Identification of Natural Compound Radicicol as a Potent FTO Inhibitor

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Identification of Natural Compound Radicicol as a Potent FTO Inhibitor Ruiyong Wang, Zhifu Han, Bingjie Liu, Bin Zhou, Ning Wang, Qingwei Jiang, Yan Qiao, Chuanjun Song, Jijie Chai, and Junbiao Chang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00522 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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Molecular Pharmaceutics

Identification of Natural Compound Radicicol as a Potent FTO Inhibitor

Ruiyong Wang1, Zhifu Han2, Bingjie Liu3, Bin Zhou2, Ning Wang1, Qingwei Jiang1, Yan Qiao4, Chuanjun Song1, Jijie Chai2*, Junbiao Chang1,3*

1

. College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, China 2 . School of Life Sciences, Tsinghua University, and Tsinghua-Peking Center for Life Sciences, Beijing 100084, China 3 . College of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang 453007, China 4 . School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China

For Table of Contents Use Only

The Recognition Mechanism of Radicicol by FTO

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The fat mass and obesity-associated protein (FTO), as an m6A demethylase, has been involved in many human diseases. Virtual screening and similarity search in combination with bioactivity assay lead to the identification of the natural compound radicicol as a potent FTO inhibitor, which exhibits a dose-dependent inhibition of FTO demethylation activity with IC50 value of 16.04 µM. Further ITC experiments show that the binding between radicicol and FTO was mainly entropy-driven. Crystal structure analysis reveals that radicicol adopts an L-shaped conformation in the FTO binding site and occupies the same position as N-CDPCB, a previously identified small molecular inhibitor of FTO. Unexpectedly, however, the 1,3-diol group conserved in radicicol and N-CDPCB assumes strikingly different orientations for interaction with FTO. The identification of radicicol as an FTO inhibitor and revelation of their recognition mechanism not only open the possibility of developing new therapeutic strategies for treatment of leukemia, but also provide clues for elucidation of the acting mechanisms of radicicol, which is a possible clinical candidate worth in-depth study.

Keywords: FTO; Radicicol; Inhibitor


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Introduction The fat mass and obesity-associated protein (FTO) has been shown to contribute to polygenic obesity in several genome wide association researches 1-2. Animal studies show that the association between FTO and obesity results from its function in energy homeostasis.3 Nevertheless, how FTO affects energy homeostasis remained elusive until the discovery that FTO acts as an RNA demethylase. FTO catalyzes the demethylation of N6-methyladenosine (m6A) depending on Fe (II)- and 2-oxoglutarate (2OG).4 As one of the most prevalent modification identified in mammalian mRNAs, m6A has been demonstrated to play critical roles in regulation of mRNA fate, development of tissue, self-renewal and differentiation of stem cell, heat shock response control, and circadian clock control.5-16 FTO, as the first m6A demethylase identified in mammals, is involved in many disease processes, including obesity, type II diabetes, Alzheimer’s disease, and cardiovascular diseases.17-18 More recently, He et al. showed that FTO functions as on oncogene in acute myeloid leukemia, and inhibition of FTO activity toward m6A demethylation may be a potential leukemia therapy. 19 In view of the importance of FTO function as an m6A demethylase in obesity and acute myeloid leukemia as well as in other diseases, development of FTO inhibitors is of great value. The structure of FTO in complex with m3T provides the structure basis for substrate binding site. Since then, structure-based design of FTO inhibitors has been pursued by pharmaceutic chemists.

20

In 2011, Yang et al. reported the first

inhibitor of FTO, i.e. rhein, through structure-based virtual screening,4 but the following studies showed that rhein is not an effective inhibitor of FTO in vivo. Afterwards, they discovered that meclofenamic acid is a selective inhibitor of FTO, because the compound displayed much higher inhibition activity to FTO than to Alkbh5,21 which is another m6A demethylase identified in mammals. In the same year, they found the bifunctional fluorescein derivatives, which can label FTO in addition to the function as FTO inhibitors.

22

Besides Yang’s research, there are several other

groups dedicating to developing FTO inhibitors. For example, Aik et al. identified several 2OG analogues as FTO inhibitors, but most of them inhibit not only FTO but ACS Paragon Plus Environment


also other 2OG oxygenases.23 Woon et al. reported the identification of a cell-active inhibitor of FTO,

which occupies both the 2OG-binding site and the

nucleotide-binding site.24 He et al. also synthesized an FTO inhibitor with anticonvulsant activity.25 Our group has also been working on the development of FTO inhibitors since we solved its crystal structure. Recently, we have identified a set of N-CDPCB analogues and CHTB (shown in Figure 1) as FTO inhibitors.26-28 CHTB interacts with FTO at a similar site to meclofenamic acid, whereas N-CDPCB (n=2) and its analogues bind with FTO at a site that has never been identified. The discovery of the novel binding site provided opportunities for the rational design of more specific inhibitors toward FTO.

Figure 1. The structures of N-CDPCB analogues and CHTB. It is of interest to note that 4-Cl-1,3-diol, the same structure motif shared by N-CDPCB and CHTB, (Figure 1) is critical for the two compounds to bind to FTO. Therefore, we reasoned that compounds containing this structural motif may be advantageous for specific interaction with FTO. Inspired by this rationale, we performed structure-based virtual screening on compounds with 4-Cl-1,3-diol group based on the novel binding site identified in N-CDPCB-FTO complex. In the end, the compounds 1a and 1b (shown in Figure 2) containing 4-Cl-1,3-diol group were selected for further studies, resulting in the identification of the natural compound radicicol as a potent FTO inhibitor.

Figure 2. Chemical structure of 1a, 1b and radicicol.


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Radicicol, a macrocyclic natural compound that was originally separated from the fungus Monosporium bonorden,29 has attracted much interest from pharmaceutic chemists. Previous research showed that radicicol is an antifungal antibiotic and a potent tranquilizer with low toxicity.30 In more recent years, radicicol was found to inhibit a wide range of tumor cell lines, and the antitumor effect is mainly attributed to its interaction with the heat shock protein 90 (Hsp90),31-32 a promising target for cancer therapy. Additionally, radicicol was also shown to interact with the bacterial sensor kinase PhoQ.33. Given the potential therapeutic effects, Radicicol was proposed to be a good candidate for further preclinical development. In the present work, we provided several lines of evidence showing that FTO is a target of radicicol in vitro. This finding opens the possibility of designing radicicol derivatives as FTO inhibitors and also, perhaps more importantly, helps elucidate radicicol biology.

2. Experimental Section 2.1 Protein Expression and Purification

The FTO protein (residues 31−505) was expressed and purified as described in the previous paper.20, 26, 28 The protein concentrations were determined by UV–VIS spectrophotometry. The protein stock solution was stored at -80 °C.

2.2 Synthesis and Characterization of Radicicol Analogues

The compounds 1a and 1b were synthesized according to literature procedures (Scheme 1) .34-35 Their structures were characterized by 1HNMR, 13C NMR, and HRMS.

Scheme 1



Reagents and conditions: (a) DIAD, PPh3, toluene, r.t.; (b) Grubbs 2nd generation catalyst, DCM, reflux, then DMSO, r.t.; (c) concd. HCl, dioxane, r.t.

Hept-6-en-2-yl 3-chloro-4,6-bis(methoxymethoxy)-2-(2-oxooct-7-en-1-yl)benzoate 3a To a stirred mixture of acid 1 (41 mg, 0.1 mmol), DIAD (22 mg, 0.11 mmol) and PPh3 (29 mg, 0.11 mmol) in dry toluene (2 mL), was added alcohol 2a (14 mg, 0.12 mmol) in 1 mL of toluene. The resulting mixture was stirred at ambient temperature for 24 h and evaporated. The residue was purified by column chromatography on silica gel (20% ethyl acetate in petroleum ether) to give 3a (42 mg, 85%) as a colorless oil: 1H NMR (400 MHz, CDCl3): δ = 1.29‒1.62 (13H, m), 2.03‒2.07 (4H, m), 2.47 (2H, t, J = 7.2 Hz), 3.46 (3H, s), 3.51 (3H, s), 3.87 (2H, s), 4.20 (1H, m), 4.92‒5.03 (4H, m), 5.15 (2H, s), 5.26 (2H, s), 5.75‒5.82 (2H, m), 7.01 (1H, s).

Oct-7-en-2-yl 3-chloro-4,6-bis(methoxymethoxy)-2-(2-oxooct-7-en-1-yl)benzoate 3b Compound 3b was synthesized according to the procedure as described for the synthesis of 3a by stirring a mixture of acid 1 (50 mg, 0.13 mmol), DIAD (27 mg, 0.14 mmol), PPh3 (36 mg, 0.14 mmol) and alcohol 2b (16 mg, 0.14 mmol) in dry toluene (3 mL) for 20 h. The crude product was purified by column chromatography on silica gel (17% ethyl acetate in petroleum ether) to give 3b (43 mg, 67%) as a colorless oil: 1H NMR (400 MHz, CDCl3): δ = 1.27‒1.65 (15H, m), 2.03‒2.08 (4H, m), 2.48 (2H, t, J = 7.2 Hz), 3.48 (3H, s), 3.52 (3H, s), 3.88 (2H, s), 4.21 (1H, m), 4.93‒5.03 (4H, m), 5.16 (2H, s), 5.27 (2H, s), 5.76‒5.84 (2H, m), 7.01 (1H, s).

(E)-15-chloro-16,18-bis(methoxymethoxy)-3-methyl-3,4,5,6,9,10,11,12-octahydro-1H-benzo[ c][1]oxacyclohexadecine-1,13(14H)-dione 4a A mixture of 3a (65 mg, 0.1 mmol), Grubbs 2nd generation catalyst (11 mg, 0.01 mmol) and dry DCM (40 mL) under nitrogen was heated at reflux for 2 h, then cooled DMSO (20 µL) was added. The mixture stirred at ambient temperature for 24 h. The bulk of solvent was evaporated


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in vacuo. The residue was purified by column chromatography on silica gel (25% ethyl acetate in petroleum ether) to give 4a (45 mg, 74%) as a colorless solid: mp 112‒115 °C; 1H NMR (300 MHz, CDCl3): δ = 1.25‒1.64 (11H, m), 2.00‒2.12 (4H, m), 2.47 (2H, t, J = 7.2 Hz), 3.46 (3H, s), 3.50 (3H, s), 3.98 (2H, s), 4.25 (1H, m), 5.15 (2H, s), 5.25 (2H, s), 5.28‒5.36 (2H, m), 6.98 (1H, s); HRMS (ESI) calcd for: C24H3335ClNaO7 [M + Na]+ 491.1813, found 491.1807. (E)-16-chloro-17,19-bis(methoxymethoxy)-3-methyl-4,5,6,7,10,11,12,13-octahydrobenzo[c][ 1]oxacycloheptadecine-1,14(3H, 15H)-dione 4b Compound 4b was synthesized according to the procedure as described for the synthesis of 4a by stirring a mixture of 3b (31 mg, 0.06 mmol), Grubbs 2nd generation catalyst (5 mg, 0.007 mmol) and dry DCM (16 mL) under nitrogen at reflux for 3 h and then cooled. DMSO (15 µL) was added. The resulting mixture was stirred at ambient temperature for 20 h. The crude product was purified by column chromatography on silica gel (25% ethyl acetate in petroleum ether) to give 4b (24 mg, 82%) as a colorless solid: 1H NMR (300 MHz, CDCl3): δ = 1.25‒1.67 (13H, m), 2.02‒2.09 (4H, m), 2.40‒2.57 (2H, m), 3.46 (3H, s), 3.50 (3H, s), 3.94 (2H, s), 4.24 (1H, m), 5.14 (2H, s), 5.25 (2H, s), 5.28‒5.33 (2H, m), 6.98 (1H, s); HRMS (ESI) calcd for: C25H3535Cl NaO7 [M + Na] + 505.1969, found 505.1994. (E)-15-chloro-16,18-dihydroxy-3-methyl-3,4,5,6,9,10,11,12-octahydro-1H-benzo[c][1]oxacyc lohexadecine-1,13(14H)-dione 1a A mixture of 4a (32 mg, 0.07 mmol), concentrated HCl (0.2 mL) and dioxane (4 mL) was stirred at ambient temperature for 20 h. Saturated aqueous NaHCO3 (4 mL) was added. The resulting mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic extracts were washed with water (3 x 10 mL) and then brine (3 x 10 mL). The separated organic phase was dried (Na2SO4), filtered and evaporated in vacuo. The residue was purified by column chromatography on silica gel (33% ethyl acetate in petroleum ether) to give 1a (22 mg, 85%) as a colorless solid: mp 161‒163 °C; 1H NMR (300 MHz, methanol-d6): δ = 1.23‒1.69 (11H, m), 2.00‒2.12 (4H, m), 2.42‒2.50 (2H, m), 4.28 (2H, s), 4.25 (1H, m), 5.00 (1H, m), 5.23‒5.27 (2H, m), 6.19 (1H, s), 6.50 (1H, s), 11.50 (1H, s); HRMS (ESI) calcd for : C20H2535ClNaO5 [M + Na] + 403.1288, found 403.1283.

(E)-16-chloro-17,19-dihydroxy-3-methyl-4,5,6,7,10,11,12,13-octahydrobenzo[c][1]oxacycloh eptadecine-1,14(3H, 15H)-dione 1b Compound 1b was synthesized according to the procedure as described for the synthesis of 1a by stirring a mixture of 4b (17 mg, 0.03 mmol), concentrated HCl (0.1 mL) and dioxane (2 mL) at ambient temperature for 18 h. The crude product was purified by column chromatography on silica gel (33% ethyl acetate in petroleum ether) to give 1b (10 mg, 88%) as a colorless solid: mp 111‒113 °C; 1H NMR (300 MHz, methanol-d6): δ = 1.25‒1.65 (13H, m), 2.05‒2.14 (2H, m),



2.30‒2.45 (2H, m), 4.19‒4.33 (2H, m), 5.24‒5.33 (3H, m), 6.18 (1H, s), 6.62 (1H, s), 11.74 (1H, s); HRMS (ESI) calcd for: C21H2735ClNaO5 [M + Na] + 417.1445, found 417.1474. 2.3 Protein Crystallization and X-ray Crystallography

Crystallization experiments were carried out with hanging-drop vapor-diffusion methods as described in the previous paper. HKL2000. model.

25

15, 16, 26

All data were indexed, integrated, and scaled using

Radicicol was built into the electron density in COOT after refinement of the initial

36

2.4 Isothermal Titration Calorimetry of Radicicol Binding to FTO Protein

The Isothermal titration calorimetry (ITC) experiments were performed with a Microcal iTC200 isothermal titration calorimeter (GE Healthcare) as described in our previous published material.

26, 28

Water was in the reference cell. In a typical ITC experiment, FTO solution was

placed in the sample cell of the calorimeter and radicicol (or analogues) solution was loaded into the injection syringe. Radicicol (or analogues) solution was titrated into the sample cell by the aid of syringes. The amount of each injection was 2.0 µL. The FTO protein and radicicol (or analogues) solutions were prepared to contain 10% DMSO. The concentration of FTO was 220 µM in the 0.2 mL sample cell. The concentration of radicicol (or analogues) was 2.0 mM in the syringe.

2.5 Enzymatic Activity Assays

The enzymatic activity assays in vitro were performed using Thermo TSQ Quantum Ultra LC-MS as described in our previous publications.

26, 28

The concentration of radicicol (or its

analogues) used in this experiments was 2.0 µM, 4.0 µM, 8.0 µM, 16.0 µM, 32.0 µM, 50.0 µM, and 100.0 µM, respectively. Dose-response curves were fitted with GraphPad Prism 6.0 (GraphPad Software) to obtain the drug concentration for 50% inhibition (IC50) of FTO.

3. Results 3.1 Virtual Screening An in-house database containing ～ 1000 compounds was selected for virtual screening against the FTO crystal structure in complex with N-CDPCB (PDB ID: 5DAB)26 in AutoDock Vina.38 Residues within 5Å of N-CDPCB were selected as the


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binding site. Through virtual screening, the compounds 1a and 1b with new structural fold containing 4-Cl-1,3-diol group were selected as required. To identify more compounds with this structural motif, we performed similarity search through the DrugBank39 database using the SHAFTS software.40-42 These studies led to the identification of the natural compound radicicol as one of the hits. Subsequent molecular docking studies were performed using AutoDock4.2,43 which has been frequently used in molecular modelling studies.27, 44-45 The docking results showed that radicicol has a higher docking score (ΔG=-7.0 kcal/mol) than compound 1a (Δ G=-6.0 kcal/mol) and 1b (ΔG=-4.9 kcal/mol). Modeling studies, however, showed that the macrocyclic rings in the three compounds adopt different conformations when interacting with FTO, despite the conserved structural fold they share.

Figure 3. The binding modes of 1a, 1b, and radicicol with FTO.

3.2 Inhibition of 1a, 1b and radicicol on FTO Demethylation To investigate the inhibitory effect of 1a, 1b and radicicol on FTO, we used the LC-MS method with the 15-mer ssRNA (5′-CUUGUCA(m6A)CAGCAGA-3′) as substrate to quantify the FTO demethylase activity in the presence of each one of the three compounds. The demethylation activity toward FTO was detected using the method described previously.26,

28

The results show that radicicol displayed a

dose-dependent inhibition of FTO with IC50 of 16.04 µM (shown in Figure 4). In contrast, no notable inhibition of FTO was detected when compounds 1a and 1b were used in the assay.



100

FTO activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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IC 50 = 16.04 µM

50

0 0.5

1.0

1.5

2.0

Log [Concentration (µM)]

Figure 4. Inhibitory potency (IC50) was measured for radicicol with FTO from the dose-response curves. Points are the means of 3 determinations, and error bars indicate the standard error of the mean. 3.3. Thermodynamic Data To further investigate the direct interaction between radicicol and FTO suggested by the enzymatic assays (Figure 4), we performed the isothermal titration calorimetry (ITC) experiment to quantify their binding affinity. As shown in Figure 5, the ITC results show that radicicol binds to FTO with a dissociation constant of ～12 µM, supporting radicicol inhibition of FTO in our demethylation assays. In further support of the data from the enzymatic assays, the ITC results show no detectable interaction between 1a/1b and FTO (data not shown). The thermodynamic parameters obtained by ITC reveal that radicicol binds to FTO with high positive entropy (∆S) and small negative enthalpy (∆H), indicating that radicicol interaction with FTO is mainly entropy-driven. The binding free energy (∆G=∆H-T∆S) of radicicol to FTO was calculated -6.71 kcal/mol, which agrees well with that from the docking studies.


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Time (min) 0

10

20

30

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µ cal/sec

-0.10 -0.20 -0.30 -0.40

0.00

-1

kcal mol of injectant

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-0.05 -0.10 -0.15 -0.20

Data: O2mM800328_NDH Model: OneSites Chi^2/DoF = 416.8 N 0.984 ±0.0322 Sites -1 K 8.55E4 ±2.48E4 M ∆H -364.0 ±17.69 cal/mol ∆S 21.3 cal/mol/deg

-0.25 -0.30 -0.35 0.0

0.5

1.0

1.5

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Molar Ratio

Figure 5. Determination of the binding affinity between radicicol and FTO by ITC. A binding dissociation constant of ∼12 µM between radicicol and FTO was provided by data fitting. The equilibrium binding constant (K), the binding stiochiometry (n), the enthalpy of complex formation (∆H) and entropy (∆S) was obtained according to the independent binding model.

3.4 Recognition Mechanism of Radicicol by FTO. To determine the structural basis for the recognition of radicicol by FTO, we solved the crystal structure of radicicol-FTO complex. In the electron density map of FTO-radicicol complex at 2.10 Å resolution, the bound radicicol is well defined (Figure 6A-6C). As seen from Figure 6C, radicicol adopts an L-shaped conformation, in which the macrocyclic lactone plane is nearly perpendicular to the aromatic ring plane. This is reminiscent of the recently reported FTO inhibitors fluorescein,22 meclofenamic acid,21 N-CDPCB26 and CHTB,28 despite their strikingly different chemical structures. Structural alignment of radicicol-FTO complex with m3T-FTO shown in Figure 6D reveals that radicicol and m3T bind at different sites of FTO, with m3T binding to a deep cavity and radicicol to an adjacent but comparatively more solvent-exposed site. Superimposition other FTO-inhibitor structures onto radicicol-FTO complex (shown in Figure 6E) shows that radicicol partially overlaps with fluorescein,22 meclofenamic acid21 and CTHB28 when binding FTO. In contrast,



radicicol almost occupies the same site as N-CDPCB upon interaction with FTO.26, suggesting that the 4-Cl-1,3-diol group shared by the two compounds is important to position them when interacting with FTO.

Figure 6. Binding of radicicol to FTO. A, The FTO- radicicol binding complex shown in cartoon. B, electrostatic potential mapped onto the surface of FTO. C, the binding mode of radicicol with FTO. D, Structure alignment of radicicol (yellow) and m3T (cyan) in FTO. D, Structural alignment of radicicol (yellow), N-CDPCB (pink), fluorescein (green), and meclofenamic in FTO (shown in surface). In the structure of radicicol-FTO complex, the small molecule forms extensive hydrophobic and polar interactions with FTO. Specifically, the phenyl hydroxyl group at C4-position in radicicol is hydrogened bond with the amide nitrogen of Lys216 and the carbonyl oxygen of Met226, while the other -OH group in the aromatic ring forms a hydrogen bond with the amide nitrogen of Ser229 (Figure 7A). Besides, there is also a hydrogen bond between the epoxy group and the sidechain of Ser229. Moreover, the Cl- group in the aromatic ring forms Cl―O bond interaction with the C=O group of Tyr214, whereas the aromatic ring packs against the sidechain of Pro93. In addition, the macrocyclic lactone plane establishes extensive hydrophobic interactions with the sidechains of Val83, Ile85, Leu90, Thr92 and Leu109. Although radicicol and N-CDPCB bind to a conserved site on FTO, notable


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differences exist between them in their mechanisms of interaction with FTO. Interestingly, the most striking difference unexpectedly occurs to the conserved 4-Cl-1,3-diol group, with its orientations varying significantly when interacting with FTO. Following interaction with FTO, Cl from radicicol is positioned to point the solvent region and establishes a Cl―O bond with the carbonyl oxygen of Tyr214. The similar positioning of the Cl was also predicted by docking, supporting robustness of our modeling studies. In sharp contrast to radicicol, the Cl from N-CDPCB is rotated clockwise around the -OH group at 4-position about 120 degrees toward FTO, forming van der Waals contacts with Met226 and Ser229. The rotation brings the -OH group from N-CDPCB at C2-position toward the solvent region, rendering the group unable to contact FTO. The same -OH group from radicicol forms a hydrogen bond with the amide nitrogen of Ser299. Despite the different orientations of the 4-Cl-1,3-diol group, both radicicol and N-CDPCB form a conserved set of hydrogen bonds with FTO via the -OH group at 4-position. The non-conserved portions of the two compounds form conserved interactions with FTO.

Figure 7. (A), Recognition Mechanism of radicicol by FTO. FTO is shown in cartoon in golden colour. Radicicol is shown in yellow and the residues interacting with radicicol from FTO are in purple. (B), Recognition mechanism of N-CDPCB by FTO. FTO is shown in cartoon and green. N-CDPCB is magenta and the residues interacting with it are light blue. Radicicol-FTO is superimposed on N-CDPCB-FTO and radicicol is shown in yellow. (C), The predicted binding conformation of radicicol shown in green is superimposed on the radicicol in crystal structure of radicicol-FTO complex shown in yellow.

4.

Discussion



In the present study, we utilized structure-based virtual screening, similarity search and activity assays in order to discover potent small-molecule inhibitors of FTO. These studies led us to identify the natural compound radicicol as a potent FTO inhibitor. Our enzymatic assays showed that radicicol exhibits a dose-dependent inhibition of FTO demethylation activity with IC50 values of 16.04 µM. Inhibition of FTO by radicicol is then validated by the ITC experiment, which showed direct interaction between them. The results from ITC also indicated that the binding between radicicol and FTO was mainly driven by entropy. Crystal structure analysis revealed the mechanism underlying radicicol binding to FTO. The structure revealed that the 4-Cl-1,3-diol group of radicicol and N-CDPCB binds to the same cavity of FTO, further supporting the idea that this site of FTO can be employed to design FTO inhibitors. Unexpectedly, however, the conserved group adopts strikingly different orientations when interacting with FTO. To accommodate the different orientations of the 4-Cl-1,3-diol group, slightly conformational changes occur to the loop region of FTO interacting with these two compounds. These structural observations indicate that the plasticity of this inhibitor binding site can be used to design FTO inhibitors carrying different derivatives of 4-Cl-1,3-diol. Taken together, our study identified radicicol as an FTO inhibitor in vitro and provided new information on designing more potent compounds to inhibit the activity of the enzyme. Multiple targets of radicicol have been identified. Our study potentially added FTO to the increasing list of radicicol targets, although many more studies are needed to test the in vivo activity of radicicol as an FTO inhibitor. Given an oncogenic role of FTO in acute myeloid leukemia, inhibition of FTO is consistent with the antitumor effect of radicicol. Our findings help probe radicicol biology, which will be important to design its derivatives to more specifically interact with cellular targets. Acknowledgements We are grateful to the National Natural Science Foundation of China (No. 81330075) and Science Foundation of the Henan Province of China (No. 19A350013) for financial support.

Reference


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