Tissue selective inhibition and activation of the carbohydrate-responsive element-binding protein (chrebp)

Abstract

The invention refers to modified carbohydrate-responsive element-binding proteins (ChREBP's) functional as tissue specific antagonist and / or agonist for use in medicine.

Description

[0001] Tissue selective inhibition and activation of the carbohydrate-responsive element-binding protein (ChREBP)

[0002] Field of the Invention

[0003] The invention refers to modified carbohydrate-responsive element-binding proteins (ChREBP’s) functional as tissue specific antagonist and / or agonist for use in medicine.

[0004] Background of the Invention

[0005] Obesity poses a globally growing threat to human health, as cases have nearly tripled since 1975. Alongside with this, the prevalence of insulin resistance and type 2 diabetes mellitus (T2D) is on the rise. One driving force behind this future pandemic is the dramatic shift in our dietary habits. Previously as hunters, human ancestors predominantly consumed protein-rich diets. However, since the advent of early settlements, human diets have become rich of carbohydrates. This rapid dietary change has outpaced body’s ability to adapt, leaving the genetic programming unequipped to handle this sugar- overload.

[0006] Especially the long-term effects of T2D are a great burden for patients and health insurances. T2D is a well-established risk factor for dyslipidemia, cardiovascular disease, chronic kidney disease and nonalcoholic fatty liver disease (NAFLD). The current approach to reduce the risk of long-term effects is combining lifestyle changes and pharmacotherapeutic strategies, often involving several drugs of various mechanisms, with a focus on good glycemic control.

[0007] However, such combinatory strategies as diabetes therapy have only limited success. Additionally, only few of the current T2D medications exert a beneficial effect on classic comorbidities, in fact many of the drugs are no longer safe to use if liver or kidney function declines. For example, organ damage caused by years of elevated blood glucose levels still cannot be prevented and leads to a wide range of secondary diseases, such as diabetic food or retinal disease, which reduces patients’ quality of life and results in considerable costs for society.

[0008] In general, insulin resistance and T2D develop over the years as a consequence of carbohydrate rich diet. The term “insulin resistance” describes that cellular insulin receptors start to wear out and because of the reduced effectiveness of the insulin receptors, even more insulin is produced by the body. If an increasing insulin resistance develops, this creates a vicious circle. Current therapies for T2D rely on exogenous insulin or oral therapeutics, like GLP-1 analogues and sulfonylureas, which also promote the release of additional insulin. However, these drugs do not break this vicious circle, but rather reinforce it.

[0009] Another approach, using metformin has no direct influence on the insulin release, but reduces gluconeogenesis. It therefore lowers blood glucose level but does not affect glucose peaks which occur with food intake. Still some other medications, such as SGLT2 inhibitors, can reduce postprandial glucose spikes by increasing the glucose excretion in kidneys, but often cause urinary tract infections and nocturnal urge to urinate, and therefore also affect the patients’ quality of life.

[0010] Especially pancreatic beta cells are at risk as they are negatively affected by insulin resistance. Hence, these insulin producing cells are often challenged during T2D, because the increasing loss of working insulin receptors leads to excessive insulin production. Despite research efforts, there is currently no medication or cure available that can protect beta cells during T2D and therefore prevent their cells death.

[0011] Thus, there is still a serious need to develop new drugs and medication to treat and / or prevent T2D, insulin resistance and obesity.

[0012] One of the key regulators of glucose turnover and de-novo fatty-acid synthesis is a regulatory transcription factor, identified as carbohydrate response element binding protein (ChREBP). This transcription factor ChREBP controls the utilization of glucose in the body and thus, plays an important role in the sugar metabolism in the whole human body.

[0013] ChREBP is one of the most important factors that regulates blood glucose levels. (Abdul-Wahed et al., 2017) The expression of ChREBP is e.g. most prominent in active sites of de novo lipogenesis such as in the liver and white adipose tissues.

[0014] Further, ChREBP is also expressed in pancreatic islets, small intestine, skeletal muscle and even in kidneys and the brain. As ChREBP functions as a central metabolic coordinator in a variety of cell types it has a wide range of effects on health and disease.

[0015] It has been found that the expression of ChREBP in white adipose tissue (WAT) leads to positive effects on insulin sensitivity, since it triggers de novo lipogenesis and therefore reduces blood glucose levels. In addition, the benefits of adipose ChREBP expression have also been associated with the initiation of valuable lipid species synthesis and the control of PPARy agonist synthesis.

[0016] In this regard, the peroxisome proliferation-activated receptor y (PPARy) is a major regulator of adipocyte function and insulin sensitivity. Activation of PPARy promotes adipocyte differentiation, leading to the synthesis of small insulin-sensitive adipocytes.

[0017] However, in the liver a ChREBP expression can lead to the accumulation of fat alongside a global decline of insulin sensitivity. Equally in the pancreas during prolonged high glucose blood levels, ChREBP activation increases expression of ChREBPfJ, the constitutively active isoform. Its action is responsible for beta cell death. In the kidney, a ChREBP knockdown contributed to a slowdown of diabetic nephropathy. Taken together, in the liver, the pancreas and the kidneys, ChREBP activity clearly contributes to the menacing long-term effects of T2D.

[0018] Thus, ChREBP function is considered not only extremely versatile by tissue, but this tissue specificity has been found to be a “double-edged sword” particularly for the problem of influencing insulin sensitivity. In summary, the ChREBP transcription factor is an extremely challenging target to employ in pharmacotherapy.

[0019] Nonetheless, targeting ChREBP is the declared aim of the present invention and bears the unique potential to develop a more complete treatment option for T2D.

[0020] It is thus, an objective of the present invention to develop a ChREBP driven strategy, which allows by activation a reduction of blood glucose levels and which at the same time, allows by inhibiting the activity e.g. in liver, kidney and pancreas to reduce the devasting long-term effects of T2D, further avoiding cell death of insulin producing cells and thus improving general health.

[0021] It is further an object of the present invention to develop a ChREBP driven strategy, which allows a tissue specific control of the carbohydrate and lipid metabolism. Such control of the sugar metabolism is particularly interesting for the control of cells with an induced or pathogenically induced high metabolism, such as cancer cells.

[0022] Beside others, this object is solved by the present invention in providing a modified carbohydrateresponsive element-binding protein (ChREBP) according to claim 1. Further advantageous details, aspects and embodiments of the present invention are represented by the embodiments of the dependent claims.

[0023] Due to the efforts of the inventors a modified carbohydrate-responsive element-binding protein (ChREBP) is provided, which proofed to act highly tissue specific as inhibitor or activator of the carbohydrate and lipid metabolism in cells.

[0024] The normal and unmodified transcription factor ChREBP is activated by binding of the endogenous activator G6P (glucose-6-phosphate) and such activation leads to a conformational change, which results in spatial separation of the functional domain GRACE and the inhibitory domain LID. Alternatively, in the absence of G6P, the proximity of LID and GRACE stabilizes a protein conformation, which leads to an inhibition of the GRACE functionality and thus also to an inhibition of transcriptional activity and an inhibition of the carbohydrate and lipid metabolism of the cell.

[0025] According to the inventors, the modified ChREBP according to the invention can now be controlled in a tissue specific manner, independently of the presence or absence of G6P for an in vivo activation or inhibition of the carbohydrate and lipid metabolism in cells.

[0026] To reach this tissue specific activation or inhibition the inventors provide a modified carbohydrateresponsive element-binding protein (ChREBP) comprising an inhibitory domain (LID), a linker, a functional domain (GRACE) and a DNA-binding domain, characterized in that at least one proteolytic site is incorporated within the sections selected from the inhibitory domain (LID), the linker, the functional domain (GRACE), the region between GRACE and DNA binding domain and the DNA-binding domain.

[0027] In the context of the present invention the term “inhibitory domain” or “LID” refers to the low glucose inhibitory domain, which suppresses in the absence of glucose the activation of GRACE, caused by an interaction of LID with the functional domain (GRACE) and the scaffolding protein 14-3-3. In other words, the glucose responsiveness of ChREBP is mediated by the dynamic intermolecular interaction between GRACE and LID.

[0028] In the context of the invention the term “linker”, which is connecting GRACE and LID is an amino acid sequence of about 20 amino acids in length. It refers to a naturally occurring amino acid sequence or to an artificially introduced amino acid sequence, which is described hereinafter as short unstructured linker. In addition, it is to be understood that the linker connecting GRACE and LID does not affect ChREBP’s mechanistic function in promoting the expression of proteins for the glucose utilization in cells.

[0029] In the context of the present invention the term “functional domain” or “GRACE” refers to the glucose- response activation conserved element of ChREBP, which can when activated promote transcriptional activity after binding of ChREBP to a DNA-motif identified as carbohydrate response element (ChoRE).

[0030] In native ChREBP, low glucose concentrations restrain transcriptional activity of GRACE, likely due to a conformational blockade, with LID. On the other hand, high glucose concentrations reverse this inhibition, likely by a conformational change and repositioning of the domains and thus by allowing after a binding of the DNA binding domain to ChoRE a transcriptional activation.

[0031] In the context of the present invention the term “transcriptional activation” refers to an enhancing of the rate of transcription for genes that are part of the cellular carbohydrate and lipid metabolism, such as genes encodingfor glycolytic and lipogenic enzymes. The transcriptional activation is further understood as binding of the DNA-binding domain to the ChoRE DNA-motif while the GRACE domain is functionally active.

[0032] In the context of the application the term “DNA-binding domain” refers to the part of the ChREBP protein that directly binds to the ChoRE DNA-motif. Binding of the DNA-binding domain to ChoRE in combination with a GRACE domain, which is notsterically inhibited by LID, promotes transcriptional activity and thus activates the carbohydrate and lipid metabolism in cells.

[0033] The modified carbohydrate-responsive element-binding protein (ChREBP) of the invention, is characterized by the modification of the protein sequence and specifically by the introduction of at least one artificially created, mutated and / or inserted proteolytic site within the sections selected from the inhibitory domain (LID), the linker, the functional domain (GRACE), the region between GRACE and DNA- binding domain as well as the DNA-binding domain of the ChREBP.

[0034] Due to the use of a proteolytic site recognized by a tissue specific expressed protease the modified ChREBP of the invention proofed to be activated or inhibited in a highly tissue specific manner.

[0035] The term “proteolytic site” in the context of the invention refers to a peptide motif which defines the binding and / or cleavage sequence for a proteolytic enzyme. Proteolytic enzymes may be e.g. proteases that cleave peptide bonds by hydrolyzation. The at least one proteolytic site incorporated into the modified ChREBP of the invention determines by which endogenous proteolytic enzyme the modified CHREBP of the invention is cleaved and thus this selection of the proteolytic site for modifying ChREBP also determines the type of tissue which is specifically targeted.

[0036] In the context of the present invention the term “at least one proteolytic site” refers to one, two, three or more proteolytic sites serving as a binding and / or recognition site for one or multiple proteases. The at least one covers particularly the situation where e.g. two or more proteolytic sides are introduced into one or several defined section such as the linker, the GRACE domain or both.

[0037] In this embodiment it is possible to tailor the modified ChREBP to e.g. be activated in one specific tissue, due to the tissue specific proteolytic site introduced into the linker sequence and at the same time act in a different specific tissue in an inhibiting manner due to the insertion of the tissue specific proteolytic site introduced into the GRACE domain.

[0038] It must be understood that with the modified ChREBP of the invention not only a one directions approach is possible, but the invention is actually leading into a platform technology, which allows on the one hand a tissue specific activation but also, depending on the position of the introduced mutation and / or the position of the incorporated proteolytic site a tissue specific inhibition of the cellular carbohydrate and lipid metabolism. Both approaches are highly specific and therapeutically useful, as will be described in the following.

[0039] Thus, on the one hand the invention provides a modified carbohydrate-responsive element-binding protein (ChREBP) comprising an inhibitory domain (LID), a linker, a functional domain (GRACE) and a DNA-binding domain, wherein at least one proteolytic site is incorporated within the sections selected from the inhibitory domain (LID), the linker, the functional domain (GRACE), the region between GRACE and DNA binding domain as well as the DNA-binding domain.

[0040] According to one exemplary embodiment of the invention the sequence comprising the inhibitory domain (LID), the linker, the functional domain (GRACE), the region between GRACE and DNA binding domain as well as the DNA-binding-domain, located within amino acids 61-79, 194-214, 215-298, 341-603, and 649-724 respectively of human ChREBP, defines the section into which the at least one proteolytic site is cloned for modifying ChREBP.

[0041] Due to the conformational properties of the transcription factor, the positioning of the cloning site is essential for determining the agonist or antagonist properties of the modified ChREBP. This means, that the placement of the proteolytic site determines whether the modified ChREBP acts as an activator or an inhibitor after proteolytic cleavage compared to the native ChREBP.

[0042] In the embodiment where the proteolytic cleavage site is positioned inside the functional GRACE domain, the GRACE domain will lose its functionality thereby causing that the associated DNA-binding domain still binds the ChoRE DNA-motif butcan no longer be activated or transcribed. In competition with native ChREBP this leads to a transcriptional reduction or inhibition of the cellular carbohydrate and lipid metabolism. Thus, the present invention is suitable to be used with incorporation of at least one but also several proteolytic sites and can thus be used as tissue specific agonistic or antagonistic tool. Proof of principle examples for successful tissue specific uses are demonstrated in the examples.

[0043] In another embodiment of the invention the at least one proteolytic site is incorporated into the linker connecting the GRACE and LID domain. The linker is about 20 amino acids long and the proteolytic site can be placed at any position within said linker. Interestingly, the placement of at least one proteolytic site in the linker between GRACE and LID causes after proteolysis with the respective proteolytic enzyme the dissociation of GRACE and LID. This further results in a strong activation of the transcriptional activity and results in a protein which is no longer dependent on the presence of glucose-6-phsosphate (G6P) for activation but binds with its attached DNA-binding domain to the ChoRE motif thereby independently activating the cellular carbohydrate and lipid metabolism.

[0044] Thus, the modified ChREBP carrying the at least one proteolytic site in the linker acts now as tissue specific activator and is in comparison to the native ChREBP independent from G6P.

[0045] According to other embodiments of the invention, the modified ChREBP carrying the at least one proteolytic site within the sequence section of the GRACE, the region between GRACE and DNA binding domain as well as the DNA-binding domain, is used as inhibitor as explained later in this specification.

[0046] According to the teaching of the present invention the cleavage of the proteolytic site is catalyzed by proteases, wherein the incorporated proteolytic site is a recognition site, binding side and / or cleavage site for one specific or multiple proteases. In general, proteases are able to cleave peptide bonds within proteins by hydrolysis. Some are highly specific and cleave only their substrates e.g. proteins, or only when a specific site for binding or cleavage is recognized, the so-called recognition or binding site.

[0047] In one embodiment of the present invention, the one or more incorporated proteolytic sites are recognition and / or binding sites preferably for tissue specific proteases and / or proteases of mammalian origin.

[0048] In the context of the present invention the term “tissue specific proteases” refers to proteases, which are expressed only in specific tissues of a mammal including a human. Known tissue specific proteases are e.g. expressed specifically in the liver, in adipose tissue or e.g. in specific tumor cells.

[0049] Proteases suitable for the modification of ChREBP according to the invention are e.g. TEV protease, cathepsin B, furin, thrombin, legumain, MT-SP1, MMP-2, MMP-9, MMP14, THOP1, KLK3 or KLK6.

[0050] With the incorporated tissue specific proteolytic sites, the modified ChREBP of the invention can act selectively as activator and / or inhibitor depending on the proteases expressed in the respective tissue and the position of the insertion of the proteolytic site.

[0051] Such tissue specific or disease specific activation or inhibition is highly specific and thus beneficial over traditional approaches. According to further embodiments the one or more incorporated proteolytic sites can be recognition sites and / or binding sites for proteases selected from, but not limited to, the group comprising TEV protease, cathepsin B, furin, thrombin, legumain, MT-SP1, MMP-2, MMP-9, MMP14, THOP1, KLK3, KLK6 and other endogenous mammalian proteases.

[0052] In the context of the present invention the incorporated proteolytic sites are selected on the basis of endogenous tissue specific proteases in order to selectively generate ChREBP activators. The subsequent Table 1 shows a selection of tissue specific proteases in relation to a clinical picture suitable in the present invention.

[0053] Table 1: Tissue specific Proteases

[0054] According to one Example of the present invention, a modified ChREBP carrying at least one proteolytic site for the protease, cathepsin B, which is predominantly found in the cytosol of obese white adipose tissue (WAT), can be suitable for use in the treatment of diabetes and / or obesity.

[0055] The in this tissue type expressed protease cathepsin B will cleave the modified ChREBP of the invention at the incorporated cleavage site e.g. in the linker and as a result the GRACE and LID domain are going to be separated spatially, thereby generating a ChREBP activator. Selective activation of ChREBP in WAT leads to an expression of ChREBP target genes, therefore to a reduction of blood stream glucose levels. It improves insulin sensitivity, promotes lipogenesis and adipocyte differentiation, and reduces inflammation. In the next section, is now described the second main embodiment, namely a modified ChREBP with a dominant negative effect.

[0056] As above described in tissues, where an activation of ChREBP is harmful or preferably avoided, such as liver and pancreatic B cells, the present invention offers a solution, which allows the modified ChREBP to be dominantly negative in said tissues.

[0057] According to another embodiment of the invention, the modified ChREBP carries at least one mutation in the LID domain, or more precisely within the G6P bindingsite in LID of ChREBP. This mutation prevents G6P from binding and thus prevents the spatial separation of GRACE and LID otherwise triggered by e.g. high glucose concentrations in the cell.

[0058] In this embodiment the modified ChREBP carrying additionally the at least one mutation disabling the binding of the endogenous activator G6P, which otherwise induces the spatial separation of GRACE and LID, will lead to a dominant negative transcription factor, which cannot activate the cellular carbohydrate and lipid metabolism and thus has protective effects for the cell.

[0059] Consequently, the presence of such dominant negative ChREBP prevents transcriptional activity in cells where the proteolytic site cannot be cleaved.

[0060] In addition, due to the inability of G6P binding to LID the modified ChREBP of the invention acts as dominant negative protein by also blocking the ChoRE binding site with an inactive GRACE domain, thereby competing with native ChREBP.

[0061] As a general proof of this principle in Example (1) of the specification, this dominant negative effect is shown for the mutations in LID, particularly but are not limited to W127A, W127L, N123A or N123D.

[0062] Due to the design of the modified ChREBP of the invention, the inventors have created a protein which can be adapted patient-specifically to the targeted tissue type and a corresponding clinical picture by selection and placement of the proteolytic site. Further, the selection and placement of the proteolytic site defines whether the modified ChREBP of the invention acts as activator or inhibitor.

[0063] Thus, in still another embodiment of the invention the at least one proteolytic site is incorporated within the sequence section of the GRACE, the region between GRACE and DNA binding domain as well as the DNA-binding domain. Interestingly, the placement of the at least one proteolytic site within the GRACE, the region between GRACE and DNA binding domain, and / or the DNA-binding domain results after cleavage in the inhibition of the transcriptional activity.

[0064] Thus, the cleavage at the proteolytic site results in a protein or peptide which hinders or competes with native ChREBP in binding to ChoRE and thus inhibits or reduces the transcriptional activation. It is crucial that the proteolytic cleavage of the modified ChREBP of the invention results in a protein or peptide, which can still bind to ChoRE, but which no longer has a functional GRACE domain or any GRACE domain, consequently no transcriptional activity can occur. According to the teaching of the invention proteases e.g. MT-SP1, which are implicated in human cancers such as those of prostate, colon or gastrointestinal tract are suitable for designing the modified ChREBP as selective inhibitor. According to the inventors the shift of the incorporated proteolytic site from the linker towards the GRACE and / or the DNA-binding domain leads to selective inhibition in cancer cells which express MT-SP1 and causes cancer cells to starve or at least stop growing.

[0065] According to still a further embodiment of the invention the at least one proteolytic site is incorporated within the sequence section of LID. In the embodiment, where the insertion of the proteolytic site destroys the G6P binding domain, the resulting modified ChREBP is also a dominant negative inhibitor, which competes with native ChREBP.

[0066] Such dominant negative LID variants can according to a further embodiment become reactivated, if a second proteolytic site is inserted. Preferably such second proteolytic site is selected to show a different tissue specificity. In this example the first proteolytic site or mutation in the LID is destroying the G6P activation and thus down regulates or blocks any ChREBP activity. If now with a second proteolytic site, which is cell specific and which is placed between LID and GRACE in the target cells and by activity of the specific protease in theses target cells, the LID and GRACE domains are separated GRACE can together with the DNA binding domain become reactivated.

[0067] Similar combinations of two or more different proteolytic sites, incorporated in different locations in ChREBP allow a very specific targeting of ChREBP activity or inhibition for therapeutic use in personalized medicine.

[0068] In still another embodiment, the invention provides a nucleic acid sequence encoding the modified CHREBP of the invention as a DNA, cDNA, mRNA and / or modified RNA molecule.

[0069] The nucleic acid sequence encoding modified ChREBP of the invention can be used as therapeutic preparation or in medicine.

[0070] The invention also encompasses vectors containing said nucleic acid sequence, which are suitable for delivery into the patient. Such vectors may include viral vectors (e.g., adenoviral, lentiviral, or adeno- associated viral vectors), non-viral vectors (e.g., plasmid DNA, lipid nanoparticles), or other gene delivery systems.

[0071] For the use in treatment the above-described modified carbohydrate-responsive element-binding protein (ChREBP) is provided in form of a pharmaceutical composition comprising an effective amount of modified ChREBP and / or a nucleic acid encoding modified ChREBP, be it pure, encapsulated or protected, and a suitable additive such as a pharmaceutically acceptable diluent, preservative, solubilizes, emulsifier, adjuvant, carrier and / or excipient.

[0072] According to the invention, an “effective amount” of the modified ChREBP and / or the nucleic acid encoding the modified ChREBP is an amount for achieving treatment or prevention of any carbohydrate and lipid metabolism related diseases or disorders. For uses in the treatment of a patient in need thereof, an effective amount of the modified ChREBP and / or a nucleic acid encoding modified ChREBP according to the invention, be it pure, encapsulated or protected or in a pharmaceutical composition, is used to treat and / or prevent pathological symptoms related to the cellular carbohydrate and lipid metabolism, or related to diseases and disorders selected from the group comprising diabetes, obesity, atherosclerosis and / or cancer.

[0073] According to the invention the term “treat or prevent”, or any lingual variation thereof, as used herein refers to preventing the manifestation of symptoms before they occur, slowing down the progression of the disease, slowing down the deterioration of symptoms, enhancing the onset of remission period, slowing down the irreversible damage caused in the progressive chronic stage of the disease, delaying the onset of said progressive stage, reducing the severity or curing the disease, improving the survival rate or a more rapid recovery, preventing the disease form occurring or a combination of two or more of the above.

[0074] Additionally, the term prevent in the context of this application is directed to a lifestyle use or an early- stage application in patients with a predisposition to develop a disorder or symptoms related to the cellular carbohydrate and lipid metabolism.

[0075] The present invention may be administered by various routes. Examples of such routes, without limitation may be intravenous, subcutaneous, orally and / or topical.

[0076] Description of the Figures

[0077] Figure 1 1 ChREBP mutants act dominant negative in D. melanogaster and cell culture.

[0078] (A) In 832 / 13 cells, transcription of ChREBP target genes is reduced in the presence of the W127L mutant vs. an empty vector control as analyzed by a qPCR experiment.

[0079] (B) ChREBP target genes (GPD1, Pklr, Acly) are involved in glycolysis and de novo lipogenesis.

[0080] (C) In D. melanogaster, the ChREBP mutant expression was initiated by a Gal4 / UAS system in WT (Mondo) background. Despite presence ofthe WT ChREBP, flies were not viable on a high glucose diet, hinting towards a dominant negative effect ofthe ChREBP mutants in vivo.

[0081] Figure 21 Dissociation of LID and GRACE domains regulates the endogenous and artificial activation mode of ChREBP.

[0082] (A) ChREBPa is organized in three major domains: the low glucose inhibitory domain (LID), the glucose-response activation conserved element (GRACE), and a DNA binding bHLH / ZIP domain. The LID and GRACE domain together are further defined as the “glucose sensing module” (GSM) of ChREBP. The LID serves as the contact point of the scaffolding protein 14-3-3. At the interface of the LID and 14-3-3, the binding site of the endogenous activator G6P is located. The ChREBPfJ isoform is lacking the inhibitory LID domain and is constitutively active. (B) In the inactivated status, ChREBP is inactive due to a direct contact of the LID and the GRACE domain. During the endogenous mode of activation, G6P binding to the LID / 14-3-3 interface triggers dissociation of the GRACE domain resulting in transcriptional activation.

[0083] (C) The protease recognition sequence (TCS) is integrated into an unstructured loop connecting the LID and GRACE domains. Proteolysis results in the dissociation of LID and GRACE, resulting in constitutively active ChREBP. This is an orthogonal approach for ChREBP activation independent of G6P, the endogenous trigger.

[0084] (D) Binding of the endogenous activator G6P triggers unique conformational changes in the GSM of the wild-type complex (GSM). This novel conformation can be visualized by limited proteolysis using Chymotrypsin digest. The G6P-specific band is marked by an asterisk (*). Limited proteolysis suggests that the introduction of a protease cleavage site in the linker between LID and GRACE domain does not alter the endogenous protein function of the GSMTCS mutant, as the G6P specific band is present.

[0085] (E) The His-tagged GSMTCS complex is coupled to Ni-beads (“bound”). Following overnight proteolysis by TEV protease (“TEV cleavage”), the LID / 14-3-3 complex is detected in the flowthrough (“wash”), while the Grace domain remains on the beads until elution by high concentrated imidazole washes (“elution”). Results were detected by SDS page and Coomassie stain.

[0086] Figure 3 | Site-directed proteolysis can induce structural changes to ChREBP comparable to activating ligand binding

[0087] (A) A nanoDSF experiment indicates that the GSM / 14-3-3 complex shows instability at the temperature range of 35 to 45°C (highlighted in grey), resulting in an infliction temperature of 48°C. Upon G6P binding, the complex takes up a novel conformation with increased stability (infliction temperature of 51.7°C). A similarly stabilizing effect was achieved upon proteolytic digest (infliction temperature of 50.5°C). A deletion construct of LID / 14-3-3 (no GRACE domain) shows also increased stability over the GSM / 14-3-3 complex (infliction temperature of 52.3°C).

[0088] (B) An ITC titration comparing GSM / 14-3-3 (“WT”) and LID / 14-3-3 (“LID”) binding to G6P suggests a 3-fold increased affinity of G6P to LID / 14-3-3 (KdwT=1.6 pM vs. KdLiD=0.5 pM).

[0089] (C) There is a strong reduction in enthalpy and more favorable entropy observed in the ITC titration in the LID vs. WT, suggesting minor structural changes upon G6P binding in the LID vs. WT construct. This shows that the removal of the GRACE domain by proteolysis initiates similar structural changes to the 14-3-3 / LID complex as binding of the activating ligand G6P to the 14-3- 3 / GSM complex.

[0090] Figure 4 | Proteolysis initiates transcription by ChREBP mutants in vitro and in cellulo.

[0091] (A) A model of the endogenous mode of activation tested by a UASG / lt-d riven luciferase reporter assay in response to glucose.

[0092] (B) The classic system was adapted to test whether protease digest could activate transcription in addition to the endogenous activation by glucose. TEV protease was used as a model system.

[0093] (C) Luminescence was detected for GSM and GSMTCS based on the endogenous activation mechanism (“-TEV protease”) in high glucose, and for GSMTCS, N123ATCS, and W127LTCSbased on the proteolytic activation mechanism under low and high glucose conditions (“+TEV protease”) in INS- 832 / 13 rat insulinoma cells.

[0094] (D) By Western blot, protein expression levels as well as proteolytic products of the GSMTCS derivatives are detected to show expression of ChREBP mutants and TEV protease in cells used for the luciferase assay.

[0095] Figure 5 | Proteolysis can reprogram fly metabolism.

[0096] (A) qPCR experiments suggest that ChREBP target gene expression is elevated in flies if they express TEV and artificial proproteins of ChREBP. Studied genes were cabut, ACC and FASN1, rp49 was used as house-keeping gene.

[0097] Figure 6 | Perspective on ChREBP dual agonist / antagonist system application in T2D therapy.

[0098] (A) The ChREBP mutant will be applied as mRNA-based treatment using non-targeted application. In liver, pancreas, muscle, kidney antagonistic effects will be mediated due to the point mutation and its dominant negative effect. In white adipose tissue, ChREBP will be digested by CatB and therefore tissue-specifically activated.

[0099] (B) The ChREBP CatB optimized mutant can be site-directed digested. Modification of the ChREBP amino acid sequence were performed in two regions of the protein. An optimized Cathepsin B site has been introduced into the linker between LID and GRACE (amino acid positions 191-198). Additionally, to avoid unspecific cleavage at amino acid residues 140-148, the mutant G144H was introduced to increase resistance to non-specific Cathepsin B digest. The mutation successfully reduced unspecific cleavage by Cathepsin B at this position, as detected by SDS page.

[0100] Figure 7 | LID and GRACE domain separate only after proteolysis, independent of G6P as the activating ligand.

[0101] (A) When performingthe same experiment as presented in Figure 2E in the absence of TEV protease, the GSM / 14-3-32construct remained intact and bound to the column until final elution using high imidazole concentrations.

[0102] (B) The presence of G6P as the activating ligand did not affect the result of the experiment shown in Figure 2E.

[0103] Figure 8 | Introduction of the TCS into GSM / 14-3-3 does not impact protein stability and ligand binding ability

[0104] (A) Performing a nanoDSF experiment, the GSMTcs / 14-3-3 complex shows a comparable melting temperature as the wt protein. Like wt, the protein is stabilized upon the addition of G6P.

[0105] (B) Quantitative cleavage of GSMTCS was achieved overnight using GST-tagged TEV protease. Prior to melting scan, the protease was removed using GSH resin. Successful cleavage as well as removal of TEV protease can be shown on SDS gel with Coomassie stain.

[0106] Figure 9 | D. melanogaster tolerates the expression of TEV protease and the protease effectively cleaves ChREBPTcs

[0107] (A) The expression of the protease by a Tubulin or CG driver barely impacted fly survival on low (black outline) and high (gray outline) glucose fly food.

[0108] (B) ChREBP mutants containing the TCS motif were successfully proteolytically digested in TEV protease-expressing D. melanogaster. The “ChREBP” band represents the full-length protein, ChREBPauo represents the proteolytic product. Tubulin was detected as loading control.

[0109] Figure 10 | Sequence alignment

[0110] Amino acid sequence alignment of mouse and human ChREBP performed with Clustal 0 (1.2.4): Multiple Sequence Alignment tool. The sequences are labelled according to their uniprot ID. An “*” (asterisk) indicates that the amino acids at the displayed position is fully conserved. A (colon) indicates that the amino acids at the displayed position have strongly similar properties. A (period) indicates that the amino acids at the displayed position have weakly similar properties. Numbers at the end of the line indicate residue numbers.

[0111] Figure 11 1 Endogenous proteases selectively cleave LID and GRACE

[0112] (A) The WT GSM construct, a Furin resistant mutant (“WTUndeav”and a GSM containing a Furin cleavage site in the linker between LID and GRACE (“WTFurin” have been digested by Furin in vitro. Cleavage of LID and GRACE is most prominently observed for the WTFurin mutant.

[0113] (B) ChREBP mutants containing recognition site for Thrombin (“WT Thrombin”) within the linker region were successfully proteolytically digested into LID and GRACE in vitro.

[0114] (C) ChREBP mutants containing recognition site for MT-SP1 (“WTMatriPtase”) within the linker region were successfully proteolytically digested into LID and GRACE in vitro.

[0115] Figure 12 | The proprotein activation system can be adapted to the human isoform of ChREBP

[0116] Luminescence as a readout for ChREBP transcriptional activity was detected for GSMhuman and GSM ics based on the endogenous activation mechanism, while W127L and N123A were inert to activation by the natural ligand. The mutants GSMTCS, N123ATCS, and W127LTCs can de activated by proteolysis independently of nutrient state in 832 / 13 cells, as previously observed for GSMm0USe (Figure 4).

[0117] Table 2 | ChREBP amino acid sequences

[0118] Examples

[0119] The following examples illustrate multiple ways of carrying out the invention as intended, without the intent of limiting the invention to said examples.

[0120] General experimental and analytical techniques as used for the subsequent Examples

[0121] All ChREBP constructs and TEV protease were expressed in BL21 (DE3) competent E. coli (ThermoFisher).

[0122] The cells were grown in TB / PBS medium at 37 °C, induced at the OD600 = 1.5 with 0.3 mM isopropyl-d-1- thiogalactopyranoside, followed by 18 h of growth at 18 °C before harvest.

[0123] Luciferase assays and qPCR experiments were performed using INS-1 832 / 13 cell line grown in RPMI supplemented by 10 mM HEPES (Thermo), 2 mM L-glutamine, 1 mM sodium-pyruvate (Thermo), 10% fetal calf serum, 0.05 mM [3-mercaptoethanol (Sigma) and under addition of Penicillin-Streptomycin.

[0124] The cells were kept in humidified incubators at 37 °C and 5% CO2 and passaged twice a week. Cell counting was performed using the Vi-CELL XR (Beckman Coulter).

[0125] Flies were kept at 25 °C under controlled humidity on medium supplemented with varying concentrations of sucrose.

[0126] Protein purification. The purification of GSM / 14-3-3 complex and mutants were performed using following buffers were used: lysis Buffer (50 mM HEPES pH 7.4, 500 mM NaCI, 20 mM imidazole, 10% glycerol, 1 mM TCEP, supplemented with complete EDTA-free protease inhibitor cocktail (Roche)), wash buffer (lysis buffer with 40 mM imidazole), elution buffer (lysis buffer with 500 mM imidazole), dialysis buffer (25 mM HEPES pH 7.4, 150 mM NaCI, 10% glycerol, 1 mM TCEP), SEC buffer (dialysis buffer with 1 mM DTT instead of TCEP).

[0127] For cell lysis, the pellet was resuspended in lysis buffer, sonicated and the lysate was centrifuged for 30 min at 4 °C, 40.000 x g. The supernatant was added to pre-equilibrated Ni-NTA beads (Qiagen). The beads were incubated for 45 min while rolling at 4 °C. The protein lysate was centrifuged at 4 °C, 1000 x g, 3 min, the supernatant discarded and the Ni-NTA beads were washed 6 x with wash buffer. The protein was then eluted. Afterwards, an overnight TEV cleavage occurred during dialysis. The next day, the His- tagged TEV protease was removed by incubating the overnight dialyzed protein in Ni-NTA beads. The supernatant was injected onto a HiLoad 26 / 60 Superdex 200 (Cytiva) column and the peak was collected, analyzed by SDS page and frozen with 20% glycerol.

[0128] Mammalian cell culture and transfection. Cells were grown in at 37 °C in a humidified atmosphere of 5 % CO2. 832 / 13 cells were maintained in 11 mM glucose RPMI (Fisher Scientific) supplemented with 10 % FCS (Sigma), 100 U / ml penicillin-streptomycin solution (Life Technologies), 2 mM L-glutamine (Thermo Fisher), 10 mM HEPES (Thermo Fisher), 1 mM sodium pyruvate (Thermo Fisher), and 0.05 mM [3- mercaptoethanol. 832 / 13 cells were transfected using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s protocol. Transfections were verified via immunoblotting. qPCR of human 832 / 13 cells expressing ChREBP mutants. RNA isolation was performed with TRIzol (Invitrogen) and was reversed transcribed. cDNA was measured using nanoDrop (ThermoFischer). QuantStudio 3 Real-Time qPCR system (ThermoFischer) was used, selecting the run-mode “fast” and “Sybr green”. Gene expression was calculated based on the delta-delta CT method. As housekeeping genes, Hmbs, Ywhaz, Actb and Alas were used. Ratios were calculated based on target gene expression in cells that were transfected with a ChREBP mutant compared to cells transfected with an empty plasmid.

[0129] Drosophila melanogaster stocks. The stocks Cy Roi balancer (BDSC#25), mondoK / K(BDSC#10562) Tub- Gal4 / TM3, Sb, Ser (BDSC#5138) were obtained from the Bloomington Drosophila Stock Center. Mondo knockout flies have previously been generated in our laboratory.

[0130] Fly food and fly maintenance. For standard fly maintenance, food containing agar 0.84% (w / v), cornmeal 7.6% (w / v), dry yeast 6% (w / v), sucrose 3.14% (w / v), glucose 6.27% (w / v), sodium potassium tartrate 0.87% (w / v), CaCb 0.07% (w / v) and nipagin (methylparaben) 0.26% (w / v) was used. Additional dried yeast was added on top of the food before use. For defined nutrient studies, larvae were grown on food containing 10% (w / v) dry yeast, 1% (w / v) agar, and 2.5% (v / v) nipagin in PBS supplemented with 20% (w / v) sucrose for the high sucrose media.

[0131] Sugar intolerance assay. The mondo mutants are heterozygous and maintained over the 2ndchromosome balancer Cy Roi. For the rescue experiments, mondomutICy Roi; tubulin Gal4 / TM3, Ser, Sb were crossed with mondomut / Cy Roi; UAS-rescue / UAS-rescue. All rescue constructs are inserted via phiC31 integrase in the attP2 landing site. The constructs were injected by Bestgene. The viability on low and high sugar was calculated based on the emergence of heterozygous mondo mutant flies, comparing the numbers of flies containing non SER, non Sb to Ser, Sb flies. The expected ratio of flies with non SER, non Sb vs. Ser, Sb flies is 1:1. The percent viability was calculated as follows: 100 x {(number of Cyo Roi; non Ser, non Sb flies) I number of Cy Roi;TM3, Ser, Sb flies}. Data visualization was performed in GraphPad Prism 9.

[0132] Limited Proteolysis assays with Chymotrypsin. The ChREBP constructs were diluted to a concentration of 10 pM in Protease Cleavage Buffer (20 mM HEPES, pH 7.4, 50 mM NaCI, 10 mM MgCb). Prior to protease digest, the samples were incubated with 0 or 1 mM of G6P for 10 min. Chymotrypsin (Sigma) was added to a final concentration of 0.8 pg / mL and the digest took place for one hour on ice. The reaction was quenched using 4 x Laemmli Sample Buffer, followed by heating the samples to 95 °C for five minutes. Next, the samples were loaded onto a 14% SDS-PAGE gel, and stained using Coomassie Stain (50% ethanol, 10% acetic acid, 0.25% Brilliant Blue R250), followed by destaining (40% ethanol, 10% acetic acid). Gels were finally scanned with the Bio-Rad ChemiDoc MP imaging system. Proteolysis on Ni NTA beads to show separation of LID and GRACE. All steps were performed at 4°C. Ni NTA beads were equilibrated with buffer A (50 mM HEPES, pH 7.4, 500 mM NaCI, 1 mM DTT, 20 mM imidazole, pH 8, 10% glycerol). The His-tagged ChREBP construct was added (0.3 mg) and binding was ensured by rolling the beads for lhr. Beads were then washed 5-fold with buffer A. The sample “beads” was taken for SDS page. TEV protease was added (0.02 mg) and the digest was incubated overnight. The sample “TEV cleavage” was taken for SDS page. The next day, the overnight solution was eluted, and the beads were washed 2 times (SDS page sample “wash”). Then 500 mM imidazole was added to buffer A and the remaining protein was eluted from the NiNTA column (SDS sample “elution”). The protein content of the collected fractions was analyzed by SDS page using Coomassie stain. As control, the same procedure was performed in the absence of TEV protease. To test the effect of the activating ligand on proteolysis, an additional experiment was performed under addition of 1 mM G6P to buffer A. nanoDSF experiments. Thermal shift assays were performed usingthe Prometheus NT.48 (NanoTemper GmbH). Proteins were diluted in GF buffer to 10 pM. D-Glucose-6-phosphate sodium salt (Cayman Chemicals, Cay20376) was dissolved in GF buffer, neutralized and added to the conditions containing G6P at a concentration of 500 pM. The experimental conditions were set to a temperature slope of 1 °C / min, covering a range from 20 °C to 85 °C using an excitation power of 30%.

[0133] Isothermal titration calorimetry. Measurements were performed on a MicroCai PEAQ-ITC instrument (Malvern). The proteins were dialyzed overnight in ITC buffer (10 mM HEPES, pH 7.4, 50 mM NaCI and 0.5 mM TCEP) and spun at 13.000*gto remove any precipitate before dilutingthe protein to 50 pM and adding it to the sample cell. G6P was dissolved in ITC buffer. The pH was adjusted by NaOH addition, and the solution was then filtered using a 0.4 pm syringe filter to remove any particles. 200 pM G6P were loaded into the syringe. The solution was equilibrated to 25°C. Each titration consisted of 18 injections a 2 pL with 4.0 s injection time, reference power 10 pCal / s, initial delay 60 s, stirring speed 750 rpm, and spacing 170 s. Kd values were fitted using MicroCai PEAQ-ITC Analysis Software.

[0134] Luciferase assay. 832 / 13 cells were plated in 11 mM glucose RPM I on a white 96-well plate (Greiner) 200uL / well. 832 / 13 cells were plated at 2x105 cells / ml on the indicated plate. 24 hours after plating, 11 mM RPMI was aspirated and replaced with low (2.5 mM) or high (27.5 mM) glucose RPMI. Cells were transfected with equimolar amounts of pRL-TK (Promega, containing a constitutive TK-promoter driven Renilla luciferase), pG5luc (Promega, containing a Gal4 UAS-promoter driven Firefly luciferase), and a pGampac vector expressing the indicated Gal4 fusion protein. Cells were transfected using Lipofectamine 2000 Transfection Reagent (Thermo) following manufacturer protocol. The constitutive TK-promoter driven Renilla luciferase (pRL-TK) was included as an internal control for transfection efficiency. 24h after RPMI change, luciferase and renilla expression were determined using the Dual-Glo Luciferase Assay System (Promega), according to the manufacturer’s protocol. Data was normalized to renilla expression and visualization was performed in GraphPad Prism 9.

[0135] Western blots. Immunoblotting was used to confirm expression of ChREBP constructs in mammalian cell culture. The protein contents of whole cell extracts were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher). Equal amounts of protein were then loaded on to 10 % SDS-PAGE gels for analysis. Trans-Blot Turbo Transfer System (was used to transfer the protein to the nitrocellulose membrane. Tubulin was used as a loading control for all samples. The antibody mouse anti-Tubulin (1:20 000, T9026, Sigma Aldrich) was used. Bands were visualized using Immobilon western HRP substrate (Merck) and a Bio-Rad ChemiDoc MP imaging system. qPCR experiments in D. melanogaster. For the RNA extraction, 10 female flies were grinded in 500 uL TRIzol (Invitrogen). Standard RNA extraction procedure was followed and RNA was reversed transcribed. cDNA was measured using nanoDrop (ThermoFischer). QuantStudio 3 Real-Time qPCR system (ThermoFischer) was used, selecting the run-mode “fast” and “Sybr green”. Gene expression was calculated based on the delta-delta CT method. As housekeeping gene, rp49 was used. Ratios were calculated relative to target gene expression in flies that were not expressing a ChREBP mutant.

[0136] CatB digest. Recombinant human Cathepsin B was obtained from R&D systems (Cat. 953-CY) and activated based on the provider’s protocol. The purified ChREBP mutants were diluted to 20 pM and digested by 0.1 ug Cathepsin B for 2 h at room temperature. The reaction was quenched by adding 12 uL SDS buffer, and boil samples at 95C for 3-5 min. An SDS gel page gel was performed (gradient gel, 20%), UV-detection and Coomassie stain was used for protein detection.

[0137] Example 1: ChREBP point mutants act dominant negative over WT protein and are ChREBP inhibitors in cellulo and in vivo

[0138] As ChREBP over-activation in obese individuals in pancreas and liver has devastating effects, it is aimed to find a ChREBP inhibitor. This inhibitor had to fulfill special requirements, as it needed to inhere the flexibility to be turned into an activator in select tissue. As tissue-selective action of small molecules is hard to archive, the focus was on the development of a protein-based approach.

[0139] Previously, ChREBP mutants were published which showed impaired binding of the endogenous activator glucose-6-phosphate (G6P). These mutations hindered ChREBP activation in vitro and in vivo. Characterizing these mutants in more detail, it became clear that they act dominant negative over WT protein (Figure 1).

[0140] When expressing the W127L or N123A mutants, a reduction in ChREBP target gene expression using qPCR despite WT background in 832 / 13 cells was detected (Figure 1A). These mutants therefore acted dominant negative; they suppressed the endogenous ChREBP function and hindered the expression of target genes belonging to glycolysis and de novo lipogenesis (Figure IB). Their action as selective ChREBP inhibitors will likely hinder the glucose-to-fat conversion, slowing down fat accumulation in target organs like the liver.

[0141] To investigate if this dominant negative effect was further relevant on the organismal level, the effect of the mutants was tested in vivo. As previously described, Mondo (CHREBP equivalent) knockout Drosophila melanogaster showed impaired survival on a high glucose diet. This phenotype could be rescued by either Mondo or mammalian ChREBP expression. In contrast, the ChREBP mutants did not rescue survival. The current study focused on analysis of flies which expressed ChREBP or ChREBP mutants in WT background (Figure 1C). This implies that the flies are able to express the endogenous protein Mondo while simultaneously expressing mammalian ChREBP or ChREBP mutants via a Gal4 driver system. Expression of ChREBP in WT background (WT; ChREBP) had no effect on survival of the flies (Figure ID). Yet, expressing the ChREBP mutants W127A / L or N123A / D hindered development of these flies on a high glucose diet, despite the presence of WT Mondo. This result shows that the mutant proteins were able to suppress WT function, therefore act dominant negative in vivo.

[0142] Altogether, these results show that the W127A / L or N123A / D ChREBP point mutants act as inhibitors and are suitable therapeutic ChREBP antagonists to slow down lipid synthesis in vital organs like the liver and kidney. Next, it was aimed to find an approach to activate these dominant negative mutants selectively in WAT, where ChREBP function is highly beneficial to improve the diabetic condition.

[0143] Example 2: An orthogonal, nutrient-independent activation mechanism for ChREBP as artificial proprotein

[0144] To find an orthogonal approach for the activation of the dominant negative ChREBP mutants, the protein domain organization, as well as the endogenous mode of activation was analyzed. ChREBP consists of three major domains (Figure 2A). N-te rm inally, the low glucose inhibitory domain (LID) is described to suppress protein activation in the absence of glucose by an interaction to the glucose-response activation conserved element (GRACE) domain and the scaffolding protein 14-3-3. Both domains are connected via a short, unstructured linker region. Rising blood glucose levels lead to an accumulation of G6P inside the cells. Binding of this glucose metabolite in the interface of the LID domain and 14-3-3 triggers a conformational change which results in the dissociation of LID and GRACE domain. The GRACE domain is now free to recruit the transcriptional machinery, while the DNA binding domain couples a sequence motif called carbohydrate response element (ChoRE) (Figure 2B).

[0145] Taken together, the key step towards activation is the dissociation of the GRACE domain from the LID. The ChREBP beta isoform is lacking the inhibitory LID domain (Figure 2A), and is reported to be constitutively active. Therefore, it seemed plausible to engineer an orthogonal activation mode for ChREBP by proteolytically removing the LID domain from the remaining ChREBP protein. By engineering a protease cleavage site into the short, disordered linker between the LID and the GRACE domain the proteolysis will activate the transcriptional activity of ChREBP (Figure 2C)

[0146] A ChREBP proprotein containing a TEV protease cleavage site (TCS) in the linker between LID and GRACE was designed. As TEV protease is a well-established protease routinely used in laboratory practice, this protease was well-suited for the proof-of-principle. The GSMTCS / 14-3-3 co-expressed construct formed a complex consisting of one GSMTCS protein and two 14-3-3 proteins, as previously observed for the WT protein.

[0147] To make sure that the introduction of the TCS sequence into the linker between the LID and GRACE domain had no negative consequences on ChREBP mechanistic function required for the endogenous activation mechanism, a limited proteolysis experiment was performed. The Chymotrypsin-based digest revealed the enrichment of the G6P characteristic band in comparison to the digest pattern observed under apo conditions, suggesting that G6P can induce the activating structural changes in the mutant protein (Figure 2D).

[0148] However, forthe artificial proprotein mechanism to work successfully, the proteolysis had to be sufficient to cause the dissociation of the LID and GRACE domains, as domain interactions are often driven by hydrophobic contacts, hydrogen bonds or Van der Waals force, and not necessarily dependent on a covalent linker between them. A C-terminal His-tag adjacent to the GRACE domain allowed to couple this construct to Ni / NTA beads. After an overnight incubation with TEV protease, the resin was washed extensively and the flowthrough was checked for protein content by SDS page (Figure 2E). Indeed, following the proteolysis, 14-3-3 and the LID domain were detected in the wash. Both co-eluted, suggesting that 14-3-3 interacted with the GSM by contacts to the LID domain.

[0149] The GRACE domain was detected in the flowthrough upon protein elution with high concentrations of imidazole buffer, showingthatthe LID and GRACE domain were successfully separated upon proteolysis. When performing the same experiment without TEV protease, the GSM / 14-3-3 construct remained on the column until final elution (Figure 7A). Further, the presence of G6P as the activating ligand did not affect the result (Figure 7B).

[0150] Example 3: Proteolysis initiates structural changes in ChREBP similar to endogenous activator binding

[0151] Proteolysis was sufficient to initiate the dissociation of the GRACE domain from the LI D / 14-3-32complex. The next step was to understand the mechanistic details of proteolysis on the GSM / 14-3-32complex. The endogenous activation mechanism relies on conformational changes within the LID domain upon binding of G6P which result in the release of the GRACE domain. It was examined whether the orthogonal approach could mimic this process and whether conformational changes occur upon digestion.

[0152] To detect the stabilization of the GSM / 14-3-32complex by G6P binding, a thermal shift assay using nanoDSF was performed. The apo GSM / 14-3-32complex showed reduced stability from 35°C, with the infliction point determined at 48°C (Figure 3A, upper part). However, as part of the endogenous activation mechanism, bi nding of G6P stabilized the complex at the lower temperature range and shifted the infliction point to 52.5°C. Clearly, G6P binding caused strong stabilization of the complex. Similar results were obtained for the GSMTCS / 14-3-32complex (Figure 8A).

[0153] The TEV-digested GSMTCs / 14-3-32complex was tested for its melting temperature. Most interestingly, proteolytic digestion had similarly stabilizing effects on the complex as ligand binding. Similar to the G6P-bound WT, the digested protein showed increased stability at lower temperature range and the infliction point shifted to 52°C (Figure 3A, lower part). Importantly, stabilization was observed in absence of the ligand, but by proteolysis alone. This suggested that proteolysis might trigger similar conformational consequences on GSM / 14-3-32as ligand binding.

[0154] The GRACE domain and the LI D / 14-3-32complex are the products of the proteolytic reaction. To untangle the effects observed by nanoDSF, the individual fragments were tested for their contribution to the melting profile. However, purification of the GRACE domain was unsuccessful, likely due to reduced stability of this domain in isolation. In contrast, the LI D / 14-3-3 complex could be purified in high yields. Finally, the thermal shift assay suggested that the apo LI D / 14-3-32complex was equally stable (infliction point 52°C) as the G6P-bound and as the proteolytically digested complex, hinting that it is the melting profile of the TEV-digested GSMTcs / 14-3-32complex was mostly caused by the LI D / 14-3-32complex.

[0155] The melting profiles suggested that the LID fragment had a more stable conformation than the full-length GSM in complex with 14-3-3. To test the hypothesis that the LI D / 14-3-3 conformation was similar to the one observed for the G6P-bound GSM, an ITC experiment was performed comparing G6P binding to GSM / 14-3-32complex VS. LI D / 14-3-32. AS previously reported, G6P bound to the GSM / 14-3-32complex with a Kd = 1.6 pM (Figure 3B). Binding was characterized by a high enthalpy reaction and unfavorable entropy (Figure 3C). In contrast, G6P bound the LID construct with increased affinity compared to the full GSM complex (Figure 3B). This tight binding came along with a decrease in binding enthalpy and reduced entropic costs (Figure 3C). It is likely that the unbound state in the LI D / 14-3-32complex is less flexible and likely closer in structure to the ligand-bound conformation, therefore, less structural rearrangements are required to fit the ligand. Asa consequence, an increased binding affinity is observed due to reduced conformational energy penalties. This finding argues that the LI D / 14-3-3 fragment, the product of proteolysis, indeed takes a similar conformation to the G6P-bound GSM in its activated state. More specifically, this shows that the dissociation of the GRACE domain, triggered either by ligand binding or by proteolysis, results in an alteration of the LID / 14-3-3 complex conformation into a more stable conformation which tightly coordinates G6P.

[0156] Example 4: Proteolysis can trigger transcription in dominant negative mutants, allowing for the design of dual antagonists / agonists

[0157] As proteolysis led to comparable conformational changes in ChREBP as the activating ligand binding, the next step was to find out if proteolysis could indeed activate transcription by the engineered ChREBP proprotein. Foran initial proof of concept, a Gal4 / Luciferase system was used in cultured cells which had previously been established as a readout for ChREBP transcriptional activity. Classically, increased glucose levels would lead to the production of G6P. This glucose metabolite would bind to the GSM / Gal4 fusion construct and activate transcription of luciferase as expected by the endogenous mechanism (Figure 4A). To test if proteolysis could indeed function as an orthogonal approach to activate ChREBP’s transcriptional activity, the TEV cleavage site was engineered into the GSM / Gal4 fusion construct (Figure 4B). Assays were performed under low glucose (2.5 mM) and high glucose (27.5 mM) conditions, allowing for the endogenous activation mechanism to occur alongside with proteolysis activated transcription.

[0158] It was found that both, the GSM-Gal4 and GSMTcs-Gal4 fusion constructs could be activated under high glucose (Figure 4C), suggesting that the introduction of the TEV cleavage site did not alter ChREBP’s endogenous activation mechanism. In contrast, the N123ATcs-Gal4 construct was inactive under high and low glucose conditions, and therefore inert to glucose-dependent activation. However, when TEV protease was co-expressed in the cells, high expression levels of luciferase were detected for both, the GSM TCS and N123ATCS constructs. This suggests that proteolytic activation can indeed serve as an orthogonal approach for ChREBP activation, and this mechanism is completely independent of nutrient levels. Moreover, this mechanism of activation is suitable for ChREBP mutants which have been described as dominant negative, suggesting that these ChREBP inhibitors can turn into activators by sitespecific proteolysis.

[0159] The results obtained by the Gal4 / Luciferase system showed the transcriptional control by proteolysis.

[0160] Example 5: Activation of ChREBP proproteins in vivo re-programs fly metabolism

[0161] To find out how proteolytic activation of ChREBP in the adipose tissue would influence metabolism of a living organism, ChREBP mutants were expressed in D. melanogaster, a well-established model system for metabolic disorders and diabetes. For proof-of-principle, TEV protease expressing flies were raised. The expression of the protease by a Tubulin or CG driver barely impacted fly survival (Figure 9A) as previously observed and ChREBP mutants containing the TCS motif were successfully cleaved in these flies (Figure 9B).

[0162] Next, ChREBP target gene expression levels were examined using qPCR on whole fly bodies (Figure 5). Under low and high glucose conditions, expression of ChREBP target genes was clearly increased in flies which were expressing TEV protease as well as the ChREBPTCs mutant compared to those that did not express the ChREBP mutant protein. This shows that, even on the organismal level, the transcriptional activity of ChREBP can be successfully regulated by proteolysis using the engineered ChREBP proproteins. The artificial activation mechanism leads to the expression of ChREBP target genes which are highly involved in glycolysis as well as de-novo lipogenesis. Therefore, the system serves as a handle to finetune metabolism in a living organism.

[0163] Example 6: Pharmaceutical application of ChREBP proproteins in the treatment of diabetes and atherosclerosis

[0164] To make the system applicable for human therapy and to introduce the possibility for cell-type specific activation of the engineered proproteins, endogenous human proteases were tested for their suitability to selectively digest and thereby activate the ChREBP mutants.

[0165] Cathepsin B is a protease that is generally located in lysosomes, yet, it has previously been reported (Mizunoe et al, 2020) that Cathepsin B translocates to the cytosol in diseased, adipose WAT (Figure 6A). As ChREBP is generally located in the cytoplasm, Cathepsin B and ChREBP would only share a common cellular compartment under the diseased state of obesity and T2D. This makes the protease Cathepsin B a suitable candidate to activate the engineered ChREBP proprotein solely in WAT.

[0166] When wild-type ChREBP was digested by recombinant Cathepsin B in vitro, there was a non-intended cleavage site within ChREBP. The ChREBP sequence was therefore optimized paying attention to ChREBP sequence conservation. Mutation of the lowly conserved 144G to H removed the non-specific cleavage product (Figure 6B). The Cathepsin B specific motif (GYGFVG) was introduced in Position 194. Cleavage of this motif was specific in vitro, as analyzed by SDS page (Figure 6B). The digest of our artificial ChREBP proprotein is therefore highly site-specific. Extending on the laboratory example using TEV protease, this result shows that activation of ChREBP proproteins can be performed in a highly specific fashion by endogenous proteases, such as Cathepsin B. To extend on this claim, additional endogenous proteases were tested, such as furin, thrombin, and MT-SP1. All produced identical, highly specific digestion patterns for our engineered ChREBP proproteins (Figure 11), underlining the feasibility of selective digestion, and therefore ChREBP activation, by endogenous proteases.

[0167] Example 7: Proteolysis can trigger transcription in dominant negative mutants, allowing for the design of dual antagonists / agonists in the human CHREBP isoform

[0168] Going forward, to apply the technology of artificial ChREBP proproteins in pharmacotherapy, the human isoform will be used. The human and mouse ChREBP isoform share high sequence similarity (Figure 10), therefore the conservation of the activation mechanism is expected.

[0169] Based on the example of the luciferase assay as a fast and reliable readout for transcriptional activity in cell culture, the applicability of the proprotein strategy on human ChREBP has been confirmed. The assay shows that, equally to the mouse isoform, the point mutants N123A and W127L are transcriptionally inactive. Upon introduction of a TEV cleavage site and co-expression of TEV protease, the activity of these mutants can be released. The artificial pro proteins can de activated under low glucose conditions by TEV protease, therefore independently of the endogenous activator. The system is therefore independent of the nutrient status of the organism.

[0170] ChREBP function is highly conserved between species and these results show that the technology is applicable across species and thus also for mammals and in particular humans.

[0171] References

[0172] Abdul-Wahed et al., 2017, “Sweet Sixteenth for ChREBP Cell Metabolism, Elsevier 2017

[0173] Mizunoe, Y., Kobayashi, M., Hoshino, S. etal. Cathepsin B overexpression induces degradation of peri lipin

[0174] 1 to cause lipid metabolism dysfunction in adipocytes. Sci Rep 10, 634 (2020)

Claims

Patent Claims1. Modified carbohydrate-responsive element-binding protein (ChREBP) comprising an inhibitory domain (LID), a linker, a functional domain (GRACE) and a DNA-binding domain, characterized in that at least one proteolytic site is incorporated within the sections selected from LID, the linker, GRACE, the sequence between GRACE and the DNA-binding domain and up to the end of the DNA-binding domain.

2. Modified ChREBP according to claim 1, wherein the at least one proteolytic site incorporated into the linker connecting the LID and GRACE domain.

3. Modified ChREBP according to claims 1-2, wherein the at least one proteolytic site is a recognition site and / or binding site for a protease.

4. Modified ChREBP according to claims 1-3, wherein the at least one proteolytic site is a recognition site and / or binding site for a tissue specific protease and / or a protease of mammalian origin.

5. Modified ChREBP according to claims 1-4, wherein the at least one proteolytic site is a recognition site and / or binding site for a protease selected from the group comprising TEV protease, cathepsin B, furin, thrombin and / or MT-SP1.

6. Modified ChREBP according to claims 1-5, wherein LID carries at least one mutation disabling binding of the endogenous activator G6P.

7. Modified ChREBP according to claims 1-6, wherein the at least one proteolytic site is incorporated within the sequence starting at the section of GRACE and up to the end of the DNA-binding domain.

8. Modified ChREBP according to claims 1-6, wherein the at least one proteolytic site is incorporated within the sequence section of LID.

9. Nucleic acid encoding the modified ChREBP according to claims 1-8.

10. Pharmaceutical composition comprising modified ChREBP according to claims 1-8 and / or nucleic acid encoding the modified ChREBP according to claim 9, and a pharmaceutical acceptable carrier, diluent or excipient.

11. Modified ChREBP accordingto claims 1-8 and / or nucleic acid encodingthe modified ChREBP according to claim 9 for use in medicine.

12. Modified ChREBP accordingto claims 1-8 and / or nucleic acid encodingthe modified ChREBP accordingto claim 9 for use in treatment of diabetes, obesity, atherosclerosis and / or cancer.

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