A Novel Inhibitor of the Obesity-Related Protein FTO - American

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A Novel Inhibitor of the Obesity-Related Protein FTO Yan Qiao, Bin Zhou, Meizi Zhang, Weijia Liu, Zhifu Han, Chuanjun Song, Wenquan Yu, Qinghua Yang, Ruiyong Wang, Shaomin Wang, Shuai Shi, Renbin Zhao, Jijie Chai, and Junbiao Chang Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00023 • Publication Date (Web): 25 Feb 2016 Downloaded from http://pubs.acs.org on February 27, 2016

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Biochemistry

A Novel Inhibitor of the Obesity-Related Protein FTO

Yan Qiao,1,▽Bin Zhou,2,3,▽Meizi Zhang,4 Weijia Liu,2,3Zhifu Han,2,3 Chuanjun Song,5 Wenquan Yu,5 Qinghua Yang,5 Ruiyong Wang,5 Shaomin Wang,5 Shuai Shi,5 Renbin Zhao,4 Jijie Chai,*, 2,3 and Junbiao Chang*,1

1

Pathophysiology Department, Basic Medical College of Zhengzhou University, Zhengzhou

450001, PR China 2

School of Life Sciences, Tsinghua University, Beijing 100084, PR China

3

Tsinghua-Peking Center for Life Sciences, Beijing 100084, PR China

4

Space Biology Research and Technology Center, Engineering Research Center of Space Biology, China Academy of Space Technology, Beijing 100190, PR China

5

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou

450001, PR China

▽

Y. Qiao and B. Zhou contributed equally to this work

To whom correspondence should be addressed: Prof. Jijie Chai, School of Life Sciences, Tsinghua University, Beijing 100084, PR China, Email: [email protected] Prof. Junbiao Chang, Professor of Basic Medical College of Zhengzhou University, Zhengzhou 450001, PR China. Email: [email protected]; Phone: +86-371-67781788 Running title: Identification of a Novel FTO Inhibitor Keywords: FTO, AlkB, Inhibitor, N6-methyl adenosine, Demethylation

1

ACS Paragon Plus Environment

Biochemistry

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ABSTRACT FeII and α-ketoglutarate-dependent fat mass and obesity associated protein (FTO)-dependent demethylation of m6A is important for regulation of mRNA splicing and adipogenesis. Developing FTO specific inhibitors can help probe the biology of FTO and unravel novel therapeutic targets for treatment of obesity or obesity-associated diseases.

In

the

present

paper,

we

have

identified

4-chloro-6-(6′-chloro-7′-hydroxy-2′,4′,4′-trimethyl-chroman-2′-yl)benzene-1,3-diol (CHTB) is an inhibitor of FTO. The crystal structure of CHTB complexed with human FTO reveals that the novel small molecule binds to FTO in a specific manner. The identification of the novel small molecule offers opportunities for further development of more selective and potent FTO inhibitors.

Introduction Cellular DNA and RNA are subjected to various forms of methylation.1 In eukaryotic mRNAs, N6-methyl adenosine (m6A) is the most prevalent modification, with more than 80% of all RNA base methylations and a frequency of 1 m6A per 2000 ribonucleotides on average. This modification has been associated with regulation of mRNA fate including metabolism, transcription, translation, splicing and their nuclear export.2-5 A recent study showed that m6A mRNA methylation affects the mammalian circadian clock.6 In support of its important roles in vivo, dysregulation of m6A modification has been suspected of involvement in human diseases.7 AlkB family members of FeII/α-ketoglutarate (αKG)-dependent dioxygenases possess both DNA and RNA demethylation activities and thus are critical mediators of nucleic acid methylation.1,

8

The Escherichia coli AlkB has been demonstrated to demethylate

N3-methylcytosine

(m3C),

N1-methyladenine

(m1A),

N3-methylthymidine

(m3T),

N1-methylguanine (m1G) and other lesions in DNA/RNA.9, 10 In mammals, nine homologs exist in the AlkB family, including ABH1-8 and fat mass and obesity associated protein (FTO).11 These AlkB family members show different substrate recognition specificity due to their unique structural characteristics. For example, ABH1 can demethylate m3C in both RNA 2


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Biochemistry

and DNA.11 ABH2 and ABH3 repair the same spectrum of lesions as AlkB, but ABH3 displays higher activities with ssDNA and ssRNA in vitro whereas ABH2 acts as the primary housekeeping enzyme in mammals for repairing endogenously formed alkylated lesions in dsDNA.12, 13 ABH8 is a tRNA methyltransferase required for wobble uridine modification and DNA damage survival.14 FTO was initially shown to possess demethylation activity toward various single-stranded methylated nucleic acids.15-17 Subsequent structural study on FTO showed that a unique extra loop confers its ability to discriminate against double-stranded nucleic acids.18 Now, only m6A in nuclear RNA was confirmed as a physiological substrate of FTO.17 More recently, another AlkB homolog in humans, ABH5, has also been shown to possess m6A demethylase activity.19-22 Nevertheless, the two enzymes function in different physiological processes likely by regulating different subsets of mRNAs.5 FTO has been identified to be associated with obesity and type II diabetes in several genome-wide-association studies.23-25 However, the mechanism by which FTO affects metabolism has been poorly understood until the recent discovery that FTO-dependent demethylation of m6A functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.5 Although FTO has for a long time been implicated in obesity, whether it is a valid drug target remains ambiguous. Thus, developing FTO-specific inhibitors is of great importance to evaluate the therapeutic potential of FTO.26 Following the FTO crystal structure resolution by our group in 2010.18 the development of FTO-specific inhibitors has drawn much attention from pharmaceutical chemists. By now, FTO inhibitors such as rhein, αKG analogues, meclofenamic acid and compounds occupying both αKG and substrate binding sites have been reported.27-31 It is worth mentioning that our group has also been dedicated to the development of FTO-specific inhibitors. In particular, we have recently identified a novel small-molecule binding site of FTO and ascertained that the small

molecule,

N-(5-Chloro-2,4-dihydroxyphenyl)-1-phenylcyclobutanecarboxamide

(N-CDPCB), is a selective inhibitor binding at this site.32 The identification of this novel binding site has offered great opportunities for the design of more specific and potent FTO inhibitors. Inspired by the previous work, we tried to screen some other potent FTO inhibitors. To

our

delight,

we

found

that

4-chloro-6-(6′-chloro-7′-hydroxy-2′,4′,4′-trimethyl-chroman-2′-yl)benzene-1,3-diol 3


compound (CHTB)

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can inhibit FTO demethylation activity. The crystal structure of CHTB complexed with FTO reveals that it binds with FTO in different manners with N-CDPCB. Intriguingly, CHTB binds a similar site with meclofenamic acid although the chemical structure of this small molecule is completely different from that of meclofenamic acid. EXPERIMENTAL PROCEDURES Protein Expression and Purification―The FTO protein (residues 31-505) was expressed and purified as described previously.18, 32 Chemical

synthesis

of

4-chloro-6-(6′-chloro-7′-hydroxy-2′,4′,4′-trimethyl-chroman-2′-yl)benzene-1,3-diol (CHTB)―The title compound was synthesized according to a literature procedure.33 A solution of Me2CO (802 mg, 13.8 mmol), 2,4-dihydroxychlorobenzene (3.0 g, 22.7 mmol) and p-toluenesulfonic acid (237 mg, 1.4 mmol) in 20 mL of CHCl3 was heated to 40 °C and stirred for 5 h until a viscous oil was produced. Then the mixture was heated to boiling, refluxed for 0.5 h and cooled. The formed precipitate was filtered off. 20 mL of H2O were added, and the contents were heated with stirring. Next, the aqueous layer was separated, the organic layer was evaporated to dryness, and the residue was crystallized from petroleum ether to afford the product as a pale yellow solid in 50% yield; 1H NMR (400 MHz, CDCl3) δ 7.69 (br, 1H), 7.15 (s, 1H), 7.05 (s, 1H), 6.59 (s, 1H), 6.49 (s, 1H), 5.61 (br, 2H), 2.53 (d, J = 14.4 Hz, 1H), 1.97 (d, J = 14.4 Hz, 1H), 1.64 (s, 3H), 1.35 (s, 3H), 1.10 (s, 3H) ppm; HRMS (ESI, m/z) calcd for C18H18Cl2NaO4 [M + Na]+: 391.0474, found 391.0488. Crystallization, Data

collection, Structure Determination

and Refinement―the

crystallization experiments in the present paper also followed the previous paper32 and thus were not described in detail here. Diffraction data were also collected on the BL17U1 beam-line of the Shanghai Synchrotron Research Facility (SSRF), and the datasets were processed using HKL2000.34 The structure of FTO complexed with CHTB was determined using isomorphous replacement as the coordinates of free FTO (PDB code: 3LFM) as initial model. After refinement of the initial model, CHTB was built into the electron density in COOT.35 The structure of CHTB-FTO complex was finally refined to a resolution of 2.0 Å with Rfactor 19.8% and Rfree 23.6%, respectively.

Isothermal Titration Calorimetry (ITC)―The Isothermal Titration Calorimetry (ITC) 4


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Biochemistry

experiments were carried out at 25 °C with a Microcal ITC200 isothermal titration calorimeter (GE Healthcare). Purified FTO protein stock solution in a buffer containing 25 mM HEPES (pH 7.5), 100 mM NaCl, and 10 mM β-mercaptoethanol was subjected to ITC experiment. The titrant was FTO at a concentration of 180 µM in 5% DMSO. 20 µM CHTB solutions were prepared from the stock solution to contain 5% DMSO. To correct the thermal effect due to mixing and dilution, a control experiment was performed by injecting the titrant into the buffer without CHTB. The 0.2 mL sample cell was filled with 20 µM CHTB and stirred constantly at 750 rpm. The syringe was filled with 180 µM FTO and titrated into the sample cell in one 0.4 µL injection, followed by 2.0 µL injections at 150 s intervals. After the background dilution heats were subtracted from the experimental data, the net titration data were analyzed with the Microcal ORIGIN V7.0 software (Microcal Software, Northampton, MA). In vitro enzymatic activity assays―In vitro enzymatic activity assays were performed as previously described.36 The 15-mer ssRNA (5′-CUUGUCA (m6A)CAGCAGA-3′) was used as a substrate to evaluate the demethylase activity of FTO. All the reactions were carried out in 50 µL of buffer containing 50 mM Tris-HCl pH 7.5, 2.0 M ssRNA, 2.0 nM FTO, 1.0 mM α-KG, 280 µM (NH4)2Fe(SO4)2 and 2 mM L-ascorbic acid. CHTB with varying concentrations (1.00 M, 4.00 M, 8.00 M, 16.00 M, 32.00 M, 40.00 M, 64.00 M, 84.00 M, 164.00 M and 328.00 M) were individually added to each reaction mixture and incubated at room temperature for 0.5 h. The reactions were quenched by heating for 5 min at 95 °C. The ssRNA was digested by nuclease P1 (1 Unit) and NH4OAc (100 µM, 5 µL) at 42 ºC for 4 h, followed by the addition of NH4HCO3 (1.0 M, 5 µL), and alkaline phosphatase (0.5 Unit). After an additional incubation at 37 °C for 3 h, 6-Cl-G(10 µg/ml, 5 µl) was added as an internal reference, then the solution was diluted to 100 µL, and then 10 µL of the solution was injected into LC-MS/MS. The nucleosides were separated by reverse phase high-performance liquid chromatography on a C18 column, with online mass spectrometry detection using Thermo TSQ Quantum Ultra LC/MS in positive electrospray ionization mode. The nucleosides were quantified using the nucleoside to base ion mass transitions of 282 to 150 (m6A), and 268 to 136 (A). Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running on the same batch of samples. 5


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Analysis of m6A levels in mRNA using dot blot―HEK293 cells were plated at 3x105 per well in six-well plates and transfected with plasmid pcDNA3.1-N-6His-3myc-FTO-1-50518 using Lipofectamine 2000 (Invitrogen, USA) following manufacturer’s instruction. Transfection media were removed after 24 hours and cells were cultured in normal growing media for 48 hours. Cells were then passed 1:10 into 10 cm dishes and grown in selective media with G418 (Sigma, cat.A1720) for 21 days when single colonies were picked from each culture. Total RNAs were isolated from HEK293 cell lines with TRIZOL reagent (Invitrogen, cat.15596-018). Cellular mRNAs were isolated with Poly(A) Purist Kit (Ambion, cat.AM1916), followed by rRNA removal using Ribo Minus Transcriptom Isolation Kit (Invitrogen, cat.K1550-01). The concentration and quality of mRNA were determined by NanoDrop (Thermo) and Agilent 2100 bioanalyzer. Purified mRNA was denatured at 95 ºC for 5 min and then chilled on ice. Two fold serial dilutions were spotted onto Amersham Hybond-N+ membranes (GE Healthcare, cat.RPN303B). After crosslinking at 80 ºC for 2 hours, the membranes were blocked with 5% nonfatmilk in TBST for 1 hour, and incubated with rabbit anti-m6A antibody (1:2000, SySy, cat.202003) overnight at 4ºC. Membranes were then incubated with peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) for 1 hour at room temperature, and visualized by ImmobilionTM western Chemoluminescent HRP Substrate (Millipore, cat. WBKL S0050). The intensity of each spot was quantified using ImageJ software(NIH, USA).

Cell-based activity―Differentiated 3T3-L1 cells from American Type Culture Collection (ATCC) were cultured in differentiating media supplemented with tested compounds at concentrations of 1-10 µM. For 3T3-L1 differentiation, post-confluent preadipocytes were incubated with a cocktail of insulin (1 µg/ml, Sigma, cat.I5500), dexamethasone (1 µM, Sigma, cat.D4902), and 3-isobutyl-1-methylxanthine (0.5 µM, Sigma, cat.I7018) in DMEM supplemented with 10% fetal bovine serum (Hyclone) for 48 hours, followed by culture with DMEM, 10% fetal bovine serum and 1 µg/ml insulin for another 48 hours. The media were then removed and replaced with DMEM plus 10% fetal bovine serum until collection for differentiation assessment. After day 8 of differentiation, Cell viability was determined by cell counting kit-8 (CCK-8) (Dojindo Laboratorise, Japan), a colorimetric assay based on the formation of water-soluble yellow formazan by mitochondrial dehydrogenase in active 6


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Biochemistry

mitochondria.

RESULTS Recognition of CHTB by FTO. The FTO inhibitor (CHTB) (Figure 1A) was identified using the same method as that of N-CDPCB, for which the detailed procedures have been described in our recently published paper.32 To validate the activity of CHTB compound in FTO inhibition, we used the 15-mer ssRNA (5′-CUUGUCA(m6A)CAGCAGA-3′) as a substrate and quantified FTO demethylase activity in the presence of CHTB using LC-MS. The results from this assay showed CHTB reduces FTO enzymatic activity in a dose-dependent manner with an IC50 ~39.24 M (Figure 1B), thus supporting the postulated competitive inhibition of FTO demethylase activity by CHTB in vitro.

Figure 1. Identification of an FTO inhibitor. A, Molecular formula of m3U (top panel) and CHTB (bottom panel). Numbers indicate the positions of the two hydroxyl groups. B, Dosage-dependent inhibition of FTO demethylase activity by CHTB. The 15-mer ssRNA, 5′-CUUGUCA(m6A)CAGCAGA-3′, was used to determine FTO demethylase activity in the presence of varied concentrations of CHTB (1.00 M, 4.00 M, 8.00 M, 16.00 M, 32.00 M, 40.00 M, 64.00 M, 84.00 M, 164.00 M and 328.00 M). FTO activity (%, vertical axis) was plotted against logarithm of CHTB concentrations (horizontal axis).

Next, to determine whether CHTB inhibits FTO demethylase activity via a direct interaction, we measured the binding affinities using isothermal titration calorimetry (ITC). Due to the limited solubility of CHTB even in the presence of 10% DMSO, initial forward 7


Biochemistry

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titration in which CHTB was used as the titrant and FTO as the titrand failed to generate an ideal fit for the thermogram. We therefore switched to reverse titration using FTO as the titrant, which produced statistically larger exothermicity than the background and gave a good fit for the thermogram. The ITC results showed that CHTB interacts with FTO with a dissociation constant of ~1.0 M (Figure 2), further supporting the biochemical assay that showed the compound displaying strong inhibition of FTO demethylation of ssRNA.

Figure 2. CHTB Directly Interacts with FTO. Measurement of the binding affinity between FTO and CHTB by isothermal titration calorimetry (ITC). Data fitting revealed a binding dissociation constant of ~1 M between CHTB and FTO. Titration profiles at 25 °C when a titrant of 180 µM FTO was mixed at 2.0 µL per injection with a solution containing 20 µM CHTB at pH~7.5. Curve in bottom figure shows the fitting of data to a one-set model by the non-linear Levenberg−Marquardt fitting algorithm supplied by the instrument.

Then, to identify the structural basis for the recognition of CHTB by FTO, we soaked native FTO crystals with the compound (~2.0 mM) for 24 h prior to data collection. The structure of the soaked FTO crystals obtained in this way was solved with isomorphous 8


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Biochemistry

replacement using the coordinates of m3T-bound FTO (PDB code: 3LFM) as the initial model. In the electron density map of this derived structure, the bound CHTB is well defined (Figure 3A-3B). The two planes defined by the chroman and the benzene ring of CHTB are nearly perpendicular to each other and nicely occupy an L-shaped cavity on the surface of FTO (Figure 3B-3C). This is reminiscent of the recently reported FTO inhibitor MA, in which the two phenyl planes connected via an imino group are also perpendicular to each other. Moreover, structural comparison shown in Figure 3C reveals that CHTB and MA are partially overlapped in FTO although with different structures. A close scrutiny of the two complex structures shows that there is no significant difference in the overall conformation of the two crystal structures except two residues, i.e. Arg96 and Tyr108, which take different orientations in order to make reasonable interactions with the inhibitor. It is noteworthy that the interaction between Cl and guanidine group of Arg96 in MA-bound structure is replaced by interaction between Cl and phenolic hydroxyl group of Tyr108 in CHTB-bound structure. In contrast to the αKG-analogue inhibitors of FTO, the small molecule in this case has no interaction with either the bound iron ion or αKG from which the minimal separation is about 5.0 Å. The structural comparison shown in Figure 3D reveals that CHTB bind at the surface area of the FTO active site while m3T bind at a relatively deeper cavity. The superposition of the three crystal structures also reveal that Arg96 and Tyr108 in the active site are flexible and can take different orientations.

9


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Figure 3. Binding of CHTB to a Basic Surface induces no Conformational Changes in FTO. A, The FTO-bound CHTB is well defined in the crystal structure. Mesh shown around CHTB (in stick) is omit electron density “Fo-Fc” (pink) in the finally refined structure contoured at 2.6 sigma. B, Surface and cartoon representations of FTO bound by CHTB. C, Structure alignment of CHTB-bound FTO (yellow) and MA-bound FTO (pink). D, Structural comparison

between

CHTB-bound

FTO

(yellow),

m3T-bound

FTO

(cyan),

and

N-CDPCB-bound FTO (purple).

Specific recognition of CHTB by FTO. The bound CHTB is sandwiched between an anti-parallel-sheet and the extended C-terminal of the long loop of FTO (Figure 4) that is important for selective recognition of ssRNA/ssDNA.18 Specific recognition of CHTB by 10


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Biochemistry

FTO is through a combination of hydrogen bonds, hydrophobic and van der Waals contacts. The benzene ring, sandwiched between His231 and Leu109, establishes strong hydrophobic contacts with FTO (Figure 4). Both the hydroxyl group and the chlorine from the chroman ring of CHTB participate in the interaction with FTO. In addition to the hydrogen bond with the carbonyl oxygen of His232, the benzene hydroxyl group OH4 also forms a water-mediated hydrogen bond with the amide nitrogen of Glu234. The chlorine mainly establishes van der Waals contacts with Tyr109. In contrast, the hydroxyl group OH2 is largely solvent-exposed and does not interact with FTO. The methyl group at the intersection point of the two planes of CHTB is less than 3.5 Å from the carbonyl oxygen of Trp230 and the hydroxyl group of Ser229, and possibly forms non-conventional hydrogen bonds with the two oxygen atoms. Besides making contacts with the first benzene ring, Leu109 forms hydrophobic interactions with the chroman ring. Four residues, Val83, Ile85, Leu90 and Thr92 from the anti-sheet, form a hydrophobic pocket that is used to recognize the chlorine atom in the chroman ring of CHTB. Additionally, Ile85 is also located within a van der Waals distance of one of the gem-dimethyl groups. Two water-mediated hydrogen bonds are apparent between the hydroxyl group of the chroman ring and the amide nitrogen of Val94, and between the chroman oxygen atom and the hydroxyl group of Ser229.

Figure 4. Recognition Mechanism of CHTB by FTO. The side chains of FTO are shown in yellow (stick). Hydrogen bonds are indicated by red and dashed lines. Red sphere represents water molecule. 11


Biochemistry

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To examine the impact of CHTB compound on FTO-mediated dememthylation of mRNAs, total mRNAs were purified from cells overexpressing FTO (FTO-2 cell lines) treated with the tested compound for 48hr. Level of m6A was determined by dot blotting with antibody specifically recognizing it.16 In agreement with our biochemical assay (Figure 2), the CTHB compound greatly increased the m6A level of the FTO-2 cells compared to the control (Figure 5).

Figure 5. Effect of CTHB on the m6A level in cells. The m6A levels of FTO-expressing cells (FTO-2 cell lines) in the presence (CHTB) or absence (control, Ctl) of CTHB compound in wt FTO-2 cell lines were determined by dot blotting and quantified by Grayscale analysis with ImageJ software. Data are represented as means±SD of four replicates.

To investigate whether CHTB is cytotoxic, we treated differentiated 3T3-L1 cells with CHTB compound at the concentration of 1～5µM. Eight days later, the cells were still healthy (Figure 6). Once the CHTB concentration was increased to 10µM, there is a slight cytotoxicity in differentiated 3T3-L1 cells.

12


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Biochemistry

Figure 6. Cell viability analysis of CHTB compound. Dif represents normal differentiating media group, Ctl-iso represents normal differentiating media only supplemented with isopropanol group, All data are represented as mean +SD of triplicates, * stands for p

A Novel Inhibitor of the Obesity-Related Protein FTO - American

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