A Review of Foxo1-Regulated Metabolic Diseases and Related Drug Discoveries

cells

Review A Review of FoxO1-Regulated Metabolic Diseases and Related Drug Discoveries

Shiming Peng 1, Wei Li 2, Nannan Hou 1 and Niu Huang 1,3,*

1 National Institute of Biological Sciences, Beijing 102206, China; [email protected] (S.P.); [email protected] (N.H.) 2 RPXDs (Suzhou) Co., Ltd., Suzhou 215123, China; [email protected] 3 Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing 102206, China * Correspondence: [email protected]

Received: 30 November 2019; Accepted: 8 January 2020; Published: 10 January 2020

Abstract: FoxO1 is a conserved transcription factor involved in energy metabolism. It is tightly regulated by modiﬁcations on its mRNA and protein and responds to environmental nutrient signals. FoxO1 controls the transcription of downstream genes mediating metabolic regulation. Dysfunction of FoxO1 pathways results in several metabolic diseases, including diabetes, obesity, non-alcoholic fatty liver disease, and atherosclerosis. Here, we summarize the mechanism of FoxO1 regulation behind these diseases and FoxO1-related drug discoveries.

Keywords: FoxO1; metabolic disease; drug discovery

1. Introduction of FoxO1 Forkhead Box O (FoxO) genes belong to a transcription factor (TF) family characterized by the presence of a conserved DNA-binding domain (the Forkhead box) in the N-terminal end of the protein [1–3] (Figure1A). FOXOs bind the consensus DNA-binding element within the promoter of their target genes and regulate their transcription in response to external signal stimulation [4–6] (Figure1B). There are four FoxO genes identified in mammals, including FoxO1, FoxO3a, FoxO4, and FoxO6 [7]. In the 1990s, the FoxO1 gene was first identified in studies of chromosomal translocations found in human tumors, including rhabdomyosarcomas and acute myeloid leukemias [1,3,8,9]. In addition to its important regulatory roles in oncogenesis [10], FoxO1 also transcriptionally mediates pathways behind many metabolic diseases, including gluconeogenesis, glycogenolysis, adipogenesis, thermogenesis, and feeding behavior [11–15], which is the focus of this review article.

2. Regulation of FoxO1

2.1. Protein Modifications FOXO1 protein is sophisticatedly and strictly regulated by modifications on its protein and mRNA, which ensures that transcription of its downstream target genes is tightly responsive to environmental signals [16] (Figure1C). Kinase-mediated phosphorylation of the FOXO1 protein in response to insulin or growth factors is a form of regulation [17] (Figure1C). Phosphoinositide 3-kinase /protein kinase B (PI3K/PKB) phosphorylates the FOXO1 protein at three conserved residues, including Threonine 24, Serine 256, and Serine 319. These modifications result in disrupted interactions between the FOXO1 protein and its target DNA and lead to the translocation of the FOXO1 protein from the nucleus to the cytoplasm, thus suppressing FOXO1-dependent transcription. The FOXO1 protein can also be phosphorylated by c-Jun N-terminal kinase (JNK) or macrophage-stimulating 1 (Mst1) [18,19]. This

Cells 2020, 9, 184; doi:10.3390/cells9010184 www.mdpi.com/journal/cells Cells 2019, 8, x FOR PEER REVIEW 2 of 10 Cells 2020, 9, 184 2 of 10 macrophage-stimulating 1 (Mst1) [18,19]. This phosphorylation results in the import of the FOXO1 protein from the cytoplasm to the nucleus, thereby antagonizing the action of PI3K/PKB. phosphorylationFOXO1 protein results activity in the is import also ofregulated the FOXO1 by proteinreversible from acetylation the cytoplasm modification to the nucleus, on therebylysine residuesantagonizing (Figure the action1C). ofThey PI3K /PKB.are acetylated by histone acetyltransferase cAMP-response element-bindingFOXO1 protein protein activity (CREB)-binding is also regulated protei by reversiblen (CBP) acetylationand deacetylated modification by NAD-dependent on lysine residues histone(Figure deacetylase1C). They are silent acetylated information by histone regulator acetyltransferase 2 (Sir2) on conserved cAMP-response residues element-binding (Lys 242, Lys 245, protein and Lys(CREB)-binding 262) [20–22]. The protein positive (CBP) charge and of deacetylated these lysine residues by NAD-dependent in FOXO1 contributes histone deacetylaseto DNA binding. silent Thus,information acetylation regulator at these 2 (Sir2) residues on conserved attenuates residues the ability (Lys of 242, FOXO1 Lys 245, to bind and LysDNA 262) and [20 suppress–22]. The transcription.positive charge Interestingly, of these lysine it was residues also found in FOXO1 that acetylation contributes regulated to DNA binding. the function Thus, of acetylation FOXO1 by at influencingthese residues its sensitivity attenuates for the phosphorylation. ability of FOXO1 to bind DNA and suppress transcription. Interestingly, it wasThe also FOXO1 found protein that acetylation is polyubiquitinated regulated the functionby E3 ligases of FOXO1 (Figure by influencing 1C) such itsas sensitivityS-phase kinase-associatedfor phosphorylation. protein 2 (SKP2) and mouse double minute 2 homolog (MDM2), and targeted for proteinThe degradation FOXO1 protein in human is polyubiquitinated primary tumors by and E3 ligases cancer (Figure cell lines1C) such[23–25]. as S-phase The ubiquitination kinase-associated and proteasomeprotein 2 (SKP2) degradation and mouse of the double FOXO1 minute protein 2 homolog are essential (MDM2), in tumorigenesis and targeted for and protein represent degradation a viable in targethuman for primary cancer treatment. tumors and cancer cell lines [23–25]. The ubiquitination and proteasome degradation of the FOXO1 protein are essential in tumorigenesis and represent a viable target for cancer treatment. 2.2. mRNA Methylation 2.2. mRNA Methylation In addition to these post-translational modifications on protein, FoxO1 is also regulated by In addition to these post-translational modifications on protein, FoxO1 is also regulated by methylation modification on its mRNA [26–28] (Figure 1D). There are two specific adenosine sites in methylation modification on its mRNA [26–28] (Figure1D). There are two specific adenosine sites in the coding sequence of the FoxO1 mRNA [26,27]. The adenosine (A) at these sites can be methylated the coding sequence of the FoxO1 mRNA [26,27]. The adenosine (A) at these sites can be methylated to N6-Methyladenosine (m6A) by methyltransferase like 3 (METTL3) protein, and m6A can be to N6-Methyladenosine (m6A) by methyltransferase like 3 (METTL3) protein, and m6A can be demethylated to A by fat mass and obesity-associated protein (FTO). These reversible m6A demethylated to A by fat mass and obesity-associated protein (FTO). These reversible m6A modifications modifications participate in regulating FoxO1 gene translation [28]. participate in regulating FoxO1 gene translation [28].

FigureFigure 1. 1. RegulationRegulation of of FOXO1FOXO1 transcriptiontranscription activity. activity. (A (A) )FOXO1 FOXO1 binds binds to to the the promoters promoters of of target target genesgenes and and stimulates stimulates their their transcription transcription in in nucleus. nucleus. (B (B) )The The complex complex structure structure of of the the FOXO1 FOXO1 protein protein andand its its target-binding target-binding DNA DNA (Protein (Protein Data Data Bank Bank id: id: 3CO6). 3CO6). (C (C) The) The post-translational post-translational modifications modiﬁcations of of thethe FOXO1 FOXO1 protein. protein. (D (D) )Methylations Methylations on on the the FoxO1FoxO1 mRNAmRNA regulate regulate its its expression. expression. This This figure ﬁgure was was createdcreated with with UCSF UCSF Chimera Chimera [29] [29 ]and and BioRender.com. BioRender.com .

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3. FoxO1-Regulating Mechanism Behind Diseases

3.1. Glucose Production in the Liver FoxO1 regulates many important metabolic pathways in the liver, fat tissue, and hypothalamus [30,31]. It is well established that FoxO1 regulates hepatic gluconeogenesis and glycogenolysis in response to an insulin signal in the blood (Figure 2). High concentration of insulin decreases blood glucose level by promoting glucose absorption after feeding and inhibits glucose production by hepatic gluconeogenesis and glycogenolysis in the fasting state. In the liver, insulin activates the PI3K/PKB signaling pathway and results in FOXO1 protein phosphorylation and degradation [32]. FOXO1 TF binds and promotes transcription of glucose 6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase (PEPCK), which are key enzymes stimulating gluconeogenesis and glycogenolysis [33,34]. When FOXO1 is suppressed, transcription of G6PC and PEPCK subsequently decreases, which consequently inhibits the rates of glucose production in the liver. It has been well established that FOXO1 is a key mediator in the signaling pathway of insulin regulating hepatic gluconeogenesis. Hepatic FOXO1 loss-of-function mutant suppresses G6PC and PEPCK expression, decreases hepatic gluconeogenesis, and improves fasting glycemia in diabetic db/db mice [32].

Cells3.2. Lipoprotein2020, 9, 184 Uptake in the Liver 3 of 10 Apolipoprotein C-III (ApoC3) is another downstream target of FoxO1 that functions directly in plasma 3.triglycerideFoxO1-Regulating metabolism Mechanism [31,35]. ApoC3 Behind is Diseasessecreted by the liver and enriched in very low-density lipoprotein (VLDL). It was reported to suppress hepatic uptake of VLDL and inhibit lipoprotein lipase 3.1. Glucose Production in the Liver [36]. ApoC3 overexpression in humans functions in atherosclerosis [37]. FoxO1FOXO1regulates binds to many the importantApoC3 promoter metabolic and pathways enhanc ines the its liver, transcription fat tissue, and[35] hypothalamus (Figure 2). FOXO1 [30,31]. Itoverexpression is well established increases that FoxO1hepaticregulates ApoC3 expression hepatic gluconeogenesis and elevates plasma and glycogenolysis triglyceride levels. in response FOXO1 to anloss-of-function insulin signal mutation in the blood interferes (Figure with2). High ApoC3 concentration expression ofin insulinresponse decreases to insulin blood stimulation. glucose level Insulin by promotingdeficiency or glucose resistance absorption results afterin unrestrained feeding and Ap inhibitsoC3 expression glucose production and impaired by hepatic triglyceride gluconeogenesis metabolism andin the glycogenolysis pathogenesis of in atherosclerosis the fasting state. and hypertriglyceridemia.

Figure 2.2. RegulationRegulation ofof glucose glucose production production and and lipoprotein lipoprotein uptake uptake by FOXO1by FOXO1in the in liverthe liver (created (created with BioRender.comwith BioRender.com).).

In the liver, insulin activates the PI3K/PKB signaling pathway and results in FOXO1 protein phosphorylation and degradation [32]. FOXO1 TF binds and promotes transcription of glucose3

6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase (PEPCK), which are key enzymes stimulating gluconeogenesis and glycogenolysis [33,34]. When FOXO1 is suppressed, transcription of G6PC and PEPCK subsequently decreases, which consequently inhibits the rates of glucose production in the liver. It has been well established that FOXO1 is a key mediator in the signaling pathway of insulin regulating hepatic gluconeogenesis. Hepatic FOXO1 loss-of-function mutant suppresses G6PC and PEPCK expression, decreases hepatic gluconeogenesis, and improves fasting glycemia in diabetic db/db mice [32].

3.2. Lipoprotein Uptake in the Liver Apolipoprotein C-III (ApoC3) is another downstream target of FoxO1 that functions directly in plasma triglyceride metabolism [31,35]. ApoC3 is secreted by the liver and enriched in very low-density lipoprotein (VLDL). It was reported to suppress hepatic uptake of VLDL and inhibit lipoprotein lipase [36]. ApoC3 overexpression in humans functions in atherosclerosis [37]. FOXO1 binds to the ApoC3 promoter and enhances its transcription [35] (Figure2). FOXO1 overexpression increases hepatic ApoC3 expression and elevates plasma triglyceride levels. FOXO1 loss-of-function mutation interferes with ApoC3 expression in response to insulin stimulation. Insulin deﬁciency or resistance results in unrestrained ApoC3 expression and impaired triglyceride metabolism in the pathogenesis of atherosclerosis and hypertriglyceridemia. Cells 2019, 8, x FOR PEER REVIEW 4 of 10

3.3. Lipogenesis in the Liver Clinically, it was observed that therapeutically decreasing blood glucose usually caused increased lipogenesis in the liver [11,38]. Lipogenesis is induced by suppressing the FoxO1-dependent inhibition of glucokinase (Gck) [39] (Figure 3). Hepatic Gck expression is associated with lipogenesis and fatty liver in Cells 2020, 9, 184 4 of 10 humans. Increased Gck activity induced fatty liver and its metabolic consequences in humans. Moreover, SIN3 Transcription Regulator Family Member A (SIN3A) was identified as an 3.3.insulin-sensitive Lipogenesis in theFOXO1 Liver corepressor of Gck. SIN3A knockout interferes with Gck expression regulated by FOXO1 in response to environmental nutrients, while not affecting expression of other Clinically, it was observed that therapeutically decreasing blood glucose usually caused increased genes targeted by FOXO1, such as G6PC. These results may provide the opportunity to develop lipogenesis in the liver [11,38]. Lipogenesis is induced by suppressing the FoxO1-dependent inhibition selective modulators of the FoxO1 pathway, which removes adverse effects of hepatic lipogenesis in of glucokinase (Gck)[39] (Figure3). Hepatic Gck expression is associated with lipogenesis and fatty liver therapeutically treating diabetes [39]. in humans. Increased Gck activity induced fatty liver and its metabolic consequences in humans.

Figure 3. Regulation of lipogenesis by FOXO1 in the liver (created with BioRender.com ).

Moreover,FigureSIN3 3. Regulation Transcription of lipogenesis Regulator by FamilyFOXO1 Memberin the liver A (created(SIN3A )with was BioRender.com). identified as an insulin- sensitive FOXO1 corepressor of Gck. SIN3A knockout interferes with Gck expression regulated by 3.4. Lipid Metabolism in Adipocytes FOXO1 in response to environmental nutrients, while not affecting expression of other genes targeted by FOXO1FoxO1, suchbinds as toG6PC the .promoter These results sites may of provideperoxisome the proliferator-activated opportunity to develop receptor selective gamma modulators (PPARγ) ofDNA the FoxO1and repressespathway, its which transcription removes adverse[40]. In e ffrespectsonse of hepatic to insulin lipogenesis stimulation, in therapeutically FOXO1 protein treating is diabetesdegraded [39 and]. subsequently unable to prevent transcription of PPARγ. Because high levels of PPARγ protein initiate adipogenesis, FoxO1 suppresses adipogenesis [41]. 3.4. LipidMoreover, Metabolism the FOXO1 in Adipocytes protein is a repressor of uncoupling protein 1 (Ucp1) gene transcription [31,42].FoxO1 UCP1binds protein to the is promoter a mitochondrial sites of peroxisomeinner memb proliferator-activatedrane proton channel receptor linked gamma to energy(PPAR useγ) DNA that andserves represses as a well-known its transcription biomarker [40]. In of response the ther tomogenesis insulin stimulation, state of adipose FOXO1 proteintissues is[43–45]. degraded FoxO1 and subsequentlyinhibition increases unable UCP1 to prevent expression, transcription thereby of PPARaugmentingγ. Because thermogenesis high levels ofand PPAR fat γloss.protein Selectively initiate adipogenesis,inhibiting FTOFoxO1 by inhibisuppressestors decreases adipogenesis FOXO1 [expression41]. and reduces body weight and fat mass in a high-fatMoreover, diet-induced the FOXO1 obesity protein (DIO) is a repressormouse mode of uncouplingl. Suppressing protein FOXO1 1 (Ucp1 )ex genepression transcription increased [31 ,42the]. UCP1energy protein expenditure is a mitochondrial of mice. Thermogenesis inner membrane in protonadipose channel tissue linkedwas induced to energy by use reduced that serves FOXO1 as a well-knownexpression, which biomarker was ofa cause the thermogenesis of the decreased state body of adipose weight tissues and increased [43–45]. FoxO1 energyinhibition expenditure increases [28]. UCP1 expression, thereby augmenting thermogenesis and fat loss. Selectively inhibiting FTO by inhibitors decreases FOXO1 expression and reduces body weight and fat mass in a high-fat diet-induced obesity (DIO) mouse model. Suppressing FOXO1 expression increased the energy expenditure of mice. Thermogenesis in adipose tissue was induced by reduced FOXO1 expression, which was a cause of the decreased body weight and increased energy expenditure [28]. 4

3.5. Feeding Behavior in the Hypothalamus FoxO1 in the central nervous system, mainly the hypothalamus, functions directly in integrating signals from peripheral tissues and mediating feeding behavior. Insulin and leptin are well-studied Cells 2019, 8, x FOR PEER REVIEW 5 of 10

3.5. Feeding Behavior in the Hypothalamus FoxO1 in the central nervous system, mainly the hypothalamus, functions directly in integrating Cellssignals2020 ,from9, 184 peripheral tissues and mediating feeding behavior. Insulin and leptin are well-studied5 of 10 nutrient signals, integrating peripheral energy status to the hypothalamus. In the arcuate nucleus nutrient(ARC) of signals, the integratinghypothalamus, peripheral two energyneuronal status populations to the hypothalamus. express specific In the arcuatefeeding-related nucleus (ARC)neuropeptides, of the hypothalamus, including pro-opiomelanocortin two neuronal populations (POMC) express and speciﬁc agouti-related feeding-related peptide neuropeptides, (AgRP) [46]. includingPOMC suppresses pro-opiomelanocortin appetite and (POMC)decreases and body agouti-related weight. AgRP peptide enhanc (AgRP)es food [46 intake]. POMC and suppresses increases appetitebody weight and decreases[47,48]. body weight. AgRP enhances food intake and increases body weight [47,48]. The PI3KPI3K/PDK1/PKB–FoxO1/PDK1/PKB–FoxO1 signalingsignaling axisaxis functionsfunctions to integrateintegrate leptin and insulin signals to regulate POMC and AgRP secretion [[49–51]49–51] (Figure4 4).). AsAs thethe downstreamdownstream targettarget ofof thesethese kinases,kinases, the FOXO1FOXO1 protein protein is phosphorylatedis phosphorylated and inactivated and inactivated in neurons, in thusneurons, promoting thus POMCpromoting transcription POMC andtranscription suppressing and AgRP suppressing transcription. AgRP In transcription. mouse models, InPDK1 mouseknockout models, in PDK1 POMC knockout neurons suppressesin POMC FOXO1neurons phosphorylation suppresses FOXO1 and degradation, phosphorylation thus suppressing and degradation, POMC transcription. thus suppressing By contrast, inPOMC AgRP neurons,transcription.PDK1 Bydeletion contrast, results in AgRP in enhancedneurons, PDK1 AgRP deletion expression results and in increased enhanced food AgRP intake. expression In the PDK1and increased-knockout food mouse intake. models, In theFoxO1 PDK1inactivation-knockout rescuesmouse thesemodels, phenotypes, FoxO1 inactivation which demonstrates rescues these the crucialphenotypes, roles ofwhichFoxO1 demonstratesin regulating the feeding crucial behavior. roles of FoxO1 in regulating feeding behavior.

Figure 4. Regulation of appetite by FOXO1 in the hypothalamus (created with BioRender.com). Figure 4. Regulation of appetite by FOXO1 in the hypothalamus (created with BioRender.com). Furthermore, in a DIO rat model, the mechanism of FoxO1 in regulating feeding behavior was conﬁrmedFurthermore, [52]. In in these a DIO rats, rat becausemodel, the of insulinmechanism resistance, of FoxO1 the inPI3K regulating/PDK1/PKB-FoxO1 feeding behaviorregulatory was axisconfirmed was damaged, [52]. In these resulting rats, in because increasing of insulin the amount resistance, of FOXO1 the proteinPI3K/PDK1/PKB-FoxO1 and food intake. regulatory However, theaxis intracerebroventricular was damaged, resulting (i.c.v.) in increasing micro-infusion the amount of FoxO1 of FOXO1-antisense protein oligonucleotides and food intake. (FoxO1 However,-ASOs) largelythe intracerebroventricular decreased the hypothalamic (i.c.v.) micro-infusionFoxO1 expression, of FoxO1 food-antisense intake, oligonucleotides and body weight (FoxO1 gain.-ASOs) These suggestedlargely decreased that pharmacological the hypothalamic inactivation FoxO1 of expression,FoxO1 may befood used intake, to suppress and body appetite weight and treatgain. obesity. These suggested that pharmacological inactivation of FoxO1 may be used to suppress appetite and treat 4.obesity.FoxO1 -Related Drug Discovery Although some biologic therapies antagonizing FOXO1 activity were reported in treating cancers, 4. FoxO1-Related Drug Discovery wound repair, and cardiovascular diseases [53–57], many eﬀorts are still focused on designing small-moleculeAlthough some compounds biologic indicated therapies for treatingantagonizing metabolic FOXO1 disorders activity [58 ].were reported in treating cancers, wound repair, and cardiovascular diseases [53–57], many efforts are still focused on 4.1.designing FOXO1 small-molecule Protein Inhibitors compounds indicated for treating metabolic disorders [58].

Selectively silencing FOXO1 has the potential to treat metabolic disorders. In a mass spectrometric5 a ffinity screening, a small-molecule compound, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (AS1842856), was identified to directly bind to the FOXO1 protein, and it selectively bound to the dephosphorylated activated form, but not the phosphorylated one Cells 2019, 8, x FOR PEER REVIEW 6 of 10

4.1. FOXO1 Protein Inhibitors Selectively silencing FOXO1 has the potential to treat metabolic disorders. In a mass spectrometric affinity screening, a small-molecule compound, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856), was identified to directly bind to the FOXO1 protein, and it selectively bound to the dephosphorylated activated form, but not the phosphorylated one (Figure 5A). Cellular assay showed that AS1842856 blocked the transcription activity of FOXO1 with an IC50 of 0.03 μM but much less potently inhibited FOXO3a and FOXO4 with IC50 larger than 1 μM, which suggested that AS1842856 could be a potent and selective FOXO1 inhibitor [59,60]. In a diabetic db/db mouse model, oral administration of AS1842856 resulted in largely decreased plasma glucose after fasting and suppressed expression of gluconeogenic genes. Focused on FOXO1 activity inhibition, one cell-based high-throughput screening of 170,000 small-molecule compounds resulted in a number of hits. Among them, FOXO1 inhibitor AS1708727 was identified with high oral activity, low clearance, and high liver distribution in mice [61] (Figure 5B). In a diabetic db/db mouse model, treatment with AS1708727 largely reduced blood glucose and blood triglyceride and decreased expression of hepatic G6PC, PEPCK, and ApoC3.

4.2. FoxO1 Pathway Modulators Considering that FOXO1 is a TF, it is not easy to manipulate FoxO1 through pharmacological approaches. Therefore, excepting the FOXO1 protein itself, researchers also pursue design modulators to selectively mediate the FoxO1 pathway. After a small-molecule screening, a series of selective inhibitors of the FoxO1 pathway were identified [39]. Interestingly, some compounds inhibited FoxO1-dependent G6PC transcription and enhanced GCK transcription in hepatocytes, while other compounds decreased G6PC transcription without significant influence on GCK transcription. These compounds inhibited glucose production without lipogenic activity. Among them, cpd-10 was reported by a group from AstraZeneca as AZ-4490, with an IC50 of 0.02 μM [62] (Figure 5C). In addition to experimental compound screening, selective inhibitors of the FoxO1 pathway Cells 2020, 9, 184 6 of 10 were designed rationally. FTO protein demethylases the m6A modifications on FoxO1 mRNA and enhances its expression. Selective inhibition of FTO protein decreases the amount of FOXO1 protein (Figurein vivo.5 A).Via Cellularstructure-based assay showed drug design, that AS1842856 entacapone blocked and its the analogues transcription were activity identified of FOXO1 as selectivewith ansmall-molecule IC50 of 0.03 µ inhibitorsM but much of the less FTO potently protein inhibited (Figure FOXO3a 5D). Entacapone and FOXO4 decreased with IC 50FOXO1larger expression, than 1 µM, whichthus suppressing suggested that gluconeogenesis AS1842856 could and be aincreasi potentng and thermogenesis selective FOXO1 in inhibitor mouse [models59,60]. In(Figure a diabetic 6). dbSelectively/db mouse suppressing model, oral FoxO1 administration activity through of AS1842856 the inhibition resulted inof largelyFTO by decreased entacapone plasma provides glucose the afterpossibility fasting to and treat suppressed type II diabetes expression and obesity of gluconeogenic [28]. genes.

Figure 5.5. InhibitorsInhibitors of of the the FOXO1 FOXO1 protein protein and andFOXO1 FOXO1pathway. pathway. (A,B )( InhibitorsA,B) Inhibitors of the FOXO1of the FOXO1 protein. (protein.C) Inhibitors (C) Inhibitors of FoxO1 -dependentof FoxO1-dependent glucose production glucose production without enhancing without lipogenesis.enhancing lipogenesis. (D) Entacapone (D) suppressingEntacapone FOXO1suppressing protein FOXO1 expression protein through expr inhibitingession fatthrough mass andinhibiting obesity-associated fat mass protein and (FTO)obesity-associated enzymatic activity. protein (FTO) enzymatic activity.

Focused on FOXO1 activity inhibition, one cell-based high-throughput screening of 170,000 small-molecule compounds resulted in a number of hits. Among them, FOXO1 inhibitor AS1708727 was identified with high oral activity, low clearance, and high liver distribution in mice [61] (Figure5B). In a diabetic db/db mouse model, treatment with AS1708727 largely reduced blood glucose and blood 6 triglyceride and decreased expression of hepatic G6PC, PEPCK, and ApoC3. 4.2. FoxO1 Pathway Modulators Considering that FOXO1 is a TF, it is not easy to manipulate FoxO1 through pharmacological approaches. Therefore, excepting the FOXO1 protein itself, researchers also pursue design modulators to selectively mediate the FoxO1 pathway. After a small-molecule screening, a series of selective inhibitors of the FoxO1 pathway were identified [39]. Interestingly, some compounds inhibited FoxO1-dependent G6PC transcription and enhanced GCK transcription in hepatocytes, while other compounds decreased G6PC transcription without significant influence on GCK transcription. These compounds inhibited glucose production without lipogenic activity. Among them, cpd-10 was reported by a group from AstraZeneca as AZ-4490, with an IC50 of 0.02 µM[62] (Figure5C). In addition to experimental compound screening, selective inhibitors of the FoxO1 pathway were designed rationally. FTO protein demethylases the m6A modifications on FoxO1 mRNA and enhances its expression. Selective inhibition of FTO protein decreases the amount of FOXO1 protein in vivo. Via structure-based drug design, entacapone and its analogues were identified as selective small-molecule inhibitors of the FTO protein (Figure5D). Entacapone decreased FOXO1 expression, thus suppressing gluconeogenesis and increasing thermogenesis in mouse models (Figure6). Selectively suppressing FoxO1 activity through the inhibition of FTO by entacapone provides the possibility to treat type II diabetes and obesity [28]. Cells 2020, 9, 184 7 of 10 Cells 2019, 8, x FOR PEER REVIEW 7 of 10

Figure 6.6.Entacapone Entacapone suppresses suppresses gluconeogenesis gluconeogenesis in the in liver the and liver increases and increases thermogenesis thermogenesis in adipocytes in throughadipocytes reducing through FOXO1 reducing expression FOXO1 (createdexpression with (created BioRender.com with BioRender.com).).

AuthorAuthor Contributions:Contributions: Conceptualization,Conceptualization, S.P. S.P. and and N.H. N.H. (Niu (Niu Huang); Huang); writing—-original writing––original draft draft preparation, preparation, S.P.; writing—-reviewS.P.; writing––review and editing,and editing, W.L., W.L., N.H. (NannanN.H. (Nanna Hou),n N.H.Hou), (Niu N.H. Huang); (Niu Huang); supervision, supervision, N.H. (Niu N.H. Huang). (Niu AllHuang). authors All have authors read have and read agreed and to agreed the published to the publishe versiond of version the manuscript. of the manuscript.

Funding: This research received no external funding.

ConﬂictsConflicts ofof Interest:Interest: TheThe authorsauthors declaredeclare nono conﬂictsconflicts ofof interest.interest.

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