Quantitative analysis of histone demethylase probes using fluorescence polarization

Article abstract of DOI:10.1021/jm3018628

We previously reported methylstat as a selective inhibitor of jumonji C domain-containing histone demethylases (JHDMs). Herein, we describe the synthesis of a fluorescent analogue of methylstat and its application as a tracer in fluorescence polarization assays. Using this format, we have evaluated the binding affinities of several known JHDM probes, as well as the native cofactor and substrate of JHDM1A. This fluorophore allowed a highly robust and miniaturized competition assay sufficient for high-throughput screening.

Full text of DOI:10.1021/jm3018628

Brief Article
pubs.acs.org/jmc
Quantitative Analysis of Histone Demethylase Probes Using
Fluorescence Polarization
Wenqing Xu,†,⊥ Jessica D. Podoll,†,⊥ Xuan Dong,†,‡,⊥ Anthony Tumber,§ Udo Oppermann,§,∥
and Xiang Wang*,†
†Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, United States
‡Department of Developmental Biology, Shandong University, Jinan, Shandong 250100, China
§Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Headington OX3 7DQ, United
Kingdom
∥The Botnar Research Center, NIHR, Oxford Biomedical Research Unit, Oxford OX3 7LD, United Kingdom
S
* Supporting Information
ABSTRACT: We previously reported methylstat as a selective inhibitor of jumonji C domain-containing histone demethylases
(JHDMs). Herein, we describe the synthesis of a fluorescent analogue of methylstat and its application as a tracer in fluorescence
polarization assays. Using this format, we have evaluated the binding affinities of several known JHDM probes, as well as the
native cofactor and substrate of JHDM1A. This fluorophore allowed a highly robust and miniaturized competition assay sufficient
for high-throughput screening.
Scheme 1. Structures of Methylstat (1), Methylstat Acid (2),
and Its Fluorescent Analogue Methylstatfluor (3)
INTRODUCTION
■
Methylation of histone proteins is one of the most important
epigenetic modifications.1 These reversible modifications
recruit effector proteins and trigger a wide range of cellular
events, including regulation of gene expression, proliferation,
and differentiation. Histone methylations are closely regulated
by histone methyltransferases and histone demethylases. Since
2004, two families of enzymes have been reported to exhibit
demethylation activities: FAD-dependent monoamine oxidases
(LSD1 and 2) and jumonji C domain-containing histone
demethylases (JHDMs).2 Compared with LSDs, JHDMs have a
much broader substrate scope and can modify lysine residues at
all methylation states.
Expression of JHDMs plays critical roles in both develop-
ment and diseases such as cancer and mental retardation.3
Overproduction of 2-hydroxy-glutarate, a natural JHDM
inhibitor, due to mutation of isocitrate dehydrogenases, has
been identified in multiple cancers.4 This development has led
to numerous efforts to develop chemical probes targeting
JHDMs. Several classes of α-ketoglutarate (αKG) mimics have
been developed to inhibit JHDM activity5 because all JHDMs
use αKG as a cofactor. In addition, a substrate-mimicking small
molecule was recently reported to selectively inhibit H3K9-
demethylase KIAA1718.6 Our group recently discovered a
selective, cell-permeable, small-molecule inhibitor methylstat
(1, Scheme 1), which was designed as a bivalent substrate−
cofactor conjugate.7 Its corresponding acid, 2 (Scheme 1),
selectively inhibits JHDMs in vitro. This bivalent strategy has
also proven successful in two very recent reports on JMJD2
class-selective peptidic inhibitors.8
various JHDM isoforms. Most established JHDM biochemical
assays are enzyme inhibition assays.5a,9 Because of the self-
destructive nature of JHDMs under biochemical reaction
conditions,10 these assays typically require optimization for
different JHDM isoforms. In addition, they do not allow for
accurate measurement of the dissociation constants of the
JHDM probes. Thus, the IC50 values derived from these assays
cannot be compared directly. Here, we report the synthesis of a
fluorescent JHDM probe, 3 (Scheme 1), and the development
of a fluorescence polarization (FP)-based binding assay. This
assay allows us not only to quantitatively measure the
dissociation constants of several JHDM probes but also to
validate the inhibitory mechanism of methylstat.
Although several classes of JHDM inhibitors have been
discovered, determining the selectivity of these inhibitors
against various JHDM classes remains a major challenge. This
is mainly due to the lack of a uniform biochemical assay for
Received: December 17, 2012
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
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inhibitors may be obtained by modifying the secondary amine
of methylstat.
RESULTS AND DISCUSSION
■
The synthesis of fluorophore 3 began with conversion of
commercially available 4-cyanobenzaldehyde to aldehyde 4
(Scheme 2),11 which then underwent a reductive amination
3 shows similar fluorescent properties to fluorescein in pH
7.5 aqueous buffer with the maximum excitation and emission
wavelengths as 492 and 517 nm, respectively. With the
fluorophore in hand, we set out to develop and optimize an
FP binding assay to determine the affinity of fluorophore 3 to
various JHDMs. Three JHDMs (JHDM1A, JMJD2A, and
JMJD3) from three different classes were evaluated in a
saturation binding experiment by following the protocol
reported by Rossi and Taylor.12 FP signals were recorded
using the Envision Multilabel plate reader (Perkin-Elmer), and
expressed as the change in millipolarization ΔmP, where ΔmP
is the mP of the JHDM/3 mixture minus the mP of 3 only in
assay buffer. To determine the dissociation constants (Kd) of 3
and the JHDMs, we plotted JHDM concentrations against
ΔmP and fitted the data using eq 1:
a
Scheme 2. Synthesis of Fluorophore 3
ΔmP = [Pmin + Pmax × (x/Kd)n]/[1 + (x/Kd)n]
(1)
where, Pmax and Pmin are the maximum and minimum observed
ΔmP values, respectively, x is the JHDM concentration, and n
is the Hill coefficient of the binding curve. Preliminary tests
indicated that 3 bound JHDM1A with the highest affinity
among these three JHDMs; therefore, JHDM1A was used for
assay optimization.
a
Reagents and conditions: (a) LiAlH4, THF, reflux, 12 h, Boc2O, Et3N,
THF, 25 °C, 12 h, Dess−Martin periodinane, CH2Cl2, 25 °C, 30 min,
72% for 3 steps; (b) AcOH, NaBH3CN, MeOH, 25 °C, 12 h, 59%; (c)
TFA, CH2Cl2, 0 °C, 30 min, LiOH, THF, H2O, 25 °C, 2 h, 94% for 2
steps; (d) fluorescein isothiocyanate, satd NaHCO3/THF, 25 °C, 12 h,
37%.
The initial assay buffer composition was adapted from
previous reports on JHDM enzyme activity assays. These
generally contain an Fe2+ salt and a reducing agent, sodium
ascorbate,2,7 as Fe2+, but not Fe3+, is a cofactor for JHDM. Our
initial results showed that fluorophore 3 binds to JHDM1A
with much higher affinity (Kd: 8.6 1.1 nM) compared with
JMJD2A and JMJD3, but the Pmax was relatively low (<50 mP,
Figure 1A). In the absence of any additional metal ions, the
Pmax increased significantly (ca. 120 mP, Figure 1A); however,
the Kd of JHDM1A for 3 also increased significantly (19 1.4
nM, Figure 1A). Taken together, these results suggested the
loss of the Fe2+ during protein purification and protein
degradation in the presence of Fe2+, a common problem for
all Fe2+- and αKG-dependent hydroxylases.5a To minimize the
effects of Fe2+ oxidation, the binding assay was evaluated in the
presence of Fe2+ under anaerobic conditions; the results were
similar to those observed under ambient conditions in the
presence of ascorbate.
with amine 57 to afford secondary amine 6. The tert-
butoxycarbonyl (Boc) protecting group of 6 was then removed
by trifluoroacetic acid (TFA) and ester hydrolyzed under basic
conditions. The resulting diamine (7) was treated with
fluorescein isothiocyanate (FITC) to afford 3 as the major
product.
We next evaluated compound 7 and the fluorescent analogue
3 in the enzyme inhibition assays against JMJD2A and JMJD3,
two of the most sensitive JHDMs to methylstat acid (2).7
Compound 7 showed half-maximum inhibitory concentrations
(IC50s) of 2.4 and 7.3 μM against JMJD2A and JMJD3,
respectively, which are comparable to those of 2 (i.e., 3.3 and
3.1 μM, respectively). Interestingly, while 3 showed similar
activity against JMJD3 with an IC50 of 9.5 μM, it is less active
against JMJD2A, with an IC50 of 23.5 μM. Furthermore, the
IC50 values of 3 and 7 against JHDM1A were determined as
0.90 and 0.42 μM. These results suggest that selective JHDM
Because JHDM substrate binding necessitates the addition of
metal ions, we next examined the binding in the presence of
Figure 1. Optimization of the binding conditions for JHDMs and 3. (A) Binding to JHDM1A was measured with the addition of Fe2+, no additional
metal, or Ni2+ in assay buffer. (B) Binding of 3 to JHDM1A, JMJD2A, and JMJD3 was assessed. Only the binding of JHDM1A and 3 reaches
saturation at the concentrations tested.
B
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Figure 2. FP competition assay optimization and results. (A) 15, 30, and 60 nM of JHDM1A were tested in the FP competition assay with PDCA.
(B) The FP competition assay was used to determine IC50 and Ki values for known JHDM probes.
Ni2+ or Co2+. Ni2+ and Co2+ were chosen because they exhibit
increased stability under ambient conditions as compared to
Fe2+. Ni2+ and Co2+ have been shown to interact with native
substrates as well as inhibitors in the JHDM active site in
crystallographic studies; furthermore, these transition metals
are capable of inhibiting enzyme activation without altering
substrate or cofactor binding in the JHDM active site.13
Replacement of Fe2+ with Ni2+ or Co2+ in the assay buffer
afforded highly stable binding with a much larger dynamic
range (ca. 300 mP, Figure 1A). The Kd of JHDM1A for 3
except that a constant concentration of protein was used in
each condition and the nonfluorescent inhibitor was titrated
into the system (5 nM−2 mM). DMSO (1 μL, 1%) was added
to each assay well as a vehicle for the inhibitor. Of the three
JHDM1A concentrations tested, 60 nM JHDM1A afforded a
large dynamic range appropriate for competition assays (Figure
2A); therefore, 60 nM JHDM1A was selected as the protein
concentration for the following FP competition studies.
A series of JHDM probes were evaluated in the optimized FP
competition assay, such as methylstat acid (2), the JHDM
cofactor, αKG, N-oxalyl glycine (NOG, an αKG mimic), and
the substrate of JHDM1A, an H3K36me2 peptide (Figure
2B).5a,13 The results showed that all of the above molecules can
displace 3 from JHDM1A. Additionally, the IC50 values
stabilized after approximately 4 h and remained stable for up
to 24 h. These results not only suggest that 3 binds both the
substrate and αKG cofactor-binding sites of JHDM1A but also
confirms that methylstat acid (2) is a competitive JHDMs
inhibitor.
calculated in the presence of Ni2+ (9.3
0.5 nM) is more
similar to the native condition than that calculated in the
presence of Co2+ (22 1.4 nM). Hence, we chose to further
optimize the FP assay in the presence of Ni2+.
We found the binding of JHDM and 3 reached equilibrium
after 4 h of incubation at room temperature, and the signals are
stable over at least 24 h (Supporting Information (SI) Figure
S1). The stabilizing effect of the Ni2+ buffer on JHDM1A was
also observed by SDS-PAGE analysis (SI Figure S2). JHDM1A
in assay buffer containing Fe2+ or no additional metal showed
significant decomposition after as little as 2 h, whereas
JHDM1A in buffer containing Ni2+ showed no observable
decomposition over the course of 24 h. In addition to the
divalent metal ion, we have also optimized the buffer and
fluorophore concentration. TRIS buffer provided more
consistent results than other buffers such as MOPS and
HEPES; a stable and reliable FP signal was achieved using 1 nM
fluorophore 3.
Once the assay conditions were optimized for JHDM1A, we
reevaluated the binding of 3 with JMJD2A and JMJD3.
Although both these enzymes bound 3 at high concentrations,
saturation of the FP signal was not achieved at up to 10 μM
protein, the highest concentration tested (Figure 1B). Because
a protein fluorophore pair with high binding affinity is required
for appropriate resolution of nonfluorescent inhibitors in an FP
competition assay, further development of the FP competition
assay was performed using only JHDM1A.14
Next, the ability of 3 to serve as a tracer for FP competitive
binding experiments was assessed and the appropriate
concentration of JHDM1A for FP competition assays
optimized. Typically, a protein concentration at which 50−
80% of the fluorescent ligand is initially bound is used for FP
competition assays but must be optimized to achieve a desirable
dynamic range.12,14,15 The competition assay was validated and
optimized using pyridine 2,4-dicarboxylic acid (PDCA), a well-
known αKG mimic, as the competitive ligand (Figure 2A). The
competition assay set up was similar to that of the binding assay
This competition assay also afforded the half inhibitory
concentrations (IC50, Figure 2B) of these JHDM-binding
molecules. The IC50 values were then used to calculate their
dissociation constants (Ki) with JHDM1A using eq 2 and the
online Ki calculator
Ki = [I]50 /([L]50 /Kd + [P]0 /Kd + 1)
(2)
where, [I]50 and [L]50 are free inhibitor concentration and free
ligand concentration at 50% inhibition, [P]0 is free protein
concentration at 0% inhibition, and Kd is the dissociation
constant between protein JHDM1A and 3.15
The ability to quantify binding of competitive inhibitors in
the JHDM active site is an unprecedented development. Only
the binding affinities of some JHDMs relative to histone
peptides have been quantified by surface plasmon resonan-
ce;5a,13c however, the binding affinity of αKG for various
JHDMs has never been reported. Competitive binding assays
designed for screening inhibitors have shown relative binding
affinities, but report IC50 values of competitive inhibitors as
opposed to Ki values. Although IC50 values are useful for
assessing the relative effectiveness of inhibitors for a single
enzyme, they are not directly comparable between enzymes or
between different assays and always depend on the individual
assay conditions.16 Further expansion of our FP binding assay
to other JHDM isoforms may be extremely useful for assessing
the specificities as well as the inhibitory mechanism of various
JHDM probes.
C
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FP Competitive Binding Assay. Known JHDM active site
binders (2, αKG, PDCA, NOG, or peptide H3K36me2) were tested
for their ability to compete the binding of 3 to JHDM1A. Then 2-fold
serial dilutions of compounds were prepared as 100× solutions in
DMSO. Then 80 μL of JHDM1A (75 nM in assay buffer) were added
to each well, to which 1 μL of the above compound solutions were
added. The mixtures were incubated at room temperature for 30 min
prior to addition of 20 μL of 3 (5 nM in assay buffer). Controls
containing 3 and JHDM1A or JHDM1A and the competing
compound were included for background subtraction. Each experi-
ment was performed in duplicate. The assay plates were incubated at
room temperature for 4 h before signals were recorded by the Envision
Multilabel plate reader. The calculated data was fitted using
KaleidaGraph (v4.1.1, Synergy Software).
The FP competition assay was further miniaturized to the
384-well plate format. The total volume for each assay was
reduced to 20 μL, while the concentrations of JHDM1A (60
nM), fluorophore 3 (1 nM), and DMSO (1%) remained the
same. For high-throughput screening purposes, we also
evaluated the influence detergent, Tween 20, to our FP assay.
We found that addition of 0.1% Tween 20 to the assay buffer
completely abolishes the binding, and 0.001% of Tween 20 is
well tolerated. To calculate the Z′ factor, we used 2 (100 μM in
DMSO) and DMSO only as positive and negative controls,
respectively. Although the dynamic range of this assay
decreased slightly (SI Figure S3), a high Z′ factor value
(0.78) was calculated,17 which further demonstrates the
robustness of this assay and its suitability for high-throughput
screening.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for the syntheses and spectroscopic
characterization of the compounds, as well as determination
of Z′ factor experiment and result in this paper. \This material
is available free of charge via the Internet at http://pubs.acs.org.
CONCLUSION
■
We have discovered a fluorescent analogue of methylstat, 3, and
have used it to develop an FP binding assay. 3 selectively binds
JHDM1A with high affinity (Kd: 9.3 nM) and a large dynamic
range (ca. 300 mP). Ni2+ ion was found not only to be a good
surrogate to the native cofactor Fe2+, but it also stabilizes the
protein. The binding of 3 to JHDM1A can be displaced by
several known JHDM probes, including its cofactor (αKG),
substrate (H3K36me2), and methylstat acid (2). These results
confirm that methylstat acid is a bivalent competitive inhibitor
of JHDMs. In addition, this FP competition assay allows
quantitative measurement of Ki values of nonfluorescent
JHDM1A active site binding molecules. It is also noteworthy
that the Ki of αKG and JHDM1A was determined, which has
previously been impossible and illustrates the utility of our FP
binding assay for quantifying the binding affinities of native
JHDM substrates quickly and easily. Furthermore, we were able
to use our FP system to develop a highly robust and
miniaturized assay appropriate for high-throughput screening
of large compound libraries (Z′: 0.78). Further optimization of
the fluorophore for the development of FP assays appropriate
for other JHDMs is ongoing and will be reported in due course.
AUTHOR INFORMATION
Corresponding Author
*Phone (303) 492-6266. E-mail: xiang.wang@colorado.edu.
■
Author Contributions
⊥These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the University of Colorado
Boulder, and by the National Institutes of Health under grant
no. R01-GM098390, and the institutional training grant
GM008732 to J.D.P. X.D. thanks the China Scholarship
Council for a Visiting Student Scholarship. The Structural
Genomics Consortium is a registered charity (no. 1097737)
that receives funds from Abbvie, Boehringer Ingelheim, the
Canadian Institutes for Health Research, the Canadian
Foundation for Innovation, Eli Lilly and Company, Genome
Canada, GlaxoSmithKline, the Ontario Ministry of Economic
Development and Innovation, Janssen, the Novartis Research
Foundation, Pfizer, Takeda, and the Wellcome Trust. U.O. is
also supported by BBSRC, Bayer Healthcare and Oxford NIHR
Biomedical Research Unit.
EXPERIMENTAL SECTION
■
Synthetic procedure for the preparation of fluorophore 3, character-
ization data, and NMR spectra of all new compounds are in SI.
Protein Expression and Purification. Recombinant JHDM1A
(1−517) and JMJD3 (1018−1590) were expressed as 6XHis fusion
proteins using the pNIC28 and the pNH-TrxT expression vectors,
respectively. The coding regions were verified by sequencing and the
plasmids were transfected into BL21 Escherichia coli. Following
expression, JHDM1A was purified using Ni SepharoseTM 6 Fast
Flow beads (GE) by gravity chromatography according to the
manufacturer’s instructions. JMJD3 was purified using cobalt (high
density) agarose beads (Gold Biotechnology) according to the
manufacturer’s protocol. JMJD2A was expressed and purified as
described previously.7 The purified proteins were exchanged into assay
buffer, flash frozen in liquid nitrogen, and stored at −80 °C.
ABBREVIATIONS USED
■
JHDM, jumonji C domain-containing histone demethylase;
αKG, α-ketoglutarate; FP, fluorescence polarization; Boc, tert-
butoxycarbonyl; TFA, trifluoroacetic acid; FITC, fluorescein
isothiocyanate; PDCA, pyridine 2,4-dicarboxylic acid; NOG, N-
oxalyl glycine; Kd, dissociation constant; IC50, half-maximum
inhibitory concentration; Ki, dissociation constant of inhibitor
FP Binding Assay. FP binding experiments were performed in
black, low-binding, half area 96-well plates (Corning 3993). Then 80
μL of JHDM1A (2.44 nM to 2.50 μM in 2-fold dilution) in assay
buffer (50 μM NiCl2, 25 mM TRIS, 100 mM NaCl, pH 7.5) were
added to experimental wells. After 10 min, 20 μL of 3 (5 nM in assay
buffer) was then added to each well. Wells containing protein only
were subtracted from assay wells as background. Plates were incubated
at room temperature and read five times at each time point (1, 2, 4, 6,
8, 10, and 24 h), and the average values at each time point were used
for the calculation of the polarization values. Binding curves were fit
using KaleidaGraph (v4.1.1, Synergy Software).
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