Targeting metabolic pathways with MNK inhibitors

Abstract

Targeting Metabolic Pathways with MNK Inhibitors The invention belongs to the field of medicine and concerns the treatment of diseases related to aberrant upregulated de novo lipid biosynthesis using a MAPK interacting kinase (MNK) inhibitor, especially for the prophylaxis or treatment of breast cancers, prostate cancers, obesity, atherosclerosis, diabetes mellitus, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, chronic kidney disease, and neurological disorders. An aspect of the invention relates to the use of an MNK inhibitor designated (4-(3-((2- (dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone, for the prophylaxis or treatment of triple-negative breast cancer and brain metastases therefrom.

Description

[0001] TARGETING METABOLIC PATHWAYS WITH MNK INHIBITORS

[0002] FIELD OF THE INVENTION

[0003] The invention belongs to the field of medicine and concerns the treatment of diseases related to aberrant upregulated de novo lipid biosynthesis using a MAPK interacting kinase (MNK) inhibitor, especially for the prophylaxis or treatment of breast cancers, prostate cancers, obesity, atherosclerosis, diabetes mellitus, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, chronic kidney disease, and neurological disorders. An aspect of the invention relates to the use of an MNK inhibitor designated (4-(3-((2-(dimethylamino)pyridin-4- yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone, for the prophylaxis or treatment of triple-negative breast cancer and brain metastases therefrom.

[0004] BACKGROUND OF THE INVENTION

[0005] Lipids, including sterols, isoprenoids, acylglycerols and phospholipids, are components of biological membranes, are used in energy metabolism and storage and have important roles as signalling molecules. It has been shown that cancer cells frequently exhibit enhanced de novo lipid biosynthesis, as well as increased uptake of exogenous lipids so as to maintain increased metabolic needs. As activation of de novo lipid synthesis is specific to cancerous tissues (as compared with normal tissues) in various cancer types, substantial efforts have been made to develop strategies to target this pathway for cancer treatment.

[0006] It has been reported that the mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) play an important role in lipid metabolism. Specifically, phosphorylation of eukaryotic initiation factor 4E (elF4E) by MNK has been shown to play a role in de novo lipid synthesis in the setting of high-fat diet-induced obesity, and that MNK inhibitors (MNKis) prevent obesity in mice. Mechanistically, MNK-dependent elF4E phosphorylation promotes the translation of genes involved in lipid processing and storage, including APOC3, and LPIN2, but not SREBP1 (sterol regulatory element-binding protein 1 ) itself. elF4E is a crucial mRNA cap-binding protein that regulates global mRNA translation in mammalian cells. Changes in elF4E levels impact specific oncogenic mRNAs, contributing to its oncogenic functions. Directly targeting elF4E is challenging, so attention has shifted to MNKs, which regulate specific aspects of elF4E's transforming function without affecting global mRNA translation. MNKs are phosphorylated by stress- and mitogen-activated pathways, leading to the activation of downstream targets including elF4E. MNK-dependent phosphorylation of elF4E at Ser209 is essential for cancer development, while its absence has no impact on normal cellular processes. This axis is involved in enhancing the recruitment of specific mRNAs with secondary structure or terminal oligopyrimidine tracts, including key regulators of survival and proliferation in cancer models. Thus the MNK-elF4E axis presents a potential therapeutic target for cancer treatment, especially since MNK deficient mice are normal, and first-in-human MNKi studies reported minimal side effects.

[0007] Thus, there remains a need for the development of new drugs which can suppress the aberrant increased de novo lipid synthesis required by the rapid cell growth, thereby achieving treatment of diseases related to aberrant upregulated de novo lipid biosynthesis.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention provides the use of a bicyclic alkyne derivative, which is a MNK inhibitor (MNKi) and is effective in suppressing the de novo lipid biosynthesis, for the prophylaxis or treatment of diseases related to aberrant upregulated de novo lipid biosynthesis.

[0010] In a first aspect of the invention, there is provided a compound of Formula I: having IUPAC name (4-(3-((2-(dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5- yl)phenyl)(morpholino) methanone [also referred to as ETC-501 in the present disclosure], or a pharmaceutically acceptable form thereof.

[0011] In a second aspect of the invention, there is provided a pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable form thereof, and at least one pharmaceutically acceptable excipient.

[0012] In some embodiments, the pharmaceutical composition may further comprises at least one cytotoxic agent and / or at least one inhibitor of an enzyme involved in de novo lipogenesis. For examples, the cytotoxic agent may be selected from a group comprising taxotere, 5- fluorouracil, doxorubicin, mitoxantrone, capecitabine, etoposide, methotrexate, doxorubicin, cyclophosphamide, docetaxel, cisplatin, and carboplatin; the enzyme is selected from a group comprising citrate / isocitrate carrier (CIC), Acetyl-CoA synthetase 2 (ACSS2), cytosolic isocitrate dehydrogenase 1 (IDH1 ), ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS).

[0013] In a third aspect of the invention, there is provided the compound of Formula I or a pharmaceutically acceptable form thereof, or a pharmaceutical composition comprising said compound for use in preventing or treating diseases related to aberrant upregulated activity of sterol regulatory element-binding protein 1 (SREBP1).

[0014] In some embodiments, the disease may be selected from the group comprising breast cancers, including triple-negative breast cancer (TNBC), more preferably a TNBC and brain metastases therefrom, prostate cancers, obesity, atherosclerosis, type II diabetes mellitus, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cardiovascular disease, hyperlipidaemia, chronic kidney disease, and neurological disorders, including neurodegenerative diseases.

[0015] In a fourth aspect of the invention, there is provided the compound of Formula I or a pharmaceutically acceptable form thereof in the manufacture of a medicament for the prophylaxis or treatment of diseases related to the aberrant upregulated activity of SREBP1.

[0016] In a fifth aspect of the invention, there is provided a use of (4-(3-((2-(dimethylamino)pyridin-4- yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone or a pharmaceutically acceptable form thereof for the prophylaxis or treatment of diseases related to the aberrant upregulated activity of SREBP1.

[0017] In a sixth aspect of the invention, there is provided a method of prophylaxis or treatment of diseases related to the aberrant upregulated activity of SREBP1 in a subject in need thereof, comprising administering to said subject an effective amount of (4-(3-((2- (dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone or a pharmaceutically acceptable form thereof.

[0018] In some embodiments, the disease is selected from the group comprising breast cancers, including triple-negative breast cancer (TNBC), more preferably a TNBC and brain metastases therefrom, prostate cancers, obesity, atherosclerosis, type II diabetes mellitus, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cardiovascular disease, hyperlipidaemia, chronic kidney disease, and neurological disorders, including neurodegenerative diseases. Preferably, the disease is a breast cancer. More preferably, the breast cancer is a triplenegative breast cancer (TNBC). Most preferably, the breast cancer is a TNBC with brain metastases.

[0019] In some embodiments, (4-(3-((2-(dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin- 5-yl)phenyl)(morpholino) methanone is used in combination with at least one second agent (such as cytotoxic agents and / or inhibitors of enzymes involved in de novo lipogenesis) either in the form of a single composition or in separate compositions.

[0020] In some embodiments, the second agent is a cytotoxic agent selected from a group comprising taxotere, 5-fluorouracil, doxorubicin, mitoxantrone, capecitabine, etoposide, methotrexate, doxorubicin, cyclophosphamide, docetaxel, cisplatin and carboplatin or a pharmaceutically acceptable form thereof.

[0021] In some embodiments, the second agent is an inhibitor of enzymes involved in de novo lipogenesis, wherein said enzyme is selected from a group comprising citrate / isocitrate carrier (CIC), Acetyl-CoA synthetase 2 (ACSS2), cytosolic isocitrate dehydrogenase 1 (IDH1 ), ATP- citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN).

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Certain embodiments of the present disclosure are described more fully hereinafter with reference to the accompanying drawings.

[0024] Figure 1 shows the synthesis scheme of ETC-501 .

[0025] Figure 2 illustrates in vitro and brain-penetrant in vivo activity of ETC-501 [(4-(3-((2- (dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone]. A. Kinome binding map of MNKis, ETC-501 and ETC-206. Kinome binding maps were generated by testing 1 pM of compound against a panel of 104 kinases (KINOMESCAN). B. PK data for ETC-501 and ETC-206 in C57BL / 6 mice treated with 10 mg / kg body weight of drug. Cmax=maximurn concentration. AUC=area under curve. B / P=brain / plasma ratio of respective C max-

[0026] Figure 3 illustrates brain penetrant activity of ETC-501 . A. Mice were treated by daily gavage for 7-days with the indicated doses of ETC-501 , following which specified tissues were obtained and analyzed for MNK-dependent phosphorylation of elF4E (p-elF4E) by immunoblot of cell lysates. B. Phospho-elF4E was detected by IHC of brain sections from mice treated with 100 mg / kg ETC-501 and radiation (RT) using an antibody to phospho-elF4E Ser209.

[0027] Figure 4 illustrates neurotropic TNBC cells upregulate the SREBP1-FASN axis in a MNK- dependent manner. Parental MDA-MB-231 cells and their neurotropic derivative, 231 -Br7 cells were grown overnight in lipid-depleted media. Thereafter, cells were harvested for RNA, and RT-qPCR was performed for SREBP1, FASN, ACC1, and MNK1 / 2. Relative values were plotted.

[0028] Figure 5 shows SREBP1 mRNA expression in Br-7 cells treated with ETC-501 at 10 pM, with or without SREBP1 inducer T0901317 and harvested at 48 hrs.

[0029] Figure 6 illustrates MNK is required for neurotropic breast cancer cells to grow in a lipid- depleted environment. Parental MDA-MB-231 cells and their neurotropic derivative, 231 -Br7 cells were grown in normal, lipid-depleted, and lipid-depleted media supplemented with 5% lipids, and treated with increasing concentrations of ETC-501. At 96 hours, colonies were stained with 1 % crystal violet and imaged using GelCount™.

[0030] Figure 7 illustrates de novo cellular lipid synthesis is dependent on MNK. A. Parental MDA- MB-231 (top row) cells and their neurotropic derivative, 231 -Br7 (bottom row) were grown overnight in lipid-depleted media. For each cell line, three representative high-power fields are shown. B. 231 -Br7 cells were treated with DMSO (Control), the SREBP1 antagonist GSK2033 10 pM, ETC-501 10 pM, T0901317 0.5 pM, or both ETC-501 and T0901317. For A and B, cells were stained with 5 pM BODIPY and fixed with 4% paraformaldehyde.

[0031] Figure 8 illustrates MNKis kill neurotropic TNBC cells. Parental MDA-MB-231 cells (blue bars) and their neurotropic derivative, 231 -Br7 cells (red bars), were grown overnight in lipid- depleted media, and treated with ETC-501 10 pM, with or without supplementation by 5% lipids. After 48 hrs, apoptotic cells were assessed by Annexin V staining using flow cytometry.

[0032] Figure 9 illustrates ETC-501 prevents TNBC BrM in vivo. In a pilot study, 231 -Br7 cells were injected into the left ventricles of anaesthesized mice. The following day, mice were treated with either vehicle control or ETC-501 100 mg / kg by gavage for 14 days. At the end of treatment, mice were imaged by real-time bioluminescent imaging. Figure 10 illustrates MNK activity is required for SREBP1 nuclear translocation in neurotropic TNBC cells. After overnight lipid depletion, 231-Br7 cells were treated with 10 pM ETC-501 , with or without 0.5 pM T0901317. After 48 hrs, cells were harvested and, following separation of the indicated sub-cellular fractions, probed on immunoblots with the indicated antibodies.

[0033] Figure 11 illustrates Genetic inactivation of MNK decreases SREBP1 activity. 231 -Br7 cells were transfected with siRNA to MNK1, MNK2, and scrambled control (Scrm. Cont.). After 48hrs, cells were harvested, and whole cell lysates probed on immunoblots with the indicated antibodies.

[0034] Figure 12 illustrates MNK2 expression is upregulated in TNBC BrM. Normalised RNA-Seq gene expression of MNK1, MNK2, FASN, and ACLY for 5 paired primary TNBC (Primary tumour) to brain metastatic (Matched brain met.) cancer patient samples were analysed from the Siegel dataset. A two-tailed paired t-test was performed with p-value as indicated.

[0035] Figure 13 illustrates MNK2 expression in invasive breast cancer correlates with enrichment of lipid metabolism pathways. Breast cancer proteomics data from TCGA was analyzed for associations between MNK1 / 2 protein expression and Hallmark MSigDB gene sets.

[0036] Figure 14 illustrates ETC-501 administration to cells causes nuclear-located SREBP1 to be translocated to the endoplasmic reticulum. Cells were exposed to empty vehicle (top row), SCAP (middle row), or 10 pM ETC-501 MNK inhibitor. Overexpression of SCAP (positive regulator of SREBP1 ) causes accumulation of nuclear SREBP1 in the nucleus (Fig. 14 middle row). However, treatment of cells expressing SCAP with 10 pM ETC-501 MNK inhibitor causes the nuclear exclusion of SREBP1 into the endoplasmic reticulum (Fig. 14 bottom row). EV: Empty vector.

[0037] Figure 15 illustrates that administration of brain metastatic breast cancer cell line 231 -Br cells to NSG mice causes brain metastases (top row, dark patches under arrow heads), whereas treatment of the mice with ETC-501 prevented brain metastases (bottom row).

[0038] Figure 16 illustrates a timeline to determine whether MNK-knockout has an effect on weight gain of mice fed a normal chow diet (NCD) or a Western Diet supplemented with Fructose (WDF). DETAILED DESCRIPTION OF THE INVENTION

[0039] Further details of the invention will now be described with reference to the following nonlimiting examples. Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art.

[0040] A. Definitions

[0041] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0042] As used herein, the term “comprising” may include the embodiments “consisting of” and “consisting essentially of”. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions, mixtures, or processes as “consisting of” and “consisting essentially of” the enumerated ingredients / steps, which allows the presence of only the named ingredients / steps, along with any impurities that might result therefrom, and excludes other ingredients / steps.

[0043] As used herein, “ETC-501” is used as shorthand to represent the compound (4-(3-((2- (dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone, which has the following structure of Formula I:

[0044] (I)-

[0045] ETC-501 has the chemical formula: C26H24N6O2; exact mass: 452.20; and molecular weight:

[0046] 452.52. Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral forms; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al, Enantiomers, Flacemates and Resolutions (Wiley Interscience, New York, 1981 ); Wilen et al, Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0047] The phrase "pharmaceutically acceptable", as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a human).

[0048] As used herein, the term "pharmaceutically acceptable form" refers to those forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable forms are well known in the art. Pharmaceutically acceptable forms of the compounds of this invention include pharmaceutically acceptable salts, solvates, hydrates, prodrugs, tautomers, isomers, enantiomers, diastereomers, and / or polymorphs of a compound of the present inventionoIn certain embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt, and salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.

[0049] In certain embodiments, the pharmaceutically acceptable form is a hydrate or solvate. The term "hydrate" as used herein refers to a compound non-covalently associated with one or more molecules of water. Likewise, the term "solvate" refers to a compound non-covalently associated with one or more molecules of an organic solvent. As used herein, the term “pharmaceutically acceptable excipient’ refers to a component that can be compatible with the other ingredients of the composition in that it can be combined with the agents and / or compositions of the present invention without eliminating the biological activity of the agents or the compositions (for example, ETC-501 ), and can be suitable for use in subjects as provided herein without undue adverse side effects (such as toxicity, irritation, allergic response, and death).

[0050] As used herein, the term “combination” refers to two agents (such as ETC-501 and a second agent), and means that said two agents are administered in conjunction. Thus, the agents may be presented (i.e. formulated) either as a combined preparation (i.e. presented as a single composition including both agents) or, alternatively, may be presented as separate compositions, wherein at least one of those compositions comprises one agent (such as ETC- 501) and at least one comprises the other agent (such as the second agent). When the two agents are in separate compositions they are administered, if not simultaneously, then sequentially within a timeframe, so that they both are available to act therapeutically within the same time frame.

[0051] As used herein, the term “simultaneously” is used to mean that the two agents are administered concurrently. The term “sequentially” means that one agent is administered within 5 minutes, 10 minutes or a matter of hours after the other agent provided the circulatory half-life of the first administered agent is such that they are both concurrently present in therapeutically effective amounts. The time delay between administrations of the two agents will vary depending on the exact nature of the agents, the interaction there between, and their respective half- lives.

[0052] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, increasing the quality of life, and / or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of the disease. The methods of the present disclosure contemplate any one or more of these aspects of treatment. As used herein, the term “effective amount” refers to an amount of an agent sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and / or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0053] As used herein, an “individual” or a “subject’ refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0054] B. ETC-501 and its use for prevention or treatment of diseases

[0055] The present invention provides a novel compound [ETC-501] of Formula I: having IUPAC name (4-(3-((2-(dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5- yl)phenyl)(morpholino) methanone, or a pharmaceutically acceptable form thereof. The present invention also provides a pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable form thereof, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt. The present invention provides a use of the compound (4-(3-((2-(dimethylamino)pyridin-4- yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone [ETC-501] or a pharmaceutically acceptable form thereof for the prophylaxis or treatment of diseases related to the aberrant upregulated activity of SREBP1. In some embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt.

[0056] In particular, the present invention provides use of the compound ETC-501 or a pharmaceutically acceptable form thereof in the manufacture of a medicament for the prophylaxis or treatment of diseases related to the aberrant upregulated activity of SREBP1. The present invention also provides use of the compound ETC-501 or a pharmaceutically acceptable form thereof for the prophylaxis or treatment of diseases related to the aberrant upregulated activity of SREBP1. The present invention still further provides a method for preventing or treating diseases related to the aberrant upregulated activity of SREBP1 in a subject in need thereof, comprising administering to said subject an effective amount of ETC- 501 or a pharmaceutically acceptable form thereof. In some embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt.

[0057] In some embodiments, the compound ETC-501 or a pharmaceutically acceptable form thereof is incorporated into a medicament or a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. Non-limiting examples of pharmaceutically acceptable excipients include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, sterile water, polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil, sesame oil, emulsions such as oil / water emulsions or water / oil emulsions, microemulsions, nanocarriers and various types of wetting agents. Additives such as alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like can also be included in the composition. In one embodiment, a pharmaceutically acceptable excipient appropriate for use in the compositions disclosed herein can be sterile, pathogen free, and / or otherwise safe for administration to a subject without risk of associated infection and other undue adverse side effects.

[0058] The inventors surprisingly found that the compound ETC-501 , as a MNK inhibitor, is able to penetrate the blood brain / tumour barrier effectively and inhibit SREBP1 activity by excluding it from the cell nucleus, thus to decrease the de novo cellular lipid synthesis in cells, and finally inducing apoptosis of the cells.

[0059] Sterol Regulatory Element Binding Protein (SREBP) is an upstream regulator of lipid synthesis controlling the expression of enzymes involved in this process. It is a family of three basic- helix-loop-helix-leucine zipper (bHLH-LZ) transcription factors: SREBPI a and SREBPI c, two splice variants of the SREBF1 gene, and SREBP2, the product of the SREBF2 gene. It has been reported that SREBP regulates several processes involved in the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH), an essential cofactor that is required in lipid biosynthesis at large amounts. SREBP1 has also been connected to the one-carbon cycle, which provides methyl groups for the synthesis of phosphatidylcholine and other membrane lipids.

[0060] Considering the important role of SREBP1 in regulating lipid homeostasis, it has been reported to act as a central regulator of a variety of diseases related to aberrant lipogenesis activity, such as certain types of cancer, some metabolic and inflammatory disorders.

[0061] Thus, in some embodiments, ETC-501 is suitable for preventing or treating a disease selected from the group comprising breast cancers, including triple-negative breast cancer (TNBC), more preferably a TNBC and brain metastases therefrom, prostate cancers, obesity, atherosclerosis, type II diabetes mellitus, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cardiovascular disease, hyperlipidaemia, chronic kidney disease, and neurological disorders, including neurodegenerative diseases.

[0062] Preferably, the disease to be prevented or treated by ETC-501 is a breast cancer. More preferably, the breast cancer is a triple-negative breast cancer (TNBC). Most preferably, the disease is a TNBC with brain metastases.

[0063] Triple-negative breast cancer (TNBC) is a clinically and biologically heterogeneous disease that represents a major unmet need because of its poor prognosis. This is largely due to the lack of effective targeted therapies, as well as the propensity of TNBC to develop brain metastases (BrM) in 46% of patients, placing them beyond the reach of most therapeutic agents due to the blood brain / tumour barrier (BTB).

[0064] Current barriers to the effective management of patients with TNBC include the following: 1 . The lack of biomarkers identifying TNBC patients at increased risk of developing BrMs necessary for prevention studies; 2. Limited treatment options; 3. The BTB which limits effective targeting; 4. Neurological toxicity of current therapeutic modalities including radiation; 5. Inter- and intra-patient tumour heterogeneity; 6. Intrinsic drug resistance; 7. Brain microenvironmental factors, including immune-privileged status of the brain. Thus, the identification of novel therapeutic targets in TNBC as well as the development of molecules that can cross the BTB would be major advances. Thus, the present invention is a major advance by providing a brain-penetrating compound ETC501 , which markedly inhibits the growth of neurotropic TNBC cells and induces apoptotic cell death in vitro, and completely prevents the establishment of BrM in a mouse model.

[0065] C. Combined therapy with ETC-501

[0066] In some embodiments, (4-(3-((2-(dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin- 5-yl)phenyl)(morpholino) methanone ETC-501 is used in combination with at least one second agent (such as cytotoxic agents and / or inhibitors of enzymes involved in de novo lipogenesis).

[0067] In some embodiments, ETC-501 and the second agent are formulated as a single composition. In other embodiments, ETC-501 and the second agent are formulated as separate compositions.

[0068] It has been reported that FASN inhibition increases the chemosensitivity of breast cancer cells to a variety of cytotoxics including taxotere, 5-fluorouracil, doxorubicin, and mitoxantrone. Since SREBP1 directly regulates FASN and ETC-501 inhibits SREBP1 function, ETC-501 may be used to resensitize cancer cells to cytotoxic agents, especially the current armamentarium of recommended agents in the adjuvant, metastatic, and brain metastatic setting.

[0069] Thus, in some embodiments, the second agent is a cytotoxic agent. Preferably, the cytotoxic agent is selected from a group comprising taxotere, 5-fluorouracil, doxorubicin, mitoxantrone, capecitabine, etoposide, methotrexate, doxorubicin, cyclophosphamide, docetaxel, cisplatin, and carboplatin or a pharmaceutically acceptable form thereof. In some embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt.

[0070] Considering that ETC-501 -mediated suppression of SREBP1 may select for activation of alternative pathways of de novo lipogenesis as a mechanism of resistance, it would be advantageous to use ETC-501 in combination with inhibitors of core enzymes of the de novo lipogenesis pathway to overcome or at least minimise the drug resistance.

[0071] Thus, in some embodiments, the second agent is an inhibitor of enzymes involved in de novo lipogenesis, wherein said enzyme is selected from a group comprising citrate / isocitrate carrier (CIC), Acetyl-CoA synthetase 2 (ACSS2), cytosolic isocitrate dehydrogenase 1 (IDH1 ), ATP- citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). It should be understood that any and all embodiments of the present disclosure can be combined with technical features in any other embodiment or multiple other embodiments to obtain additional embodiments under the premise of no conflict. The invention includes such combinations resulting in further embodiments.

[0072] EXAMPLES

[0073] The examples and exemplary embodiments below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way.

[0074] Example 1 : Synthesis of MNK inhibitor ETC-501

[0075] The synthesis scheme of ETC-501 is shown in Figure 1 and described below.

[0076] Step 1: Preparation of morpholino(4-(oyrazolof1,5-aloyrimidin-5-yl)phenyl)methanone (compound 3):

[0077] To a solution of morpholino(4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl) methanone (compound 1 , 4 g, 12.61 mmol), 5-chloropyrazolo[1 ,5-a]pyrimidine (compound 2, 2.1 g, 13.88 mmol) and K3PO4 in dioxane-water (50 mL 4:1) under argon was successively added Pd(PPh3)4 (728 mg, 0.63 mmol). The reaction mixture was heated at 80 °C for 6 h under argon, and was concentrated under reduced pressure. The residue was diluted with water, and the precipitate was isolated by filtration and dried. The crude product was purified by column chromatography (silica gel, eluent dichloromethane / methanol 98:2) to afford morpholino(4-(pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)methanone (3 g, LC-MS 75%) as a pale yellow solid.

[0078] 1H NMR (400 MHz, DMSO-d6): 53.60-3.61 (m, 8H), 6.79 (s, 1 H), 7.59-7.69 (m, 2H), 7.76 (d, J = 5.9 Hz, 1 H), 8.27-8.32 (m, 3H), 9.24 (d, J = 7.4 Hz, 1 H).

[0079] MS (ESI) m / z: 309.42 [Ci7HiSN4O2+H]+.

[0080] Step 2: Preparation of (4-(3-iodooyrazolo[ 1,5-alpyrimidin-5-yl)phenyl)(morpholino)methanone (compound 4):

[0081] To a solution of morpholino(4-(pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)methanone (compound 3, 3 g, 9.74 mmol) im DMF (15 mL) at 0 °C, was added A / -lodosuccinimide (2.8 g, 12.66 mmol). The reaction mixture was stirred at room temperature for 1 h and poured into crushed-ice. The precipitate was isolated by filtration and dried to afford (4-(3-iodopyrazolo[1 ,5-a]pyrimidin-5- yl)phenyl)(morpholino)methanone (3.8 g, LC-MS 85%) as a pale yellow solid.1H NMR (400 MHz, CDCI3) 6 (ppm): 8.71 (d, J = 7.2 Hz, 1 H), 8.22 (d, J = 8.0 Hz, 2H), 8.16 (s, 1 H), 7.59 (d, J = 8.4 Hz, 2H), 7.33 (d, J= 7.2 Hz, 1 H), 3.92-3.41 (m, 8H).

[0082] MS (ESI) m / z: 435.29 [C17H15IN4O2 +H]+.

[0083] Step 3: Preparation of (4-(3-((2-(dimethylamino)pyridin-4-yl)ethynyl)pyrazolo(1:5-a]pyrimidin- 5-yl)ohenyl)(morphohno)methanone (ETC-501)

[0084] To a solution of (4-(3-iodopyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino)methanone (compound 4, 3.8 g, 8.75 mmol) and / V, / -diisopropylethylamine (3.1 mL, 17.51 mmol) in DMF (15 mL) were successively added Pd(PPhs)4 (505 mg, 0.437 mmol), Cui (249 mg, 1 .31 mmol) and 4-ethynyl-M,M-dimethylpyridin-2-amine (compound 5, 1.4 g, 9.63 mmol). The reaction mixture was stirred at room temperature for 2 h under argon, then was diluted with water. The precipitate was isolated by filtration and dried. The crude product was purified by flash column chromatography (silica gel, eluent dichloromethane / methanol 99:1) to afford ETC-501 (2.5 g, 44% over 3-steps AUC-HPLC 98.98%) as a yellow solid, mp: 200-202 °C.

[0085] 1H NMR (400 MHz, CDCI3) 5 (ppm): 8.74 (d, J = 7.2 Hz, 1 H), 8.33 (s, 1 H), 8.26 (d, J = 8.4 Hz, 2H), 8.17 (d, J= 5.2 Hz, 1 H), 7.60 (d, J = 10.0 Hz, 2H), 7.39 (d, J= 6.8 Hz, 1 H), 6.73-6.71 (m, 2H), 3.92-3.41 (m, 8H), 3.12 (s, 6H).

[0086] MS (ESI) m / z: 453.34 [C2SH24N5O2+H]+.

[0087] Example 2: Specificity and activity of ETC-501

[0088] The specificity of ETC-501 and a similar compound ETC-206 was tested by using 1 pM of the compounds against a panel of 104 kinases (KINOMESCAN). ETC-206 was synthesized according to the method described in Yang H, et al. J Med Chem. 2018 May 24;61 (10):4348- 4369. The structure of ETC-206 is shown as follows.

[0089] (ETC-206)

[0090] As shown, in addition to MNKs, ETC-501 inhibited only one other kinase out of 104 tested (Fig. 2A, 98.7% inhibition towards DRAK1 ). Meanwhile, ETC-501 showed very high inhibition efficiency towards MNK1 (87% inhibition) and MNK2 (95% inhibition). Comparably, ETC-206 showed very high inhibitory activity towards MNK1 and MNK2, although it also inhibited MEK1 . The in vitro activity of ETC-501 and ETC-206 was then determined. Specifically, IC50was determined according to the method described in Cherian J et al. J Med Chem. 2016 Apr 14; 59(7)3063-3078, and PK parameters such as CmaS, AUC and B / P ratios were determined according to the method described in Yang et al. J Med Chem. 2018 May 24; 61 (10): 4348- 4369. As shown in Fig. 2B, ETC-501 exhibits comparable IC50 concentrations with ETC-206 against MNK1 and MNK2. However, the capability of ETC-501 to penetrate brain is much superior over that of ETC-206, the B / P ratio of the former is around 7 times that of the latter (0.75 vs 0.1 ).

[0091] To confirm in vivo MNK kinase inhibition, the inventors also treated mice with increasing doses of ETC-501 (0, 50, 75 or 100 mg / kg) by gavage over one week. 7 days later, mice were sacrificed to obtain brain, blood and spleen, which were analysed for MNK-dependent phosphorylation of elF4E (p-elF4E) by immunoblot of cell lysates. The inventors also detected phospho-elF4E by immunohistochemistry (IHC) of brain sections from mice treated with 100 mg / kg ETC-501 for 7 days and radiation (RT) using an antibody to phospho-elF4E Ser209. Both immunoblot results of brain, blood, and spleen and immunohistochemistry (IHC) of the brain tissue demonstrated inhibition of elF4E phosphorylation, a canonical read-out for MNK activity (Zhang M, et al. Mol Cell Biol 2008; 28(20): 6496-6509; Lim S, et al. Blood, 201 1 ; 118(21):963; Cherian J, et al. J Med Chem 2016; 59(7): 3063-3078), at 100 mg / kg dosing (Fig. 3A and 3B).

[0092] Example 3: TNBC cells upreaulate the SREBP1-FASN axis in a MNK-dependent manner

[0093] The recent development of brain- / neuro-tropic breast cancer (BC) cell lines have been an important advance to study the molecular biology of brain-tropism in BC brain metastases (BrM). The inventors tested a pair of TNBC cell lines, comprising the original parental, non- neurotropic cell line, MDA-MB-231 (ATCC Accession No. HTB-26), and its neurotropic derivative, 231 -Br7 (generous gift of Dr. Patricia Steeg, US NIH) (Valiente M, et al. Cancer Res 2020; 80(20): 4314-4323). These two cell lines were grown overnight in lipid-depleted condition (DMEM (high glucose) + 10% lipid depleted FBS (Biowest S181 L-500) + 1 % penicillin + streptomycin). Then, the cell lines were harvested for RNA analysis by RT-qPCR. The inventors observed that SREBP1 , as well as two of its canonical direct transcriptional targets FASN and ACC1 , were upregulated in the neurotropic line compared to the parental line (Fig. 4). Importantly, both MNK1 and MNK2 transcripts were upregulated in parallel with the SREBP1 -FASN axis. To assess if SREBP1 -FASN axis was dependent on MNK activity, the inventors also treated 2.5 x 105231 -Br7 cells with 10 pM ETC-501 , with and without the SREBP1 inducer T0901317 (0.5 uM, Sigma Aldrich) and found that ETC-501 decreased SREBP1 expression in both instances (Fig. 5).

[0094] Example 4: MNK is required tor neurotropic breast cancer cells to grow in a lipid- depleted environment

[0095] To determine whether MNK kinase activity is important to the growth of TNBC cells under the lipid-poor conditions found in the brain, 1 .5 x 104TNBC cells were treated with various doses of ETC-501 (0, 5 and 10 pM) under normal condition (DMEM (high glucose) + 10% FBS) and lipid-depleted condition (DMEM (high glucose) + 10% lipid depleted FBS (Biowest S1 SI L- 500)). As expected, the growth of parental non-neurotropic TNBC cells (MDA-MB-231 ) was significantly impaired compared to their paired neurotropic lines (231 -Br7), and demonstrated the striking adaptation of the latter to low lipid conditions. Importantly, the growth advantage of 231 -Br7 cells in lipid-depleted media was abrogated by MNKis, an effect which could be rescued by the addition of 5% (w / v) exogenous lipids Chemically Defined Lipid Concentrate (Cat No. 1 1905031 , Thermo Fisher Scientific) (Fig. 6). Taken together, these findings indicate that MNK is important for the growth of neurotropic TNBC cells under conditions which mimic the brain microenvironment, and that this is likely due to the ability of MNKis to prevent de novo lipid synthesis under conditions of limiting exogenous lipids.

[0096] Example 5: Increased de novo cellular lipid synthesis in neurotropic TNBC is dependent on MNK

[0097] Next, to demonstrate the effect of MNKis on de novo lipid synthesis in vivo, the inventors evaluated the formation of lipid droplets in parental and neurotropic TNBC cells.

[0098] Specifically, 2.5 x 104parental MDA-MB-231 cells and neurotrophic TNBC cells 231 -Br7 cultured in lipid-depleted conditions (DMEM (high glucose) + 10% lipid depleted FBS (Biowest S181 L-500)) were incubated with 5 pM of the fluorescent lipid dye, 4,4-difluoro-1 , 3, 5,7,8- pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) and the DNA dye, Hoescht 33342 for 5 minutes at 37 °C. Cells were then washed twice in 1 X phosphate buffered saline, fixed with 4% paraformaldehyde for 10 minutes at room temperature then imaged for lipid droplets.

[0099] Under lipid-depleted conditions, neurotropic TNBC cells were able to form lipid droplets, whereas parental TNBC cells were not (Fig. 7A). Whether lipid droplet formation in neurotropic TNBC cells was dependent on MNK activity was evaluated. 2.7 x 104neurotrophic TNBC cells cultured in lipid-depleted conditions (DMEM (high glucose) + 10% lipid depleted FBS (Biowest S181 L-500)) were treated with DMSO (Control), 10 M SREBP1 antagonist GSK2033 (Tocris), 10 pM ETC-501 , 0.5 pM SREBP1 inducer T0901317 (Sigma Aldrich), or both ETC-501 and T0901317 for 48 hours and assayed for lipid droplet formation as above. The inventors found that both established SREBP1 antagonists GSK2033 and ETC-501 prevented the formation of lipid droplets, and that ETC-501 prevented lipid droplet formation even following SREBP1 induction by T0901317 (Fig. 7B). These results confirm that MNK activity is required for de novo lipid synthesis in vivo under lipid-depleted conditions.

[0100] Example 6: MNKis kill neurotropic TNBC cells

[0101] Given that lipid droplet formation is essential for TNBC BrM establishment the inventors determined whether MNKis induced apoptotic cell death in TNBC cells. 4 x 104parental and neurotrophic TNBC cells cultured overnight in lipid-depleted conditions (DMEM (high glucose) + 10% lipid depleted FBS (Biowest S181 L-500)) were treated with 10 pM of ETC-501 , with or without supplementation by 5% lipids, for 48 hours. Apoptosis was subsequently assessed through flow cytometry, utilizing positive Annexin-V staining. It was found that MNK inhibition resulted in a 193% increase in apoptotic cell death in neurotropic cell lines (231 -Br7) vs a 43% increase in parental cells (MDA-MB-231 ). Notably, the addition of exogenous lipids almost completely rescued the pro-apoptotic effect of MNKis in TNBC cells, suggesting that MNKis kill TNBC cells via inhibiting de novo lipid synthesis (Fig. 8).

[0102] Example 7: MNKis prevent the establishment of TNBC BrM in vivo

[0103] The in vitro data shows that MNKis prevent the growth of neurotropic TNBC cells (Fig. 6). To determine if this was also the case in vivo, a pilot study was performed in a mouse model of systemic BC metastases (Jin X, et al. Nature 2020; 588(7837): 331 -336). Following intracardiac injection (IC) of 1 .75 x 105231 -Br7 cells, mice were treated for 14-days with 100 mg / kg ETC-501 (dissolved in 0.1 % Tween 80 + 99.9% of 0.5% methylcellulose) via oral gavage, after which they were imaged. Strikingly, the inventors found that ETC-501 treatment prevented metastases in all 3 mice, whereas mice treated with vehicle control all developed brain (arrow heads) as well as systemic metastases (Fig. 9).

[0104] Example 8: MNK activity is required for SREBP1 nuclear translocation in neurotropic TNBC cells As a master regulator of lipid metabolism, SREBP activity is tightly regulated at multiple levels. In brief, when cells initiate lipid synthesis, SREBP1 transcription is induced and nuclear translocation is facilitated by movement of SREBP1 from the endoplasmic reticulum (ER) to the Golgi, where it undergoes cleavage by a set of two proteases (site-1 protease (S1 P), and site-2 protease (S2P)). Upon cleavage, a mature form of SREBP1 is then free to enter the nucleus and function as a transcription factor. Under lipid-replete conditions, SREBP1 is retained in the ER by a cholesterol-sensing protein called SREBP cleavage-activating protein (SCAP), preventing the transport of SREBP1 from the endoplasmic reticulum (ER) to the Golgi apparatus. In addition, SREBP1 nuclear localization is also regulated by phosphorylation, including by the AMPK and PKA kinases (Li Y, et al. Cell Metab 201 1 ; 13(4): 376-388; Dong Q, et al. Biochem Biophys Res Common 2014; 449(4): 449-454).

[0105] Prior work suggests that the canonical MNK-elF4E translation-regulating axis does not affect SREBP1 translation (Conn CS, et al. Nat Metab 2021 ; 3(2): 244-257). However, since SREBP1 cleavage, followed by nuclear translocation are necessary steps in de novo lipid synthesis, the inventors investigated whether ETC-501 inhibits either of these processes under conditions of SREBP1 induction, e.g. with lipid depletion, as well as exposure to SREBP1 inducers.

[0106] 1 million 231 -Br7 cells cultured in lipid-depleted media were treated with 10 pM ETC-501 , with or without 0.5 pM T0901317 for 48 hours. Cells were harvested and subjected to subcellular fractionation. 2 hours prior to harvesting cells, a final concentration of 25 pg / ml N-acetyl-leucyl- leucyl-norleucinal (ALLN) was added to cells in culture. Cells were then harvested by scraping in medium, and the cell suspension was centrifuged at 1000 x g for 5 minutes at 4°C to obtain original supernatant and cell pellet. The resulting cell pellet was washed by recentrifugation in PBS at 4°C, after which the cell pellet was resuspended in Buffer A containing 250 mM sucrose, 10 mM Hepes-KOH at pH 7.6, 10 mM KCI, 1.5 mM MgCh, 1 mM sodium EDTA, 1 mM sodium EGTA, and a mixture of protease inhibitors that include 2.8 pg / ml aprotinin, 10 pg / ml leupeptin, 25 pg / ml ALLN, 5 pg / ml pepstatin A, and 0.5 mM Pefabloc. The cell suspension was passed through a 23-gauge needle 20 times and centrifuged at 1000 x g for 5 minutes at 4°C. The pellet was then resuspended in 0.05-0.1 ml of Buffer B (20 mM Hepes- KOH at pH-7.6, 0.42 M NaCI, 2.5% (v / v) glycerol, 1 .5 mM MgCI2, 1 mM sodium EDTA, 1 mM sodium EGTA and the above mixture of protease inhibitors). This suspension was rotated at 4°C for 1 hour and centrifuged at 10,000 x g for 15 minutes at 4°C. The resulting supernatant is the nuclear fraction. The original supernatant was centrifuged at 100,000 x g for 15 minutes at 4°C, after which the pellet was dissolved in 0.05-0.1 ml of SDS lysis buffer (10 mM Tris-HCI at pH 6.8, 100 mM NaCI, 1% (w / v) SDS, 1 mM sodium EDTA, 1 mM sodium EGTA and the above mixture of protease inhibitors) and designated as the membrane fraction. Protein concentration was determined by the BCA Protein Assay Kit (Thermo Scientific). 20 pg of protein lysates was run on a 10% SDS-PAGE gel and immunoblotting was performed with anti-SREBP (MABS1987, Merck-Millipore) and anti-Calnexin (ADI-SPA-865-D,Enzo) antibodies.

[0107] Results showed that MNKi treatment resulted in near-complete exclusion of mature SREBP1 from the nucleus (compare lanes 5 and 6, Fig. 10). At the same time, there was increased membrane retention (i.e. in the ER and / or Golgi) of both precursor and mature forms of SREBP1 (compare lanes 1 and 2, Fig. 10). Next, to mimic the lipid-poor brain microenvironment, SREBP1 was activated with T0901317 prior to MNKi treatment. As expected, T0901317 increased nuclear mature SREBP1 (compare lanes 5 and 7, Fig. 10), while the addition of MNKi almost completely prevented this (compare lanes 7 and 8). Here, MNKis also greatly increased the amount of SREBP1 retained in membrane (compare lanes 3 and 4). Together, these results demonstrate that MNKis profoundly inhibit SREBP1 processing and activation in neurotropic TNBC.

[0108] Example 9: Genetic inactivation of MNK1 and MNK2 decreases SREBP1 expression and activity

[0109] To confirm the connection between MNK and SREBP1 , the inventors performed genetic knockdown of MNK1 and MNK2 r\ 231 -Br7 cells and measured total cellular proteins. 1 million 231 -Br7 cells were transfected using electroporation (Amaxa Nucleofector, Lonza) with siRNAs targeting MNK1 and MNK2. Non-targeting pool siRNA (used as a scrambled control): 5’-UGGUUUACAUGUCGACUAA-3’ (SEQ ID NO: 1 ), 5’-UGGUUUACAUGUUGUGUGA-3’ (SEQ ID NO: 2), 5’-UGGUUUACAUGUUUUCUGA-3’ (SEQ ID NO: 3), 5’- UGGUUUACAUGUUUUCCUA-3’ (SEQ ID NO: 4); MNK1 siRNA: 5’- GGAGUAGGGUGUUUCGAGA-3’ (SEQ ID NO: 5); MNK2 siRNA: 5’-GCC UUG GAC UUU CUG CAU-3’ (SEQ ID NO: 6). Cells were then washed twice with 1X phosphate buffered saline (PBS) and cultured in lipid-depleted media for 14 hours in 37°C, 5% CO2. 2 hours later, the cells were harvested by scraping in the medium, and the cell suspension was centrifuged at 1000 x g for 5 minutes at 4°C. The resulting cell pellet was washed by recentrifugation in PBS at 4°C, after which the cell pellet was resuspended in Buffer A containing 250 mM sucrose, 10 mM Hepes-KOH at pH 7.6, 10 mM KCI, 1 .5 mM MgCh, 1 mM sodium EDTA, 1 mM sodium EGTA, and a mixture of protease inhibitors that included 2.8 pg / ml aprotinin, 10 pg / ml leupeptin, 25 pg / ml ALLN, 5 pg / ml pepstatin A, and 0.5 mM Pefabloc. The cell suspension was passed through a 23-gauge needle 20 times. Protein concentration of the lysate was determined by the BOA Protein Assay Kit (Thermo Scientific). 15 g of protein lysates was run on a 10% SDS-PAGE gel and immunoblotting was performed with the following antibodies: anti-MNK1 (2195, Cell Signaling Technology), anti-MNK2 (M0696, Merck), anti-phospho- EIF4E (9741 , Cell Signaling Technology), anti-EIF4E (9742, Cell Signaling Technology), anti- SREBP (MABS1987, Merck-Millipore), anti-FASN (3180, Cell Signaling Technology) and anti- GAPDH (21 18, Cell Signaling Technology).

[0110] As expected, knockdown of MNK1 or MNK2 individually decreased elF4E phosphorylation, while knockdown of both extinguished elF4E phosphorylation completely (Fig. 11 ). Interestingly, MNK2 knockdown partially decreased both precursor and mature SREBP1 , while knockdown of both MNKs completely inhibited SREBP1 expression. Here, FASN expression, a direct transcriptional target of SREBP1 was also completely inhibited, demonstrating functional SREBP1 inhibition. While these experiments support the general notion that MNK regulates SREBP1 function, they also suggest that MNKs regulate SREBP1 expression, adding a layer of complexity.

[0111] Example 10: MNK2 is uprequlated in primary TNBC BrM and correlates with enrichment of lipid pathways

[0112] To determine whether MNK expression would be upregulated in TNBC BrM compared to their matched primary tumours, the Siegel et al. dataset of primary TNBC and their paired brain metastases were interrogated (Siegel MB, et al. J Clin Invest 2018 2; 128(4): 1371 -1383). Remarkably, in all five pairs, MNK2, but not MNK1 expression, was increased in the matched BrM compared to the original primary tumour, and was also associated with increased expression of direct transcriptional targets of SREBP1 , including FASN and ACLY (Fig. 12).

[0113] Next, the TCGA database (http:www.cbioportal.org) was interrogated to test the functional relevance of MNK2 in activating lipid pathways in primary breast cancers. As expected, both MNK1 and MNK2 expression correlated with enrichment of ‘Myc Targets’, confirming an established relationship between MNK and Myc (Fig. 13) (Knight JRP, et al. Cancer Discov 2021 ; 11 (5): 1228-1247; Shi Y, et al. Oncogene 2016; 35(8): 1015-1024; Shi Y, et al. Oncogene 2013; 32(2): 190-197). However, it was only for MNK2 that ‘Fatty Acid Metabolism’ and ‘Adipogenesis’ pathways were enriched (Fig. 13). These results provide strong external validation of an important association between MNK2 and lipid metabolism in TNBC BrM.

[0114] Example 11 : ETC-501 causes exclusion of SREBP1 from the nucleus into the ER 0.01 % Poly-L-Lysine solution (P4707, Sigma) was used to coat glass cover slips for 1 hour. Subsequently, Poly-L-Lysine solution was aspirated and washed with sterile water twice before left to dry. 35,000 HeLa cells were seeded on Poly-L-Lysine coated glass cover slips in 24-well plates and left to adhere overnight. The YFP-SREBP1c-CFP plasmid was a kind gift from Professor Hitoshi Shimano. The pCDH-SCAP plasmid was generated by cloning hamster SCAP into a pCDH plasmid. HeLa cells are transfected with YFP-SREBP1c-CFP using Jetprime ® (101000046, Polyplus Sartorius). Media containing the transfection reagent was replaced with fresh medium 4 hours post-transfection. Drug treatment was done using lipid- depleted media with / without ETC-501 MNK inhibitor for 4 hours before harvesting the cells for immunofluorescence. To harvest the cells, the HeLa cells was washed with 1 x PBS before fixing with 4% paraformaldehyde for 10 minutes at room temperature. Following fixation, the cells were washed thrice with 1x PBS. Cells were permeabilised using 0.3% Triton-X for 10 minutes in room temperature, which is then followed by three washes of PBS & 0.1 % Triton- X (PBS-T) solution. Cells were blocked for 1 hour at room temperature with 3% bovine serum albumin (BSA) in phosphate-buffered saline with 0.1% Triton-X (PBS-T). Subsequently, primary antibodies diluted in 3% BSA-PBS-T were incubated for 2 hours at room temperature. The following primary antibodies were used, GM130 (1 :3200, Cell Signalling Technology, 12480), Calnexin (1 :50, Santa Cruz Biotechnology, sc23954). After three washes with PBS-T, secondary antibodies diluted in 3% BSA-PBS-T were incubated for 1 hour at room temperature in the dark. The following secondary antibodies were used, Alexa Fluor 594 / 647 secondary antibodies (1 :500, Invitrogen). Following three additional washes with PBS-T, cells were stained with 4',6-diamidino-2-phenylindole (DAPI) at a 1 :1000 dilution in PBS-T. Finally, cells were mounted with Dako mounting medium (S302380-2, Agilent) on glass slides and allowed to dry before it was sealed with clear nail polish.

[0115] Overexpression of SCAP (positive regulator of SREBP1 ) causes accumulation of nuclear SREBP1 in the nucleus (Fig. 14 middle row). However, treatment of with 10 pM ETC-501 MNK inhibitor causes the nuclear exclusion of SREBP1 into the endoplasmic reticulum (Fig. 14 bottom row). EV: Empty vector.

[0116] Example 12: ETC-501 reduces tumour burden in mouse BCBM model

[0117] ETC-501 was tested for its ability to reduce breast cancer brain metastases in an in vivo mouse model. All procedures involving animals were reviewed and approved by Singhealth Institutional Animal Care & Use Committee (IACUC). Female 12-16-week-old NOD scid gamma (NSG) were randomly assigned to be pre-treated with vehicle (99.9% 0.5% methylcellulose & 0.1 % tween-80) and 100 mg / kg ETC-501 administered via oral gavage once daily. To develop the brain metastasis models, female NOD scid gamma (NSG) mice were injected with 100,000 brain metastatic breast cancer cell line 231 -Br cells tagged with Nano luciferase in 0.05 mL into the left common carotid artery. After a day of surgery recovery, mice were treated with vehicle (99.9% 0.5% methylcellulose & 0.1% tween-80) and 100 mg / kg ETC- 501 administered via oral gavage once daily for two weeks. Animals were imaged by injecting 50 pL Fluorofurimazine (Ffz, N4100, Promega) into the tail vein and imaged using the MS Lumina machine (Revvity). Whole brains were excised 23 days post-implantation for imaging and histologic examination. The whole brain was fixed in 4% paraformaldehyde for overnight at 4°C, transferred to 70% ethanol. Brain sections (5 pm thick) were serially cut and stained with haematoxylin and eosin (H&E) according to standard procedures. Ten serial sagittal sections (50 pm thick) every 300 microns throughout the brain were analyzed for presence of metastatic lesions using a Zeiss AxioScan 7 Slide Scanner.

[0118] Treatment with 100 mg / kg ETC-501 via oral gavage in NSG mice reduces tumour burden in an adjuvant setting (Fig. 15). Regions of greatest luciferase were seen in the vehicle-treated control mice (top panel) indicated by arrows.

[0119] Example 13: Comparison between normal and MNK-knockout mice fed a NCD or WDF diet.

[0120] Dav 0: Prior to start of diet

[0121] Body weight measurements will be taken for each mouse. Body composition analysis of fat and lean mass will be conducted using magnetic resonance imaging (MRI) using the EchoMRI system.

[0122] Dav 1 : Start mice on NCD or WDF

[0123] Ten-week-old male wild type (WT) or MNK knockout (KO) mice will be fed a Western diet (D12079B; Research Diets) and provided with drinking water supplemented with 15% weight / volume fructose to model metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) conditions similar to those in humans (Baena, M. 2017, Machado, M. V. 2015) or a normal chow diet (SF00-100; Specialty Feeds) and tap water for a duration of either 8 weeks or 16 weeks. Body weight measurements will be conducted on a bi-monthly basis.

[0124] Week 7 and 15: Week prior to terminal endpoints

[0125] One week prior to reaching terminal endpoints, intraperitoneal glucose tolerance tests (ipGTT) and intraperitoneal insulin tolerance tests (ipITT) will be performed on mice fasted for 6 hours to assess glucose intolerance and insulin resistance. These assessments will occur at week 7 for the cohort on 8-week regimen and at week 15 for those on the 16-week regimen.

[0126] Intraperitoneal glucose tolerance tests (ioGTT)

[0127] To prepare for the glucose tolerance tests, mice are fasted for 6 hours by transferring them to clean cages without food, while ensuring they have access to drinking water. The procedure begins with cleaning the tail using gauze soaked in 70% alcohol, followed by drying with dry gauze. The tip of the tail is then scored, approximately 1 -2 mm from the end, using sterile scissors. Blood is collected by milking the tail, and a small drop, less than 5 pl, is placed on a glucose test strip in the Accu-Check glucose meter to record the baseline fasting glucose level. Subsequently, the mice are injected intraperitoneally with a 20% glucose solution, at a dose of 2 g glucose / kg lean mass. Blood glucose levels are measured at intervals of 15, 30, 45, 60, 75, 90, 105, and 120 minutes post- injection by placing a small drop of blood on a new test strip for each measurement, ensuring accurate monitoring of glucose levels over time.

[0128] Intraperitoneal insulin tolerance tests (ipITT)

[0129] For the intraperitoneal insulin tolerance test (IpITT), the procedure is similar to the glucose tolerance test, with the key difference being the injection of an insulin solution at 1 unit / kg lean mass. Blood glucose levels are then measured at specified intervals up to 120 minutes to assess the response to insulin.

[0130] Week 8 and 16: Endpoint analysis

[0131] At weeks 8 and 16 of WDF treatment, body weight measurements and body composition analyses will be conducted. Following these assessments, the mice will be euthanized. Euthanasia will be performed by gradually increasing carbon dioxide to displace 30-70% of the chamber volume per minute over a minimum duration of 5 minutes, followed by cervical dislocation to ensure death. Blood samples will be collected via cardiac puncture. Tissues including the liver, heart, pancreas, muscle, kidney, and adipose tissue will be harvested. These tissues will be preserved in 4% formalin for subsequent histopathological analysis and snap-frozen in liquid nitrogen for expression analysis of genes and proteins related to lipid metabolism, inflammation, and fibrosis.

[0132] Expected results

[0133] We anticipate that MNK KO mice will exhibit significantly reduced weight gain compared to wild-type mice when subjected to a WDF. Additionally, MNK KO mice are expected to demonstrate improved glucose tolerance, as evidenced by a less pronounced increase in glucose levels following a glucose challenge and a more rapid return to baseline. Furthermore, these mice are likely to show enhanced insulin sensitivity, with blood glucose levels decreasing more significantly during insulin tolerance tests (ITT) in response to insulin administration. We also expect the MNK KO mice to exhibit reduced hyperlipidaemia, characterized by lower levels of serum triglycerides and free fatty acids, compared to wildtype mice.

[0134] In the livers of WDF-fed KO mice, we expect to observe reduced hepatic lipid and triglyceride accumulation, indicating protection against fatty liver (hepatic steatosis). This may be reflected in lower liver weight and less pale livers with fewer fat droplets. Additionally, we anticipate lower levels of liver enzymes such as serum alanine transaminase (ALT) and aspartate aminotransferase (AST), signifying reduced liver damage. Additionally, lobular inflammation and hepatocyte ballooning are anticipated to be diminished, with the liver likely maintaining a normal lobular structure that exhibits less inflammatory cell infiltration and non-enlarged hepatocytes. We further expect a downregulation in the expression of genes and proteins associated with de novo lipogenesis, inflammation and fibrosis.

[0135] In the adipose tissue of KO mice, we may observe reduced adipocyte hypertrophy and diminished inflammation with decreased influx of inflammatory infiltrates such as macrophages.

[0136] In mice on the WDF, the pancreas may initially exhibit an increase in islet area, indicative of compensatory islet cell hyperplasia similar to what is observed in pre-diabetes. With prolonged exposure to the diet, the islet size may decrease compared to control mice on a NCD, reflecting a deterioration in islet mass likely due to sustained insulin resistance. Additionally, nephromegaly accompanied by increased immune infiltration and signs of fibrosis may be observed, along with indications of diabetic nephropathy, such as remarkable mesangial expansion characterized by significant thickening of the mesangial matrix in the kidney glomeruli. Cardiac effects may include increased heart mass and remodelling (Low, Z. S. 2024). These adverse effects might be attenuated or less pronounced in WFD-fed KO mice. Furthermore, as mice on the WFD progress from MASLD to MASH, they exhibit altered serum cytokine profiles compared to those on NCD. These cytokine profile alterations may not be as pronounced in MNK KO mice.

[0137] For one skilled in the art, various modifications and changes may be made to the present disclosure. Those skilled in the art should understand that any amendments, equivalent replacements, improvements, and so on, made within the spirit and principle of the present disclosure, should be covered within the scope of protection of the present disclosure. Bibliography

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Claims

Claims1 . A compound of Formula (I):having IUPAC name (4-(3-((2-(dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5- a]pyrimidin-5-yl)phenyl)(morpholino) methanone, or a pharmaceutically acceptable form, preferably a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising a compound of claim 1 and at least one pharmaceutically acceptable excipient.

3. The pharmaceutical composition of claim 2, further comprising at least one cytotoxic agent and / or at least one inhibitor of an enzyme involved in de novo lipogenesis.

4. The pharmaceutical composition of claim 3, wherein: a) the cytotoxic agent is selected from a group comprising taxotere, 5-fluorouracil, doxorubicin, mitoxantrone, capecitabine, etoposide, methotrexate, doxorubicin, cyclophosphamide, docetaxel, cisplatin, and carboplatin and / or b) the enzyme is selected from a group comprising citrate / isocitrate carrier (CIC), ATP- citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN).

5. A compound of claim 1 , or a pharmaceutical composition of any one of claims 2 to 4, for use in preventing or treating diseases related to aberrant upregulated activity of sterol regulatory element-binding protein 1 (SREBP1 ).

6. A compound or a pharmaceutical composition of claim 5, wherein the disease is selected from the group comprising breast cancers, including triple-negative breast cancer (TNBC), more preferably a TNBC and brain metastases therefrom, prostate cancers, obesity, atherosclerosis, type II diabetes mellitus, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cardiovascular disease, hyperlipidaemia,chronic kidney disease, and neurological disorders, including neurodegenerative diseases.

7. Use of (4-(3-((2-(dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5- yl)phenyl)(morpholino) methanone or a pharmaceutically acceptable form thereof according to claim 1 in the manufacture of a medicament for the prophylaxis or treatment of diseases related to the aberrant upregulated activity of SREBP1 .

8. Use of (4-(3-((2-(dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5- yl)phenyl)(morpholino) methanone or a pharmaceutically acceptable form thereof for the prophylaxis or treatment of a disease related to aberrant upregulated activity of SREBP1.

9. The use according to claim 7 or 8, wherein the disease is selected from the group comprising breast cancers, including triple-negative breast cancer (TNBC) and brain metastases therefrom, prostate cancers, obesity, atherosclerosis, type II diabetes mellitus, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cardiovascular disease, hyperlipidaemia, chronic kidney disease, and neurological disorders, including neurodegenerative diseases.

10. The use according to claim 9, wherein the disease is a breast cancer, preferably a triple-negative breast cancer (TNBC), more preferably a TNBC with brain metastases.

11. The use according to any one of claims 7 to 10, wherein (4-(3-((2- (dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone is used in combination with at least one second agent.

12. The use according to claim 1 1 , wherein the second agent is a cytotoxic agent or an inhibitor of an enzyme involved in de novo lipogenesis.

13. The use according to claim 12, wherein the cytotoxic agent is selected from a group comprising taxotere, 5-fluorouracil, doxorubicin, mitoxantrone, capecitabine, etoposide, methotrexate, doxorubicin, cyclophosphamide, docetaxel, cisplatin, and carboplatin.

14. The use according to claim 12, wherein the enzyme is selected from a group comprising citrate / isocitrate carrier (CIG), Acetyl-CoA synthetase 2 (ACSS2), cytosolicisocitrate dehydrogenase 1 (IDH1 ), ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN).

15. The use according to any one of claims 1 1 to 14, wherein (4-(3-((2- (dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone and the second agent are in a single composition.

16. The use according to any one of claims 1 1 to 14, wherein (4-(3-((2- (dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone and the second agent are in separate compositions.

17. A method of preventing or treating diseases related to aberrant upregulated activity of SREBP1 in a subject in need thereof, comprising administering to said subject an effective amount of (4-(3-((2-(dimethylamino)pyridin-4-yl)ethynyl)pyrazolo[1 ,5- a]pyrimidin-5-yl)phenyl)(morpholino) methanone or a pharmaceutically acceptable form thereof.

18. The method according to claim 17, wherein the disease is selected from the group comprising breast cancers, including triple-negative breast cancer (TNBC) and brain metastases therefrom, prostate cancers, obesity, atherosclerosis, type II diabetes mellitus, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cardiovascular disease, hyperlipidaemia, chronic kidney disease, and neurological disorders, including neurodegenerative diseases.

19. The method according to claim 18, wherein the disease is a breast cancer, preferably a triple-negative breast cancer (TNBC), more preferably a TNBC with brain metastases.

20. The method according to any one of claims 17 to 19, wherein the method further comprising administering to said subject at least one second agent.21 . The method according to claim 20, wherein (4-(3-((2-(dimethylamino)pyridin-4- yl)ethynyl)pyrazolo[1 ,5-a]pyrimidin-5-yl)phenyl)(morpholino) methanone and the second agent are administered in combination.

22. The method according to claim 20 or 21 , wherein the second agent is a cytotoxic agent or an inhibitor of an enzyme involved in de novo lipogenesis.

23. The method according to claim 22, wherein the cytotoxic agent is selected from a group comprising taxotere, 5-fluorouracil, doxorubicin, mitoxantrone, capecitabine, etoposide, methotrexate, doxorubicin, cyclophosphamide, docetaxel, cisplatin, and carboplatin.

24. The method according to claim 22, wherein the enzyme is selected from a group comprising citrate / isocitrate carrier (CIC), Acetyl-CoA synthetase 2 (ACSS2), cytosolic isocitrate dehydrogenase 1 (IDH1 ), ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN).

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