Structural basis for inhibition of the fat mass and obesity associated protein (FTO)

Article abstract of DOI:10.1021/jm400193d

The fat mass and obesity associated protein (FTO) is a potential target for anti-obesity medicines. FTO is a 2-oxoglutarate (2OG)-dependent N-methyl nucleic acid demethylase that acts on substrates including 3-methylthymidine, 3-methyluracil, and 6-methyladenine. To identify FTO inhibitors, we screened a set of 2OG analogues and related compounds using differential scanning fluorometry-and liquid chromatography-based assays. The results revealed sets of both cyclic and acyclic 2OG analogues that are FTO inhibitors. Identified inhibitors include small molecules that have been used in clinical studies for the inhibition of other 2OG oxygenases. Crystallographic analyses reveal inhibition by 2OG cosubstrate or primary substrate competitors as well as compounds that bind across both cosubstrate and primary substrate binding sites. The results will aid the development of more potent and selective FTO inhibitors.

Full text of DOI:10.1021/jm400193d

Article
pubs.acs.org/jmc
Structural Basis for Inhibition of the Fat Mass and Obesity Associated
Protein (FTO)
WeiShen Aik, Marina Demetriades, Muhammad K. K. Hamdan, Eleanor. A. L. Bagg, Kar Kheng Yeoh,†
Clarisse Lejeune, Zhihong Zhang, Michael A. McDonough,* and Christopher J. Schofield*
Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom
S
* Supporting Information
ABSTRACT: The fat mass and obesity associated protein (FTO) is a
potential target for anti-obesity medicines. FTO is a 2-oxoglutarate
(2OG)-dependent N-methyl nucleic acid demethylase that acts on
substrates including 3-methylthymidine, 3-methyluracil, and 6-methyl-
adenine. To identify FTO inhibitors, we screened a set of 2OG
analogues and related compounds using differential scanning fluorom-
etry- and liquid chromatography-based assays. The results revealed sets
of both cyclic and acyclic 2OG analogues that are FTO inhibitors.
Identified inhibitors include small molecules that have been used in
clinical studies for the inhibition of other 2OG oxygenases. Crystallo-
graphic analyses reveal inhibition by 2OG cosubstrate or primary
substrate competitors as well as compounds that bind across both
cosubstrate and primary substrate binding sites. The results will aid the
development of more potent and selective FTO inhibitors.
FTO is a member of the Fe(II) and 2-oxoglutarate (2OG)-
dependent oxygenase family, of which there are >60 members
predicted in humans. Human nucleic acid oxygenases
(NAOXs) include the AlkB homologues (ABH 1−8), and the
ten−eleven translocation enzymes (TETs 1−3). Some human
NAOXs are known to have “biochemical roles” in DNA repair,
RNA-hydroxylation, and 5-methylcytosine oxidation.7−10 2OG
oxygenases are being targeted for therapeutic intervention.
These include γ-butyrobetaine hydroxylase, the hypoxia-
inducible transcription factor (HIF) prolyl- and asparaginyl-
hydroxylases, and the Nε-methyllysine histone demethylase
(JmjC) enzymes.11 However, there have been few reports on
the inhibition of NAOXs.12 Here, we report biochemical and
crystallographic studies on FTO inhibition. The results will aid
in the development of potent and selective inhibitors suitable
for validation of FTO as an obesity target.
INTRODUCTION
■
There is an unmet medical need for new approaches to treating
obesity. Mutations to the fto gene are associated with increased
body mass, as shown by genome-wide association and mouse
model studies.1−3 The fto gene product, FTO, is an N-methyl
nucleic acid demethylase acting on both single-stranded DNA
and RNA substrates, including 3-methylthymine, 3-methylur-
acil, and 6-methyladenine (Scheme 1).4−6 How the modulation
Scheme 1. Reactions Catalyzed by FTO
RESULTS
■
We initially employed a differential scanning fluorometry
(DSF)-based assay to screen >150 2OG analogues and metal-
chelators consisting of 10 structural series. Where the DSF
assay was not suitable due to compound fluorescence (e.g., 8-
hydroxyquinolines), we assayed FTO inhibition using liquid
chromatography (LC). The LC assay was also used to
determine IC50 values for hits. These studies led to the
identification of structurally different FTO inhibitors, including
the well-characterized 2OG oxygenase inhibitors N-oxalylgly-
of nucleic acid methylation status by FTO relates to increased
body mass is unknown. Possible mechanisms include altered
metabolism, appetite regulation, or both. Inhibition of FTO
activity by small molecules has been proposed as a potential
treatment for extreme obesity,7 but validation of this approach
requires the development of selective inhibitors.
Received: February 8, 2013
© XXXX American Chemical Society
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Figure 1. Wall-eyed stereo views from crystal structures of FTO in complex with (a) 1 (green), (b) 2 (cyan), and (c) 3 (light green). Residue side
chains C (white), O (red), N (blue), Ni (green ball), Zn (gray ball), and hydrogen bonds are indicated as black dashes. mFo-DFc ligand omit maps
(contour level, σ = 3.0) are indicated as lime green mesh.
cine and pyridine-2,4-dicarboxylate, as well as hydroxyquino-
line-, pyridyl-, and isoquinoline-based compounds. We then
performed structural studies with representatives of different
classes of the identified inhibitors.
in the 2OG binding pocket, with the C-5 carboxylate of 1 and
C-4 carboxylate of 2 positioned to form electrostatic and
hydrogen-bonding interactions with the side chains of Arg-316,
Ser-318, and Tyr-295. Both 1 and 2 are positioned to chelate in
a bidentate manner to the metal. In each case, the C-1
carboxylate is positioned trans to the His-307 imidazole and the
other chelating group (amide carbonyl for 1, and pyridyl
nitrogen for 2) trans to the Asp-233 carboxylate.
We also determined a structure (2.5 Å resolution) for
FTO·Zn in complex with 317 (Figure 1c) (PDB ID: 4IE4), a
relatively potent FTO inhibitor (IC50 3.3 μM), which also
inhibits other 2OG oxygenases.14 Two derivatives of 3, in
which the carboxylate was substituted with a sulfonamide, were
less potent (IC50 23 μM for 4 and 18 μM for 5). The
FTO·Zn·3 structure reveals the C-5 carboxylate in a position
similar to that observed for the side chain carboxylates of 1 and
2, rationalizing the reduced potencies of the C-5 sulfonamides,
4 and 5. In the FTO·Zn·3 complex, the relative metal
coordination positions of the hydroxyl and nitrogen of the
hydroxyquinoline are swapped relative to the active site
A crystal structure of FTO in complex with N-oxalylglycine
(NOG) (a 2OG analogue) and 3-methylthymidine (3meT) has
been reported.13 Initially, we determined structures for FTO in
complex with two broad-spectrum 2OG oxygenase inhibitors,
NOG, 1, (2.4 Å resolution) (Figure 1a) (PDB ID: 4IDZ) and
pyridine-2,4-dicarboxylate, 2, (2.5 Å resolution) (Figure 1b)
(PDB ID: 4IE0). As observed for some of the JmjC
demethylases,14−16 2 (IC50 8.3 μM) was a more potent
inhibitor than 1 (IC50 44 μM, Table 1). Although the structures
were obtained under different conditions (Ni and Zn
substituting for Fe with 1 and 2, respectively, and in the
presence of N6-methyladenosine for 1), in each case, a molecule
of glycerol (likely from the cryoprotectant) was observed in the
previously identified 3meT substrate binding site (Supporting
Information Figure S2a).13 Consistent with observations of
other 2OG oxygenase-inhibitor complexes, 1 and 2 are located
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Table 1. Structures of Selected FTO Inhibitors from This Study and the Tm Shift Values (ΔTm/°C), Residual FTO Activity at
200 μM Compound Concentration (SPA/%), and IC50 Values for Hydroxyquinoline-, Pyridyl-, and Isoquinoline-Based
a
Inhibitors
a
For compounds 1, 2, 19, and 20, IC50 values are in parentheses. Standard deviations were calculated from triplicate experiments.
HxD···H metal binding motif when compared with a
JMJD2A·Ni·3 structure14 (JMJD2A is a histone demethylase).
JMJD2A structures show a shift in the metal position upon 3
binding;14 however, no such difference in metal position was
observed for FTO.
suggesting that 6 may sterically hinder binding of FTO’s nucleic
acid primary substrate.
Compound 11 (FG-2216/IOX3)20 is a known inhibitor of
the HIF-prolyl hydroxylases (PHD1−3) that is active in cells
and animals.11 It gave the lowest IC50 value against FTO (2.8
μM). Substitution of the glycine side chain21 of 11 led to
reduced potency; however, the D-amino acid derivatives (D-
phenylalanine, 12, IC50 38 μM; D-alanine 13, IC50 60 μM; D-
valine, 14, IC50 120 μM) were generally more potent than the
L-derivatives (L-phenylalanine, 15, IC50 84 μM; L-alanine, 16,
IC50 110 μM; L-valine, 17, IC50 160 μM). Notably, a previous
study shows that 13 is relatively less active than 16 against
PHD2,22 suggesting that selectivity for FTO over PHD2 may
be achieved using the isoquinoline scaffold. Moreover, the PHD
inhibitor 18 (FG-4592 as identified by SelleckBio),21 displayed
relatively potent FTO inhibition (IC50 9.8 μM).
The observed binding modes of 1 and 2 prompted the
investigation of pyridyl glycinamides (Table 1; 6,12 7, 8,12 9,18
and 1018). Although these compounds were less potent than
the parent templates, the results reveal that in this series, the
presence of a C-3 hydroxy group increases potency, and
substitution at the glycyl Cα position is possible (see Table 1).
We determined a structure (2.0 Å resolution) of FTO·Zn in
complex with 6 (Figure 2a) (PDB ID: 4IE5), which also
inhibits the alkylated-DNA repair enzyme AlkB (Escherichia
coli) (IC50 3.4 μM).12 A structure of the AlkB·Fe·6 complex
reveals 6 bound in the 2OG binding site with its pyridine ring
positioned in a hydrophobic pocket buried within the core of
the double-stranded β helix (DSBH) fold (Supporting
Information Figure S2c),12 leading to the prediction that 6
may bind similarly to FTO without disrupting nucleotide
binding.19 To our surprise, the FTO·Zn·6 complex shows that
6 binds to FTO differently from that observed in AlkB. In both
FTO and AlkB, the side chain carboxylate of 6 is positioned to
bind in the 2OG pocket; however, the hydrophobic pocket of
AlkB in which the pyridine ring of 6 binds is apparently larger;
more flexible; and, thus, more accommodating than that of
FTO. As a result, the pyridine ring of 6 is positioned closer to
the substrate binding site in FTO. Superimposition of the
FTO·Zn·6 and FTO·Fe·NOG·3meT structures (Supporting
Information Figure S2b) shows that the C5 of the pyridine ring
of 6 is positioned ∼1.4 Å from the 3meT carbonyl oxygen,
A crystal structure of FTO in complex with 11 was
determined (2.50 Å resolution) (Figure 2b) (PDB ID: 4IE6)
and shows 11 binds FTO similarly to 6. The chlorine of 11 sits
in the nucleic acid substrate binding site. An unusual metal
coordination geometry was observed for 11; the side chain
amide carbonyl deviates from planarity with respect to the
isoquinoline ring by ∼60°, making the isoquinoline ring
nitrogen-to-metal coordination skewed. This may be due to
the position of the side chain of Arg-96 with respect to the
bicyclic rings, sterically forcing a non-ideal metal coordination
by the compound.
A recent study has identified the natural product rhein as a
reversible and competitive FTO inhibitor and led to the
prediction that rhein (19) binds to the 2OG, Fe(II), and 3meT
binding sites, also interacting with Arg-316.19 Consistent with
the reported work, we found that 19 is an FTO inhibitor, with
C
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Figure 2. Wall-eyed stereo views from crystal structures of FTO in complex with (a) 6 (pink); (b) 11 (blue), Cl (green); (c) 19 (yellow), and citrate
(light red). Labeling and color as in Figure 1.
an IC50 of 9.0 μM. Compound 20 was found to be similarly
potent (IC50 11 μM) despite its different substitution pattern.
We determined a structure of the FTO·Zn·19 complex (2.6 Å
resolution) (Figure 2c and Supporting Information Figure S2d)
(PDB ID: 4IE7). Under our crystallization conditions, 19 is
observed bound to the nucleic acid substrate binding site, as
opposed to the predicted 2OG binding site. The C-2
carboxylate of 19 is positioned to hydrogen bond with the
Ser-229 side chain OH (2.7 Å). The A-ring of 19 is positioned
to form partial π−π interactions with His-231 and forms a
hydrogen bond between the 4-hydroxyl group of the A-ring and
the side chain of Arg-96.
A citrate molecule from the crystallization buffer is observed
in the 2OG binding site in the FTO·19 structure. One of the
citrate carboxylates is positioned to interact with the active site
metal, similar to succinate binding observed in other 2OG
oxygenase structures,23 and another carboxylate is interacting
with the side chain of Arg-316. The third citrate carboxylate is
positioned to hydrogen-bond to the side chain of Asn-205. The
IC50 for citrate was found to be 300 μM; fumarate (IC50 150
μM) and L-2-hydroxyglutarate (IC50 320 μM) were also shown
to inhibit FTO weakly (Supporting Information Table S1).
Although these values are relatively high, given the links of
FTO to obesity, it raises the possibility that FTO inhibition by
endogenous small molecules (e.g., tricarboxylic acid (TCA)
cycle intermediates and related compounds such as L-2-
hydroxyglutarate) may be of physiological or pathophysio-
logical relevance, as proposed to occur for other 2OG
oxygenases.15,24 IC50 values for FTO inhibition by other TCA
cycle intermediates were all >1 mM.
DISCUSSION AND CONCLUSIONS
■
Overall, we have identified inhibitor templates that will provide
starting points for the development of selective inhibitors to
probe the physiological role of FTO-mediated nucleic acid
demethylation. Most of the inhibitors investigated in this study
utilize a metal chelating group for enhanced binding affinity and
are 2OG competitors. Work with other 2OG oxygenases has
shown that optimization of such compounds can lead to highly
potent and selective inhibitors.11 The structural analyses with
compound 19 imply that efficient inhibition via substrate
competition should also be possible. The results with the
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broad-spectrum inhibitors are important because they suggest
that some of the previously observed biological effects of these
compounds may be in part mediated via FTO (or other
NAOX) inhibition. A cell-penetrating form of 125 has been
widely used as a hypoxia mimic.11,26 It may be that some of the
observed effects of 1 are due to the inhibition of 2OG
oxygenases other than the HIF hydroxylases. Notably, among
the most potent FTO inhibitors we identified, compounds 11
and 18 are in clinical trials as prolyl-hydroxylase inhibitors.
Thus, the potential for FTO- and other 2OG oxygenase-
mediated inhibition should be considered, and care should be
taken when interpreting the physiological effects of these
inhibitors.
incubated at room temperature for 1 h. The reaction was
quenched with methanol (50 μL) then centrifuged to remove
precipitated protein. The supernatant was then dried using an
Eppendorf Speedvac concentrator and reconstituted with (50
μL) water. The product (thymidine) and substrate (3meT)
were separated using a Waters Acquity Ultra Performance
Liquid Chromatography (UPLC) BEH C18 column (130 Å,
1.7 μm, 2.1 mm × 50 mm) with a gradient of 95% A (H2O with
1% formic acid) to 80% B (methanol with 0.1% formic acid)
over 5 min. The UV detection wavelength was set to 266 nm
room temperature (thymidine) = 0.95 min, room temperature
(3meT) = 1.48 min. (The liquid chromatography method was
modified from that previously reported.13) The peaks were
identified using a positive ion mode in the ESI-TOF mass
spectrometry (Waters LCT Premier XE machine). The UV
peaks were integrated using MassLynx software (Agilent), and
the percentage conversion of 3meT to thymidine was used to
quantify the activity of FTO in the presence of inhibitors. For
IC50 determinations, assays were carried out in triplicate with a
range of compound concentrations (0, 10, 30, 100, and 300 μM
and 1, 3, and 10 mM). IC50 values were calculated from dose−
response curves plotted using GraphPad Prism 5.
Protein Crystallization and X-ray Crystallography.
Crystals of the hFTOΔ31 in complex with 1, 2, 3, 6, 11, or
19 were grown in hanging drops at 293 K by the vapor
diffusion method using 24 well pregreased VDX plates and
round plastic coverslips (HR8-082, Hampton Research). The
protein solution and reservoir solution (200 μL) conditions are
listed in Table 2 (see below). Hanging drops were set up by
adding protein solution to reservoir solution at a 2:1 ratio (total
volume, 3 uL). The resultant crystals were cryoprotected using
the well solution diluted with 15−20% v/v glycerol
(cryoprotectant solution for the FTO·19 crystal contains 1
mM 19) then flash cooled in liquid nitrogen. Data were
collected at 100 K for single crystals of FTO·1, FTO·3, and
FTO·6 complexes using a Rigaku FR-E+ Superbright copper
rotating anode diffractometer equipped with an osmic HF
optics, and a Saturn 944+ CCD detector. Data were collected at
100 K for FTO·2, FTO·11, and FTO·19 using a single crystal
at Diamond Beamline I03 equipped with a Pilatus 6M-F
detector.
All data were indexed, integrated, and scaled using
HKL2000.28 The structures were solved by molecular
replacement using the MR-PHASER29 subroutine in PHE-
NIX30 using FTO:Fe:NOG:3meT (PDB ID: 3LFM) as a
search model. Iterative cycles of model building and refinement
were performed using COOT31 and PHENIX until decreasing
R and Rfree no longer converged. A data collection and
refinement statistics table can be found in Supporting
Information Table S2.
Organic Synthesis Procedures. General Information.
Reagents and solvents were from Aldrich, Alfa Aesar, or Acros.
Compounds 1 and 2 were from Sigma Aldrich. Compounds
3,17 6,12 8,12 9,18 10,18 11,20 and (13, 14, 16, 17)21,22 were
prepared as described. Compound 18 was from Selleckbio,
compounds 19 and 20 were from TCI UK, and 1-chloro-4-
hydroxyisoquinoline-3-carboxylic acid was from Reddy Chem-
tech. Reactions were monitored by TLC, which was performed
on precoated aluminum-backed plates (Merck, silica 60 F254).
Flash column chromatography was carried out using a Biotage
SP1 Purification system. Melting points were determined using
a Leica Galen III hot-stage melting point apparatus and
microscope. Infrared spectra were recorded from KBr discs, on
EXPERIMENTAL SECTION
■
Purification of hFTO and hFTOΔ31. Protein purification
methods were modified from methods previously reported for
full length mouse FTO.4 E. coli BL21 (DE3) transformed with
the pET28a_hFTO plasmid (encoding for N-terminally
hexahistidine-tagged full-length human FTO without the
thrombin cleavage site between the hexahistidine tag and the
protein) or pET28a_hFTOΔ31 (encoding for N-terminally
hexahistidine-tagged human FTO with residues 2−31 deleted
from the N terminus) were grown (37 °C; 180 rpm) to an
OD600 of 0.6. FTO production was induced by the addition of
0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and
growth was continued at 16 °C for 16 h (hFTO) or 18 °C for 8
h (hFTOΔ31). The resultant cell pellets were stored at −80
°C. The cell pellets were thawed and resuspended in 20 mM
Tris−HCl, pH 7.5; 500 mM NaCl; and 10 mM imidazole, then
lysed by sonication on ice. The lysate was cleared by
centrifugation, and the supernatant was loaded onto a 5 mL
HisTrap FF column (GE Healthcare) and purified using an
AKTA FPLC system. The column was treated with 20 mM
Tris−HCl, 500 mM NaCl, and 40 mM imidazole, then eluted
with 20 mM Tris−HCl, pH 7.5; 500 mM NaCl; and 500 mM
imidazole. The eluted hFTOΔ31 protein solution was then
treated with 200 mM EDTA. hFTOΔ31 was further purified
using a (5 mL) HiTrap Heparin column (GE Healthcare)
followed by a (20 mL) MonoQ column; in both cases, a
gradient of buffer A (25 mM Tris−HCl, pH 7.5) and 50%
buffer B (25 mM Tris−HCl, pH 7.5; 1 M NaCl) was used.
Both proteins were then buffer-exchanged into 25 mM Tris−
HCl, pH 7.5, and concentrated to 20 mg/mL for storage.
Dynamic Scanning Fluorometry Assays.27 For dynamic
scanning fluorometry (DSF) assays, a mixture (50 μL)
containing 2 μM full-length FTO protein; 1× SYPRO orange
dye (Invitrogen) (from commercial stock that is 5000×); 50
μM diammonium iron(II) sulfate complex; 200 μM test
compound (or 2% DMSO control); and 50 mM HEPES, pH
7.5, was prepared. Assays were performed using a MiniOpticon
Real-Time PCR Detection System (Bio-Rad). The sample plate
was inserted into the machine, and fluorescence readings were
taken between 25 and 90 °C, increasing the temperature
linearly over 1 h 20 min. The excitation and emission
wavelengths were 470 and 570 nm, respectively. Melting
curves were analyzed using GraphPad Prism.
In Vitro Demethylation Assay. For catalytic assays, a 50
μL reaction mixture containing final concentrations of 3 μM
FTO; 70 μM 3meT nucleoside; 160 μM 2OG; 500 μM L-
ascorbate; 100 μM diammonium iron(II) sulfate complex; 200
μM test compound (2% DMSO in control reaction); and 50
mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.3, was
E
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a Bruker Tensor 27 FT-IR spectrometer. NMR spectra were
acquired using a Bruker Avance II 500 MHz spectrometer
equipped with a 5 mm 13C(1H) dual cryoprobe or Avance 400
MHz spectrometer equipped with a 5 mm z-gradient
1H/13C/19F/31P quadnucleus probe. Chemical shifts (δ) are
given in parts per million, and the multiplicities are given as
singlet (s), doublet (d), doublet doublet (dd), triplet (t),
quartet (q), multiplet (m), or broad (br). Coupling constants, J,
are given in hertz ( 0.5 Hz). High resolution mass spectra
(HRMS) were recorded using a Bruker MicroTOF. The
purities of all compounds synthesized were ≥98%, as
determined by analytical reverse-phase LC/MS or elemental
analysis.
General Procedures. General Procedure A. A mixture of 8-
hydroxyquinoline-5-sulfonic acid (2.4 g, 10 mmol) and
chlorosulfonic acid (8 mL, excess) was stirred at room
temperature overnight. The reaction was poured into ice
water, and the formed yellow precipitate was filtered off and
dried to obtain the desired sulfonyl chloride derivative (2.3g,
95%), which was kept under nitrogen at −20 °C.
To a solution of 8-hydroxyquinoline-5-sulfonic chloride (0.21
mmol, 50 mg) in acetonitrile (1 mL), the appropriate amine
was added (1.05 mmol), and the reaction mixture was heated in
a sealed tube. After evaporation in vacuo of the solvent, the
mixture was dissolved in methanol, and the resulting crude
product was purified using semipreparative reverse-phase
HPLC performed on a Waters Sunfire C18 column (150 mm
× 10 mm, 5 mm). Separation was achieved using a gradient of
0% B over 2 min and 0−50% B over 30 min, solvent A eluting
at a flow rate of 5 mL/min, and monitoring at 254 nm. Solvent
A: water +0.1% CF3CO2H. Solvent B: acetonitrile +0.1%
CF3CO2H.
General Procedure B. To a solution of phenylalanine methyl
ester hydrochloride (151 mg, 0.7 mmol, 1.2 equiv) and
triethylamine (0.2 mL, 1.5 mmol, 2.5 equiv) in CH2Cl2 (10
mL) was added 1-chloro-4-hydroxyisoquinoline-3-carboxylic
acid (130 mg, 0.6 mmol, 1 equiv), followed by benzotriazol-1-
yl-oxytripyrrolidinophosphonium hexafluorophosphate (332
mg, 0.6 mmol, 1.1 equiv). The resulting mixture was then
stirred at room temperature until completion (TLC). After 20
h, the organic solvents were removed, and the residue was
purified by flash chromatography (hexane/EtOAc, 4:1) to
afford the desired methyl-2-(1-chloro-4-hydroxyisoquinoline-3-
carboxamido)propanoate derivative.
General Procedure C. A mixture of the methyl-2-(1-chloro-
4-hydroxyisoquinoline-3-carboxamido)propanoate (110 mg, 0.3
mmol) in 1 N NaOH/MeOH (1:1) (10 mL, excess) was stirred
at room temperature. TLC analysis (hexane/EtOAc, 4:1)
indicated complete consumption of the starting material (Rf =
0.4) after 2 h. The reaction mixture was then washed with
EtOAc (3 × 10 mL). The aqueous layer was acidified to pH 1
with 1 N HCl and then extracted with EtOAc (3 × 10 mL),
dried (MgSO4), filtered, and concentrated in vacuo to give the
corresponding 2-(1-chloro-4-hydroxyisoquinoline-3-carboxami-
do)-3-phenylpropanoic acid derivative.
N-3-Methylthymidine. The synthesis was modified from
the previously reported method.32 To a solution of thymidine
(0.79 g, 3.26 mmol) and potassium carbonate (0.83 g, 6.01
mmol) in MeOH (50 mL), methyl iodide (0.38 mL, 6.1 mmol)
was added dropwise at room temperature. The reaction was
heated at 37 °C for 24 h, then 5 mL of acetic acid was added to
neutralize the mixture. The solvent was evaporated in vacuo,
and the crude compound was redissolved in water. The product
F
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was purified using preparative reversed-phase HPLC (tR = 25
min) (Vydac 218TP C18, 250 mm × 22 mm, 10−15 μm, Grace
Davison, USA) using a gradient of 2% B for 5 min, 2−98% B in
A over 30 min, and 98% B for 5 min. Solvent A: 0.1% formic
acid v/v water. Solvent B: 0.1% formic acid v/v in acetonitrile.
Product (24%) mp 129−133 °C; IR (neat) ν/cm−1 3500
(OH), 3412 (OH), 3300 (OH), 1683 (CO), 1665 (CO),
1622 (CO); 1H NMR (400 MHz, DMSO) δ 7.77 (1H, s, 6-
H), 6.20 (1H, t, J = 6.5, 1′-H), 5.04 (1H, br. s, 5′-OH), 4.24
(1H, m, 3′-H), 3.78 (1H, app. q., J = 4.0, 4′-H), 3.58 (2H, m,
5′-H), 3.16 (3H, s, N−CH3), 2.09 (2H, m, 2′-H), 1.82 (3H, s,
CH3); 13C NMR (400 MHz, DMSO) δ 162.9 (CO), 150.6
(CO), 134.5 (6C), 108.3 (5C), 87.4 (4′C), 84.9 (1′C), 70.3
(3′C), 61.2 (5′C), 27.5 (N−CH3), 13.0 (CH3). HRMS calcd for
C11H16N2NaO5 [M + Na]+ 279.0951; Found 279.0951.
8-Hydroxyquinoline-5-sulfonamide (4). Following gen-
eral procedure A, the reaction mixture was heated at 45 °C for
1 h. Compound 4 was isolated as a pale yellow solid (36.7 mg,
78%), mp dec 250 °C. tR = 6.12; IR (neat) ν/cm−1 3345, 3097,
2942, 2832, 1427, 1171, 1021; 1H NMR (500 MHz, CD3OD) δ
9.20 (1H, d, J = 9.0, Ar CH), 8.86 (1H, d, J = 4.5, Ar CH), 8.11
(1H, d, J = 8.0, Ar CH), 7.64 (1H, dd, J = 9.0, 4.0, Ar CH), 7.09
(1H, d, J = 8.0, Ar CH); 13C NMR (500 MHz, CD3OD) δ
155.6, 148.3, 138.8, 135.8, 131.7, 127.7, 125.8, 122.3, 108.7.
HRMS calcd for C9H8NO4S (corresponding sulfonic acid)+
226.0169; Found 226.0161.
CH3O), 3.31 (1H, dd, J = 14.0, 6.0, PhCH2), 3.25 (1H, dd, J =
14.0, 7.0, PhCH2); 13C NMR (125 MHz, CDCl3) δ 171.1 (C
O), 168.2 (CO), 154.5 (ArC), 139.3 (ArC), 135.4 (PhC),
130.5 (2 × PhCH), 130.0 (ArC), 129.2 (ArC), 128.9 (2 ×
PhCH), 127.0 (PhCH), 126.2 (ArCH), 123.1 (ArCH), 120.1
(ArC), 52.9 (CH), 52.2 (CH3O), 38.0 (PhCH2). HMRS calcd
for C20H16ClN2O4 [M − H]− 383.0804; Found 383.0804.
(R)-2-(1-Chloro-4-hydroxyisoquinoline-3-carboxami-
do)-3-phenylpropanoic Acid (12). Following general
procedure C, 12 (100 mg, 95%) was isolated as a pale yellow
22
solid; mp 184−186 °C; [α]D −13.0 (c, 1.0 in MeOH); IR
(KBr) ν/cm−1 3448 (OH), 3362 (NH), 2930 (C−H), 1736
1
(NCO), 1637 (HOCO); H NMR (500 MHz, DMSO-
d6) δ 13.49 (1H, s, ArOH), 8.93 (1H, d, J = 8.0, NH), 8.29−
8.27 (1H, m, Ar-H), 8.22−8.24 (1H, m, Ar-H), 7.93−7.96 (2H,
m, 2 × Ar-H), 7.26−7.27 (4H, m, 4 × Ph-H), 7.16−7.20 (1H,
m, Ph-H), 4.79−4.84 (1H, m, CH), 3.28−3.33 (2H, m,
PhCH2); 13C NMR (125 MHz, DMSO-d6) δ 172.1 (CO),
168.4 (CO), 154.3 (ArC), 138.5 (ArC), 137.5 (PhC), 131.8
(PhCH), 131.7 (PhCH), 129.4 (ArC), 129.1 (2 × ArCH),
128.6 (ArC), 128.3 (2 × PhCH), 126.6 (PhCH), 126.1
(ArCH), 123.0 (ArCH), 120.3 (ArC), 53.2 (CH), 35.9
(PhCH2). HMRS calcd for C19H14ClN2O4 [M − H]−
369.0648; Found 369.0651. Anal. (C19H15ClN2O4) requires:
C, 61.55; H, 4.08; N, 7.56%; found C, 61.45; H, 4.00; N, 7.63%.
(S)-2-(1-Chloro-4-hydroxyisoquinoline-3-carboxami-
do)-3-phenylpropanoic Acid (15). Following general
procedure C, 15 (90 mg, 92%) was isolated as a pale yellow
8-Hydroxy-N-methylquinoline-5-sulfonamide (5). Fol-
lowing general procedure A, the reaction mixture was heated at
70 °C for 2 h. Compound 5 was isolated as a pale yellow solid
(35.5 mg, 71%), mp dec 190 °C. tR = 5.89 min; IR (neat) ν/
22
solid; mp 185−188 °C; [α]D +12.9 (c, 1.0 in MeOH); IR
(KBr) ν/cm−1 3448 (OH), 3362 (NH), 2929 (C−H), 1736
1
cm−1 3346, 2943, 2832, 1152, 1021; H NMR (500 MHz,
1
(NCO), 1637 (HOCO); H NMR (500 MHz, DMSO-
CD3OD) δ 9.18 (1H, dd, J = 8.5, 1.5, Ar CH), 8.84 (1H, br s,
Ar CH), 8.10 (1H, d, J = 8.0, Ar CH), 7.61 (1H, dd, J = 8.5, 4.0,
Ar CH), 7.08 (1H, d, J = 8.0, Ar CH), 2.54 (3H, s); 13C NMR
(500 MHz, CD3OD) δ 155.9, 148.3, 139.0, 135.6, 131.5, 127.7,
125.8, 122.3, 108.6, 24.5 (CH3). HRMS calcd for C9H8NO4S
(corresponding sulfonic acid)+ 226.0169; Found 226.0161.
(R)-2-(1-Chloro-4-hydroxyisoquinoline-3-carboxami-
do)-3-phenylpropanoate (25). Following general procedure
B, 25 (147 mg, 66%) was isolated as a yellow oil (hexane/
d6) δ 13.50 (1H, s, ArOH), 8.94 (1H, d, J = 8.0, NH), 8.30−
8.32 (1H, m, Ar-H), 8.26−8.28 (1H, m, Ar-H), 7.97−7.99 (2H,
m, 2 × Ar-H), 7.26−7.27 (4H, m, 4 × Ph-H), 7.17−7.21 (1H,
m, Ph-H), 4.79−4.83 (1H, m, CH), 3.27−3.32 (2H, m,
PhCH2); 13C NMR (125 MHz, DMSO-d6) δ 172.1 (CO),
168.4 (CO), 154.3 (ArC), 138.5 (ArC), 137.4 (PhC), 131.8
(PhCH), 131.7 (PhCH), 129.5 (ArC), 129.1 (2 × ArCH),
128.6 (ArC), 128.3 (2 × PhCH), 126.6 (PhCH), 126.1
(ArCH), 123.0 (ArCH), 120.3 (ArC), 53.2 (CH), 35.9
(PhCH2). HMRS calcd for C19H14ClN2O4 [M − H]−
369.0648; Found 369.0651. Anal. (C19H15ClN2O4) requires:
C, 61.55; H, 4.08; N, 7.56%; found C, 61.61; H, 4.01; N, 7.62%.
22
EtOAc, 4:1 Rf = 0.4); [α]D −16.8 (c, 1.0 in CH2Cl2); IR
(KBr) ν/cm−1 3384 (N−H), 2925 (C−H), 1745 (HNCO),
1638 (MeOCO); 1H NMR (500 MHz, CDCl3) δ 12.85 (1H,
s, ArOH), 8.34−8.37 (1H, m, Ar−H), 8.23−8.26 (1H, m, Ar−
H), 8.21 (1H, d, J = 8.0, NH), 7.77−7.81 (2H, m, 2 × Ar−H),
7.23−7.34 (5H, m, 5 × Ph-H), 5.02−5.06 (1H, m, CH), 3.77
(3H, s, CH3O), 3.31 (1H, dd, J = 14.0, 6.0, PhCH2), 3.25 (1H,
dd, J = 14.0, 7.0, PhCH2); 13C NMR (125 MHz, CDCl3) δ
171.0 (CO), 168.2 (CO), 154.5 (ArC), 139.3 (ArC), 135.9
(PhC), 130.5 (2 × PhCH), 130.0 (ArC), 129.1 (ArC), 128.9 (2
× PhCH), 128.5 (2 × ArCH), 127.0 (PhCH), 126.1 (ArCH),
123.1 (ArCH), 120.4 (ArC), 52.9 (CH), 52.2 (CH3O), 37.9
(PhCH2). HMRS calcd for C20H16ClN2O4 [M − H]−
383.0804; Found 383.0805.
ASSOCIATED CONTENT
■
S
* Supporting Information
Details of synthesis, IC50 curves and crystallography. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Accession Codes
The coordinates of crystal structures of FTO in complex with 1,
2, 3, 6, 11, and 19 have been deposited in RCSB Protein Data
Bank as PDB IDs 4IDZ, 4IE0, 4IE4, 4IE5, 4IE6, and 4IE7,
respectively.
(S)-2-(1-Chloro-4-hydroxyisoquinoline-3-carboxami-
do)-3-phenylpropanoic Acid (26). Following general
procedure B, 26 (130 mg, 59%) was isolated as a yellow oil
(hexane/EtOAc, 4:1 Rf = 0.4); [α]D22+17.5 (c, 1.0 in CH2Cl2);
IR (KBr) ν/cm−1 3384 (N−H), 2925 (C−H), 1745 (NCO),
1639 (MeOCO); 1H NMR (500 MHz, CDCl3) δ 12.86 (1H,
s, ArOH), 8.35−8.37 (1H, m, Ar-H), 8.25−8.26 (1H, m, Ar-H),
8.21 (1H, d, J = 8.0, NH), 7.77−7.81 (2H, m, 2 × Ar-H), 7.23−
7.34 (5H, m, 5 × Ph-H), 5.02−5.06 (1H, m, CH), 3.77 (3H, s,
AUTHOR INFORMATION
■
Corresponding Author
*(M.A.McD.) Phone: +44(0)1865 275 629; E-mail: michael.
mcdonough@chem.ox.ac.uk. (C.J.S.) Phone: +44(0)1865 275
625; E-mail: christopher.schofield@chem.ox.ac.uk
G
dx.doi.org/10.1021/jm400193d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(6) Jia, G.; Fu, Y.; Zhao, X.; Dai, Q.; Zheng, G.; Yang, Y.; Yi, C.;
Lindahl, T.; Pan, T.; Yang, Y. G.; He, C. N6-methyladenosine in
nuclear RNA is a major substrate of the obesity-associated FTO. Nat.
Chem. Biol. 2011, 7, 885−887.
(7) Thalhammer, A.; Aik, W.; Bagg, E. A. L.; Schofield, C. J. The
potential of 2-oxoglutarate oxygenases acting on nucleic acids as
therapeutic targets. Drug Discovery Today Ther. Strateg. 2012, 9, e91−
e100.
(8) Fu, Y.; Dai, Q.; Zhang, W.; Ren, J.; Pan, T.; He, C. The AlkB
domain of mammalian ABH8 catalyzes hydroxylation of 5-methox-
ycarbonylmethyluridine at the wobble position of tRNA. Angew.
Chem., Int. Ed. 2010, 49, 8885−8888.
(9) Fu, Y.; He, C. Nucleic acid modifications with epigenetic
significance. Curr. Opin. Chem. Biol. 2012, 16, 516−524.
(10) van den Born, E.; Vagbo, C. B.; Songe-Moller, L.; Leihne, V.;
Lien, G. F.; Leszczynska, G.; Malkiewicz, A.; Krokan, H. E.; Kirpekar,
F.; Klungland, A.; Falnes, P. O. ALKBH8-mediated formation of a
novel diastereomeric pair of wobble nucleosides in mammalian tRNA.
Nat. Commun. 2011, 2, 172.
(11) Rose, N. R.; McDonough, M. A.; King, O. N.; Kawamura, A.;
Schofield, C. J. Inhibition of 2-oxoglutarate dependent oxygenases.
Chem. Soc. Rev. 2011, 40, 4364−4397.
(12) Woon, E. C.; Demetriades, M.; Bagg, E. A.; Aik, W.; Krylova, S.
M.; Ma, J. H.; Chan, M.; Walport, L. J.; Wegman, D. W.; Dack, K. N.;
McDonough, M. A.; Krylov, S. N.; Schofield, C. J. Dynamic
combinatorial mass spectrometry leads to inhibitors of a 2-
oxoglutarate-dependent nucleic acid demethylase. J. Med. Chem.
2012, 55, 2173−2184.
(13) Han, Z.; Niu, T.; Chang, J.; Lei, X.; Zhao, M.; Wang, Q.; Cheng,
W.; Wang, J.; Feng, Y.; Chai, J. Crystal structure of the FTO protein
reveals basis for its substrate specificity. Nature 2010, 464, 1205−1209.
(14) King, O. N.; Li, X. S.; Sakurai, M.; Kawamura, A.; Rose, N. R.;
Ng, S. S.; Quinn, A. M.; Rai, G.; Mott, B. T.; Beswick, P.; Klose, R. J.;
Oppermann, U.; Jadhav, A.; Heightman, T. D.; Maloney, D. J.;
Schofield, C. J.; Simeonov, A. Quantitative high-throughput screening
identifies 8-hydroxyquinolines as cell-active histone demethylase
inhibitors. PLoS One 2010, 5, e15535.
Present Address
†School of Chemical Sciences, Universiti Sains Malaysia, 11800
USM, Pulau Pinang, Malaysia.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
■
We thank the Biotechnological and Biological Sciences
Research Council, and Pfizer (U.K.) for funding our NAOX
work, as well as the Malaysian government and Universiti Sains
Malaysia (scholarship for K.K.Y.) for financial support. We
thank Jacob Bush, Marcus Sims, and Luc Henry for help with
organic synthesis, Martin Munzel for advice on protein
̈
purification, Ian Clifton and Rasheduzzaman Chowdhury for
advice on crystallography, Akane Kawamura and Richard
Hopkinson for discussions, and the beamline staff at Diamond
Light Source for assistance.
ABBREVIATIONS USED
■
FTO, fat mass and obesity associated protein; 2OG, 2-
oxoglutarate; NAOX, nucleic acid oxygenase; ABH, AlkB
human homologue; HIF, hypoxia-inducible transcription factor;
JmjC, Jumonji C domain containing histone demethylase; DSF,
dynamic scanning fluorometry; ΔTm, melting temperature shift;
LC, liquid chromatography; NOG, N-oxalylglycine; 3meT, 3-
methylthymidine; 6meA, 6-methyladenosine; DSBH, double-
stranded β helix; PHD, HIF-prolyl hydroxylase; TCA cycle,
tricarboxylic acid cycle/Krebs cycle; IPTG, isopropyl β-D-1-
thiogalactopyranoside; DMSO, dimethyl sulfoxide; MES, 2-(N-
morpholino)ethanesulfonic acid; ESI-TOF, electrospray ioniza-
tion time-of-flight
REFERENCES
■
(15) Chowdhury, R.; Yeoh, K. K.; Tian, Y. M.; Hillringhaus, L.; Bagg,
E. A.; Rose, N. R.; Leung, I. K.; Li, X. S.; Woon, E. C.; Yang, M.;
McDonough, M. A.; King, O. N.; Clifton, I. J.; Klose, R. J.; Claridge, T.
D.; Ratcliffe, P. J.; Schofield, C. J.; Kawamura, A. The oncometabolite
2-hydroxyglutarate inhibits histone lysine demethylases. EMBO Rep.
2011, 12, 463−469.
(16) Rose, N. R.; Ng, S. S.; Mecinovic, J.; Lienard, B. M.; Bello, S. H.;
Sun, Z.; McDonough, M. A.; Oppermann, U.; Schofield, C. J. Inhibitor
scaffolds for 2-oxoglutarate-dependent histone lysine demethylases. J.
Med. Chem. 2008, 51, 7053−7056.
(1) Frayling, T. M.; Timpson, N. J.; Weedon, M. N.; Zeggini, E.;
Freathy, R. M.; Lindgren, C. M.; Perry, J. R.; Elliott, K. S.; Lango, H.;
Rayner, N. W.; Shields, B.; Harries, L. W.; Barrett, J. C.; Ellard, S.;
Groves, C. J.; Knight, B.; Patch, A. M.; Ness, A. R.; Ebrahim, S.;
Lawlor, D. A.; Ring, S. M.; Ben-Shlomo, Y.; Jarvelin, M. R.; Sovio, U.;
Bennett, A. J.; Melzer, D.; Ferrucci, L.; Loos, R. J.; Barroso, I.;
Wareham, N. J.; Karpe, F.; Owen, K. R.; Cardon, L. R.; Walker, M.;
Hitman, G. A.; Palmer, C. N.; Doney, A. S.; Morris, A. D.; Smith, G.
D.; Hattersley, A. T.; McCarthy, M. I. A common variant in the FTO
gene is associated with body mass index and predisposes to childhood
and adult obesity. Science 2007, 316, 889−894.
(17) Rotili, D.; Altun, M.; Kawamura, A.; Wolf, A.; Fischer, R.;
Leung, I. K.; Mackeen, M. M.; Tian, Y. M.; Ratcliffe, P. J.; Mai, A.;
Kessler, B. M.; Schofield, C. J. A photoreactive small-molecule probe
for 2-oxoglutarate oxygenases. Chem. Biol. 2011, 18, 642−654.
(18) Demetriades, M.; Leung, I. K.; Chowdhury, R.; Chan, M. C.;
McDonough, M. A.; Yeoh, K. K.; Tian, Y. M.; Claridge, T. D.;
Ratcliffe, P. J.; Woon, E. C.; Schofield, C. J. Dynamic combinatorial
chemistry employing boronic acids/boronate esters leads to potent
oxygenase inhibitors. Angew. Chem., Int. Ed. 2012, 51, 6672−6675.
(19) Chen, B.; Ye, F.; Yu, L.; Jia, G.; Huang, X.; Zhang, X.; Peng, S.;
Chen, K.; Wang, M.; Gong, S.; Zhang, R.; Yin, J.; Li, H.; Yang, Y.; Liu,
H.; Zhang, J.; Zhang, H.; Zhang, A.; Jiang, H.; Luo, C.; Yang, C. G.
Development of Cell-Active N(6)-Methyladenosine RNA Demethy-
lase FTO Inhibitor. J. Am. Chem. Soc. 2012, 134, 17963−17971.
(20) Yan, L.; Colandrea, V. J.; Hale, J. J. Prolyl hydroxylase domain-
containing protein inhibitors as stabilizers of hypoxia-inducible factor:
small molecule-based therapeutics for anemia. Expert Opin. Ther. Pat.
2010, 20, 1219−1245.
(2) Church, C.; Lee, S.; Bagg, E. A.; McTaggart, J. S.; Deacon, R.;
Gerken, T.; Lee, A.; Moir, L.; Mecinovic, J.; Quwailid, M. M.;
Schofield, C. J.; Ashcroft, F. M.; Cox, R. D. A mouse model for the
metabolic effects of the human fat mass and obesity associated FTO
gene. PLoS Genet. 2009, 5, e1000599.
(3) Church, C.; Moir, L.; McMurray, F.; Girard, C.; Banks, G. T.;
Teboul, L.; Wells, S.; Bruning, J. C.; Nolan, P. M.; Ashcroft, F. M.;
Cox, R. D. Overexpression of FTO leads to increased food intake and
results in obesity. Nat. Genet. 2010, 42, 1086−1092.
(4) Gerken, T.; Girard, C. A.; Tung, Y. C.; Webby, C. J.; Saudek, V.;
Hewitson, K. S.; Yeo, G. S.; McDonough, M. A.; Cunliffe, S.; McNeill,
L. A.; Galvanovskis, J.; Rorsman, P.; Robins, P.; Prieur, X.; Coll, A. P.;
Ma, M.; Jovanovic, Z.; Farooqi, I. S.; Sedgwick, B.; Barroso, I.; Lindahl,
T.; Ponting, C. P.; Ashcroft, F. M.; O’Rahilly, S.; Schofield, C. J. The
obesity-associated FTO gene encodes a 2-oxoglutarate-dependent
nucleic acid demethylase. Science 2007, 318, 1469−1472.
(5) Jia, G.; Yang, C. G.; Yang, S.; Jian, X.; Yi, C.; Zhou, Z.; He, C.
Oxidative demethylation of 3-methylthymine and 3-methyluracil in
single-stranded DNA and RNA by mouse and human FTO. FEBS Lett.
2008, 582, 3313−3319.
(21) Arend, M. P.; Flippin, L. A.; Volkmar, G. P.; Ho, W. B.; Turtle,
E. D.; Du, X. Novel nitrogen-containing heteroaryl compounds and
methods of use thereof. WO 2004 108681 A1, 2004.
H
dx.doi.org/10.1021/jm400193d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(22) Stubbs, C. J.; Loenarz, C.; Mecinovic, J.; Yeoh, K. K.; Hindley,
N.; Lienard, B. M.; Sobott, F.; Schofield, C. J.; Flashman, E.
Application of a proteolysis/mass spectrometry method for investigat-
ing the effects of inhibitors on hydroxylase structure. J. Med. Chem.
2009, 52, 2799−2805.
(23) Hausinger, R. P. FeII/alpha-ketoglutarate-dependent hydrox-
ylases and related enzymes. Crit. Rev. Biochem. Mol. Biol. 2004, 39, 21−
68.
(24) Frezza, C.; Pollard, P. J.; Gottlieb, E. Inborn and acquired
metabolic defects in cancer. J. Mol. Med. (Berlin) 2011, 89, 213−220.
(25) Mole, D. R.; Schlemminger, I.; McNeill, L. A.; Hewitson, K. S.;
Pugh, C. W.; Ratcliffe, P. J.; Schofield, C. J. 2-Oxoglutarate analogue
inhibitors of HIF prolyl hydroxylase. Bioorg. Med. Chem. Lett. 2003, 13,
2677−2680.
(26) Elvidge, G. P.; Glenny, L.; Appelhoff, R. J.; Ratcliffe, P. J.;
Ragoussis, J.; Gleadle, J. M. Concordant regulation of gene expression
by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: the
role of HIF-1alpha, HIF-2alpha, and other pathways. J. Biol. Chem.
2006, 281, 15215−15226.
(27) Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential
scanning fluorimetry to detect ligand interactions that promote protein
stability. Nat. Protoc. 2007, 2, 2212−2221.
(28) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. Method Enzymol. 1997, 276, 307−326.
(29) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.
D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl.
Crystallogr. 2007, 40, 658−674.
(30) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis,
I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-
Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R.
J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H.
PHENIX: a comprehensive Python-based system for macromolecular
structure solution. Acta Crystallogr. D 2010, 66, 213−221.
(31) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and
development of Coot. Acta Crystallogr. D 2010, 66, 486−501.
(32) Balzarini, J.; Celen, S.; Karlsson, A.; de Groot, T.; Verbruggen,
A.; Bormans, G. The effect of a methyl or 2-fluoroethyl substituent at
the N-3 position of thymidine, 3′-fluoro-3′-deoxythymidine and 1-
beta-D-arabinosylthymine on their antiviral and cytostatic activity in
cell culture. Antivir. Chem. Chemother. 2006, 17, 17−23.
I
dx.doi.org/10.1021/jm400193d | J. Med. Chem. XXXX, XXX, XXX−XXX