 24 hours after transfection for immunoblotting and immunofluorescence analysis.
Total RNA was isolated with TRIzol (Invitrogen). Reverse transcription was carried out with random hexanucleotides. Quantitative PCR assays were performed using the Lightcycler SYBR Green Master mix on a Lightcycler apparatus (Roche). All used primers were intron-spanning. The relative amount of target cDNA was obtained by normalization by geometric averaging of multiple internal control genes.
Methanol-fixed CHL-1 cells and mouse cortical neurons were analyzed using a Zeiss Axioimager Apotome microscope after standard immunofluorescence experiments. Briefly, samples were incubated with primary antibodies overnight, washed three times in PBS before 1 h incubation with the following secondary antibodies: Alexa Fluor 488-conjugated goat anti-rat, goat anti-mouse and Alexa Fluor 555-conjugated goat anti-rabbit (Invitrogen). After incubation, samples were washed three times in PBS, stained with DAPI, and mounted.
Microscopy image acquisition was performed at the Montpellier RIO Imaging facility and images were processed using OMERO.
Peptide stability in serum Human serum reaction sample contained 250 μl of heat inactivated Human Serum (Sigma Aldrich) and 750 μL of RPMI Medium 1640 (Sigma Aldrich). The reactions were initiated by addition of 50 μl compound (in mM stock solution in DMSO). Assays were performed in a shaking water bath at 37° C. 100 μl samples were collected at known time intervals and added to 200 μl of acetonitrile 1% % TFA to precipitate serum proteins. Cloudy sample was cooled to 4° C. for 15 min and then submitted to centrifugation for 10 min at 12,000 rpm to pellet the precipitated serum proteins. 150 μl of the clear supernatants were collected and peptides were finally analyzed by RPHPLC and LC-MS.
We used human CHL-1 cells in which tubulin detyrosination is catalyzed exclusively by VASHs. In these cells, detyrosination is predominantly VASH-dependent (Nieuwenhuis J. et al. Science, 2017, 358 (6369): 1453-1456). Cells were pre-treated with taxol before incubation with Epo-Y. Samples were immunoblotted using the indicated antibodies.
By using CHL-1 cells, we analyzed the use of Epo-Y to reduced taxol induced detyrosination. Cells were routinely cultured in a standard humidified tissue culture incubator at 37° C. in presence of 5% CO2 and plated in a 6-wells culture dish. The cells were treated for 2 hours with Taxol in absence or presence of Epo-Y.
The cells were collected in a RIPA buffer (of 50 mM Tris HCl, 150 mM NaCl, 1.0% (v/v) NP-40, 0.5% (w/v) Sodium Deoxycholate, 1.0 mM EDTA, at a pH of 7.4), and quantitation of total protein performed using BCA kit (Thermo Fisher Scientific). A 20 μg protein sample of a total cell extract was run on 10% SDS-PAGE, transferred to nitrocellulose, and probed with each antibody. Western blot analysis showed a striking decrease of taxol treated (2 hours) and consequent tubulin detyrosination in CHL-1 cells. As illustrated in
So this in cellulo screening assay with CHL-1 cells and taxol is a relevant screening assay for identification of putative inhibitor of VASH-mediated tubulin detyrosination.
2.3.2 Putative Inhibitor Parthenolide does not Inhibit VASH-Mediated Tubulin Detyrosination
To study the inhibitory effects of parthenolide (PTL) on VASH-mediated tubulin detyrosination, we used the previously developed in vitro detyrosination assay disclosed in WO2020/012002. Reactions were incubated with inhibitor, stopped and analysed by immunoblotting with the indicated antibodies. As illustrated in
A small, highly reactive epoxide-based molecule such as Epo-Y might interact with free thiols present on proteins other than the VASHs or with nucleophilic lysine residues in the active sites of tyrosine kinases. Therefore, through iterative chemical variations and analysis with our standardized in vitro assay, we identified a compound, which we termed LV-43 (
To test the LV-80 potency on the endogenous detyrosination activity, we compared wild type CHL-1 cells incubated with LV-80 to double knockout CHL-1 cells (2KOs) that lack both VASH1 and VASH2. Cells were collected and analysed by immunoblotting with the indicated antibodies. After 24-hour incubation with LV-80, the level of detyrosination was comparable in treated CHL-1 and CHL-1 2KOs cells (
We next choose a widely used MTT-based colorimetric assay to assess the potential metabolic toxicity and cell viability alterations in the presence of LV-80. The MTT-based assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) was performed 48 hours after incubation with the indicated compounds to monitor cell viability.
Incubation for 24 hours with 0.1 μM taxol and 10 μM PTL resulted in a significant reduction of cell viability. Conversely, incubation with a much higher concentration of LV-80 (100 μM) had no significant effect on cell viability, which was similar to that of cells incubated with vehicle (
Next, we tested LV-80 inhibitory activity towards cytosolic carboxypeptidases (CCPs), another family of proteases that target tubulin C-terminal tails and act as tubulin deglutamylases. The inhibitor did not affect CCP1 and CCP5 activities. Samples were immunoblotted using the indicated antibodies. When polyglutamylated brain tubulin was incubated in the presence of protein lysates from HEK293 cells transfected with GFP-CCP1, we observed that CCP1-dependent deglutamylation activity towards long glutamate chains was not affected by addition of LV-80 (
Since detyrosination levels are particularly high in brain, we tested whether LV-80 fused to biotin could be used to pull down VASH1 from human brain extracts. Cells were pre-treated with taxol before incubation with biotinylated LV-80. Samples were immunoblotted using the indicated antibodies First, we confirmed that biotinylated LV-80 retained its inhibitory properties towards VASH1- and VASH2-mediated detyrosination in vitro (
2.3.9 Strong Reduction of Tubulin Detyrosination in Primary Cortical Neurons Treated with VASH Inhibitor
We decided to assess the functional role of tubulin detyrosination in differentiating primary cortical neurons purified from cerebral cortical tissue of mouse embryos. After dissociation and filtration, we differentiated cells in the presence or absence of LV-80 for 7 days. Cells were collected and analysed by immunoblotting with the indicated antibodies. Samples were analysed by immunoblotting and the relative optical density measured in three independent assays (n=3 and error bars represent SEM). P-values were calculated with the multiple t-test (n=3, *<0.05, **<0.01, ***<0.001, ****<0.0001). Analysis of the level of tubulin posttranslational modifications using specific antibodies (
Samples were analysed by immunoblotting and the relative optical density measured in three independent assays (n=3 and error bars represent SEM). P-values were calculated with the multiple t-test (n=3, *<0.05, **<0.01, ***<0.001, ****<0.0001).
Strikingly, while acetylation levels remained unaffected in treated neurons (
To study the role of tubulin detyrosination in regulating the interaction of MTs with MT associated proteins (MAPs), we first completed differentiation of the cortical neurons for 5 days in absence of VASH inhibitor and followed by a treatment of 3 days. This approach allows for monitoring the cell distribution of tau protein independently of cell differentiation. Cells were collected and analysed by immunoblotting with the indicated antibodies. Incubation of cortical neurons with LV-80 for 3 days efficiently reduced tubulin detyrosination level and significantly increased tyrosinated tubulin level (
We designed a set of highly effective prodrugs efficient at low nM IC50 in cellulo (including examples as LV-104 and LV-111) and are cell penetrant, as illustrated in
The compounds were added prior Taxol-treatment and abolished VASH-mediated detyrosination in low nM ranges in CHL-1 cells. Quantification shows LV-104 and LV-111 (IC50 of 8 nM and 20 nM, respectively) are much more efficient on cells than LV-80 (IC50 of 500 nM). Two examples of prodrug compounds (LV-104 and LV-111) and a direct VASH inhibitor. Both prodrugs did not have any effect on VASH1 and VASH2 in the context of in vitro assays.
Considering all these results, we propose that targeting VASH peptidase activity is a safe and well tolerated mechanism for treating neurodegenerative diseases like Alzheimer's disease but also cancer, muscular dystrophy and ciliopathies. Interestingly these compounds have strong effect on tubulin glutamylation and may also be used for reducing TTLL-dependent modifications of tubulin in e.g. infantile onset neurodegeneration or glaucoma.
We showed that parthenolide, a molecule extensively used as an inhibitor of detyrosination, does not inhibit VASH activity neither in vitro nor in cellulo. This is in agreement with a recently published study, which demonstrated that the effect of parthenolide on detyrosination is indirect and most likely results from changes in microtubule dynamics caused by its covalent binding to tubulin (Hotta et al. Curr Biol. 2021, Volume 31, Issue 4, Pages 900-907). More importantly, we described medicinal chemistry-based optimization of the Epo-Y compound, which led to the development of a new highly specific and cell-penetrant VASH inhibitor. We showed that our newly developed compounds (active ingredients) have the ability to completely inhibit VASH activity and, in contrast to parthenolide, shows no detectable toxicity. Furthermore, by applying the VASH inhibitor to primary neuronal cultures, we have discovered previously unknown crosstalk between tubulin detyrosination and another important tubulin modification called polyglutamylation. The newly identified link between these two modifications has far-reaching consequences for the establishment of neuronal polarity and the localization of the main neuronal microtubule associated protein called tau. Overall these compounds hold strong potential for the use in pharmaceutical development and for research purposes.
The in vitro inhibition activity of IBMT11 is tested by adding the compound to the reaction medium as described above (see part 2.2) and then detyrosination was analysed by immunoblotting with the indicated antibodies.
While LV-104 did not inhibit VASH in vitro, we found that IBMT11 inhibited VASH1-mediated tubulin detyrosination more potently than Epo-Y, as indicated by their in vitro IC50 (0.6 μM for IBMT-11 versus 10 μM for Epo-Y) (
When tested in a cell-based assay involving taxol treated CHL-1 cells, IBMT11 was found to have a weak inhibition activity (
2.3.14. Conversion of LV104 into IBMT11 Inside Human Cells
The bioanalysis of the penetration and the conversion of the compound LV-104 has been achieved using the above described protocol on human cells (see part 2.2). The results are illustrated on
They confirm the total conversion of the prodrug LV-104 into IBMT11 inside human cells. Some conversion occurs in cell's culture medium (supernatant), however LV104 was undetectable inside cells (Cells). These data confirm our hypothesis according to which the presence of less polar groups, such as esters, in particular on the position corresponding to R3, increase significantly their cell penetration. Once inside the cell, the compound is hydrolysed thus converting esters to carboxylic acids for example and provides a potent VASH inhibitor.
Thus, IBMT11 is the corresponding drug of the prodrug LV-104. Indeed, groups corresponding to R3 and R6 are ethyl esters in LV-104 whereas they are carboxylic acid in IBMT11. That is why LV-104 cannot disclose an in vitro activity and IBMT-11 has a poor in cellulo activity; And, in the other way round, IBMT-11 is very efficient in vitro, while LV-104 is potent VASH inhibitor in cellulo.
IBMT23 is found to be inactive when tested in an in vitro detyrosination assay (using the protocol as described above) (
IBMT34 is the sodium salt of LV80. It is found to have same activity in vitro and in cellulo as the corresponding base LV-80 but it possesses a dramatically increased solubility in water or PBS solution (see
IBMT28 is found to be inactive when tested in an in vitro detyrosination assay (using the protocol as described above) (
IBMT38hydro has been tested in an in vitro detyrosination assay, as described above. It is found to have an in vitro activity as good as LV80 (see
The following compounds have been tested in in vitro detyrosination assays as described above and their IC50 have been calculated and compared to the IC50 of the compound LV-80. The column “fold change” corresponds to the ratio IC50(compound)/IC50(LV80) for each compound.
2.3.17. Comparative Example with SD-139
SD139 was synthesized as previously described by Aillaud, C. et al. Science, 2017, 358, p. 1448-1453.
SD139hydro was obtained after incubation with 150 mM NaOH for 5 min, then neutralization with 150 mM HCl.
SD-139 and SD-139hydro (also named SD-139sapo) were tested in in vitro detyrosination assays and compared to the activities of LV-80 and EPO-Y (see
| Number | Date | Country | Kind |
|---|---|---|---|
| 21306141.9 | Aug 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/073625 | 8/24/2022 | WO |
