A binding site to prevent endocannabinoid cellular reuptake

Abstract

The present invention relates to a peptide having the following amino acid sequence: AKKK (SEQ ID NO: 3), wherein the peptide can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor, to a fusion protein comprising the of the present invention and a transmembrane domain, wherein the fusion protein can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor, to a composition comprising said peptide or said fusion protein, an endocannabinoid and / or an endocannabinoid reuptake inhibitor, and optionally a pharmaceutically acceptable carrier, to a binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein, wherein the binding sequence can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor, wherein the sequence comprises the following amino acid sequence: AKKK (SEQ ID NO: 3), a cell comprising the peptide of the present invention, the fusion protein of the present invention, or the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to the present invention, to a lipid bilayer comprising the peptide of the present invention, the fusion protein of the present invention or the binding sequence of the present invention, also provided for use in the treatment of a condition or a disease associated with an altered function of the endocannabinoid system, preferably for use in the treatment of a disorder of the central nervous system, such as neuropsychiatric, neuroinflammatory or neurodegenerative disorders, an inflammatory disease or an immune disease, as well as use of the same for facilitating plasma membrane trafficking of an endocannabinoid, for facilitating the reuptake of an endocannabinoid, and / or for modulating cellular immune processes.The present invention also relates to a method for facilitating plasma membrane trafficking of an endocannabinoid of the present invention, to a method for sustaining endocannabinoid signaling of the present invention, and to a method for identifying an endocannabinoid reuptake inhibitor of the present invention.

Description

[0001] A binding site to prevent endocannabinoid cellular reuptake

[0002] Field of the invention

[0003] The present invention relates to a peptide having the following amino acid sequence: AKKK (SEQ ID NO: 3), wherein the peptide can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor, to a fusion protein comprising the of the present invention and a transmembrane domain, wherein the fusion protein can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor, to a composition comprising said peptide or said fusion protein, an endocannabinoid and / or an endocannabinoid reuptake inhibitor, and optionally a pharmaceutically acceptable carrier, to a binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein, wherein the binding sequence can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor, wherein the sequence comprises the following amino acid sequence: AKKK (SEQ ID NO: 3), a cell comprising the peptide of the present invention, the fusion protein of the present invention, or the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to the present invention, to a lipid bilayer comprising the peptide of the present invention, the fusion protein of the present invention or the binding sequence of the present invention, also provided for use in the treatment of a condition or a disease associated with an altered function of the endocannabinoid system, preferably for use in the treatment of a disorder of the central nervous system, such as neuropsychiatric, neuroinflammatory or neurodegenerative disorders, an inflammatory disease or an immune disease, as well as use of the same for facilitating plasma membrane trafficking of an endocannabinoid, for facilitating the reuptake of an endocannabinoid, and / or for modulating cellular immune processes. The present invention also relates to a method for facilitating plasma membrane trafficking of an endocannabinoid of the present invention, to a method for sustaining endocannabinoid signaling of the present invention, and to a method for identifying an endocannabinoid reuptake inhibitor of the present invention.

[0004] Background of the invention

[0005] Membrane proteins function in the diverse environments of the phospholipid bilayer. While experimental evidence suggests that some lipid molecules bind tightly to specific sites on the membrane protein surface acting as co-factors, other membrane-associated protein domains help the trafficking of lipids between the outer and inner leaflet of the phospholipid bilayer, constituting a mechanism to counterbalance lipid asymmetry, or between the membrane and intracellular proteins [1].

[0006] The trafficking of endocannabinoids between the different cellular compartments remains poorly understood. It remains unclear how endocannabinoids are removed from the cannabinoid receptor binding site upon activation of the receptor to terminate signaling. Endocannabinoids are neuromodulators, which are engaged in neuronal signaling. Known endocannabinoids are 2- arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA). Endocannabinoids interact with cannabinoid receptors, namely CBI and CB2, which are G protein-coupled receptors (GPCRs) in the plasma membrane of diverse cell types. There is experimental evidence that endocannabinoids can reach the orthosteric binding site (agonist site) in CB receptors by lateral diffusion in the outer leaflet of the membrane [2], Upon activation of these GPCRs by binding of the endocannabinoid to the receptor, the endocannabinoid is removed from the plasma membrane environment by facilitated diffusion, which is driven by hydrolysis involving different enzymes that are located in the intracellular space [3]. Yet there is a discrepancy between the low amounts required to activate cannabinoid receptors and passive diffusion kinetics [4], Besides the receptors, the endocannabinoids 2-arachidonoylglycerol (2- AG) and N-arachidonoylethanolamine (anandamide, AEA) also have intracellular targets. To date, the mechanism of endocannabinoid trafficking across the phospholipid bilayer to terminate CB receptor signaling remains unknown. Understanding the transport of endocannabinoids can provide further related strategies for treating diseases or disorders, which are associated with an altered function of the endocannabinoid system, such as endocannabinoid deficiency.

[0007] In addition to current strategies to block endocannabinoid hydrolysis, there remains a need to identify mechanisms of endocannabinoid reuptake in order to modulate them in the treatment of diseases or disorders associated with altered function of the endocannabinoid system. Thus, the problem of the present invention can be considered the provision of a further system of endocannabinoid reuptake. The problem could surprisingly be solved by the provision of the claimed target site (binding site) for endocannabinoids and / or endocannabinoid reuptake inhibitors.

[0008] Document US 2009 / 9155241 discloses the full sequence of human ACLS4 isoforms 1 and 2, as well as compounds inhibiting activity of such enzymes and uses thereof for treating diseaess, like cancer. Document WO 2020 / 170192 discloses certain inhibitors of ACSL4.

[0009] Chicca et al. (“Chemical probes to potently ans selectively inhibit endocannabinoid cellular reuptake:, PNAS, vol 114, no 25, pages E5006-5015) disclose certain chemical probes to inhibit endocannabinoid cellular uptake.

[0010] Summary of the invention

[0011] The inventors identified an amino acid sequence ALIFIPWYFLTNAKKK (SEQ ID NO: 1) (subsequently referred to as the "binding site", "binding sequence", "target site" or "target sequence") in an acyl-CoA synthetase protein, which can be bound by endocannabinoids and / or endocannabinoid reuptake inhibitors.

[0012] Acyl-coenzyme A synthetase proteins (ACS) are membrane-associated or membrane bound enzymes that catalyze the first step activation of fatty acids by conversion into their respective acyl-coenzyme A (acyl-CoA) forms. The substrate specificity among the ACS enzymes differs, and the length of the acyl chain mediates the substrate recognition. Long-chain acyl-CoA synthetase (ACSL) proteins prefer acyl groups with a length between C12 to C20. The conversion of the substrate by ACSL proteins ocurrs at the catalytic site, which is integrated by the lipid binding tunnel YIGYLPLA (SEQ ID NO: 5) and the neighbouring ATP binding site which corresponds to the conserved amino acid sequence: TSG.

[0013] The amino acid sequence of the binding site that facilitates endocannabinoid membrane transport was identified by the inventors in the long-chain acyl-CoA synthetase protein 4 (ACSL4) isoform 2. Based on NCBI Blast enquiries and mutation studies, the "binding site" in the mammalian ACLS4 isoform 2 is ALIFIPWYFLTNAKKK (SEQ ID NO: 1). The binding site of the present invention was not detected in ACSL1 , ACSL3, ACSL5 and ACSL6.

[0014] The inventors found that the binding sequence ALIFIPWYFLTNAKKK (SEQ ID NO: 1) in said acyl-CoA synthetase protein 4 (ACSL4) can be targeted and blocked by molecules in order to inhibit the reuptake of endocannabinoids, which is a non-obvious moonlighting function, in particular prior to their hydrolysis to arachidonic acid and ethanolamine (AEA) or glycerol (2- AG). This represents a novel strategy compared to the known blockage of degradation of endocannabinoids. The inhibition of endocannabinoid reuptake can have major therapeutic implications in different disease areas [4], particularly in conditions or diseases associated with endocannabinoid deficiency [5]. The novel endocannabinoid binding site in the acyl-CoA synthetase protein disclosed herein is independent of the conserved catalytic domain and / or the proposed lipid tunnel of the acyl-CoA synthetase protein and, thus, represents a non-obvious protein function. Reuptake of endocannabinoids by acyl-CoA synthetase proteins has not been described before.

[0015] The binding sequence disclosed herein is preferably located in an alpha helix within said acyl-CoA synthetase protein. ACSL4 isoform 2 contains one transmembrane alpha helix domain, which is a membrane-spanning sequence, which is typically 40 amino acids long and whose peptide backbones preferentially oligomerize into a four-helix bundle, packed in a coiled-coil arrangement with a sterically close-packed hydrophobic core in the center.

[0016] The occurrence of ACSL4 isoform 2 as either integral membrane proteins or membrane-associated proteins can be rationalized by the hydrophobic character of the alpha helix or by events in which a four- helix bundle terminal is inserted.

[0017] The binding sequence disclosed herein, ALIFIPWYFLTNAKKK (SEQ ID NO: 1), is not present on the acyl-CoA synthetase protein ACSL3, despite conserved sequence homology and association with the plasma membrane. Therefore, ACSL3 is incapable of endocannabinoid binding.

[0018] The inventors showed that binding of endocannabinoids to the binding site in the acyl-CoA synthetase protein can be inhibited or blocked by endocannabinoid reuptake inhibitors. Thus, the binding site represents a target site for endocannabinoid reuptake inhibitors (such as those disclosed in WO2015181337-A1 , or such as for example WOBE437, disclosed in US 15 / 996764, which is included by reference in its entirety).

[0019] The enzymatic function of ACSL4 has been extensively characterized by its conversion of arachidonic acid (its main substrate) into arachidonoyl-CoA. This previously known catalytic function is independent of endocannabinoid binding; therefore, this invention represents a novel moonlighting role of ACSL4 isoform 2 in the context of endocannabinoid reuptake, as a new druggable target with therapeutic potential

[0020] The binding sequence in ACSL4 isoform 2 serves as an endocannabinoid interaction domain likely associated to membranes but does not directly interfere with the catalytic activity of the ACSL4 enzymatic function. This is substantiated by the fact that free fatty acids that are substrates for these enzymes do not critically depend on the "binding site". Specific endocannabinoid reuptake inhibitors (SERIs) do not block the enzymatic activity of ACSL proteins, i.e. the conversion of arachidonic acid to arachidonoyl- CoA. While fatty acids may traverse membranes by simple diffusion, esterification with coenzyme A leads to amphipathic fatty acyl-CoA molecules which are membrane impermeable and remain therefore inside the cell. This process has been named "vectorial acylation" or "metabolic trapping".

[0021] The binding site in ACSL4 isoform 2 at the plasma membrane facilitates endocannabinoid diffusion (i.e. plasma membrane trafficking or endocannabinoid reuptake) prior to any enzymatic conversion such as hydrolysis of the endocannabinoid. ACLS 4 isoform 2 is membrane associated and preferable expressed in the brain. The binding site in ACLS4 isoform 2 is assumed to play a role in the context of plasma membrane trafficking in neurons.

[0022] A transmembrane spanning domain KLKLNVLTIILLPVHLLITIYSALIFIPWYFLTNAKKKNA (SEQ ID NO: 4) of ACLS4 isoform 2 likely assists the association of the binding site to the plasma membrane, thus facilitating the trafficking of endocannabinoids from the outer leaflet to the inner leaflet of the lipid bilayer of the plasma membrane. In the case of 2-arachidonoylglycerol (2-AG) reuptake, this leads to a subsequent interaction of monoacylglycerol lipase (MAGL) and hydrolysis of 2-AG, generating arachidonic acid (AA) (Fig. 1). The formed arachidonic acid then interacts with the catalytic site of ACSL4 to form arachidonoyl-CoA to enter the Lands cycle. In summary, the novel binding site of ACSL4 isoform 2 is involved in transport of endocannabinoids by facilitated diffusion of an endocannabinoid such as 2- AG from the outer to the inner leaflet of the lipid bilayer of the plasma membrane of neurons, whereas the catalytic site of ACSL4 isoform 2 is involved in the metabolic trapping of the arachidonic acid.

[0023] ACSL proteins act as acyl-CoA synthetase (ligase) converting fatty acids into acyl-CoA species and share a common lipid binding tunnel (YIGYLPLA (SEQ ID NO: 5)) and an ATP binding sequence: TSG, in the catalytic site for fatty acids such as arachidonic acid (AA). The described amino acid domain ("binding site") in certain ACSL proteins described herein is involved in the facilitated diffusion of the endocannabinoids AEA and 2-AG and is independent of the catalytic function.. The endocannabinoid binding domain is predicted to be an alpha helix domain in ACSL4 isoform 2 involved in trafficking of endocannabinoids from phospholipid membranes to other sites, including the serine hydrolase MAGL (in the case of 2-AG). Blocking the binding site in ACSL4 isoform 2 by small molecules leads to a decreased plasma membrane trafficking (facilitated diffusion) of endocannabinoids across the plasma membrane and inhibits endocannabinoid reuptake, which changes the relative amounts of incorporated endocannabinoids in the cell. This occurs independently of the enzymatic function of the protein. Thus, targeting this domain can lead to sustained endocannabinoid signaling in pathophysiological conditions and diseases related to endocannabinoid deficiency.

[0024] Brief description of figures

[0025] The invention is further illustrated by the appended drawings, described in the following.

[0026] Figure 1. Model of the involvement of ACSL4 in the transport of 2-AG and AEA: (1) 2-AG and AEA sequestrate into the outer leaflet of the plasma membrane and accumulate there (no diffusion). (2) 2-AG and AEA bind the ACSL4 domain spanning the inner leaflet - which facilitates the diffusion (transport). (3) MAGL in proximity (in a complex?) hydrolyses 2-AG to arachidonic acid (AA) and glycerol. (4) The generated AA interacts with the catalytic site of ACSL4 and is converted to arachidonoyl-CoA.

[0027] Figure 2. RX-237 is the clickable variant of RX-055 with the WOBE437 molecular scaffold and linker containing the diazirine moiety that specifically interacts with the active site in ACSL3 and ACSL4. RX-237 can spontaneously react with the catalytic site in ACSL3 and ACSL4 proteins without UV irradiation.

[0028] Figure 3 Specific labeling with RX-237 of ACSL4 (upper band) and ACSL3 (lower band) in Neuro2A cells.

[0029] Figure 4. Blots showing the only specific bands (ACSL3 and ACSL4), the other bands come from the click reaction. These bands can be competed with RX-055. The ACSL3 and 4 knock-out (KO), respectively, lead to the abolishment of the labeling. (This Figure is a representative image of numerous independent experiments).

[0030] Figure 5. Effect of SYT-510 and SYT-520 in HAPI wild type (WT) cells (left) and ACSL3 / 4 (T3 / T4) knock out (KO) (right) on intracellular and extracellular 2-AG levels (measured by LC-MS / MS) without adding 2-AG apart from what is present in the medium containing FBS.

[0031] Figure 6. AEA uptake is not taking place in ACSL4 knock out (KO) when fatty acid amidehydrolase (F AAH) is co-transfected at 5 min. Figure 7. The formation of arachidonoyl-CoA (AA-CoA) upon addition of 10 pM of arachidonic acid (AA) (control) to HAPI cells after 15 min is inhibited by the ACSL inhibitor traicsin C and the probes RX-055 and RX-237. However, the SERIs SYT-510, SYT-520 and SYT-530 do not inhibit this enzymatic function. WOBE437 only partially (15-20%) inhibited the enzymatic function at 10 pM.

[0032] Figure 8. The formation of arachidonoyl-CoA (AA-CoA) upon addition of 10 pM of arachidonic acid (AA) (control) to HAPI cells after 15 min is blocked by ACSL4 (T4) and ACSL3 / ACSL4 (T3 / T4) knockout. Since triacsin C cannot further inhibit the effects in either experiment, ACSL4 seems to be primarily responsible for this enzymatic activity in HAP 1 cells.

[0033] Figure 9. The formation of intracellular arachidonoyl-CoA (AA-CoA) upon addition of 10 pM of arachidonic acid (AA) to HAPI cells after 15 min in ACSL3 knockout cells is not blocked. A full inhibition by triacsin C in control (no addition of AA) and upon addition of AA can be seen in cells. WOBE437 cannot inhibit the enzymatic function of ACSL4 in neither of the two conditions. Moreover, these data show that ACSL3 does not have a major role in AA conversion to arachidonoyl-CoA when added to the cells through the medium.

[0034] Figure 10. Model derived from AlphaFold software of the proposed tetrameric complex. The alpha helix bundle spans throughout the plasma membrane and brings the proposed binding site into proximity to the inner leaflet.

[0035] Figure 11. qPCR data showing the mRNA expression of the ACSL4 (T4) isoform 1 variants 2 and 3 (T4- V2 and T4-V3) in kidney and liver and the expression of the isoform 2 (T4-VI) in different regions of the brain. Of note, the isoform 2 is the ACSL4 protein containing the membrane spanning domain and is the gene expressed primarily in the brain. All ACSL4 isoforms contain the "binding site".

[0036] Figure 12. qPCR data showing expression of ACSL1 (isoform b, Tl) in brain and kidney and liver.

[0037] Figure 13. qPCR data on the expression of ACSL4 isoforms 1 and 2 in different human cell lines.

[0038] Figure 14. Representative chromatograms of a given sample containing the internal standard (Cl 7-CoA or HeptaCoa) and arachidonoyl-CoA (AraCoAJ). Figure 15. Photoactivation of RX-055 is not a prerequisite for covalent blocking of AEA cellular uptake. While WOBE437 mediated AEA uptake inhibition can be washed out, RX-055 AEA uptake inhibition cannot be washed out (Chicca et al., 2017 [7]). This figure shows that photoactivation is not necessary and RX-055 like RX-237 act like ACSL3 / 4 selective mechanism-based activity probes at the catalytic site, thus keeping the molecule in place (covalent attachment adjacent to "binding site"). This appears to simultaneously block the neighboring "binding site" through the WOBE437 moiety in the molecule. RX-055 thus acts like a dual probe with an estimated EC50 for AEA cellular uptake inhibition of about 20 nM.

[0039] Figure 16. Sequence alignment of the human ACSL4 isoform 1 & isoform 2. The proposed endocannabinoid and inhibitor binding sites, the conserved P-loop and the conserved Gate domain appear illustrated. The binding site is only present in ACSL4 isoform 2.

[0040] Figure 17. An AEA and 2-AG binding assay was conducted in the BL21 (DE3) E. coli expression system, where the expression of either ACSL4 isoform 1 , isoform 2, or ACSL3 was induced. Whole cells were incubated with radioligands for 1 hour, washed, and total counts quantified. The expression of the protein was confirmed by Western blotting. Membrane preparations of ACSL4 isoform 2 and ACSL3 indicate expression at the plasma membrane. Binding of both endocannabinoids is only observed with the ACLS4 isoform 2.

[0041] Figure 18. Table of content indicating the name of the mutations, their specific amino acid substitution and the target of such mutations.

[0042] Figure 19. An AEA, 2-AG, and SYT-510 binding assay was conducted in the BL21 (DE3) E. coli expression system, where the expression of either ACSL4 isoform 2 or ACSL4 isoform 2 mutant AC1 (His16Ala) was induced. Whole cells were incubated with radioligands for 1 hour, washed, and total counts quantified. The expression of the protein was confirmed by Western blotting. Substituting His 16Ala potentially disrupts the helices, impairs complex formation, and renders the binding site inaccessible.

[0043] Figure 20. The AlphaFold in silico model depicts the residue Histidine-16, which plays a critical role in stabilizing the alpha helix bundle proposed in the tetrameric structure. This substitution potentially disrupts the assembly of the oligomer, which is essential for endocannabinoid binding.

[0044] Figure 21. A binding assay for AEA, 2-AG, SYT-510 and WOBE437 was conducted in the BL21 (DE3) E. coli expression system, where either ACSL4 isoform 2, ACSL4 isoform 2 mutant LF1 (Lys37Ala, Lys38Ala, Lys39Ala), or ACSL4 isoform 2 mutant LF2 (Lys37Gly, Lys38Gly) expression was induced. Whole cells were incubated with radioligands for one hour, washed, and total counts were quantified. The expression of the protein was confirmed by Western blotting. Substitution of the lysine residues for glycine potentially hinders the complex formation (depicted by the lack of an observable dimer band in the Western blot) and impedes binding. In comparison, substituting the lysine residues for alanine avoids binding seemingly without altering the complex formation. This mutation thus pinpoints a specific amino acid residue that can be targeted to inhibit endocannabinoid reuptake.

[0045] Figure 22. The AlphaFold in silico model illustrates Lysine-37, 38, and 39 residues. Substitution of these residues for alanine inhibits the binding without disrupting the tetrameric structure, whereas substituting them with glycine potentially destabilizes the protein complex, hindering endocannabinoid binding.

[0046] Figure 23. A binding assay for AEA and 2-AG was conducted in the BL21 (DE3) E. coli expression system, where the expression of either ACSL4 isoform 2, ACSL4 isoform 2 C mutant (Tyr315Ala, Gly317Ala, Tyr318Ala), or ACSL4 isoform 2 D mutant (Ser279Ala, Ser281 Ala) was induced. Whole cells were incubated with radioligands for one hour, washed, and total counts were quantified. Both mutations retain endocannabinoid binding with no difference compared to WT. The lack of catalytic activity was confirmed by incubating whole cells with 10 pM arachidonic acid for 10 minutes at 37°C, followed by quantification of arachidonoyl-CoA using LC-MS / MS.

[0047] Detailed description of the invention.

[0048] The peptide of the present invention comprises the following amino acid sequence: AKKK (SEQ ID NO: 3). In the present invention, this sequence is referred to as binding sequence, binding site, target sequence or target site. The peptide preferably has a length of 16 amino acids, more preferably 9 amino acids, even more preferably 4amino acids. Preferably, the peptide comprises the amino acid sequence LTNAKKKNA (SEQ ID NO: 2), or ALIFIPWYFLTNAKKK (SEQ ID NO: 1). The peptide may also consist of the amino acid sequence of SEQ ID NO: 1, 2 or 3, in other word the peptide may have the amino acid sequence ALIFIPWYFLTNAKKK (SEQ ID NO: 1), LTNAKKKNA (SEQ ID NO: 2), or AKKK (SEQ ID NO: 3). Accordingly, in one embodiment, the peptide has the amino acid sequence ALIFIPWYFLTNAKKK (SEQ ID NO: 1). In one embodiment, the peptide has the amino acid sequence LTNAKKKNA (SEQ ID NO: 2). In one embodiment, the peptide has the amino acid sequence AKKK (SEQ ID NO: 3).

[0049] The binding sequence of the present invention can be bound by an endocannabinoid and / or endocannabinoid reuptake inhibitor. The peptide of present invention can be bound by an endocannabinoid. The peptide of the present invention can also be bound by an endocannabinoid reuptake inhibitor. "Can be bound by" in the present invention means that the binding sequence "is capable of binding to". In one embodiment, the term "can be bound by" means that the binding sequence "is bound to". In one embodiment of the present invention, the peptide is bound to an endocannabinoid. In another embodiment of the present invention, the peptide is bound to an endocannabinoid reuptake inhibitor. In these embodiments, the peptide is either bound to an endocannabinoid or to an endocannabinoid reuptake inhibitor, but preferably the peptide is not bound to an endocannabinoid and an endocannabinoid reuptake inhibitor at the same time. In the present invention, the amino acid sequence of the peptide having the sequence of SEQ ID NO: 1 , preferably of SEQ ID NO: 2 or 3 is also referred to as binding sequence. All embodiments described for the peptide also apply to the binding sequence, where applicable.

[0050] The peptide or binding sequence of the present invention can originate from an acyl-CoA synthetase protein (ACSL) in particular a human acyl-CoA synthetase protein. The acyl-CoA synthetase protein is an enzyme, which can catalyze the formation of (long chain) fatty acyl-CoA by ligation of CoA to their substrate, (long chain) fatty acids. The substrate, the (long chain) fatty acids, binds to the catalytic site of the acyl-CoA synthetase protein.

[0051] The catalytic site of the acyl-CoA synthetase protein comprises the ATP binding sequence: TSG and the lipid tunnel sequence: YIGYLPLA (SEQ ID NO: 5) to which the (long chain) fatty acids can bind. The peptide or binding sequence of the present invention does not overlap with the catalytic site and / or the lipid tunnel.

[0052] In a preferred embodiment, the peptide or binding sequence of the present invention originates from ACSL4 isoform 2. The peptide or binding sequence preferably does not originate from ACSLI, ACSL4, ACSL5 or ACLS6. More preferably the peptide or binding sequence with the amino acid sequence LTNAKKKNA (SEQ ID NO: 2) or AKKK (SEQ ID NO: 3) originates from ACSL 4 isoform 2.

[0053] The binding sequence or peptide of the present invention can form an alpha helix. In this case, the binding sequence or peptide preferably has a length of 3-16 amino acids, more preferably 9-16, most preferably 3 amino acids. In a preferred embodiment, the binding sequence of the present invention can form an alpha helix.

[0054] The peptide or binding sequence of the present invention can be bound by an endocannabinoid. "Can be bound by an endocannabinoid" means the peptide or binding sequence is capable of binding to an endocannabinoid. In other words, the peptide or binding sequence can bind to an endocannabinoid but is not necessarily actually bound to an endocannabinoid. The peptide or binding sequence of the present invention can also be bound to an endocannabinoid. "Can be bound to an endocannabinoid" means that the endocannabinoid is actually bound to the endocannabinoid. Binding of the peptide or binding sequence of the present invention to an endocannabinoid is preferably specific. Specific binding of an endocannabinoid means that endocannabinoid preferentially binds to the peptide or binding sequence in a membrane environment, in particular a phospholipid membrane environment. "Preferentially binds to" means that there is more binding to the peptide or binding sequence than to the phospholipid membrane environment. "Preferentially binds" can also be referred to as "selectively binds". Binding can be measured in a competition assay as described herein. The binding of the peptide or binding sequence of the present invention to the endocannabinoid is preferably reversible.

[0055] An endocannabinoid is a neuromodulator, in particular an endogenous lipid-based retrograde neuromodulator in vertebrates, preferably mammals, more preferably humans. Endocannabinoids can be expressed in the central nervous system including the brain and the peripheral nervous system. In the present invention, the endocannabinoid can be 2-arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA). An endocannabinoid can bind to an endocannabinoid receptor, such as cannabinoid receptor type 1 (CBI) and endocannabinoid receptor type 2 (CB2). The CBI receptor is preferably located in the brain, nervous system, peripheral organs or tissues. N- arachidonoylethanolamine (anandamide, AEA) preferably binds to CB 1. The CB2 receptor is preferably located in immune cells and peripheral tissues. Peripheral tissues can be immune cells. 2- arachidonoylglycerol (2-AG) and / or N-arachidonoylethanolamine (anandamide, AEA) can bind to CB2.

[0056] As shown in the present invention, an endocannabinoid can also bind to the peptide or binding sequence of an acyl-CoA synthetase as described herein. The binding of the peptide or binding sequence of the present invention to an endocannabinoid, such as 2-arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA) is preferably independent of the binding of the endocannabinoid to an endocannabinoid receptor, such as CB1 and / or CB2. CB1 and / or CB2 can be activated by blocking the binding site of the present invention. Activation of CB1 and / or CB2 is that case is preferably indirectly and not directly by binding. In other words, when an endocannabinoid reuptake inhibitor binds to the peptide or binding sequence, thus inhibiting the reuptake of the endocannabinoid, the endocannabinoid can bind to the CB1 and / or CB2 receptor and active said receptor.

[0057] The peptide or binding sequence of the present invention can be bound by an endocannabinoid reuptake inhibitor. "Can be bound by an endocannabinoid reuptake inhibitor" means that the endocannabinoid is "capable of binding to an endocannabinoid". In other words, the peptide or binding sequence can bind but is not necessarily actually bound to an endocannabinoid reuptake inhibitor. The peptide or binding sequence of the present invention can also be bound to an endocannabinoid reuptake inhibitor. "Can be bound to an endocannabinoid reuptake inhibitor" or "is bound to an endocannabinoid reuptake inhibitor" means that the endocannabinoid reuptake inhibitor is actually bound to the endocannabinoid reuptake inhibitor. Binding of the peptide or binding sequence of the present invention to an endocannabinoid reuptake inhibitor is preferably specific. Specific binding of an endocannabinoid means that endocannabinoid preferentially binds to the peptide or binding sequence in a membrane environment, in particular a phospholipid membrane environment. "Preferentially binds to" means that there is more binding to the peptide or binding sequence than to the phospholipid membrane environment. "Preferentially binds" can also be referred to as "selectively binds". Binding can be measured in a competition assay as described herein. The binding of the peptide or binding sequence of the present invention to the endocannabinoid reuptake inhibitor can be reversible or irreversible, preferably reversible. An endocannabinoid reuptake inhibitor according to the present invention can inhibit the uptake of an endocannabinoid, the reuptake of an endocannabinoid or the release of an endocannabinoid, preferably the reuptake of an endocannabinoid. The endocannabinoid reuptake inhibitor of the present invention can also be referred to as "endocannabinoid uptake inhibitor or endocannabinoid release inhibitor", "endocannabinoid uptake inhibitor" or endocannabinoid release inhibitor". The endocannabinoid reuptake inhibitor may also be referred to as "endocannabinoid trafficking inhibitor". Inhibiting the reuptake of an endocannabinoid means that trafficking of an endocannabinoid across a plasma membrane or across an intracellular membrane is inhibited. A plasma membrane is preferably a lipid bilayer. The lipid bilayer is preferably a phospholipid bilayer. Trafficking can be the diffusion of the endocannabinoid in a lipid bilayer from the outer layer to the inner layer. Such trafficking may comprise trafficking from an extracellular fluid into an intracellular environment, from an intracellular environment into an organelle, such as the endoplasmic reticulum (ER), mitochondria, or a lipid vesicle. Trafficking of an endocannabinoid across the plasma membrane can also be the uptake of the endocannabinoid into the plasma membrane, reuptake of the endocannabinoid into the plasma membrane, release of the endocannabinoid from the plasma membrane and / or integration of the endocannabinoid into the plasma membrane. Inhibition of the reuptake of an endocannabinoid can for example mean that the endocannabinoid reuptake inhibitor inhibits uptake of the endocannabinoid from the synaptic cleft after the endocannabinoid has been released into the synaptic cleft by a releasing neuron. Inhibition of the reuptake can increase the amount of an endocannabinoid in the synaptic cleft. The inhibition can therefore increase neurotransmission. This is particularly useful in conditions or diseases associated with endocannabinoid deficiency, i.e. conditions or diseases, wherein the subject suffers from an endocannabinoid deficiency. Such conditions or diseases include disorders of the central nervous system (such as neuropsychiatric, neuroinflarnmatory or neurodegenerative disorders), inflammatory diseases or immune diseases.

[0058] An endocannabinoid reuptake inhibitor is preferably a compound, more preferably a small molecule, which inhibits the reuptake of the endocannabinoid. A small molecule has a molecular weight of less than 800 g / mol or 800 Da. An endocannabinoid reuptake inhibitor can inhibit uptake or release of an endocannabinoid at a concentration of 300-3000 nM, preferably 30-300 nM.

[0059] The endocannabinoid reuptake inhibitor can be a first or second generation endocannabinoid reuptake inhibitor. Preferably endocannabinoid reuptake inhibitor has the formula wherein in formula (I) R is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, aryl, and heteroaryl, and wherein Ri is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri is optionally substituted, wherein in formula (II) Ri, R2 and R3 are independently from each other selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri, R2 and R3 are optionally independently from each other substituted. Endocannabinoid reuptake inhibitors having formula (I) are also referred to as first generation reuptake inhibitors. Endocannabinoid reuptake inhibitors having formula (II) are also referred to as second generation inhibitors, or SYT inhibitors.

[0060] First generation endocannabinoid reuptake inhibitors comprise dodeca-2E,4E-dienoyl N-alkylamides (for example WOBE437, RX-055, guineesine and RX-237), second generation reuptake inhibitors comprise 2-immino-5-ene-thiazolidin-4-ones (for examples SYT-510, SYT-520 and SYT-530).

[0061] Examples of endocannabinoid reuptake inhibitors having formula (I) are:

[0062] Guineensine.

[0063] The structures of RX-055 and RX-237 are also shown in Fig. 2. Guineensine is disclosed in Nicolussi et al., Pharmacol Res. 2014 Feb; 80:52-65.

[0064] Examples of endocannabinoid reuptake inhibitors having formula (II) comprises: SYT-510, SYT-520 and SYT-530. Preferably the endocannabinoid reuptake inhibitor is selected from the group consisting of WOBE437, RX-055, guineensine, RX-237, SYT-510, SYT-520 and SYT-530. The most preferred endocannabinoid reuptake inhibitor is WOBE437. WOBE437 can bind to the peptide or sequence of the present inventions and does not inhibit the function of the acyl-CoA synthetase protein. RX-055 and RX- 237 can bind to the peptide or sequence of the present inventions and inhibit the function of the acyl-CoA synthetase protein.

[0065] In one embodiment of the present invention, the peptide with the amino acid sequence LTNAKKKNA (SEQ ID NO: 2) can be bound by WOBE437 or SYT inhibitors such as the compounds disclosed in WO 2015 / 181337). In another embodiment, the peptide with the amino acid sequence AKKK (SEQ ID NO: 3) can be bound by WOBE437 or SYT inhibitors (as defined hereinabove).

[0066] The present invention also relates to a fusion protein comprising a peptide as described herein (including each specific embodiment of said peptide) and a transmembrane domain, wherein the fusion protein can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor. The fusion protein may additionally comprise a linker, located between the peptide and the transmembrane domain. The transmembrane domain of the fusion protein can be integrated into a lipid layer (also referred to herein as lipid leaflet) or lipid bilayer. The transmembrane domain is not particularly limited as long as it can be integrated into a lipid layer or lipid bilayer and thus enable the fusion protein to be colocalized with said lipid layer or lipid bilayer. The lipid bilayer can for example be the lipid bilayer of the plasma membrane. In such case, said transmembrane domain is preferably integrated into the inner layer of a plasma membrane lipid bilayer. The lipid layer can for example be comprised a lipid vesicle. The peptide of the fusion protein preferably faces the plasma side of a plasma membrane lipid bilayer or the outside of a lipid vesicle. Then, the peptide of the fusion protein is exposed to and can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor.

[0067] Preferably, the fusion protein as described herein, is not ACSL4 isoform 2. Accordingly, the fusion protein preferably does not include sequence according to SEQ ID NO.: 6 or 7.

[0068] A further aspect of the present invention relates to a composition comprising the peptide of the present invention or the fusion protein of the present invention, an endocannabinoid and / or an endocannabinoid reuptake inhibitor, and optionally a pharmaceutically relevant carrier. In one embodiment, the composition comprises the peptide of the present invention and the endocannabinoid. In another embodiment, the composition comprises the fusion protein of the present invention and the endocannabinoid. In a further embodiment, the composition comprises the peptide of the present invention and the endocannabinoid reuptake inhibitor. In a yet further embodiment, the composition comprises the fusion protein of the present invention and the endocannabinoid reuptake inhibitor. All embodiments of the composition of the present invention can optionally comprise a pharmaceutically relevant carrier. Pharmaceutically acceptable carriers are known to the skilled person. Preferably, the composition of the present invention is a pharmaceutical composition. All embodiments described above with respect to the peptide and / or fusion protein also apply to the composition comprising the same, where applicable. The composition of the present invention can be used as a medicament.

[0069] Another aspect of the present invention is a binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein, which binding sequence can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor and wherein the sequence comprises the following the amino acid sequence: AKKK (SEQ ID NO: 3). In other words, the binding sequence is located in the acyl-CoA synthetase protein or a fragment of said protein. Preferably, the binding sequence comprises the following amino acid sequence: LTNAKKKNA (SEQ ID NO: 2), or ALIFIPWYFLTNAKKK (SEQ ID NO: 1). The binding sequence can also be of the amino acid sequence according to SEQ ID NO: 1 , 2, or 3. The binding sequence can also be in a fragment of an acyl-CoA synthetase protein, wherein the fragment is not particularly limited as long as it retains the capability of binding to a endocannabinoid or an endocannabinoid reuptake receptor. The fragment of an acyl-CoA synthetase protein preferably has a length of 12 to 720 more preferably a length of 50 to 500 amino acids, even more preferably 50-250. The fragment is preferably longer than the peptide. The fragment as defined can be a functional fragment, which comprises the binding sequence and optionally the catalytic site.

[0070] The acyl-CoA synthetase protein in which the binding sequence is located, is preferably an integral membrane protein or membrane associated protein." Integral membrane protein" means that the acyl- CoA synthetase protein is integrated into the membrane, preferably by a transmembrane domain of the acyl-CoA synthetase protein. The acyl-synthetase protein is preferably integrated into the plasma membrane lipid bilayer or intracellular lipid bilayer. "Membrane associated protein" means that the acyl- CoA synthetase protein is associated with the membrane by a lipophilic or amphiphilic domain. The acylsynthetase protein is preferably associated to the inner layer of a plasma membrane. The acyl-synthetase protein can be associated or integrated to the inner layer of a plasma membrane lipid bilayer or intracellular lipid bilayer.

[0071] The binding sequence is located in the acyl-CoA synthetase protein or a fragment thereof. The acyl-CoA synthetase protein or fragment thereof, in which the binding sequence is located, is preferably a long- chain acyl-CoA synthetase protein, more preferably ACLS4 isoform 2. The acyl-Coa synthetase protein is preferably not ACSLI, ACSL3, ACSL5 or ACSL6. ACSL4 isoform 2 is preferably expressed in the brain, preferably the human brain. ACSL4 isoform 2 can also be expressed in peripheral tissues, preferably immune cells. ACSL4 isoform 2 can be expressed in the brain, and / or immune cells. ACLS4 isoform 2 is preferably human ACLS4, for example human ACSL4 isoform 2 (SEQ ID NO: 6; NCBI Reference Sequence: NP_001305438.1).

[0072] ACSL4 Isoform 2 (T4) is preferably expressed on a cell membrane and / or an intracellular membrane, such as the membrane of the endoplasmatic reticulum (ER), mitochondria. The binding sequence comprising or having the amino acid sequence of LTNAKKKNA (SEQ ID NO: 2) is preferably located in ACLS4 isoform 2, for example in human ACLS4 isoform 2, or a fragment thereof. The binding sequence of the present invention can be an alpha helix of the acyl-CoA synthetase protein.

[0073] The binding sequence of the present invention is preferably independent of the catalytic domain of the acyl-CoA synthetase protein and / or the lipid tunnel of the acyl-CoA synthetase protein, preferably independent of the catalytic domain and the lipid tunnel of the acyl-CoA synthetase protein. In other words, the binding sequence of the present invention does not overlap with the catalytic site and / or the lipid tunnel of the acyl-CoA protein, more preferably, binding sequence does not overlap with the catalytic site and the lipid tunnel.

[0074] The binding sequence of the present invention is independent from the catalytic site and / or the lipid tunnel of the acyl-CoA synthetase protein. Meaning, the enzymatic reaction of acyl-CoA formation, which is catalyzed by the acyl-CoA synthetase protein is independent from binding to the endocannabinoid and / or the endocannabinoid reuptake inhibitor to the binding sequence of the present invention. Binding of an endocannabinoid reuptake inhibitor or endocannabinoid to the binding sequence of the present invention does preferably not inhibit the enzymatic function of the acyl-CoA synthetase protein. The enzymatic function is also referred to as activity or enzymatic activity. Binding of the binding sequence of the present invention to an endocannabinoid and / or an endocannabinoid reuptake inhibitor is not inhibited by substrates of the acyl-CoA synthetase protein, such as eicosatetraenoic acids. Examples of such eicosatetraenioic acids include aracidonic acid (AA) and / or docosahexaenoic acid (DHA).

[0075] Binding of the binding sequence of the present invention to an endocannabinoid and / or an endocannabinoid reuptake inhibitor is preferably not inhibited by aracidonic acid (AA) and / or docosahexaenoic acid (DHA), more preferably by aracidonic acid (AA). "Is not inhibited" in this context means that binding is preferably not directly inhibited. Direct inhibition is mediated by binding to the binding sequence. Thus, in a preferred embodiment, eicosatetraenoic acids such as aracidonic acid (AA) and / or docosahexaenoic acid (DHA) do not bind to the peptide or binding sequence of the present invention. It is important that the catalytic site of the acyl-CoA synthetase protein to which substrates of the acyl-CoA synthetase bind, is independent of the binding site of the acyl-CoA synthetase protein to which an endocannabinoid and / or endocannabinoid reuptake inhibitors can bind. The enzymatic activity of the acyl- CoA synthetase can be measured by an appropriate method. Such methods include, for example, measuring the amount of a product, which is produced by the acyl-CoA synthetase. Arachidonic acid is a substrate of the acyl-CoA synthetase and the produced product is arachidonoyl-CoA. The amount of arachidonoyl-CoA produced by the acyl-CoA synthetase can be determined by LC-MS / MS. The enzymatic activity of the acyl-CoA synthetase protein can for example be determined by measuring the amount of arachidonoyl-CoA by LC-MS / MS.

[0076] There is no enzymatic activity when the amount of arachidonoyl-CoA is equal to the amount of arachidonoyl-CoA in the absence of functional acyl-CoA synthetase protein. Functional acyl-CoA synthetase protein is absent when the protein is inhibited by its inhibitor triacsin C or in cell, in which the protein has been knocked-out. Knock-out can be done by any method known to the skilled person, for example by using the CRISPR / Cas9 system. Inhibition by triacsin C is preferably complete. Inhibition is complete, when there is no activity left.

[0077] The endocannabinoid, which can bind to the binding sequence in an acyl-CoA synthetase protein or a fragment thereof is preferably is 2-arachidonoylglycerol (2-AG) and / or N-arachidonoylethanolamine (anandamide, AEA). When the binding sequence comprises the sequence LTNAKKKNA (SEQ ID NO: 2) or AKKK (SEQ ID NO: 3) the endocannabinoid is preferably 2-arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA). Other lipids containing a polyunsaturated acyl chain derived from arachidonic acid, like noladin ether, can bind to the binding sequence.

[0078] The endocannabinoid reuptake inhibitor, which can bind to the binding sequence in an acyl-CoA synthetase protein is an endocannabinoid reuptake inhibitor as defined above with respect to the peptide of the present invention. When the endocannabinoid reuptake inhibitor is bound to the binding sequence in an acyl-CoA synthetase protein the reuptake of the endocannabinoid is inhibited. Inhibiting the reuptake of an endocannabinoid is defined as described herein. As described above, inhibition of the reuptake of an endocannabinoid is particularly useful in conditions or diseases associated with endocannabinoid deficiency, i.e. conditions or diseases, wherein the subject suffers from an endocannabinoid deficiency. Such conditions or diseases include disorders of the central nervous system ( such as neuropsychiatric, neuroinflammatory or neurodegenerative disorders), inflammatory diseases or immune diseases.

[0079] All embodiments described above with respect to the peptide, the fusion protein and / or the composition also apply to the binding sequence in an acyl-CoA synthetase protein and a fragment thereof, where applicable.

[0080] The present invention also relates to an acyl-CoA synthetase protein or fragment thereof comprising the binding sequence described herein. Thus, in one embodiment, the acyl-CoA synthetase protein or fragment thereof comprises a binding sequence, which binding sequence can be bound by an endocannabinoid and / or endocannabinoid reuptake inhibitor, and wherein the sequence comprises the following ammo acids sequence: ALIFIPWYFLTNAKKK (SEQ ID NO: 1). All embodiments described above with respect to the peptide, the composition and / or the binding sequence also apply to the acyl- CoA synthetase protein or fragment thereof comprising the binding sequence, where applicable.

[0081] Another aspect of the present invention relates to a cell comprising the peptide of the present invention, the binding sequence of the present invention in an acyl-CoA synthetase protein or in a fragment of said protein or an acyl-CoA synthetase protein comprising the binding sequence of the present invention. The cell is preferably a eukaryotic cell, more preferably, a neuronal or immune cell. The cell preferably has a plasma membrane as defined herein. The peptide or binding sequence of the present invention can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor in the cell.

[0082] To this end, the binding sequence is preferably located in the outer or inner layer of the plasma membrane lipid bilayer, more preferably the inner layer of the plasma membrane. The binding sequence in the cell of the present invention is preferably bound to an endocannabinoid or an endocannabinoid reuptake inhibitor as defined herein. The cell may further comprise at least one additional protein and / or enzyme, which is preferably selected from the group consisting of: MAGL (monoacylglycerol lipase), FAAH (fatty acid amide hydrolase), ABHD4 ((Lyso)-N-acylphosphatidylethanolamine lipase), ABDH6 (alpha / beta-Hydrolase domain containing 6), ABHD12 (Abhydrolase Domain Containing 12), DAGL (Diacylglycerol lipase), NAPE-PLD (N-acyl phosphatidylethanolamine-specific phospholipase D), CBI (cannabinoid receptor 1), CB2 (cannabinoid receptor 2), and GABAA receptors (gamma-aminobutyric acid receptors). MAGL is a membrane-associated member of the serine hydrolase superfamily, which hydrolyses 2-AG to arachidonic acid and glycerol.

[0083] In a further aspect, the present invention relates to a lipid bilayer comprising the peptide of the present invention, the fusion protein of the present invention, the binding sequence of the present invention in an acyl-CoA synthetase protein or in a fragment of said protein or an acyl-CoA synthetase protein comprising the binding sequence of the present invention. The lipid bilayer is preferably a phospholipid bilayer. The acyl-CoA synthetase protein in this embodiment is preferably ACSL4 isoform 2, i.e. the peptide preferably originates from ACSL4 isoform 2 or the binding sequence is preferably in ACSL4 isoform 2 or a fragment thereof. Thus, the peptide or binding sequence preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2. A lipid bilayer comprises two layers, also referred to as leaflets, an outer layer (or outer leaflet) and an inner layer (or inner leaflet). The inner layer is preferably facing the inside (i.e. plasma side) of a cell, the outer layer is preferably facing the outside of a cell. The binding sequence is preferably located in the outer or inner layer of the plasma membrane lipid bilayer, more preferably the inner layer of the plasma membrane. The lipid bilayer can be a plasma membrane, a membrane of a cell organelle, a liposome, a lipid bilayer vesicle or a lipid bilayer sheet. The plasma membrane is the membrane between the inside and the outside of a cell. A membrane of a cell organelle is preferably a membrane of the endoplasmic reticulum (ER) or mitochondria, preferably the endoplasmic reticulum. A liposome is an artificial vesicle with two lipid layers. A lipid bilayer vesicle has two lipid bilayers. The lipid bilayer can be in a natural environment in a cell, or in non-natural environment outside a cell. The lipid bilayer can be obtained from a cell, preferably by cell disruption and differential centrifugation or reconstitution. A lipid bilayer (preferably in form of a vesicle) can also be prepared by reconstitution. A lipid bilayer vesicle or liposome preferably has a diameter of between 50 and 1000 nm.

[0084] The diameter of a lipid bilayer vesicle or liposome can be measured by a zetasizer. In the lipid bilayer, the peptide or binding sequence can preferably be bound by or bound to an endocannabinoid and / or an endocannabinoid reuptake inhibitor as defined herein. The location of the peptide or binding sequence or fusion protein can be mediated by a transmembrane domain, such as the transmembrane domain of ACSL4 (SEQ ID NO: 4). To this end, the peptide or binding sequence preferably comprises a transmembrane domain. The lipid bilayer may further comprise at least one additional protein and / or enzyme, which is preferably selected from the group consisting of: MAGL (monoacylglycerol lipase), FAAH (fatty acid amide hydrolase), ABHD4 ((Lyso)-N-acylphosphatidylethanolamine lipase), ABDH6 (alpha / beta-Hydrolase domain containing 6), ABHD12 (alpha / beta Hydrolase Domain Containing 12), DAGL (Diacylglycerol lipase), NAPE-PLD (N-acyl phosphatidylethanolamine-specific phospholipase D), CB 1 ( cannabinoid receptor 1 ), CB 2 ( cannabinoid receptor 2), and GABAA receptors (gamma- aminobutyric acid receptors). The endocannabinoid, which can be bound by or bound to the peptide or binding sequence in the lipid bilayer is preferably 2-arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA). Figure 1 shows an example of such proteins in a lipid bilayer according to the present invention.

[0085] The peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl- CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, the lipid bilayer of the present invention.

[0086] Such uses include, but are not limited to, use in the treatment of a condition or a disease associated with an altered function of the endocannabinoid system, preferably use in the treatment of a disorder of the central nervous system ( such as neuropsychiatric, neuroinflammatory or neurodegenerative disorders), an inflammatory disease or an immune disease. The condition or a disease associated with an altered function of the endocannabinoid system can be a disease or condition associated with an endocannabinoid deficiency. The peptide of the present invention, the fusion protein of the present invention, the acyl-CoA synthetase protein of the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, the lipid bilayer of the present invention can be administered to a subject in need thereof. The subject is preferably a human patient, more preferably a human patient suffering from a condition or disease with an altered function of the endocannabinoid system, most preferably a human patient suffering from a disease or condition associated with an endocannabinoid deficiency.

[0087] In a further embodiment, the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, the lipid bilayer of the present invention can be for use in the modulating of a cellular process, such as for modulating the levels of an endocannabinoid, and / or for modulating cellular immune processes.

[0088] In one embodiment, modulation of a cellular process is facilitating plasma membrane trafficking of an endocannabinoid, facilitating the reuptake of an endocannabinoid or facilitating the release of an endocannabinoid, preferably facilitating plasma membrane trafficking of an endocannabinoid, facilitating the reuptake of an endocannabinoid or facilitating the release of an endocannabinoid, more preferably facilitating plasma membrane trafficking of an endocannabinoid or facilitating the reuptake of an endocannabinoid, most preferably facilitating the reuptake of an endocannabinoid. In this embodiment, the peptide or binding sequence is preferably bound by or bound to an endocannabinoid, more preferably in the absence of an endocannabinoid reuptake inhibitor.

[0089] In further embodiment, modulation of a cellular process is decreasing plasma membrane trafficking, inhibiting the reuptake of an endocannabinoid, inhibiting the release of an endocannabinoid or sustaining endocannabinoid signaling, preferably decreasing plasma membrane trafficking, inhibiting the reuptake of an endocannabinoid, inhibiting the release of an endocannabinoid or sustaining endocannabinoid signaling, more inhibiting the reuptake of an endocannabinoid, inhibiting the release of an endocannabinoid, most preferably inhibiting the reuptake of an endocannabinoid. In this embodiment, the peptide or binding sequence is preferably bound by or bound to an endocannabinoid reuptake inhibitor. Binding of the peptide or binding sequence by or to an endocannabinoid reuptake inhibitor inhibits binding of an endocannabinoid to the peptide or binding sequence.

[0090] All embodiments described below with respect to methods also apply to the above uses, where applicable.

[0091] The present invention also relates to a method for facilitating plasma membrane trafficking of an endocannabinoid using the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, or the lipid bilayer of the present invention. For this method, the peptide or binding sequence is preferably bound by or bound to an endocannabinoid. Plasma membrane trafficking is the diffusion of the endocannabinoid in a lipid bilayer from the outer layer to the inner layer. Such trafficking may comprise trafficking from extracellular fluid into intracellular environments, from intracellular environment into organelles, such as the endoplasmic reticulum (ER), mitochondria or vesicles. Trafficking of an endocannabinoid across the plasma membrane can also be uptake of the endocannabinoid into the plasma membrane, reuptake of the endocannabinoid into the plasma membrane, release of the endocannabinoid from the plasma membrane and / or integration of the endocannabinoid into the plasma membrane. Facilitating the plasma membrane trafficking means that the trafficking across the plasma, preferably the uptake of an endocannabinoid into the plasma membrane or the release of an endocannabinoid from a plasma membrane is increased in the presence of the peptide of the present invention, the fusion protein of the present invention. The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention or the lipid bilayer of the present invention. In other words, there is more trafficking across the plasma membrane in the presence of the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, the lipid bilayer of the present invention. The endocannabinoid in this method is preferably 2-arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA). The acyl-CoA synthetase protein in this method is preferably ACSL4 isoform 2. The method is illustrated in Figure 1 .

[0092] The present invention is also directed to a method for decreasing plasma membrane trafficking of an endocannabinoid using the peptide of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, the lipid bilayer of the present invention, and / or an endocannabinoid reuptake inhibitor as described herein. Decreasing plasma membrane trafficking means that there is less trafficking across the plasma membrane in the presence of the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, the lipid bilayer of the present invention and / or endocannabinoid reuptake inhibitor as described herein. Plasma membrane trafficking is as defined herein. The endocannabinoid reuptake inhibitor is preferably WOBE437 or a second generation reuptake inhibitor like SYT510, SYT-520 or SYT-530, disclosed in WO2015181337.

[0093] The term "substituted" refers to the addition of a substituent group to a parent moiety.

[0094] "Substituent groups" can be protected or unprotected and can be added to one available site or to many available sites in a parent moiety. Substituent groups may also be further substituted with other substituent groups and may be attached directly or by a linking group such as an alkyl, an amide or hydrocarbyl group to a parent moiety. "Substituent groups" amenable herein include, without limitation, halogen, oxygen, nitrogen, sulphur, hydroxyl, alkyl, alkenyl, alkynyl, acyl, carboxyl, aliphatic groups, alicyclic groups, alkoxy, substituted oxy, aryl, aralkyl, amino, imino, amido fluorinated compounds etc.

[0095] As used herein the term "alkyl," refers to a saturated straight or branched hydrocarbon moiety containing in particular up to 12 carbon atoms. Examples of alkyl groups include, without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, and the like. Alkyl groups typically include from 1 to about 12 carbon atoms (C1-C12 alkyl).

[0096] As used herein the term "cycloalkyl" refers to an interconnected alkyl group forming a saturated or unsaturated (or partially unsaturated) ring or polyring structure containing 3 to 10, particularly 5 to 10 carbon atoms. Examples of cycloalkyl groups include, without limitation, cyclopropane, cyclopentane, cyclohexane, norbornane, decaline or adamantan (Tricyclo[3.3.1.1]decan), and the like. Cycloalkyl groups typically include from 5 to 10 carbon atoms (C5-C10 cycloalkyl).

[0097] Alkyl or cycloalkyl groups as used herein may optionally include further substituent groups. A substitution on the cycloalkyl group also encompasses an aryl, a hetreocylce or a heteroaryl substituent, which can be connected to the cycloalkyl group via one atom or two atoms of the cycloalkyl group.

[0098] As used herein the term "alkenyl," refers to a straight or branched hydrocarbon chain moiety containing in particular up to 12 carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include, without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1 ,3-butadiene and the like. Alkenyl groups as used herein may optionally include further substituent groups

[0099] As used herein the term "alkynyl," refers to a straight or branched hydrocarbon moiety containing in particular up to 12 carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups as used herein may optionally include further substituent groups.

[0100] As used herein the term "alkoxy," refers to an oxygen alkyl moiety containing in particular 1 to 12 carbon atoms comprising at least one oxygen moiety instead of a CH2 moiety. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups. Furthermore, "alkoxy" groups include straight or branched ether groups (e. g. -CH2- CH2-O-CH3) or polyether groups, which comprise several interconnected momomere alkoxy groups (e. g. -O-CH2-CH2-O-CH3).

[0101] As used herein the term "heterocycle" refers to an interconnected alkyl group forming a saturated or unsaturated ring or polyring structure containing 3 to 10, particularly 5 to 10 carbon atoms in which at least one carbon atom is replaced with an oxygen, a nitrogen or a sulphur atom forming a nonaromatic structure. Heterocyclic groups as used herein may optionally include further substituent groups. A substitution on the heterocyclic group also encompasses an aryl, a cycloalkyl or a heteroaryl substituent, which can be connected to the heterocyclic group via one atom or two atoms of the heterocyclic group (comparable to indole). As used herein the term "aryl" refers to a hydrocarbon with alternating double and single bonds between the carbon atoms forming an aromatic ring structure, in particular a six (Ge to ten (C10) membered ring or polyring structure. The term "heteroaryl" refers to aromatic structures comprising a five to ten membered ring or polyring structure, comparable to aryl compounds, in which at least one member is an oxygen or a nitrogen or a sulphur atom. Due to simplicity reasons they are denominated Cs to Ci o heteroaryl, wherein at least one carbon atom is replaced with an oxygen, a nitrogen or a sulphur atom forming an aromatic structure. For example a Cs heteroaryl comprises a five membered ring structure with at least one carbon atom being replaced with an oxygen, a nitrogen or a sulphur atom. Aryl or hetero aryl groups as used herein may optionally include further substituent groups. A substitution on the hetero aryl group also encompasses an aryl, a cycloalkyl or a heterocycle substituent, which can be connected to the hetero aryl via one atom or two atoms of the hetero aryl group (comparable to indole). The same applies to an aryl group

[0102] For this method, the endocannabinoid is 2- arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA). The acyl-CoA synthetase protein in this method is preferably acyl-CoA synthetase protein is ACSL4 isoform 2.

[0103] The present invention also relates to a method for facilitating the reuptake of an endocannabinoid using the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl- CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, or the lipid bilayer of the present invention. The method can also be for facilitating the uptake of an endocannabinoid. Reuptake or uptake of an endocannabinoid can be the reuptake or uptake of an endocannabinoid into the plasma membrane. Facilitating the reuptake or uptake means that more endocannabinoid is taken up into the plasma membrane in the presence of the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, the lipid bilayer of the present invention. In this method, the peptide or binding sequence is preferably bound by or bound to an endocannabinoid. For this method, the endocannabinoid can be 2-arachidonoylglycerol (2-AG) and / or N-arachidonoylethanolamine (anandamide, AEA). For this method, the acyl-CoA synthetase protein can be ACSL4 isoform 2. The present invention also relates to a method for inhibiting the reuptake of an endocannabinoid using the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl- CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, or the lipid bilayer of the present invention, and an endocannabinoid reuptake inhibitor as described herein. Inhibiting the reuptake or uptake means that less endocannabinoid is taken up into the plasma membrane in the presence of the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, the lipid bilayer of the present invention and an endocannabinoid reuptake inhibitor as described herein. The endocannabinoid reuptake inhibitor is preferably WOBE437. For this method, the endocannabinoid can be 2-arachidonoylglycerol (2-AG) and / or N arachidonoylethanolamine (anandamide, AEA). For this method, the acyl-CoA synthetase protein can be ACSL4 isoform 2.

[0104] The present invention further also relates to a method for sustaining endocannabinoid signaling using the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl- CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, the lipid bilayer of the present invention, and an endocannabinoid reuptake inhibitor as defined herein. Sustaining endocannabinoid signaling means that the endocannabinoid signaling is maintained in the presence of the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, the lipid bilayer of the present invention, and an endocannabinoid reuptake inhibitor as defined herein. Sustaining endocannabinoid signaling can also mean that the endocannabinoid signaling is continues in the presence of the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, the lipid bilayer of the present invention, and an endocannabinoid reuptake inhibitor as defined herein. In this method, the endocannabinoid reuptake inhibitor is preferably WOBE437. For this method, the endocannabinoid can be 2- arachidonoylglycerol (2-AG) and / or N-arachidonoylethanolamine (anandamide, AEA). For this method, the acyl-CoA synthetase protein can be ACSL4 isoform 2. In a further embodiment, the present invention relates to the peptide of the present invention, the fusion protein of the present invention, the acyl-CoA synthetase protein of the present invention, the cell the present invention, or the lipid bilayer of the present invention, for use in the treatment of a condition or a disease associated with an altered function of the endocannabinoid system, preferably for use in the treatment of a disorder of the central nervous system (such as neuropsychiatric, neuroinflammatory or neurodegenerative disorders), an inflammatory disease or an immune disease. The condition or disease associated with an altered function of the endocannabinoid system is preferably a disease or condition associated with an endocannabinoid deficiency. The diseases are as defined herein. The use preferably comprises administering the peptide of the present invention, the fusion protein of the present invention, the acyl-CoA synthetase protein of the present invention, the cell of the present invention, the lipid bilayer of the present invention to a patient in need thereof.

[0105] In again a further embodiment, the present invention relates to use of the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, or the lipid bilayer of the present invention, for modulating of a cellular process, such as for modulating the levels of an endocannabinoid, and / or for modulating cellular immune processes.

[0106] In one specific embodiment, modulating a cellular process, as defined hereinabove, is preferably facilitating plasma membrane trafficking of an endocannabinoid, facilitating the reuptake of an endocannabinoid or facilitating the release of an endocannabinoid, preferably facilitating plasma membrane trafficking of an endocannabinoid or facilitating the reuptake of an endocannabinoid, more preferably facilitating the reuptake of an endocannabinoid. In this embodiment, the peptide or binding sequence is preferably bound by or bound to an endocannabinoid, more preferably in the absence of an endocannabinoid reuptake inhibitor.

[0107] In a further specific embodiment, modulating of a cellular process is decreasing plasma membrane trafficking, inhibiting the reuptake of an endocannabinoid, inhibiting the release of an endocannabinoid or sustaining endocannabinoid signaling, preferably, more preferably inhibiting the reuptake of an endocannabinoid, or inhibiting the release of an endocannabinoid, even more preferably inhibiting the reuptake of an endocannabinoid. In this embodiment, the peptide or binding sequence is preferably bound by or bound to an endocannabinoid reuptake inhibitor. Binding of the peptide or binding sequence by or to an endocannabinoid reuptake inhibitor inhibits binding of an endocannabinoid to the peptide or binding sequence.

[0108] The present invention further relates to a method for screening potential endocannabinoid reuptake inhibitors. The endocannabinoid reuptake inhibitor as defined herein can also be referred to as "endocannabinoid uptake inhibitor or endocannabinoid release inhibitor", "endocannabinoid uptake inhibitor" or endocannabinoid release inhibitor". The endocannabinoid reuptake inhibitor may also be referred to as "endocannabinoid trafficking inhibitor". The method for identifying an endocannabinoid reuptake inhibitor comprises using the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell the present invention, the lipid bilayer of the present invention, wherein the endocannabinoid reuptake inhibitor inhibits trafficking of an endocannabinoid across the plasma membrane or intracellular membrane. Trafficking of an endocannabinoid across the plasma membrane or across an intracellular membrane is also referred facilitated diffusion. Trafficking of an endocannabinoid across the plasma membrane can be uptake of the endocannabinoid into the plasma membrane, reuptake of the endocannabinoid into the plasma membrane, release of the endocannabinoid from the plasma membrane and / or integration of the endocannabinoid into the plasma membrane. Such trafficking may for example comprise trafficking from extracellular fluid into intracellular environments, from intracellular environment into organelles, such as the endoplasmic reticulum (ER), mitochondria or vesicles.

[0109] The method of the present invention for identifying an endocannabinoid reuptake inhibitor may comprise analyzing the binding of an endocannabinoid reuptake inhibitor candidate to the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, or the lipid bilayer of the present invention. Preferably, the endocannabinoid reuptake inhibitor does not inhibit the enzymatic function of the acyl-CoA synthetase protein.

[0110] In a preferred embodiment, the method for identifying an endocannabinoid reuptake inhibitor comprises the steps: a) incubating the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, the lipid bilayer of the present invention, with an endocannabinoid reuptake inhibitor candidate under appropriate conditions. b) Measuring the binding of the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, the lipid bilayer of the present invention, of the present invention to an endocannabinoid reuptake inhibitor candidate by an appropriate method. c) Selecting an endocannabinoid reuptake inhibitor candidate with an appropriate binding affinity as endocannabinoid reuptake inhibitor.

[0111] Appropriate methods for measuring the binding in step b) are known to the skilled person. Such methods include, for example, a radiometric assay, fluorescent-based assay, Scintillation proximity assay, fluorescent-polarization assay, FRET assay (Fluorescence Resonance Energy Transfer), chemiluminescence-based assay, and analytical chemistry assays such as HPLC (high-performance liquid chromatography), GC-MS (gas chromatography-mass spectrometry), LC-MS (liquid chromatography-mass spectrometry). A radiometric assay measures the incorporation of radioactivity by competitive binding of an endocannabinoid, a chemical probe or an endocannabinoid reuptake inhibitor (preferably an endocannabinoid reuptake inhibitor) to the peptide or bindings sequence of the present invention. The endocannabinoid or the endocannabinoid reuptake inhibitor (preferably the endocannabinoid reuptake inhibitor) are radioactively labeled with a radioactive isotope. Preferred radioactive isotopes are3H,14C,32P,35S and125l. Radioactive isotopes can allow the specific labelling of a single atom of a substrate, which makes the radiometric assay sensitive and specific. Radioactivity is usually measured using a scintillation counter. The skilled person knows how to select appropriate conditions for measuring the bindings, such as salt, temperature, and pH. The salt concentration is preferably 1-500 mM, more preferably 1-250 mM. The temperature is preferably 20 to 40°C, more preferably 25 to 40 °C, even more preferably 35 to 40°C, most preferably 37°C. The pH is preferably between 6 and 8, more preferably between 6.5 and 7.5.

[0112] The cell in step b) is preferably selected from the group of a human, mouse, rat, hamster, monkey, and dog cell. However, the cell in step b) may also be a prokaryotic cell. The cell in step b) can be a cell, which expresses ACSL4 isoform 2. Expression is herein understood to encompass any expression, including recombinant expression and overexpression. Cells which do not express ACSL4 isoform 2, which overexpress ACSL4 isoform 2 with a mutated binding sequence that is not capable of binding an endocannabinoid reuptake inhibitor can be used as a negative control.

[0113] The method for identifying an endocannabinoid reuptake inhibitor of the present invention can optionally further comprise a step of confirming the binding measured in step b), wherein it is determined if the binding as measured in step b) is abolished in the absence of a functional binding sequence in the acyl- CoA synthetase protein. A functional binding sequence in the acyl-CoA synthetase protein can be absent if the binding sequence of the acyl-CoA synthetase protein has been modified, or if the entire acyl-CoA synthetase protein is absent. Modification of the binding sequence of the acyl-CoA synthetase protein can include a mutation or deletion of the whole binding sequence or part of it. The acyl-CoA synthetase can be absent, for example, in a cell in which the acyl-CoA synthetase has been knocked-out. Knock-out can be done by methods known to the skilled person, for examples by using the CRISPR / Cas9 system. The cell can be preferably a eukaryotic cell, more specifically a neuronal or immune cell. However, in an embodiment of the invention, the cell can be alternatively be a prokaryotic cell. The step of confirming the binding is preferably carried out after step b) and / or before step c).

[0114] The method for identifying an endocannabinoid reuptake inhibitor of the present invention may further comprise the following steps: d) measuring the activity of the acyl-CoA synthetase protein in the presence of the endocannabinoid reuptake inhibitor selected in step c ), and e) selecting an endocannabinoid reuptake inhibitor, which does not inhibit the activity of the acyl-CoA synthetase protein.

[0115] The method for identifying an endocannabinoid reuptake inhibitor of the present invention may optionally further comprise steps of measuring AEA and / or 2-AG update in cellular system(s) that overexpress(es) the acyl-CoA synthetase protein and selecting an endocannabinoid reuptake inhibitor which inhibits the AEA and / or 2-AG update in cellular system(s) that overexpress(es) the acyl-CoA synthetase protein.

[0116] The activity of the acyl-CoA synthetase can be measured by an appropriate method known to the skilled person. Such methods include, for example, measuring the amount of a product, which is produced by the acyl-CoA synthetase, such as arachidonoyl-CoA. Arachidonic acid is a substrate of the acyl-CoA synthetase. The amount of arachidonoyl-CoA produced by the acyl-CoA synthetase can be determined by LC-MS / MS in vitro or ex vivo, or by using a fluorescent substrate in vitro, preferably LC-MS / MS in vitro or ex vivo.

[0117] The present invention further provides a method for producing a pharmaceutical composition comprising mixing the endocannabinoid reuptake inhibitor, which has been selected by the method for identifying an endocannabinoid reuptake inhibitor, with a pharmaceutically acceptable carrier. Preferably, the endocannabinoid reuptake inhibitor has been selected in step c) or e) or the method identifying an endocannabinoid reuptake inhibitor of the present invention. Pharmaceutically acceptable carriers are known to the skilled person.

[0118] A further embodiment of the present invention provides the use of the peptide of the present invention, the fusion protein of the present invention, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according the present invention, the acyl-CoA synthetase protein comprising the binding sequence of the present invention, the cell of the present invention, or the lipid bilayer of the present invention, for identifying an endocannabinoid reuptake inhibitor. The endocannabinoid reuptake inhibitor is as defined herein. The endocannabinoid reuptake inhibitor as defined herein can also be referred to as "endocannabinoid uptake inhibitor or endocannabinoid release inhibitor", "endocannabinoid uptake inhibitor" or “endocannabinoid release inhibitor". The endocannabinoid reuptake inhibitor may also be referred to as "endocannabinoid trafficking inhibitor". In particular, the endocannabinoid reuptake inhibitor inhibits trafficking of an endocannabinoid across the plasma membrane or intracellular membrane. Trafficking of an endocannabinoid across the plasma membrane or across an intracellular membrane is also referred to as facilitated diffusion. Trafficking of an endocannabinoid across the plasma membrane can be uptake of the endocannabinoid into the plasma membrane, reuptake of the endocannabinoid into the plasma membrane, release of the endocannabinoid from the plasma membrane and / or integration of the endocannabinoid into the plasma membrane. Such trafficking may comprise trafficking from extracellular fluid into intracellular environments, from intracellular environment into organelles, such as the endoplasmic reticulum (ER), mitochondria.

[0119] In a further embodiment, the present invention relates to a compound, which is capable of binding to the amino acid sequence AKKK (SEQ ID NO: 3), preferably selected from: ALIFIPWYFLTNAKKK (SEQ ID NO: 1) and LTNAKKKNA (SEQ ID NO: 2) and wherein the compound inhibits trafficking of an endocannabinoid across the plasma membrane or intracellular membrane. The compound can be an endocannabinoid reuptake inhibitor as defined herein. The compound can inhibit trafficking of an endocannabinoid across the plasma membrane or intracellular membrane, wherein trafficking of an endocannabinoid across the plasma membrane can be uptake of the endocannabinoid into the plasma membrane, reuptake of the endocannabinoid into the plasma membrane, release of the endocannabinoid from the plasma membrane and / or integration of the endocannabinoid into the plasma membrane. The amino acid sequence can be part of an acyl-CoA synthetase protein or a fragment thereof, preferably of ACSL4 isoform 2. Binding of the binding sequence to the compound can be measured by methods known to the skilled person and / or methods described herein. In a preferred embodiment, the compound does not inhibit the enzymatic function of an acyl-CoA synthetase protein. The enzymatic function of the acyl- CoA synthetase protein can be measured by methods known to the skilled person and / or methods described herein. The compound preferably inhibits binding of an endocannabinoid to the binding sequence, when the compound is bound to the binding sequence. Binding of the compound to the binding sequence therefore inhibits trafficking of an endocannabinoid across the plasma membrane or intracellular membrane, preferably wherein trafficking of an endocannabinoid across the plasma membrane is uptake of the endocannabinoid into the plasma membrane, reuptake of the endocannabinoid into the plasma membrane, release of the endocannabinoid from the plasma membrane and / or integration of the endocannabinoid into the plasma membrane. The endocannabinoid is preferably 2- arachidonoylglycerol (2-AG) and / or N-arachidonoylethanolamine (anandamide, AEA).

[0120] The skilled person is capable of determining if the compound binds to a binding sequence having the amino acid sequence according to SEQ ID NO.: 3, or SEQ ID NO 1 or 2. The binding can be determined using the compound uptake assay in the BL21 (DE3) E. coli expression system, where either ACSL4 isoform 2, ACSL4 isoform 2 mutant LF1 (Lys37Ala, Lys38Ala, Lys39Ala), or ACSL4 isoform 2 mutant LF2 (Lys37Gly, Lys38Gly) expression was induced. Accordingly, the skilled person can use a binding assay conducted in the BL21 (DE3) E. coli expression system, where either ACSL4 isoform 2, ACSL4 isoform 2 mutant LF1 (Lys37Ala, Lys38Ala, Lys39Ala), or ACSL4 isoform 2 mutant LF2 (Lys37Gly, Lys38Gly) expression was induced and wherein the whole cells are incubated with radioligand for one hour, washed, and the amount of bound compound is quantified (i.e. total counts are quantified). The expression of the protein can be confirmed by Western blotting. Substitution of the lysine residues for glycine potentially hinders the complex formation (depicted by the lack of an observable dimer band in the Western blot) and impedes binding. In comparison, substituting the lysine residues for alanine avoids binding seemingly without altering the complex formation. It will be apparent to the skilled person that quantification of the compound may also be done in a way that does not require a radioligand, for example by using LC-MS methods. It is to be understood that the mutants are used for comparison with the WT protein. The compound as described herein is particularly useful in the treatment of a condition or a disease associated with an altered function of the endocannabinoid system. Accordingly, the present invention relates to a compound which binds to a binding sequence having the following amino acid sequence AKKK (SEQ ID NO: 3), preferably ALIFIPWYFLTNAKKK (SEQ ID NO: 1) or LTNAKKKNA (SEQ ID NO: 2), wherein the compound inhibits the uptake of an endocannabinoid, for use in the treatment of a condition or a disease associated with an altered function of the endocannabinoid system. Preferred condution or a diseases associated with an altered function of the endocannabinoid system is a disorder of the central nervous system, such as neuropsychiatric, neuroinflammatory or neurodegenerative disorders, an inflammatory disease or an immune disease.

[0121] Preferred compound as described herein is a second generation endocannabinoid inhibitor, according to formula (1), as described hereinabove.

[0122] The compound is useful as a medicament, for example for use in the treatment of a condition or a disease associated with an altered function of the endocannabinoid system, in particular a condition or disease associated with an endocannabinoid deficiency. Preferably, the compound of the present invention is for use in the treatment of a disorder of the central nervous system (such as neuropsychiatric, neuroinflammatory or neurodegenerative disorders), an inflammatory disease or an immune disease.

[0123] In a further embodiment, the present invention provides a composition comprising the compound and a pharmaceutically acceptable carrier.

[0124] The present inventors have demonstrated the following in the experiments described in the Examples section below.

[0125] 1) First generation SERIs, such as WOBE437 and RX-055 may inhibit AEA cellular uptake at low nM concentrations.

[0126] 2) WOBE437 does not inhibit the enzymatic activity of ACSL3 / 4, while its diazirine derivative RX-055 does inhibit enzymatic function. The enzymatic function is the acyl-CoA ligation of fatty acids, in particular polyunsaturated long chain fatty acids such as arachidonic acid.

[0127] 3) RX-055 and RX-237 are WOBE437-analogues with a linker and additional diazirine head group. 4) RX-237 and arachidonic acid compete for the catalytic site in ACSL3 and ACSL4. This means that RX-237 also inhibits the enzymatic function of the acyl-CoA synthetase.

[0128] 5) Even though ACSL6 is primarily expressed in brain and thought to mediate the metabolic pathways of full name (PUFAs), RX-237 does not target this protein which is also expressed in U937 and HAPI cells. Overall, the homology with ACSL4 is poor and the "binding site" is missing.

[0129] 6) If the "binding site" is expressed as a mutant with a deficient catalytic site, RX-237 can still efficiently label this protein, but only upon photoactivation (see point 8). Labeling means that the endocannabinoid reuptake inhibitor binds to the acyl-CoA synthetase protein or a fragment thereof. Labeling only upon photoactivation means that the endocannabinoid reuptake inhibitor does not bind to the catalytic site.

[0130] 7) Native ACSL3 and ACSL4 proteins are spontaneously labeled even without photoactivation, pointing towards a mechanism-based activity probe (i.e. targeting the catalytic site). Labelling without photoactivation means that the endocannabinoid reuptake inhibitor binds to the catalytic site of the proteins via an activity-based mechanism.

[0131] 8) Knockout of ACSL4 leads to a reduction of endocannabinoid uptake, supporting a role of the "binding site" in this role.

[0132] 9) While triacsin C can inhibit arachidonoyl-CoA formation, SERIs of first generation (WOBE437) and second generation (SYT-510, SYT-520) cannot. Even in ACSL4 knockout cells, triacsin C still inhibits the remaining (likely ACSL3 mediated) formation of arachidonoyl-CoA.

[0133] 10) ACSL4 knockout significantly affects 2-AG trafficking across the plasma membrane as shown by lipidomics analyses.

[0134] 11) While RX-237 can efficiently label ACSL3 and ACSL4 isoform 1 & 2 proteins, endocannabinoid binding of 2-AG and AEA is only observed for the ACSL4 isoform 2 when expressed in BL21 (DE3) E.coli expression system. 12) Catalytically inactive mutants of ACSL4 isoform 2, C mutant (Tyr315Ala, Gly317Ala, Tyr318Ala) and D mutant (Ser279Ala, Ser281 Ala), expressed in the BL21 (DE3) E. coli expression system, retain endocannabinoid binding of 2-AG and AEA. The lack of catalytic activity was confirmed by quantification of arachidonoyl-CoA using LC-MS / MS.

[0135] 13) The expression of the ACSL4 isoform 2 mutant, AC1 (His16Ala), in the BL21 (DE3) E. coli expression system inhibits endocannabinoid binding of 2-AG and AEA, as well as binding of the second- generation SERI SYT-510, by destabilizing oligomer formation.

[0136] 14) The expression of the ACSL4 isoform 2 mutant, LF1 (Lys37Ala, Lys38Ala, Lys39Ala), in the BL21 (DE3) E. coli expression system inhibits endocannabinoid binding of 2-AG and AEA, as well as binding of the second-generation SERI SYT-510, without destabilizing oligomer formation.

[0137] 15) The expression of the ACSL4 isoform 2 mutant, LF2(Lys37Gly, Lys38Gly, Lys39Gly), in the BL21 (DE3) E. coli expression system inhibits endocannabinoid binding of 2-AG and AEA, as well as binding of the second-generation SERI SYT-510, by destabilizing oligomer formation.

[0138] Examples

[0139] Using the previously reported probe RX-237 [6] (clickable version of RX-055 published by Chicca et al., 2017 [7]) we identified two proteins of about 70-75 kDa in mouse brain tissue and different cell lines (such as Neuro2A, U937, HAPI). As confirmed later, the probe RX- 237 spontaneously and specifically reacted with the catalytic site of ACSL3 and ACSL4 proteins already at low nanomolar concentrations (ECso <10nM), even without photoactivation, thus acting like a novel ACSL3 / 4 selective mechanism-based activity probe.

[0140] The calculated ECso of this reaction is 2-5 nM. As summarized in Table 1 , the obtained bands (i.e. ACSL3 and ACSL4) in SDS PAGE from cellular labeling could be competed with RX-055 but not with WOBE437 and other SERIs including SYT510, pointing towards distinct or overlapping binding sites. Moreover, the currently only available pan-ACSL inhibitor) triacsin C (with reported selectivity towards ACSL3 / 4 proteins, ICso = 6 pM |Tomoda et al., 1991]) [8] inhibited RX-237 labeling (50 nM of probe) of ACSL3 and ACSL4 at 2.5-10 pM, being more potent or selective towards ACSL3 inhibition. Moreover, the band of ACSL4 could be readily competed with arachidonic acid (estimated IC50 = 1-2 pM), anandamide and 2-AG. Of note, 2-AG did not compete with ACSL3. To test whether endocannabinoid hydrolysis and subsequent generation of arachidonic acid accounts for the 2- AG and AEA competitions, we used methoxy arachidonyl fluorophosphonate (MAFP) to block endocannabinoid degradation (MAFP inhibited both MAGL (monoacylglycerol lipase) and FAAH (fatty acid amide hydrolase)). 2-AG preferentially competed with ACSL4 (isoform assumed to be expressed in the plasma membrane) but does not compete ACSL3 labeling up to 10 pM, unlike AEA that competed with both. MAFP itself did not interfere with the labeling of RX-237 but fully blocked the competition of 2-AG with the probe, indicating that 2-AG itself does not bind to the catalytic site of ACSL4 but arachidonic acid. We assume that cell membrane associated MAGL is in the proximity of ACSL4 but not ACSL3. Rather interestingly, MAFP could not abolish the competition of AEA with RX- 237 labeling. This strongly suggested a direct interaction of AEA with the catalytic site of ACSL3 / 4 proteins. Since RX-055 can block both endocannabinoid uptake into cells and enzymatic function of ACSL3 / 4, we assume that RX-055 and RX-237 differentially compete with the "binding site" and the catalytic site (where it covalently reacts). Of note, the SAR (structure-activity relationships) of RX- 055 / 237 suggest that the length of the acyl chain is critical for this dual action (if the acyl chain or the linker are shorter, the endocannabinoid reuptake is not blocked anymore). Currently, the relationship between the targeting of the "binding site" by SERIs and the catalytic site (arachidonic acid, triacsin C) remain only partly understood. An in-silica model suggests that the "binding site" and the catalytic site, which are on different domains, are close enough (adjacent) to allow inter-domain interactions by RX-055 and RX-237. The WOBE437 structural similarity in RX-055 and RX-237 is thought to direct the specific binding of the probes to ACSL3 / 4 via interaction with "the binding site".

[0141] This is supported by the fact that a truncated ACSL3 fragment that does not contain the catalytic site but includes a terminal "binding site" is efficiently labeled by RX-237.

[0142] Using chemical proteomics performed in Neuro2A cells, a list of approximately 50 possible target proteins were generated. Data were filtered for size (55-75 kDa) and biochemical associations (transport, membrane biochemistry and endocannabinoid system). Since most of the targets described were expressed in HAPI cells, we used this cell line to systematically knock -out the putative targets by the CRIS PR Cas9 method to see the bands disappear upon hitting the target gene. Several genes were knocked out (not disclosed herein) but did not lead to the disappearance of probe binding (disappearance of the bands at ca. 73 and / or 70 KDa in the gels). As shown in Fig. 4, the ACSL4 knockout deleted the major band (about 72 kDa) and the ACSL3 knockout deleted the second band (about 69 kDa), thus unambiguously associating the gene with the target(s) of the RX-055 / RX-237 probes. Table 1 summarizes the competition experiments from RX-237 labeling visualized by TAMRA azide click reaction.

[0143] Table 1 . Summary of RX-237 labeling (40 nM) experiments in whole cells to illustrate completion between different molecules. Full labeling ( ++++ ), strong labelling ( +++ ), moderate labelling(++), poor labelling(+), no labeling(-). The pan-ACSL inhibitor triacsin C more potently competed with RX-237 labeling of ACSL3 (estimated IC50 = 1-3 pM) over ACSL4 (estimated IC50 = 5-10 pM). Arachidonic acid (AA) fully competed with both ACSL3 and ACSL4 (estimated IC50 = 500-1000 nM), but 2-AG selectively competed only with ACSL4 (where conversion to AA takes place via MAGL) with an estimated IC50 value of 1-2 pM. AEA (10 pM) competed with ACSL4 / 3 but not fully and this competition could not be inhibited with MAFP or URB597 (inhibitor of F AAH). MAFP and URB597 were tested at 4-10 pM.

[0144] To associate endocannabinoid transport with the target proteins, we have started to perform AEA and 2- AG uptake and transport assays in HAPI WT versus ACSL3, ACSL4 knockout cells, respectively, and ACSL3 / 4 double knockout cells. HAPI cells express MAGL but they do not express F AAH. Therefore, to better measure AEA facilitated diffusion we also transfected cells with F AAH (generating a metabolic sink and an inward concentration gradient). ACSL3 and ACSL4 are both involved in AEA uptake as their knockout strongly affects AEA transport into the cells. On the other hand, ACSL4 seems to be more associated with the uptake of 2-AG. In order to assess whether this effect is related to the catalytic site, we tested the ACSL3 / 4 enzyme inhibitor triacsin C for AEA uptake inhibition. Unlike WOBE437 and SYT- 510 (second generation SERI with different chemical scaffold as WOBE437, RX-055 and RX-237) that significantly inhibited AEA uptake, at 10 pM (concentration fully effective to block the enzymatic function and to compete the RX-237 labeling) triacsin C did not affect AEA uptake. However, triacsin C effectively inhibited ACSL3 and ACSL4 labeling by RX-237. In order to confirm that SERIs (WOBE437, SYT- 510 and SYT-520) do not target the catalytic site in ACSL3 / 4 proteins, we established a new quantitative LC- MS / MS method in which we can measure arachidonoyl-CoA. Upon addition of arachidonic acid to the cells, arachidonoyl-CoA is formed and can be measured as a metabolic intermediate. Triacsin C (10 pM) fully blocked this enzymatic activity but SYT5-10 and SYT-520 did not show any effect. At 10 pM, WOBE437 (which has an IC50 for AEA cellular uptake of 10 nM) only very inefficiently inhibited the enzymatic function of ACSL3 / 4 (5-20% partial inhibition) and below 10 pM did not inhibit the function of ACSL3 / 4.

[0145] Targeted lipidomics in HAPI versus HAPI ACSL3 / 4 knockout cells indicates that ACSL4 regulates the amount of 2-AG in the cell via membrane trafficking.

[0146] Methods

[0147] ACSL4 modelling

[0148] The quaternary structure of the transmembrane region (residues: M1 - K47) was modelled as a tetramer with Alphafold2. The cytosolic domain (residues: A36 - K711) was modeled separately as a monomer with Alphafold2. The full tetrameric model of ACSL4 was obtained by aligning sequentially the cytosolic domain to each transmembrane (residues: A36 - K44). The final structure was symmetrized (C4) and relaxed with Rosetta FastRelax procedure. The relaxed tetrameric model of ACSL4 was embedded into a lipidic membrane and further refined with all-atom molecular dynamics simulations.

[0149] ACSL3 / ACSL4 enzymatic activity assay

[0150] HAPI cells (WT, ACSL3 K.O, ACSL4 K.O, ACSL3 / ACSL4 K.O) were seeded in a 6-well plate (0.8 mio per well) and experiments were performed 24 h later.

[0151] Prior to experimentation, full media was exchange for 1 mL of media supplemented with 0.5% FBS, after washing with 2 mL of PBS. Immediately after media exchange, vehicle (DMSO) or tested compounds were added to the culture and incubated for 20 min at 37 °C, shaking at 300 rpm only during the first minute. Thereafter, cells were incubated for 15 min with either vehicle (DMSO) or 10 pM arachidonic acid, shaking at 300 rpm only during the first minute. Finally, media was recovered and cells were washed with 1 % BSA in PBS. Cells were kept in the 6 well-plate at -20°C until extraction. DMSO final concentration was 1 %. Intracellular arachidonoyl-CoA levels were quantified by LC-MS / MS after extraction, which was performed following either the modified Minkler-Abranko protocol or methanol precipitation.

[0152] For data normalization, basal arachidonoyl-CoA levels were quantified in cells non stimulated with arachidonic acid, these values were subtracted from the levels found in the arachidonic acid-treated cells.

[0153] Arachidonoyl-CoA extraction Modified Minkler-Abranko protocol (Minkler PE, Kerner J, Ingalls ST, Hoppel CL. Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue. Anal Biochem. 2008 May 15; 376(2): 275-276, Laszlo Abranko, Gary Williamson, Samantha Gardner, Asimina Kerimi. Comprehensive quantitative analysis of fatty-acyl-Coenzyme Aspecies in biological samples by ultra-high performance liquidchromatography-tandem mass spectrometry harmonizinghydrophilic interaction and reversed phase chromatography. J. Chromatogr. A 1534 (2018) 111-122).

[0154] For the extraction, 0.2 mL of PBS are added to each well and the cells are allowed to freeze over dry ice for around 6 min. To guarantee total cell disruption, the plates are sonicated for 5 min in cold water. After this, cell suspensions are transferred to 2.0 mL tubes containing 0.1 mL 0.1 M phosphate buffer (pH 6.7). Each well is then washed with 0.4 mL acetonitrile: isopropanol (3:1 , v / v) and this solution is added to the 2.0 mL tubes containing the corresponding samples. Immediately after, 20 pL of the internal standard solution is added to each sample (Cl 7-CoA in 10 mM TEAA in MeOH) and samples are vortex for 30s, followed for 5 min sonication in a cold-water bath. Thereafter, samples are centrifuged for 5 min at 16,000 x g and 4°C and the supernatant is collected in 2.0 mL tubes containing 150 pL glacial acetic acid. Acyl- CoAs from the supernatant are isolated in a SPE with 2-(2-pyridyl)ethyl-fu nctionali zed silica sorbent. Each SPE column is previously equilibrated with 1 mL ACN:isopropanol:water:acetic acid (9:3:4:4) and, after sample loading, washed with 1 mL of the same solution. Samples are afterwards eluted with 1 mL MeOH:250 mM ammonium formate (4:1) pH 7 and the eluate is evaporated to dryness in a speed-vacuum at RT, in case is necessary, dried samples are stored at -80°C until analysis. Then, dried samples are first reconstituted in 10 pL water, vortexed for 30 s and sonicated in a cold-water bath for 5 min. Thereafter, 40 pL MeOH are added to the samples and vortex-sonication steps are repeated. Finally, samples are centrifuged at 16,000 x g and 4°C for 5 min and transferred into LC-MS vials.

[0155] Methanol precipitation protocol

[0156] Cell disruption was achieved by adding 0.2 mL of PBS to each well and freezing over dried ice for 6 min. After thawing, plates were sonicated in a cold-water bath for 5 min. For extraction, only 50 pL of each cell lysate was mixed with 250 pL of methanol. Samples were vortex for 30 s and centrifuged for 10 min at 4°C and 9,300 x g. From this supernatant, 90 pL of the sample were transferred into LC-MS vials and mixed with 10 pL of internal standard (C17-CoA).

[0157] Arachidonoyl-CoA quantification by LC-MS / MS

[0158] LC-MS / MS analysis was performed using a ExionLC AC UFLC coupled to a TripleQuad 5500 QTRAP mass spectrometer (AB Sciex, Canada). The LC column was a Reprosil-PUR Cl 8 column (1 .9 pm particle size; 2 x 75 mm; Dr. A. Maisch HPLC GmbH, Germany) maintained at 40° C with a mobile phase flow rate of 0.3 mL / min. The mobile phase composition was a mixture of (A) 10 mM ammonium bicarbonate pH 8.5(adjusted with ammonium hydroxide) and (B) acetonitrile. A gradient elution was used, starting with 95% of phase A, which was linearly decreased from minute 0.20 until minute 1 .00, reaching 55% of phase A. followed by another linear decrease of phase a, reaching 35% at minute 3.00. Finally, to flush the column, phase B was increase to 100 % at minute 3.20 and kept for 3 min with a subsequent reequilibration by decreasing phase B down to 5%, which was achieved at minute 8.80. The total analysis time was 11 min. Arachidonoyl-CoA was analyzed using the Turbo-Ion Spray interface operated in positive mode. The MS parameters of the ESI source were as follows: curtain gas, 20 psig; Ion Spray voltage, 4.5 kV; temperature, 600 °C; ion source gas, GS 1 20 psig and GS2 60 psig. The following MRM transition was mainly monitored for arachidonoyl-CoA: m / z 1054.4— +547.4 (QI— +03); RT was 3.17 min. For the internal standard Cl 7-CoA, following MRM transition was monitored: m / z 1020.4— + 1020.4 (QI-- -+Q3); RT was 3.59 min. For quantification, an 11 -points calibration curve was analyzed, determining the slope, intercept, and regression coefficient, and arachidonoyl-CoA concentration in the samples was calculated applying the model previously described [Chicca et al 2017 [7]]. The values were normalized to the amount of cells.

[0159] Labelling protocol with RX-237

[0160] Stock solutions:

[0161] CuSO4 was used at 12.5 mg / mL in MiliQ water

[0162] Tris-(2-carboxyethyl)-phosphin (TCEP) is used at 15 mg / mL in MiliQ water

[0163] TAMRA-Azide (Alexa 545nm) (MWTAMRA-Azide= 631 .70 g / mol) was prepared from 5 mM stock solution in DMSO by mixing equal volumes of DMSO and 5 mM stock solution (final concentration 2.5 mM). A void repeated freeze-thaw cycles.

[0164] RX237 is prepared at a concentration of 0.1 mM in DMSO (MWRX234= 652. 708 g / mol) 83.3mM TBTA (MWTBTA= 530.63 g / mol) stock solution in DMSO (e.g. 8.85 mg TBTA, 200 pLDMSO)

[0165] 1.7 mM TBTA working solution must be prepared freshly from 83.3 mM stock solution: Mix 80 pL tert- Butanol with 18 pL DMSO and add 2 pL of 83.3 mM TBTA stock solution.

[0166] 1x Lammli loading buffer (0.2 M Tris, pH 6.8, 2% SDS, 10% Glycerol, 5 % -Mercaptoethanol, 0.05% Bromophenol blue).

[0167] Consumables:

[0168] TPP 6 well plate 92006

[0169] TPP 96 well plate 92096 Pierce BCA Protein Assay Kit Thermo Fisher Scientific 23227

[0170] Equipment:

[0171] Incubator Panasonic MC0-19AIC(UV)

[0172] Centrifuge Eppendorf 5804R

[0173] Camag UV-lamp

[0174] Digital Sonifier 450 (Branson Ultrasonics Corporation)

[0175] Eppendorf Thermomixer comfort

[0176] Eppendorf Centrifuge 5415R

[0177] Vmax kinetic microplate reader (Molecular Devices) Typhoon FLA 9500 gel analyzer

[0178] Experimental Procedure

[0179] Competing compounds (e.g. substrates such as 2-AG or inhibitors e.g. MAFP) were diluted in serum-free RPMI1640 to a final concentration in the assay (10 pM) and pre-warmed to 37°C. RX237 (Cstock = 0.1 mM) was diluted in serum-free RPMI1640 to a final concentration in the assay (10 nM) and pre-warmed to 37°C in the dark. 10""6 cells (U937, human monocytic cell line, ATCC CRL-1593 .2) were seeded in each well of a 6 well culture plate in a final volume of 2mL ofRPMU 640 complete medium and incubated for 24h at 37°C, 5% CO2. After 24h, the cells were collected from each well and put in a 50 mL plastic tube. Each well was washed with 2 mL pre-warmed PBS per well; the washing solution was collected and added to the cells in the 50 mL plastic tube. The cells were spin down for 5 minutes at 260 rpm, 25°C. The supernatant was discarded, and the pellet was disrupted by flicking the tube. To wash the cells, 7 mL pre-warmed PBS was added to the tube. The cells were spin down for 5 minutes at 260 g, 25°C. The cells were re-suspended in serum-free RPMI1640 and re-distributed to the wells in a final volume of 0.5 mL per well. 0.5 mL per well of the 4x solution of the competing compound was added to the cells. Immediately after, 1 mL per well of the 2x solution of RX237 was added to the cells. The plate was incubated for 5 minutes at 37°C, 5% CO2. The plate was put on ice and UV irradiated for 10 minutes at 366 nm. Cells were collected and transferred to 1.5 mL Eppendorf micro-centrifuge tubes and were centrifuged for 5 minutes at 100 g, 4°C. The supernatant was discarded, and the cell-pellets were washed with 0.5 mL of ice-cold PBS. The cell suspensions were centrifuged for 5 minutes at 100 g, 4°C. The supernatant was discarded, and the cell-pellets were re-suspended in 120 pL ice-cold PBS. The cells were ultra-sonicated 3 times for 10 seconds on ice. Bicinchoninic acid assay (BCA) was performed to estimate protein content of the crude lysate: 20 pL of BSA protein standards and blank were added in duplicates to the wells of a 96 well plate. 10 pL of the samples and 10 pL MiliQ water per well were added in duplicates to the wells of the 96 well plate. 200 pL per well of the BCA reaction solution (50 parts reagent A; 1 part reagent B) was added to the wells of the 96 well plate. The plate was incubated for 25 minutes at room temperature before measuring absorbance at 450 nm. Total protein concentration was calculated by linear regression. 50 pg of total protein per sample was transferred to fresh tubes and kept on ice. The volume was adjusted with ice-cold PBS to 43.3 pL. The click reaction was performed in a final volume of 50 pL per sample. The reagents were added in the following order: 1 .1 pL TAMRA-Azide (2.5 mM), 1.1 pL TCEP (15 mg / mL), 3.4 pL 1.67 mM TBTA in 80% tert-Butanol, 18% DMSO, 1.1 pL CuSO4*5H2O. After each addition the reaction mixture was vortexed to ensure equal distribution of the reagent mixtures. The reaction tubes were incubated for 60 minutes at 21 °C, under shaking at 300 rpm in a Thermomixer with the lid on. To stop the reaction, tubes were centrifuged at 6600 g at 4 °C for 5 minutes and the supernatant was discarded. The pellets were re-suspended in 30 pL per sample 1x Lammli buffer and incubated at 70°C for 10 minutes. Samples were stored at -80°C. The obtained samples were run in 10% acrylamide SDS-PAGE and the fluorescent signals were recorded.

[0180] Plasmid transformation of Escherichia co / / BL2KDE3) pLysS strain Consumables

[0181] Ampicillin. A 100 mg / ml solution was prepared in type 1 water.

[0182] Chloramphenicol. A 34 mg / ml solution was prepared in type 1 water.

[0183] E. coli BL21 (DE3) pLysS strain

[0184] Glucose. A 30% solution was prepared in type 1 water.

[0185] Isopropyl P-D-1 -thiogalactopyranoside (IPTG). A 1 M solution was prepared in type 1 water.

[0186] Lysogeny broth (LB) medium. pET-21 a (+) C-6His vectors

[0187] Phosp hate-buffered saline (PBS). Buffer containing 137 mM NaCI, 2.7 nM KCI, 10 mM Na2HPO4 and 1.8 mM KH2PO4.

[0188] Culturing medium preparation

[0189] A) Culture of non-transformed cells. LB medium supplemented with 0.3% glucose and 34 g / ml chloramphenicol.

[0190] B) Culture of transformed cells. LB medium supplemented with 0.3% glucose, 34 g / ml chloramphenicol and 100 pg / ml ampicillin.

[0191] C) Mock induction of non-transformed cells. LB medium supplemented with 34 g / ml chloramphenicol.

[0192] D) Induction of transformed cells. LB medium supplemented with 34 g / ml chloramphenicol and 100 g / ml ampicillin. Transformation

[0193] A 100 pl vial of competent E coli PlysS cells is thawed on ice for 5 min. Bacteria are then transformed by adding 100 ng of the corresponding pET-21 a (+) C-6His vector, the vial is then flicked, placed at 42 °C for 75 seconds and immediately after is placed on ice for 5 min. Then, 500 pl of LB medium are added to the vial and bacteria are incubated for 45 min at 37 °C. Subsequently, the bacteria are transferred into larger culture volume containing 20 ml of either A or B medium, and incubated overnight (-14 hours) at 37 °C and 180 rpm. The cell suspension is then collected into a 50 ml conical tube and spun-down at 3300 x g for 10 min at 4 °C. The supernatant is discarded and cells are resuspended in 20 ml of PBS at room temperature. The amount of bacteria is calculated by measuring the optical density at 600 nm.

[0194] Induction

[0195] The culture is normalized to a concentration of 0.1 ODeoo by diluting the bacteria in either C or D medium and incubated for 2 hours at 37 °C. Afterwards, induction occurs by adding 1 mM IPTG and incubating the cells for 2 hours at 25 °C. The cell suspension is then centrifuged at 3300 x g for 10 min at 4 °C, the supernatant is discarded and bacteria is resuspended in 20 ml of PBS. The concentration of the cell suspension is calculated by measuring the optical density at 600 nm.

[0196] Binding / uptake assay in E. coli

[0197] Consumables

[0198] Anandamide [ethanolamine-1-3H]

[0199] 2-Arachidonyl [5, 6, 8, 9,11 ,12,14,15-3H] glycerol

[0200] SYT-510 [thiazolidinone -4-14C].

[0201] Fatty Acid-free bovine serum albumin (BSA)

[0202] Fetal Bovine Serum (FBS)

[0203] Testing compounds. Prepare a 200X stock in dimethyl sulfoxide (DMSO).

[0204] Ultima Gold liquid scintillation cocktails (LSC)

[0205] Phosp hate-buffered saline (PBS). Buffer containing 137 mM NaCI, 2.7 nM KCI, 10 mM Na2HPO4 and 1.8 mM KH2PO4.

[0206] Washing buffer. PBS buffer supplement with 1% BSA; BSA should be added with before use.

[0207] Assay buffer. PBS buffer supplement with 0.5% FBS; FBS should be added with before use.

[0208] Radioligand preparation A 200X mixture of radioligand and non radioligand is prepared in DMSO for each of the tested AEA concentrations. For the mixture, a range between 0.3 - 1 nM of [3H]AEA, [3H]2-AG and [14C]SYT-510 is considered as final concentration regardless the total amount of ligand.

[0209] Procedure

[0210] Cell suspension is prepared to a cell density of 0.5 ODeoo in assay buffer and then aliquots of 297 l are distributed in 1.5 ml plastic tubes. Non-transform cells are always included for comparison against transformed cells. Each condition is tested in triplicates. Then, 1.5 pL of vehicle or testing compound is added to the corresponding tubes. Immediately after, 1.5 pL of radioligand mix is added to each tube. Samples are briefly vortexed after each addition at low speed. Samples are incubated at 30 °C for 60 min. Afterwards, samples are centrifuged at 2000 x g for 10 min at 4 °C. The supernatant (extracellular fraction) of each sample is collected and transfer into a plastic scintillation vial containing 3 mL of LSC. The cell pellet is then resuspended in 300 pl of ice-cold washing buffer and spun-down at 2000 x g for 10 min at 4 °C. The supernatant is collected and transfer to the plastic scintillation vial already containing the previous extracellular fraction. The cell pellet is then resuspended in 300 pl of ice-cold PBS (intracellular fraction) and transfer into a plastic scintillation vial containing 3 mL of LSC. Radioactivity is measured as counts per minute (CPM) using a Tri-Carb scintillation counter.

[0211] Calculations

[0212] Per each sample, total CPM is calculated as the sum of CPM in the intracellular fraction (CPMic) and CPM the extracellular fraction (CPMEC). Then, the percentage of ligand present in the intracellular fraction is calculated against the total [% Intracellular AEA = CPMic / (CPMic + CPMEC) * 100].

[0213] Western blotting

[0214] Consumables

[0215] GAPDH Monoclonal Antibody (GA1 R)

[0216] 6x-His Tag Monoclonal Antibody (HIS.H8)

[0217] Loading buffer: 2x Laemmli buffer

[0218] 4% SDS

[0219] 10% 2-mercaptoethanol 20% glycerol

[0220] 0.004% bromophenol blue

[0221] 0.125 M Tris-HCI

[0222] Running buffer

[0223] Tris / Glycine / SDS 25mM Tris

[0224] 190 mM glycine 0.1 % SDS

[0225] Transfer buffer

[0226] 25 mM Tris

[0227] 190 mM glycine 20% methanol

[0228] Tris-buffered salinewith Tween 20 (TBST) buffer

[0229] 20 mM Tris, pH 7.5 150 mM NaCI

[0230] 0.1 % Tween 20

[0231] Blocking buffer

[0232] 3% bovine serum albumin (BSA) in TBST

[0233] Procedure

[0234] After homogenizing the samples, the total protein concentration is determined, and 30 pg of total protein is resuspended in a final volume of 24 pl by adjusting the final volume with 4X Laemmli. The samples are loaded into a 12% SDS-PAGE chamber along with molecular weight markers. The gel is run for 30 minutes at 90 V, and then the voltage is increased to 120 V to complete the run in 90 minutes.

[0235] For the transfer, the gel is placed in 1X transfer buffer to equilibrate. The transfer sandwich is then assembled and placed in the cassette in the transfer tank. The transfer is done for 100 minutes at 90 V. After the transfer, the nitrocellulose membrane is removed and briefly rinsed with water before adding the blocking buffer. The membrane is then incubated overnight at 4 degrees in the blocking buffer.

[0236] After incubation, the membranes are rinsed 3 times with TBST buffer and incubated for 2 hours at room temperature with the primary antibody (1 : 3000 dilution). Then, the membranes are washed again 3 times with TBST, and the secondary antibody (1 :5000 dilution) that is conjugated with a fluorophore is added. The membranes are incubated for 2 hours at room temperature. Finally, the membranes are imaged by a laser scan, and the fluorophore signal is quantified with the Image J software for protein expression. The data is then normalized to GADPH loading control.

[0237] References

[0238] [1] Fujimoto T, Parmryd I. Interleaflet Coupling, Pinning, and Leaflet Asymmetry-Major Players in Plasma Membrane Nanodomain Formation. Front Cell Dev Biol. 2017 Jan 10;4:155. [2] Tian X, Guo J, Yao F, Yang DP, Makriyannis A. The conformation, location, and dynamic properties of the endocannabinoid ligand anandamide in a membrane bilayer. J Biol Chem. 2005 Aug 19;280(33):29788-95.

[0239] [3] Fezza F, Bari M, Florio R, Talamonti E, Feole M, Maccarrone M. Endocannabinoids, related compounds and their metabolic routes. Molecules. 2014 Oct 24; 19(11): 17078-106.

[0240] [4] Nicolussi S, Gertsch J. Endocannabinoid transport revisited. Vitam Honn. 2015;98:441- 85.

[0241] [5] Russo EB. Clinical Endocannabinoid Deficiency Reconsidered: Current Research Supports the Theory in Migraine, Fibromyalgia, Irritable Bowel, and Other Treatment-Resistant Syndromes. Cannabis Cannabinoid Res. 2016 Jul 1 ; 1 (1 ): 154-165.

[0242] [6] Chicca A, Rubin, M, Reynoso Moreno, I, Bartholomaus, R, Nicolussi, S, Adibekian, A, Altmann, KH, Gertsch, J. Identification of proteins involved in the cellular membrane trafficking of endocannabinoids using RX-237. 8Th NCCR TransCure Site Visit, 8. October 2018, University of Bern, Switzerland.

[0243] [7] Chicca A, Nicolussi S, Bartholomaus R, Blunder M, Aparisi Rey A, Petrucci V, Reynoso-Moreno I DC, Viveros-Paredes JM, Dalghi Gens M, Lutz B, Schioth HB, Soeberdt M, Abels C, Charles RP, Altmann KH, Gertsch J. Chemical probes to potently and selectively inhibit endocannabinoid cellular reuptake. Proc Natl Acad Sci U S A. 2017 Jun 20; 114(25): E5006- E5015

[0244] [8] Tomoda, H., Igarashi, K., Cyong, J.C., et al. Evidence for an essential role of long chain acyl-CoA synthetase in animal cell proliferation. Inhibition oflong chain acyl-CoA synthetase by triacsin C caused inhibition of Raji cell proliferation. The Journal of Biological Chemisty 266, 4214-4219 (1991)

[0245] [9] Nicolussi S, Viveros-Paredes JM, Gachet MS, Rau M, Flores-Soto ME, Blunder M, Gertsch J. Guineensine is a novel inhibitor of endocannabinoid uptake showing cannabimimetic behavioral effects in BALB / c mice. Pharmacol Res. 2014;80:52-65

Claims

New PCT-Patent Application based on EP 23 174 402.0 Universitat BernVossius Ref.: AG1449 PCT BSClaims1 . A peptide consisting of the amino acid sequence AKKK (SEQ ID NO: 3), which can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor.

2. A peptide consisting of the the amino acid sequence LTNAKKKNA (SEQ ID NO: 2), or the amino acid sequence ALIFIPWYFLTNAKKK (SEQ ID NO: 1).

3. The peptide of claim 1 to 2, wherein the endocannabinoid Is 2-arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA).

4. The peptide of any of the preceding claims, wherein the endocannabinoid reuptake inhibitor has the formula:wherein in formula (I) R is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, aryl, and heteroaryl, and wherein Ri is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri is optionally substituted, wherein in formula (II) Ri, R2 and R3 are independently from each other selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri, R2 and R3 are each optionally substituted.

5. Fusion protein comprising a peptide as defined in any of claims 1 to 4 and a transmembrane domain, wherein the fusion protein can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor.

6. The fusion protein of claim 5, wherein the fusion protein is integrated into the inner layer of a plasma membrane lipid bilayer.

7. Composition comprising the peptide of any of claims 1 to 5 or the fusion protein of any of claims 5 or 6, an endocannabinoid and / or an endocannabinoid reuptake inhibitor, and optionally a pharmaceutically relevant carrier.

8. A binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein, which binding sequence can be bound by an endocannabinoid and / or an endocannabinoid reuptake inhibitor and wherein the sequence comprises the following the amino acid sequence: AKKK (SEQ ID NO: 3).9 The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to claim 8, wherein the binding sequence comprises the amino acid sequence LTNAKKKNA (SEQ ID NO: 2), or the amino acid sequence ALIFIPWYFLTNAKKK (SEQ ID NO: 1).

10. The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to claim 8 or 9, wherein the acyl-CoA synthetase protein is an integral membrane protein or membrane associated protein.11 . The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any one of claims 8 to 10, wherein the acyl-CoA synthetase protein is a long-chain acyl-CoA synthetase, preferably ACLS4 isoform 2.

12. The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to claim 9, wherein the acyl-CoA synthetase protein of claim 9 is ACLS 4 isoform 2.

13. The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of the claims 8 to 12, wherein the binding sequence in the acyl-CoA synthetase protein is independent of the catalytic domain of the acyl-CoA synthetase protein and / or the lipid tunnel of the acyl-CoA synthetase protein, preferably independent of the catalytic domain and the lipid tunnel of the acyl-CoA synthetase protein.

14. The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 13, wherein the enzymatic reaction of acyl-CoA formation by the acyl-CoA synthetase protein is independent from binding to the endocannabinoid and / or the endocannabinoid reuptake inhibitor.

15. The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of the claims 8 to 14, wherein the endocannabinoid is 2-arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA) bind.

16. The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to claim 8, wherein the endocannabinoid is 2-arachidonoylglycerol (2-AG) and / or N-arachidonoylethanolamine (anandamide, AEA).

17. The binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of the claims 8 to 16, wherein the endocannabinoid reuptake inhibitor has the formula:wherein in formula (I) R is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, aryl, and heteroaryl, and wherein Ri is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri is optionally substituted, wherein in formula (II) Ri, R2 and R3 are independently from each other selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri, R2 and R3 are each optionally substituted.

18. A cell comprising the peptide of any of claims 1 to 5, the fusion protein of claims 6 or 7, or the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17.

19. A lipid bilayer comprising the peptide of any of items 1 and 2a) b) to 5, the fusion protein of claims 7 or 8, or the binding sequence in an acyl-CoA synthetase protein or in a fragment of said proteinaccording to any of claims 8 to 18, wherein the acyl-CoA synthetase protein is preferably ACSL4 isoform 2.

20. The peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19.21 . The peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19 for use in the treatment of a condition or a disease associated with an altered function of the endocannabinoid system, preferably for use in the treatment of a disorder of the central nervous system (such as neuropsychiatric, neuroinflammatory or neurodegenerative disorders), an inflammatory disease or an immune disease.

22. The peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19 for facilitating plasma membrane trafficking of an endocannabinoid, for facilitating the reuptake of an endocannabinoid, for modulating the levels of an endocannabinoid, and / or for modulating cellular immune processes, wherein the peptide or binding sequence is preferably bound by an endocannabinoid.

23. The peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19 for inhibiting the reuptake of an endocannabinoid, for sustaining endocannabinoid signaling, for modulating the levels of an endocannabinoid and / or for modulating cellular immune processes, wherein, the peptide or binding sequence is preferably bound by an endocannabinoid reuptake inhibitor.

24. A method for facilitating plasma membrane trafficking of an endocannabinoid using the peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19 wherein the peptide or binding sequence is preferably bound by an endocannabinoid.

25. The method of claim 24, wherein plasma membrane trafficking is the diffusion of the endocannabinoid in a lipid bilayer from the outer layer to the inner layer.

26. The method of claim 24, wherein endocannabinoid is 2 arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA) and the acyl-CoA synthetase protein is ACSL4 isoform 2.

27. A method for decreasing plasma membrane trafficking of an endocannabinoid, using the peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19 and an endocannabinoid uptake inhibitor.

28. The method of claim 24, wherein the endocannabinoid reuptake inhibitor has the formula:wherein in formula (I) R is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, aryl, and heteroaryl, and wherein Ri is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri is optionally substituted, wherein in formula (II) Ri, R2 and R3 are independently from each other selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri, R2 and R3 are each optionally substituted.

29. The method of claim 24, wherein the endocannabinoid is 2-arachidonoylglycerol (2-AG) and / or N- arachidonoylethanolamine (anandamide, AEA) and / or the acyl-CoA synthetase protein is ACSL4 isoform 2.

30. A method for facilitating the reuptake of an endocannabinoid using the peptide of any of claims 1 to 4, the fusion protein of claim 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19 wherein the peptide or binding sequence is preferably bound by an endocannabinoid.31 . A method for inhibiting the reuptake of an endocannabinoid using the peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19 and an endocannabinoid reuptake inhibitor.

32. The method of claim 31 , wherein the reuptake inhibitor has the formula:wherein in formula (I) R is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, aryl, and heteroaryl, and wherein Ri is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri is optionally substituted, or wherein in formula (II) Ri, R2 and R3 are independently from each other selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri, R2 and R3 are each optionally substituted.33 A method for sustaining endocannabinoid signaling using the peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19 and an endocannabinoid reuptake inhibitor.

34. The method of claim 33, wherein the reuptake inhibitor has the formula:(I) (H) wherein in formula (I) R is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, aryl, and heteroaryl, and wherein Ri is selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri is optionally substituted, wherein in formula (II) Ri, R2 and R3 are independently from each other selected from the group consisting of alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein Ri, R2 and R3 are each optionally substituted.

35. Use of the peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, the lipid bilayer of claim 19 for the treatment of a condition or a disease associated with an altered function of the endocannabinoid system, preferably for the treatment of a disorder of the central nervous system (such as neuropsychiatric, neuroinflammatory or neurodegenerative disorders), an inflammatory disease or an immune disease.

36. Use of the peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, or the lipid bilayer of claim 19 for facilitating plasma membrane trafficking of an endocannabinoid, for facilitating the reuptake of an endocannabinoid, and / or for modulating cellular immune processes, wherein the peptide or binding sequence is preferably bound by an endocannabinoid.

37. Use of peptide of any of claims 1 to 4, the fusion protein of claims 5 or 6, the binding sequence in an acyl-CoA synthetase protein or in a fragment of said protein according to any of claims 8 to 17, the cell of claim 18, or the lipid bilayer of claim 19 for decreasing plasma membrane trafficking, for inhibiting the reuptake of an endocannabinoid, for sustaining endocannabinoid signaling and / or for modulating cellular immune processes, wherein, the peptide or binding sequence is preferably bound by a endocannabinoid reuptake inhibitor.

38. A method for identifying an endocannabinoid reuptake inhibitor using the peptide of any one of claims 1 to 4, wherein the endocannabinoid reuptake inhibitor inhibits trafficking of an endocannabinoid across the plasma membrane or intracellular membrane.

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