Compounds that inhibit the interaction between tristetraprolin and CNOT1 and methods of use

Abstract

Compounds that inhibit the interaction between CNOT1 and TTP and the methods of using the same are disclosed. The methods comprise administering a compound that inhibits the interaction between CNOT1 and TTP to a subject in need thereof. Additionally, methods of identifying a compound that inhibits the interaction between CN0T1 and TTP are also disclosed.

Description

[0001] COMPOUNDS THAT INHIBIT THE INTERACTION BETWEEN TRISTETRAPROLIN

[0002] AND CNOT1 AND METHODS OF USE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This patent application claims the benefit of priority of United States Provisional Patent Application No. 63 / 650,885, filed May 22, 2024, which is incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under grant number HL 155953 awarded by National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND OF THE INVENTION

[0008] Heart failure (HF), non-alcoholic fatty liver disease (NAFLD), and insulin resistance are associated with a high number of mortality and morbidity. For instance, HF is one of the leading causes of death. However, the molecular mechanisms responsible for these diseases and disorders are not completely understood. There is an unmet need for the treatment of heart failure and insulin resistance because the current treatment modalities are not effective. A protein called tristetraprolin (TTP) regulate several metabolic processes, including fatty acid (FA) metabolism, branched-chain amino acid (BCAA) metabolism, and insulin sensitivity, which are associated with the aforementioned diseases and disorders. Therefore, there exists a need for new compositions and methods that modulate the function of TTP.

[0009] BRIEF SUMMARY OF THE INVENTION

[0010] Disclosed herein are compounds that inhibit the interaction between CNOT1 and TTP and the methods of using and identifying the same. One aspect of the invention provides a method for the treatment of a subject in need of a compound that inhibits the interaction between CNOT1 and tristetraprolin (TTP), the method comprising administering a compound that inhibits the interaction between CNOT1 and TTP to the subject, such as a compound as described herein.

[0011] Another aspect of the invention provides a method of identifying a compound for the treatment of a subject in need of a compound that inhibits the interaction between CNOT1 and TTP, the method comprising assaying a candidate compound to determine an indicia of inhibition of the interaction between CN0T1 and TTP and comparing the indicia of inhibition of the interaction between CONTI and TTP to an indicia of inhibition of the interaction between CN0T1 and TTP of a comparator compound that inhibits the interaction between CN0T1 and TTP, wherein the candidate compound is identified for treatment of the subject if the indicia of inhibition of the interaction between CNOT1 and TTP is superior to the indicia of inhibition of the interaction between CONTI and TTP of the comparator compound.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0014] Fig. 1 shows that TTP regulates lipid metabolism. (A-C) Effective KD of TTP in HL1 cells using siRNA approach. (D) Lipid uptake (n=5), and (E) oxidation (n=5-6) with control or TTP siRNA. (F) Lipid uptake (n=6) and (G) oxidation (n=9-10) with empty vector (EV) or TTP adenovirus. *P<0.05 by ANOVA.

[0015] Fig. 2 shows FA uptake (A) and oxidation (B) in adult CM isolated from csTTP-KO mice.

[0016] Fig. 3 shows the strategy to identify FA metabolism target genes of TTP.

[0017] Fig. 4 shows that TTP regulates PPARa. PPARa mRNA in (A) HL1 cells treated with TTP siRNA (n=6), (B) hearts from TNFR1 / 2 and TNFR1 / 2 / '7TTPAmice (n=3), and (C) in HL1 cells treated with TTP adenovirus (n=12). (D) PPARa protein in HL1 cells treated with TTP siRNA (n=3). (E) PPARa mRNA stability after addition of actinomycin D in HL1 cells treated with control or TTP siRNA (n=4). (F) Computational analysis of PPARa 3’UTR revealing multiple putative AREs. Triangles represent AREs of various lengths. (G) Phylogenic conservation of AREs in PPARa 3’-UTR. Each dot is an ARE site and a positive score indicates a site under evolutionary constraint. *P<0.05 by ANOVA. Data presented as mean + / - SEM.

[0018] Fig. 5 shows PPARa mRNA (A) and protein (B) levels in csTTP-KO hearts. *P<0.05 by ANOVA.

[0019] Fig- 6 shows the proposed model for TTP regulation of cardiac FA metabolism. Fig. 7 shows BCAA levels in TTP KO MEFs (A, n=3) and HL1 cells treated with TTP siRNA (B, n=6). *P<0.05 by ANOVA. Data presented as mean + / - SEM.

[0020] Fig. 8 shows the strategy to identify BCAA metabolism target genes of TTP.

[0021] Fig. 9 shows that TTP regulates BCKDC-E2. BCKDC-E2 mRNA in (A) HL1 cells treated with TTP siRNA (n=6), and (B) hearts from TNFR1 / 2' ' and TNFR I / 2 / ' 7TTP" ' mice (n=6-8). (C) BCKDC-E2 mRNA in HL1 cells with TTP overexpression (n=12). (D) BCKDC-E2 mRNA stability assay in HL1 cells treated with TTP siRNA (n=4). (E) Computational analysis of BCKDC-E2 3’UTR showed multiple AREs. *P<0.05 by ANOVA. Data presented as mean + / - SEM.

[0022] Fig. 10 shows BCAA oxidation (A), and BCKDDC-E2 mRNA (B) and protein (C) in csTTP-KO hearts. *P<0.05 by ANOVA.

[0023] Fig. 11 shows the proposed model for TTP regulation of cardiac BCAA metabolism.

[0024] Fig. 12 shows mRNA levels of other mRNA binding proteins in the hearts of csTTP-KO mice.

[0025] Fig. 13 shows iron (A) and mRNA of inflammatory markers (B) in the hearts of WT and csTTP-KO mice. n=6.

[0026] Fig. 14 shows inflammatory mRNAs in the hearts of WT and csTTP-KO mice after TAC. n=6.

[0027] Fig. 15 shows the schematic of TTP interaction with CNOT1. Developed drugs target the site of interaction (red arrow).

[0028] Fig. 16 shows echo images (A), ejection fraction (B), left ventricular developed pressure (C), and FA oxidation (D) in hearts from WT and csTTP-KO mice. N=3. *P<0.05 by ANOVA.

[0029] Fig. 17 shows RNA co-IP of TTP and BCKDC-E2 mRNA. N=3. *P<0.05 by ANOVA.

[0030] Fig. 18 shows the proposed TTP pathways.

[0031] Fig. 19 depicts the model for interactions between TTP and CNOT1

[0032] Fig. 20 depicts the in vitro ELISA assay results.

[0033] Fig. 21 depicts the fluorescent polarization method. The polarization is low (0.14-17) when only the FL peptide is in solution and is higher (0.32-34) when the solution contains the peptide bound to the protein. Compounds that are able to "free" some or most of the peptide from the protein will bring the polarization down, the ones that are not effective will maintain the polarization around or above 0.3. The compound that aggregates the protein (without freeing the peptide) makes the polarization value even larger - 0.5 or so. Basically, polarization reflect the size of the molecule or molecular complex the fluorophore is in.

[0034] Fig. 22 depicts the lead compounds that show a strong ability to prevent the binding of CNOT and TTP.

[0035] Fig. 23 depicts the cell-based Nano-BiT PPI assay.

[0036] Fig. 24 depicts the luminescent signals collected from the Nano-BiT PPI assay.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] Disclosed herein are compounds that inhibit the interaction between tristetraprolin (TTP) and CCR4-NOT Transcription Complex Subunit 1 (CNOT1) and methods of use. The compounds that inhibit the interaction between TTP and CNOT1 may be used for the treatment of a subject. In some embodiments, the compounds may be used to treat subjects for heart disease, diabetes, and / or non-alcoholic fatty liver disease.

[0039] TTP is involved in cellular iron regulation and alters cellular metabolism by binding to and degrading the mRNAs of proteins involved in metabolic processes. Studies suggest a link between TTP and metabolism. First, TTP was originally discovered as an insulin-responsive gene. Second, there is a link between reduced TTP levels with obese individuals with insulin resistance. Third, the yeast homologs of TTP, Cthl / 2p, have been shown to regulate the expression of metabolic and mitochondrial proteins. These studies were conducted in the absence of an inflammatory process or changes in iron levels, indicating that the effects of TTP on cellular metabolism are independent of iron or inflammation.

[0040] TTP regulation of Lipid Metabolism

[0041] TTP is a regulator of fatty acid (FA) metabolism. Experimental results show that TTP levels can be reduced using an siRNA approach in HL1 cells (Fig. 1, panels A-C), which was associated with a significant increase in lipid uptake and oxidation (Fig. 1, panels D, E), while overexpression of TTP using an adenovirus resulted in lower lipid uptake and oxidation (Fig. 1, panels F, G). Additionally, isolated cardiomyocytes (CMs) from csTTP-KO mice displayed increased FA uptake and oxidation compared to wild type (WT) mice (Fig. 2). These results suggest that one of the core functions of TTP in CMs is to lower FA uptake and oxidation. Preliminary studies have also been conducted to assess FA oxidation in the hearts of WT and csTTP-KO mice. Furthermore, as shown in Fig. 16, although csTTP-KO mice display similar cardiac function as WT mice at baseline, FA oxidation is significantly increased in the hearts of these mice, suggesting that cardiac-specific TTP deletion alters FA oxidation under physiological conditions.

[0042] The following steps have been carried out to determine the targets responsible for changes in lipid metabolism (Fig. 3): 1) proteins with human and mouse genes containing AREs were identified, 2) the list was narrowed to those with high affinity AREs, and 3) further narrowed down by cross-referencing the list with another published TTP RIP-Seq dataset. These screens led to one lipid metabolism gene: PPARa and indicates that PPARa is targeted by TTP.

[0043] To assess the effects of TTP modulation on PPARa levels, Treatment of CMs with TTP siRNA or hearts from mice with global TTP KO (in TNFR1 / 2' ' background) showed a significant increase in PPARa mRNA levels, while TTP overexpression had the opposite effect (Fig. 4, panels A-C). Furthermore, TTP KD in HL1 cells resulted in a significant increase in PPARa protein (Fig. 4, panel D). Assessments also showed that the mRNA of PPARa is stabilized with TTP downregulation in cultured CMs treated with the transcriptional inhibitor actinomycin D (Fig. 4, panel E). Additionally, computational analysis revealed multiple AREs in the 3’-UTR of PPARa (Fig. 4, panel F), consistent with direct regulation of PPARa stability by interaction with TTP. A genomic evolutionary rate profiling (GERP) was also profiled. A positive GERP score indicates that the site is under evolutionary constraint, and a GERP score greater than 2 is considered significantly conserved. Using these criteria, several conserved AREs in the 3’ UTR of PPARa were identified, with three of them significantly conserved (Fig. 4, panel G).

[0044] The ~1.5-2.5-fold increase in steady state mRNA levels of TTP targets with TTP KO, likely due to the fact that TTP levels are low at baseline but are induced by specific stimuli, such as insulin. This causes robust effects on TTP’s targets to be restricted to the appropriate physiologic context. Thus, PPARa mRNA and protein in the hearts of csTTP-KO mice were measured after overnight fast and 8 hours of feeding (to synchronize their metabolic status) and showed that both the mRNA and protein are increased compared to WT littermates (Fig. 5). These results suggest that TTP regulates FA metabolism through regulation of PPARa (Fig. 6).

[0045] The disclosed compounds that inhibit the interaction between CN0T1 and TTP may increase lipid uptake and oxidation in the subject. In some embodiments, the disclosed compounds increase lipid uptake and oxidation in the subject through regulation of PPARa. In some embodiments, the disclosed compounds increase PPARa mRNA in the subject. TTP regulation of BCAA

[0046] TTP is a regulator of branched-chain amino acid (BCAA) metabolism. BCAA refers to amino acids having an aliphatic sidechain with a branch.

[0047] BCKDC is the rate-limiting enzyme in the catabolism of three essential BCAAs (leucine, isoleucine, and valine), and catalyzes the oxidation of BCAAs to their respective ketoacids. The BCKDC is composed of three subunits (El, E2 and E3), and its activity is regulated post- translationally via phosphorylation. When BCAA levels are low, BCKDC-E1 is phosphorylated and inhibited by a BCKDC kinase, resulting in an increase in BCAA levels. On the other hand, when the BCAA levels are high, BCKDC-E1 is dephosphorylated and activated by protein phosphatase 2Cm (PP2Cm), leading to a reduction in cellular BCAA. PP2Cm knockdown (KD) in cardiomyocytes (CM) results in decreased BCKDC activity and increased apoptosis, and PP2Cm-K0 mice display an increase in plasma BCAA and accelerated HF under stress. Additionally, circulating and tissue levels of BCAAs are elevated in patients and rodent models of diabetes, and an association between high circulating BCAA levels and heart disease is demonstrated. Defects in BCAA catabolism exists in failing hearts through Kriippel-like factor 15 (Klfl5)-mediated transcription. At baseline, BCAA oxidation is not a major source of energy in the heart, but alterations in BCAA oxidation can have a major effect on myocardial signaling and response to stress.

[0048] TTP KO MEFs and HL1 cells treated with TTP siRNA displayed significantly lower BCAA levels (Fig. 7), suggesting higher BCAA catabolism. It was observed that among mRNAs involved in BCAA metabolism, BCKDC-E2 is a potential target of TTP (Fig. 8). In addition, the steady state level of BCKDC-E2 mRNA is increased in CMs treated with TTP siRNA (Fig. 9, panel A) and in hearts from global TTP KO mice (Fig. 9, panel B). Additionally, BCKDC-E2 mRNA levels were reduced in HL1 cells with TTP overexpression (Fig. 9, panel C), further showing that TTP regulates BCKDC-E2 mRNA levels. mRNA stability studies in HL1 cells using actinomycin D also revealed that the mRNA of BCKDC-E2 is stabilized with TTP KD (Fig. 9, panel D). Computational analysis of the 3’ UTR of the genes involved in BCAA catabolism showed that BCKDC-E2 mRNA contains multiple AREs with a high likelihood of TTP binding (Fig. 9, panel E), and phylogenetic conservation among various species. Additionally, CMs isolated from csTTP-KO mice displayed higher BCAA oxidation (Fig. 10, panel A), and the hearts had higher BCKDC-E2 mRNA and protein after overnight fasting and 8 hours of feeding to synchronize their metabolic status (Fig. 10, panels B-C). These results indicate that TTP regulates BCAA levels by binding to and degrading BCKDC-E2 mRNA. See Fig. 11. Furthermore, it was demonstrated that TTP binds to BCKDC-E2 mRNA using RNA co-IP experiments (Fig. 17).

[0049] The disclosed compounds that inhibit the interaction between CNOT1 and TTP may reduce BCAA in the subject. In some embodiments, the disclosed compounds increase the level of BCAA catabolism in the subject. In some embodiments, the disclosed compounds reduce BCAA in the subject through regulation of BCKDC-E2. In some embodiments, the disclosed compounds increase BCKDC-E2 mRNA in the subject.

[0050] TTP Regulation of Insulin Sensitivity

[0051] TTP can also regulate insulin sensitivity through regulation of fibroblast growth factor 21 (FGF21). FGF21 is a hormone abundantly expressed in the liver, whose synthesis and secretion are highly responsive to nutrient intake in mice and humans. Hepatic FGF21 plays a key role in glucose and lipid metabolism, insulin sensitivity, and ketogenesis at both the hepatic and systemic levels. Liver-derived FGF21 acts on adipocytes to stimulate insulin-dependent glucose uptake, modulate lipolysis, and increase mitochondrial oxidative capacity. Administration of FGF21 to obese and diabetic mouse models stimulates the uptake of glucose in adipose tissue and improves systemic insulin sensitivity.

[0052] The coordination of these hepatic regulatory factors occurs on several levels to ensure an appropriate systemic metabolic response, although little is known about the regulation that occurs at the post transcriptional level by RNA-binding proteins. After binding to the ARE of a target tran-script, TTP promotes mRNA deadenylation and subsequent transcript decay. TTP has been well studied in the field of inflammation, where TNF-a is an established target of TTP. This is reflective of whole-body Ttp-KO mice, which develop a severe systemic autoimmune inflammatory syndrome shortly after birth due to elevated TNF-a accumulation in macrophages. TTP has also been implicated as a tumor suppressor gene, and we have shown that TTP is a critical regulator of cellular iron homeostasis.

[0053] Hepatic TTP is a potentially novel player in metabolism that regulates the mRNA stability of Fgf21, which has functional consequences for hepatic and systemic insulin sensitivity. Furthermore, hepatic TTP levels are induced by insulin and reduced in the livers of diabetic mice and humans. Liver-specific Ttp-KO (IsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity compared with littermate controls. Analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is post- transcriptionally repressed by TTP. Consistent with increased FGF21, IsTtp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Downregulation of hepatic Fgf21 via an adeno-associated virus-driven shRNA in mice fed HFD reverses the insulin-sensitizing effects of hepatic Ttp deletion. Thus, hepatic TTP post-transcriptionally regulates systemic insulin sensitivity in diabetes through liver-derived FGF21. See Sawicki KT, et al., Hepatic tristetraprolin promotes insulin resistance through RNA destabilization of FGF21. JCI Insight. 2018 Jul 12;3(13):e95948. doi: 10.1172 / j ci.insight.95948. The contents of which are incorporated by reference in its entirety.

[0054] The disclosed compounds that inhibit the interaction between CNOT1 and TTP may increase insulin sensitivity of the subject. In some embodiments, the disclosed compounds increase insulin sensitivity of the subj ect through regulation of FGF21. In some embodiments, the disclosed compounds increase the level of FGF21 in the subject.

[0055] Effects of TTP Inhibition

[0056] Given TTP’s effects on lipid and BCAA metabolism in the heart, investigation into whether its deletion would alter cardiac response to injury and the development of HF was carried out. Experimental data suggests that TTP deletion protects against the development of HF. Furthermore, in FA and BCAA metabolic pathways, PPARa and BCKDC are the major players that mediate the effects of TTP in HF. Finally, deletion of TTP does not alter the levels of other mRNA-binding proteins (Fig. 12), thus the metabolic features in TTP KO mice are specific to the deletion of TTP.

[0057] Since TTP deletion has an effect on inflammation and iron homeostasis, it is important to consider and exclude these potential side effects if TTP is targeted for treatment of HF. As for iron homeostasis, studies were conducted in the absence of iron deficiency and have shown that iron does not change in the hearts of TTP KO mice (Fig. 13, panel A), confirming that the effects of TTP in the development of HF is independent of iron. For the inflammation issue, TTP'fgf21' / TNFR1 / 2' ' mice (which lack the systemic inflammation that is present in mice with global TTP KO) was used for preliminary studies. Cardiac cells do not express TNFa at baseline, and deletion of TTP did not alter the levels of TNFa and its receptor and other inflammatory cytokines in csTTP-KO mice at baseline (Fig. 13, panel B) and after TAC (Fig. 14), indicating that the role of TTP in the development of HF is independent of its role in inflammation. Additionally, ~50% reduction of TTP is generally achieved using siRNA (Fig. 1, panels A-C), and still get significant increase in PPARa and BCKDC-E2. However, this degree of reduction in TTP is not sufficient to induce systemic inflammation, as heterozygous TTP KO mice do not display an inflammatory phenotype. Thus, there is a window where TTP can be reduced in a clinical setting, either by using an siRNA approach or by using drugs to inhibit its mRNA degradation activity and induce the desired metabolic effects without triggering a systemic inflammatory response. The effects of TTP on metabolism can be independent of its role in the regulation of iron and inflammation.

[0058] The disclosed compounds that inhibit the interaction between CN0T1 and TTP may exert an effect on the metabolic regulation by TTP without inducing undesirable responses in the subject. In some embodiments, the disclosed compounds inhibit the interaction between CN0T1 and TTP but do not induce systemic inflammatory in the subject. In some embodiments, the inflammation may be caused by increased levels of TNF-a. Examples of systemic inflammation responses include but are not limited to, fever, fatigue, insomnia, hypothermia, tachycardia, tachypnoea, damage to organs, systemic shock, possible death, and / or change in blood leucocyte count.

[0059] It is understood by those skilled in the art that the term “do not induce systemic inflammation” may indicate that the compound does not induce any systemic inflammation response, or the compound only induces acceptable levels of inflammation response. In some embodiments, the compound that inhibits the interaction between CN0T1 and TTP does not alter the levels of TNFa in the subject. For example, the compound does not increase the level of TNFa. In some embodiments, the compound induces fewer systemic inflammation response compared to a subject with complete deletion of TTP.

[0060] In some embodiments, the disclosed compounds inhibit the interaction between CNOT1 and TTP but do not affect iron homeostasis in the subject. It is understood by those skilled in the art that the term “do not affect iron homeostasis” may indicate that the compound does not induce any changes in the level of iron, or the compound only induces acceptable level of changes in the level of iron.

[0061] Binding of TTP to CNOT1

[0062] CCR4-NOT Transcription Complex Subunit 1 (CNOT1) is a central component of the CCR4-N0T complex. The C-terminal tail of TTP contains 13 highly conserved amino acids, which function as a binding domain for CN0T1 (CNBD), which causes the removal of poly-(A) tails and subsequent mRNA decay. Deletion of this domain is sufficient to prevent recruitment of the CCR4- NOT complex and stabilize target transcripts by abolishing the interaction between TTP and CNOT1. Mice with homozygous deletion of the CNBD of TTP do not display elevated levels of TNFa, nor do they exhibit autoimmune disease in contrast to global TTP KO mice. Thus, inhibition of TTP function by disrupting its binding to CN0T1 may modulate TTP-mediated mRNA decay without causing intolerable inflammatory side effects.

[0063] The disclosed compounds may inhibit the interaction between CN0T1 and TTP in the subject. In some embodiments, the disclosed compounds inhibit the interaction between CNOT1 and TTP by binding to the CN0T1 cleft. In some embodiments, the disclosed compounds disrupt the binding between CNOT1 and TTP. In some embodiments, the disclosed compounds show a reduction in binding between CNOT1 and TTP.

[0064] Inhibition of the interaction between CNOT1 and TTP may be assessed by assessing the compound’s ability to bind to the CNOT1 cleft.

[0065] Inhibition of the interaction between CN0T1 and TTP may be assessed by in silico screening. In some embodiments, the disclosed compounds show in silico evidence of having the structure and the ability to get inserted into the cleft where the two proteins interact.

[0066] Inhibition of the interaction between CNOT1 and TTP may be assessed by enzyme-linked immunosorbent assay (ELISA) assay. In some embodiments, the disclosed compounds result in changes in the color of the reaction that can be detected by calorimetric assays.

[0067] Inhibition of the interaction between CNOT1 and TTP may be assessed by fluorescence polarization. In some embodiments, the disclosed compounds result in a polarization value of less than 1.0, than 0.75, less than 0.6, less than 0.5, less than 0.4, less than 0.35, less than 0.3, less than 0.25, less than 0.2, less than 0.15, less than 0.1, or less than 0.05.

[0068] Inhibition of the interaction between CNOT1 and TTP may be assessed by Bio Layer Interferometry (BLI) assay. In some embodiments, the disclosed compounds interfere with the patterns of white light reflected from the surface of a biosensor tip, allowing measurements of the interaction between CNOT1 and TTP.

[0069] Inhibition of the interaction between CNOT1 and TTP may be assessed by a cell-based assay, such as by using a Nano-BiT PPI assay kit. In some embodiments, the disclosed compounds result in luminescent signals between 2,000,000 RLU to 1000,000,000 RLU. As disclosed by the Examples, one or more of these methodologies may be utilized to identify compounds that inhibit the interaction between CNOT1 and TTP. Exemplary compounds include, but are not limited to, , or ; or a pharmaceutically acceptable salt thereof.

[0070] Methods of Treatment

[0071] One aspect of the technology provides for a method for the treatment of a subject in need of a compound that inhibits the interaction between CNOT1 and tristetraprolin (TTP), the method comprising administering a compound that inhibits the interaction between CNOT1 and TTP to the subject. The compound that inhibits the interaction between CNOT1 and TTP may be any of the compounds disclosed herein.

[0072] As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0073] A “subject in need thereof’ as utilized herein refers to a subject in need of a compound that inhibits the interaction between CN0T1 and TTP. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. In some embodiments, the treated subject may be a mammalian subject. Although the methods disclosed herein are particularly intended for the treatment of proliferative disorders in humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs.

[0074] As used herein, the term "disorder" refers to a condition in which there is a disturbance of normal functioning. A "disease" is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts these may be considered distinguishable categories. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein.

[0075] The subject in need of a compound that inhibits the interaction between CN0T1 and TTP may be in need of the compound to treat diseases and disorders associated with TTP-regulated processes, such as FA metabolism, BCAA metabolism, and / or insulin sensitivity. The diseases and disorders may include, but are not limited to, heart diseases, such as ischemic heart disease; diabetes, such as type I or type II diabetes; or non-alcoholic fatty liver disease.

[0076] The compounds for use according to the methods of disclosed herein may be administered as a single compound or a combination of compounds. For example, a compound that inhibits the interaction between CN0T1 and TTP may be administered as a single compound or in combination with another compound that inhibits the interaction between CN0T1 and TTP or that has a different pharmacological activity.

[0077] In some embodiments, the compound that inhibits the interaction between CN0T1 and TTP binds to the binding cleft of CNOT1.

[0078] In some embodiments, the compound that inhibits the interaction between CN0T1 and TTP is selected from

[0079] or a pharmaceutically acceptable salt thereof. In some embodiments, the compound that inhibits the interaction between CNOT1 and TTP is or a pharmaceutically acceptable salt thereof.

[0080] In some embodiments of the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subj ect. Minimal and / or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg - 200 mg).

[0081] In some embodiments of the disclosed treatment methods, a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. In some embodiments, a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. Minimal and / or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as endpoints any of these disclosed dose levels (e.g., 500 - 2000 ng / kg body weight of the subject).

[0082] As used herein the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g, as present in a pharmaceutical composition) for treating a subject in need of a compound that inhibits the interaction between CNOT1 and TTP.

[0083] An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0084] A typical daily dose may contain from about 0.01 mg / kg to about 100 mg / kg (such as from about 0.05 mg / kg to about 50 mg / kg and / or from about 0.1 mg / kg to about 25 mg / kg) of each compound used in the present method of treatment.

[0085] Compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and / or about 25 mg. The term “unit dosage form” refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.

[0086] Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver.

[0087] The compounds and compositions disclosed herein may be administered in methods of treatment as known in the art. Accordingly, various such compounds and compositions can be administered in conjunction with such a method in any suitable way. For example, administration may comprise oral, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.

[0088] Method of Compound Identification

[0089] Another aspect of the present invention provides a method of identifying a compound for the treatment of a subject in need of a compound that inhibits the interaction between CNOT1 and TTP. The method may be applicable to identify any of the compounds disclosed herein. In some embodiments, the published crystal structure of CNOT1 binding to the CNBD of TTP may be used as the basis for computational-modeling to identify small molecules which could bind the CN0T1 cleft and disrupt its interaction with TTP (Fig. 15). A library of compounds may be screened and candidate compounds identified with potential to disrupt TTP-CNOT1 binding. To test these compounds by exemplary methods, the protein coding sequence of human CN0T1 can be cloned into the pET-21d to facilitate bacterial expression and purification of HIS-tagged CN0T1 protein. Similarly, both GST-tagged full length human TTP 1-326, and C-terminal truncated TTP 1-313 (CNBD-deletion) can cloned into the pGEXl vector. Further screening may be performed using methods disclosed herein to identify compounds with superior efficacy in disrupting TTP- CN0T1 interaction.

[0090] In vitro and / or in vivo experiments may be performed on the candidate compounds to identify one or more compounds for the treatment of a subject in need of a compound that inhibits the interaction between CN0T1 and TTP.

[0091] In some embodiments, the disclosed method comprises assaying a candidate compound to determine an indicia of inhibition of the interaction between CNOT1 and TTP and comparing the indicia of inhibition of the interaction between CONTI and TTP to an indicia of inhibition of the interaction between CN0T1 and TTP of a comparator compound that inhibits the interaction between CN0T1 and TTP.

[0092] “Comparator compound” refers to any compound capable of inhibiting the interaction between CN0T1 and TTP. The comparator compound may be any compound disclosed herein, or any compound identified using the methods disclosed herein. The comparator compound may be any compound that can bind the CNOT1 cleft. In some embodiments, the comparator compound may be

[0093]

[0094] “Candidate compound” refers to any compound believed to be capable of inhibiting the interaction between CN0T1 and TTP. The candidate compound may be any compound disclosed herein, or any compound identified using the methods disclosed herein. The candidate compound may be any compound that can bind the CNOT1 cleft.

[0095] In some embodiments, the candidate compound inhibits the interaction between CNOT1 and TTP and increases lipid uptake and oxidation. In some embodiments, the candidate compound inhibits the interaction between CNOT1 and TTP and increases PPARa mRNA. In some embodiments, the candidate compound inhibits the interaction between CNOT1 and TTP and reduces branched-chain amino acid (BCAA). In some embodiments, the candidate compound inhibits the interaction between CNOT1 and TTP and increases BCKDC-E2 mRNA in the subject. In some embodiments, the candidate compound that inhibits the interaction between CNOT1 and TTP and increases insulin sensitivity. In some embodiments, the candidate compound increases the level of fibroblast growth factor 21 (FGF21) in the subject. In some embodiments, the candidate compound inhibits the interaction between CNOT1 and TTP does not increase the level of TNFa.

[0096] “Indicia of inhibition” refers to a quantitative and / or qualitative result obtained from assaying the candidate compound. The indicia of inhibition may qualitatively and / or quantitatively assess the candidate compound’s ability to inhibits the interaction between CNOT1 and TTP, such as the candidate compound’s ability to disrupt the binding between CNOT1 and TTP. The indicia of inhibition may also qualitatively and / or quantitatively assess the candidate compound’s ability to bind to the CNOT1 cleft.

[0097] In some embodiments, the method comprises assaying the candidate compound using in silico screening. The indicia of inhibition may be the predicted binding affinity between TTP and CNOT1 from the in-silico screening. In some embodiments, the method comprises assaying the candidate compound using enzyme-linked immunosorbent assay (ELISA) assay. The indicia of inhibition may be the change in color detected by calorimetric assays. In some embodiments, the method comprises assaying the candidate compound using fluorescence polarization. The indicia of inhibition may be the polarization value. In some embodiments, the method comprises assaying the candidate compound using Bio Layer Interferometry (BLI) assay. The indicia of inhibition may be the change in patterns of white light reflected from the surface of a biosensor tip. In some embodiments, the method comprises assaying the candidate compound using a cell-based assay. For example, the candidate compound may be assayed by using a Nano-BiT PPI assay kit. The indicia of inhibition may be the value of the luminescent signals.

[0098] In some embodiments, the method comprises comparing the indicia of inhibition of the interaction between CONTI and TTP to an indicia of inhibition of the interaction between CNOT1 and TTP of a comparator compound. The candidate compound may be identified for treatment of the subject if the indicia of inhibition of the interaction between CNOT1 and TTP is superior to the indicia of inhibition of the interaction between CONTI and TTP of the comparator compound.

[0099] The term “superior” is context dependent and can be evaluated by those of skill in the art. For example, if the comparator compound increases expression of a protein, translation of RNA, and / or transcription of a gene, superior in this context may include increasing expression of the protein, translation of RNA, or transcription of the gene to a greater extent than the comparator compound. For another example, if the comparator compound decreases expression of a protein, translation of RNA, and / or transcription of a gene, superior in this context may include decreasing expression of the protein, translation of RNA, or transcription of the gene to a greater extent than the comparator compound. In some instances, the increase or decrease may be one of statistical significance.

[0100] The candidate compound need not have superior indicia of inhibition for all indicia evaluated to be identified for treatment. In some cases, the candidate compound will have one or more superior indicia when compared to the comparator and other indicia may be substantially similar or possibly inferior. Substantially similar may refer to a statistically insignificant difference between the candidate compound and the comparator for an evaluated indicia.

[0101] In some embodiments, comparison of the indicia of inhibition may comprise comparing the candidate compound’s and the comparator compound’s ability to bind to the CN0T1 cleft.

[0102] In some embodiments, comparison of the indicia of inhibition may comprise comparing the in silico screening results between the candidate compound and the comparator compound. For example, the candidate compound may display superior indicia of inhibition of the interaction between CNOT1 and TTP compared to the comparator compound, if the candidate compound results in lower predicted binding affinity between CONTI and TTP.

[0103] In some embodiments, comparison of the indicia of inhibition may comprise comparing the enzyme-linked immunosorbent assay (ELISA) assay results between the candidate compound and the comparator compound. For example, the candidate compound may display superior indicia of inhibition of the interaction between CNOT1 and TTP compared to the comparator compound, if a change in color of the assay is detected with calorimetric assays.

[0104] In some embodiments, comparison of the indicia of inhibition may comprise comparing the fluorescence polarization results between the candidate compound and the comparator compound. For example, the candidate compound may display superior indicia of inhibition of the interaction between CN0T1 and TTP compared to the comparator compound, if the candidate compound results in a lower polarization value.

[0105] In some embodiments, comparison of the indicia of inhibition may comprise comparing the Bio Layer Interferometry (BLI) assay results between the candidate compound and the comparator compound. For example, the candidate compound may display superior indicia of inhibition of the interaction between CNOT1 and TTP compared to the comparator compound, if there is a change in the pattern of white light reflected from the surface of a biosensor tip. In some embodiments, comparison of the indicia of inhibition may comprise comparing the cell-based assay results, such as the Nano-BiT PPI assay results between the candidate compound and the comparator compound. For example, the candidate compound may display superior indicia of inhibition of the interaction between CNOT1 and TTP compared to the comparator compound, if there is a change in luminescence.

[0106] In some embodiments, the method further comprises preparing a composition comprising the candidate compound identified for treatment of the subject, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier and administering the composition to the subject.

[0107] Pharmaceutical Compositions

[0108] The compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.

[0109] In some embodiments, the compounds disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg / kg body weight (preferably about 0.5 to about 500 mg / kg body weight, more preferably about 50 to about 100 mg / kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.5 pM, 0.1 pM, 1.0 pM, 10 pM, and 100 pM (e.g., 0.1 pM - 1.0 pM).

[0110] It is understood by those skilled in the art that dosage amount will vary with the activity of a particular inhibitor compound, disease state, route of administration, duration of treatment, and like factors well-known in the medical and pharmaceutical arts. In general, a suitable dose will be an amount which is the lowest dose effective to produce a therapeutic or prophylactic effect. If desired, an effective dose of such a compound, pharmaceutically acceptable salt thereof, or related composition may be administered in two or more sub-doses, administered separately over an appropriate period of time.

[0111] In some embodiments, a pharmaceutical composition comprising the compound of as disclosed herein and a pharmaceutically suitable carrier, diluent, or excipient is provided.

[0112] The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 0.5 to 500 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 1 to 100 mg. The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg / kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.5 to about 500 mg / kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 50 to about 100 mg / kg body weight. In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours postadministration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.5 pM, 0.1 pM, 1.0 pM, 10 pM, and 100 pM (e.g., 0.1 pM - 1.0 pM).

[0113] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste. The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and crosslinked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PHI 01 and Avicel® PHI 02, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0114] Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0115] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

[0116] Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0117] The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.

[0118] Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0119] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or nonaqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0120] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis.

[0121] Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

[0122] For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are in some embodiments applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

[0123] Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

[0124] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

[0125] Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

[0126] Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.

[0127] Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0128] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0129] Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0130] Optionally, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior to, concurrently with, or after administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds. In some embodiments, the disclosed pharmaceutical composition is formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cell proliferative diseases and disorders.

[0131] Methods of preparing pharmaceutical formulations or compositions include the step of bringing an inhibitor compound into association with a carrier and, optionally, one or more additional adjuvants or ingredients. For example, standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

[0132] Regardless of composition or formulation, those skilled in the art will recognize various avenues for medicament administration, together with corresponding factors and parameters to be considered in rendering such a medicament suitable for administration. Miscellaneous

[0133] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0134] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0135] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0136] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0137] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0138] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0139] EXAMPLES

[0140] TTP regulates several metabolic processes, including fatty acid (FA) update oxidation (through the regulation of PPARa) and branched chain amino acid (BCAA) metabolism (through the regulation of branched-chain a-ketoacid dehydrogenase (BCKDC)-E2). Additionally, TTP is reduced in the livers of diabetic mice and humans and is transcriptionally induced with insulin treatment. Liver-specific Ttp-KO (IsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity and analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is post-transcriptionally repressed by TTP. Consistent with increased FGF21, IsTzp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Thus, TTP regulates systemic insulin sensitivity in diabetes through liver-derived FGF21. See Sawicki KT, et al., Hepatic tristetraprolin promotes insulin resistance through RNA destabilization of FGF21. JCI Insight. 2018 Jul 12;3(13):e95948. doi: 10.1172 / jci.insight.95948. The contents of which are incorporated by reference in its entirety.

[0141] The C-terminal tail of TTP contains 13 highly conserved amino acids, which function as a binding domain for CNOT1 (CNBD), a central component of the CCR4-NOT complex, which causes the removal of poly-(A) tails and subsequent mRNA decay. See Fig. 18. Deletion of this domain is sufficient to prevent recruitment of the CCR4-NOT complex and stabilize target transcripts by abolishing the interaction between TTP and CNOT1. Interestingly, mice with homozygous deletion of the CNBD of TTP do not display elevated levels of TNFa, nor do they exhibit autoimmune disease in contrast to global TTP KO mice. Thus, inhibition of TTP function by disrupting its binding to CNOT1 is a potential means of modulating TTP-mediated mRNA decay without causing intolerable inflammatory side effects.

[0142] Identification of Lead Compounds

[0143] The published crystal structure of CNOT1 binding to the CNBD of TTP was utilized as the basis for computational modeling to identify small molecules which could bind the CNOT1 cleft and disrupt its interaction with TTP. An in silico assay was used to identify 64 molecules that can potentially disrupt the interaction between TTP and CN0T1. Among those, there were 42 molecules commercially available to be purchased (Fig. 19).

[0144] To assess the potential of these compound to disrupt the binding between these two proteins by immunologic methods, an in vitro ELISA assay was employed to assess the effects of these compounds on the interaction between TTP and CNOT1 with purified proteins. Full length human TTPi -326 and C-terminal truncated TTP1-313 (CN0T1 -Binding-Domain Deleted; negative control) were cloned into the pGEXl bacterial expression vectors containing MBP and GST tags. The TTP binding domain of human CNOT1679-1266 was cloned into the pET-21d bacterial expression vector containing a HIS tag. Plasmids were transformed into Rosetta E. coli competent cells to facilitate expression and purification. (Fig. 20). To perform the ELISA assay, purified CNOT1 protein was coated onto 96-well anti-HIS antibody conjugates plates, after blocking with 5% milk and washing with TBS-T, the plate was incubated with MBP-tagged TTP in the presence of candidate compounds. The plate was then washed and incubated with HRP-conjugated anti-MBP antibody. Subsequently, TMB substrate was added, which generates a chromogenic product in presence of HRP. The reaction was stopped by the addition of sulfuric acid and absorbance was measured at 450 nm.

[0145] 22 out of 42 showed a significant reduction in the binding between these two proteins. Since the number of potential compounds were higher than what's expected (usually 5% of the compounds show efficacy after in-silico screening), a more sensitive screening was performed to narrow down the number of compounds.

[0146] In the next step, a fluorescent polarization method was used to find the most likely compounds. In this method, one of the proteins (CNOT) is coated to a surface and there's a peptide (CNOT binding domain of TTP) fused to a FITC tag (Fluorescein isothiocyanate) to measure the tumbling speed of the fluorescent tagged CBD (CNOT Binding Domain) of TTP. If the compound can disrupt the binding, then the tumbling speed is going to be higher due to the lower weight of the particles, and it will be recorded by a sensitive sensor (Fig. 21).

[0147] The FP assays led to 4 lead compounds showing a strong ability to prevent the binding of CNOT and TTP (Fig. 22). To narrow down the number of potent compounds more, Bio Layer Interferometry (BLI) assay was used with the full-length proteins instead of the TTP peptide. After finishing the BLI assay, a protein complementation cell-based assay was designed to investigate the effect of compounds in a eukaryotic cellular system to assess the activity of the compound in physiologic condition (live cells). For this purpose, the Nano-BiT PPI assay kit developed by ProMega was purchased. Each of CNOT and TTP proteins was fused to one compartment of Luciferase enzyme (Fig. 23). Proximity of these two compartments leads to activation of the enzyme and gives luminescent signals which are detectable by luminometer. The compounds with the ability of the binding disruption, reduce the proximity of the luciferase compartments and activity of the enzyme and the signals are detectible (Fig. 24).

[0148] The lead compounds will be tested for bioavailability, toxicity, and their antidiabetic effects in mice and their effects on the levels of FGF21 will be assessed. The hypothesis is that the lead compounds that interrupt the interaction between TTP and CNOT will have a major effect on metabolic regulation by TTP without causing the inflammatory response noted through TNFa activation. The identified compounds can be used in the following conditions: 1) improving insulin sensitivity in diabetes through increased FGF21 production, 2) non-alcoholic fatty liver disease (NAFLD) through FGF21, and 3) ischemic heart disease through its effects on FA and BCAA metabolism.

Claims

CLAIMSWhat is claimed is:

1. A method for the treatment of a subject in need of a compound that inhibits the interaction between CNOT1 and tristetraprolin (TTP), the method comprising administering a compound that inhibits the interaction between CNOT1 and TTP to the subject.

2. The method of claim 1, wherein the subject is in need of a compound to treat ischemic heart disease.

3. The method of claim 1, wherein the subject is in need of a compound to treat diabetes.

4. The method of claim 1, wherein the subject is in need of a compound to treat nonalcoholic fatty liver disease.

5. The method of any one of claims 1-4, wherein the compound that inhibits the interaction between CNOT1 and TTP binds to the binding cleft of CNOT1.

6. The method of any one of claims 1-4, wherein the compound that inhibits the interaction between CN0T1 and TTP is selected from7. The method of claim 6, wherein the compound that inhibits the interaction between CNOT1 and TTP isor a pharmaceutically acceptable salt thereof.

8. The method of any one of claims 1-7, wherein the compound that inhibits the interaction between CNOT1 and TTP does not induce systemic inflammation in the subject.

9. The method of any one of claims 1-8, wherein the compound that inhibits the interaction between CNOT1 and TTP does not increase the level of TNFa in the subject.

10. The method of any one of claims 1-9, wherein the compound that inhibits the interaction between CNOT1 and TTP does not affect iron homeostasis in the subject.

11. The method of any one of claims 1-10, wherein the compound that inhibits the interaction between CNOT1 and TTP increases lipid uptake and oxidation in the subject.

12. The method of claims 11, wherein the compound increases PPARa mRNA in the subject.

13. The method of any one of claims 1-12, wherein the compound that inhibits the interaction between CNOT1 and TTP reduces branched-chain amino acid (BCAA) in the subject.

14. The method of claims 13, wherein the compound increases BCKDC-E2 mRNA in the subject.

15. The method of any one of claims 1-14, wherein the compound that inhibits the interaction between CNOT1 and TTP increases insulin sensitivity of the subject.

16. The method of claim 15, wherein the compound increases the level of fibroblast growth factor 21 (FGF21) in the subject.

17. A method of identifying a compound for the treatment of a subject in need of a compound that inhibits the interaction between CNOT1 and TTP, the method comprising: assaying a candidate compound to determine an indicia of inhibition of the interaction between CNOT1 and TTP and comparing the indicia of inhibition of the interaction between CONTI and TTP to an indicia of inhibition of the interaction between CNOT1 and TTP of a comparator compound that inhibits the interaction between CNOT1 and TTP, wherein the candidate compound is identified for treatment of the subject if the indicia of inhibition of the interaction between CNOT1 and TTP is superior to the indicia of inhibition of the interaction between CONTI and TTP of the comparator compound.

18. The method of claim 17, wherein the method comprises assaying the candidate compound using in silico screening.

19. The method of any one of claims 17-18, wherein the method comprises assaying the candidate compound using enzyme-linked immunosorbent assay (ELISA) assay.

20. The method of any one of claims 17-19, wherein the method comprises assaying the candidate compound using fluorescence polarization.

21. The method of any one of claims 17-20, wherein the method comprises assaying the candidate compound using Bio Layer Interferometry (BLI) assay.

22. The method of any one of claims 17-21, wherein the method comprises assaying the candidate compound using a cell-based assay.

23. The method of any one of claims 17-22, wherein the candidate compound inhibits the interaction between CNOT1 and TTP and increases lipid uptake and oxidation.

24. The method of any one of claims 17-23, wherein the candidate compound inhibits the interaction between CNOT1 and TTP and increases PPARa mRNA.

25. The method of any one of claims 17-24, wherein the candidate compound inhibits the interaction between CNOT1 and TTP and reduces branched-chain amino acid (BCAA).

26. The method of any one of claims 17-25, wherein the candidate compound inhibits the interaction between CNOT1 and TTP and increases BCKDC-E2 mRNA in the subject.

27. The method of any one of claims 17-26, wherein the candidate compound that inhibits the interaction between CNOT1 and TTP and increases insulin sensitivity.

28. The method of claim 27, wherein the candidate compound increases the level of fibroblast growth factor 21 (FGF21) in the subject.

29. The method of any one of claims 17-28, wherein the candidate compound inhibits the interaction between CNOT1 and TTP does not increase the level of TNFa.

30. The method of any one of claims 17-29, wherein the comparator compound is, or31. The method of any one of claims 17-30 further comprising preparing a composition comprising the candidate compound identified for treatment of the subject, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier and administering the composition to the subject.

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