Journal of Medicinal Chemistry
Article
crystallization drops contained 200 nL of Notum protein and 100 nL
of reservoir solution of 1.5 M ammonium sulphate and 0.1 M sodium
citrate, pH 4.2.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
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Thermal Shift Assay. Thermal shift assays were carried out in a
semiskirted 96-well PCR plate (4-Titude). Each well contains 3 μg of
glycosylated Notum protein, 3× SYPRO Orange dye (Thermo Fisher
Scientific), and compounds at various concentrations and adjusted to
a final volume of 50 μL with assay buffer (10 mM Hepes, pH7.4, 150
mM NaCl, and 2% DMSO). The samples were heated in an Mx3005p
qPCR machine (Stratagene, Agilent Technologies) from room
temperature at a rate of 1 °C/min for 74 cycles. Fluorescence
changes were monitored with excitation and emission wavelengths at
492 and 610 nm, respectively.
Electrospray Mass Spectrometry. Reversed-phase chromatog-
raphy was performed in-line prior to mass spectrometry using an
Agilent 1290 uHPLC system (Agilent Technologies inc. USA).
Concentrated protein samples were diluted to 0.02 mg/mL in 0.1%
formic acid and 50 μL was injected on to a 2.1 mm × 12.5 mm
Zorbax 5 μm 300SB-C3 guard column housed in a column oven set at
40 °C. The solvent system used consisted of A: 0.1% formic acid in
ultrahigh-purity water (Millipore) and B: 0.1% formic acid in
methanol (LC−MS grade, Chromasolve). Chromatography was
started in 90% A and 10% B at a flow rate of 1.0 mL/min. A linear
gradient from 10% B to 80% B was applied over 35 s. Elution then
proceeded isocratically at 95% B for 40 s followed by equilibration
under initial conditions for further 15 s. Protein intact mass was
determined using a 6530 ESI-QTOF mass spectrometer (Agilent
Technologies Inc. USA). The ion source was operated with the
capillary voltage at 4000 V, nebulizer pressure at 60 psig, drying gas at
350 °C, and drying gas flow rate at 12 L/min. The instrument ion
optic voltages were as follows: fragmentor 250 V, skimmer 60 V, and
octopole RF 250 V.
Data collection and refinement statistics, chemical
synthesis, and UPLC traces for lead compounds
Molecular formula strings (CSV)
Accession Codes
Coordinates for X-ray structures of Notum crystallized with 1
(PDB code 7ARG), 2 (PDB code 7B37), 3 (PDB code 7B2V),
4 (PDB code 7B2Y), 6 (PDB code 7B2Z), and Notum S232A
mutant with 2 (PDB code 7B3F) have been deposited in the
Protein Data Bank. Authors will release the atomic coordinates
upon article publication.
AUTHOR INFORMATION
Corresponding Authors
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Paul V. Fish − Alzheimer’s Research UK UCL Drug Discovery
Institute, University College London, London WC1E 6BT,
E. Yvonne Jones − Division of Structural Biology, Wellcome
Centre for Human Genetics, University of Oxford, Oxford
Authors
Yuguang Zhao − Division of Structural Biology, Wellcome
Centre for Human Genetics, University of Oxford, Oxford
Fredrik Svensson − Alzheimer’s Research UK UCL Drug
Discovery Institute, University College London, London
David Steadman − Alzheimer’s Research UK UCL Drug
Discovery Institute, University College London, London
Sarah Frew − Alzheimer’s Research UK UCL Drug Discovery
Institute, University College London, London WC1E 6BT,
U.K.
Notum OPTS Activity Assay. The OPTS activity assay has been
described in previous reports.24,25 Briefly, the test compounds, the
reporter substrate OPTS (Sigma), and the recombinant Notum
protein were dispensed into 384-well plates (Greiner) using a Labcyte
Echo 550 acoustic liquid handler and incubated 40 min in room
temperature. The endpoint fluorescence was measured on a
PheraSTAR FSX microplate reader with an excitation wavelength of
485 nm and an emission wavelength of 520 nm. The compound IC50
values were calculated from curves using a 4PL fit.
Cell-based TCF/LEF Reporter (Luciferase) Assay. The cellular
Wnt signaling functional assay has been described in previous
reports.24,25 Briefly, the reporter cell plate containing stable HEK293
STF cells53 (1 × 104 cells per well in 384-well microplates) carrying
the Super Top Flash firefly luciferase reporter was prepared
(overnight at 37 °C). Compounds and Notum protein were mixed
for 10 min before the recombinant Wnt-3A was added and incubated
for 1 h at room temperature. Then, the mixture from the compound
plates was added to reporter cell plates and incubation overnight at 37
°C. For luciferase assay, steady-glo luciferase assay buffer (20 μL,
Promega) was applied to the cell plates using the CyBio, the
luminescence was measured on a PHERAstar FSXmicroplate reader
with an excitation wavelength of 458 nm and an emission wavelength
of 520 nm.
Amy Monaghan − Alzheimer’s Research UK UCL Drug
Discovery Institute, University College London, London
WC1E 6BT, U.K.
Magda Bictash − Alzheimer’s Research UK UCL Drug
Discovery Institute, University College London, London
WC1E 6BT, U.K.
Tiago Moreira − Centre for Medicines Discovery, University of
Oxford, Oxford OX3 7DQ, U.K.
Rod Chalk − Centre for Medicines Discovery, University of
Oxford, Oxford OX3 7DQ, U.K.
Weixian Lu − Division of Structural Biology, Wellcome Centre
for Human Genetics, University of Oxford, Oxford OX3
7BN, U.K.
Crystal Soaking, Data Collection, and Structural Analysis.
For crystal soaking, compounds were dissolved in dimethyl sulfoxide
(100 mg/mL) and then diluted into reservoir solution with 40%
ethylene glycol at a concentration about 5 mg/mL. Equal amounts of
compound solutions at concentrations of around 20 mM were applied
to crystal drops for 30 min at room temperature. Crystals were flash-
frozen in liquid nitrogen. Data sets were recorded from crystals at 100
K at the Diamond Light Source (I03), processed using Xia2,54 and
refined with Refmac.55 The pymol Molecular Graphics System
Complete contact information is available at:
Author Contributions
∥Y.Z., F.S., and DS contributed equally.
Funding
This work was supported by Cancer Research UK (C375/
A17721). The ARUK UCL Drug Discovery Institute is core
funded by Alzheimer’s Research UK (520909). The Wellcome
Trust funds the Wellcome Centre for Human Genetics,
̈
(Schrodinger, LLC, Cambridge, Cambridgeshire, UK) was used to
prepare the figures.
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J. Med. Chem. XXXX, XXX, XXX−XXX