Identification of synthetic inhibitors for the DNA binding of intrinsically disordered circadian clock transcription factors

Article abstract of DOI:10.1039/d0cc04861e

Essential components of the human circadian clock, BMAL1 and CLOCK, which are intrinsically disordered transcription factors, were expressed and subjected to a fluorescentin vitrobinding assay using an E-box DNA fragment. Screening of a chemical library identified 5,8-quinoxalinedione (1), which was found to inhibit binding of the heterodimer BMAL1/CLOCK to E-box at low micromolar concentrations.

Full text of DOI:10.1039/d0cc04861e

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Identification of synthetic inhibitors for the DNA binding of
intrinsically disordered circadian clock transcription factors
Yusuke Hosoya,a Wataru Nojo,c Isao Kii,a Takanori Suzuki,c Miki Imanishi,b and Junko Ohkandaa*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Essential components of the human circadian clock, BMAL1 and
CLOCK, which are intrinsically disordered transcription factors,
were expressed and subjected to a fluorescent in vitro binding
assay using an E-box DNA fragment. Screening of a chemical library
identified 5,8-quinoxalinedione (1), which was found to inhibit
binding of the heterodimer BMAL1/CLOCK to E-box at low
micromolar concentrations.
Intrinsically disordered proteins and regions (IDPs and IDRs) are
unfolded proteins that lack well-defined tertiary structures and
are predicted to be present in about 40% of all human proteins.1
Significant attention has been given to these disordered proteins
due to their crucial roles in controlling cell signalling
transduction through weak protein–protein interactions.2-7
Binding to their partner proteins with dissociation constants (Kd)
in the micromolar range and lifetimes of seconds triggers
transient structural changes in IDPs and IDRs, allowing for
dynamic assembling of weak and fast signalling transmissions.8
Recent studies have shed light on their central role in liquid–
liquid phase separation,9,10 a process that generates non-
membrane liquid-like cellular compartments. These include
signalling clusters,11 RNA granules,12 and nucleoli,13
demonstrating that reversible assembly of IDPs and IDRs
mediates diverse biological processes.10,14-15
Because IDPs and IDRs have been implicated in many illnesses,
including cancers and neurodegenerative diseases,2 there is
tremendous interest in developing therapeutic agents for this
untapped class of drug targets.1-7 However, their structural
flexibility and complex regulatory mechanisms involving
posttranslational modifications16 complicate a rational approach
to the development of IDP inhibitors. In addition, because of a
tendency for aggregation, the experimental handling of
recombinant disordered proteins remains challenging.17 Recent
efforts involving yeast two-hybrid assay,18 surface plasmon
Fig. 1 a) Domain organization of truncated BMAL1 and CLOCK. b) Change in the
fluorescence polarisation of FAM-labelled oligo dsDNA fragment (10 nM) upon
addition of either BMAL1 or CLOCK protein (500 nM) or an equimolar mixture of
BMAL1 and CLOCK; E-box DNA (5'-GCGCGGTCACGTTTTCCACT-3'), control DNA (5'-
GCGCGGTGCTAGTTTCCACT-3'). FP was measured in 10 mM Tris HCl (pH 8.0), 150 mM
NaCl, and PEG200 (10% v/v) and excited at 488 nm and detected at 526 nm. n=3. Inset:
Titration curves of the equimolar mixture of BMAL1 and CLOCK (0–300 nM, solid line
with red circles) to dsDNA (10 nM, dash line with white triangles) at 25°C. n = 3. The
mean value ±SEM are presented.
resonance,19 NMR,20 thermal shift assay,21 and virtual
screening,22 have successfully led to several low-molecular-
weight inhibitors of c-Myc, EWS-FLI1, p27, NUPR1, and -
synuclein, respectively. In addition, a designed short helical
peptide was reported to bind disordered Id proteins.23 Although
these studies have highlighted the promise of IDP-directed drug
discovery, examples are still limited.
We envisioned that an in vitro fluorescence polarisation (FP)
assay using recombinant IDPs might be useful for exploration
and functional evaluation of inhibitors via high-throughput
a. Academic Assembly, Institute of Agriculture, Shinshu University, 8304 Minami-
Minowa, Kami-Ina, Nagano 399-4598, Japan.
b. Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011,
Japan.
c. Department of Chemistry, Faculty of Science, Hokkaido University, N10 W8, North-
ward, Sapporo 060-0810, Japan.
†Electronic Supplementary Information (ESI) available: See DOI: 10.1039/x0xx00000x
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DOI: 10.1039/D0CC04861E
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Fig. 3 Concentration-dependent inhibitory activity of 1 and 4 (0.005–50 M) against
binding of BMAL1/CLOCK (500 nM) to 5'-FAM-labelled E-box DNA fragment (10 nM).
The reaction mixture was incubated at 25°C for 20 min prior to measurement of the r
value. r0: the r value of the oligo DNA alone. n = 3. The mean value ±SEM are presented.
Fig.
2
Chemical structures of the compounds evaluated in this study. a) TfO-
(trifluoromethanesulfonate) salt.
screening (HTS) of chemical libraries. To this end, we focused
on brain and muscle ARNT-like 1 (BMAL1)24 and circadian
locomotor output cycles kaput (CLOCK),25 the highly
disordered circadian transcription activators.26 These proteins
form a heterodimer and bind to E-box regulatory elements in
Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2)
genes, activating their transcription during daytime. At night, the
protein products PER and CRY accumulate, dimerise,
translocate to the nucleus, and bind to BMAL1/CLOCK to
reduce its transcriptional activity.27,28 This transcriptional
feedback loop is key to the 24-hour circadian clock of mammals.
Recent studies have shown that disturbances in the circadian
rhythm mechanism are linked to psychiatric disorders29 and
tumorigenesis.30 Thus, synthetic molecules that can modulate the
heterodimer formation and its binding to E-box DNA are
desirable for the development of new therapeutic agents. Here,
we report a new fluorescent in vitro assay platform, in which
truncated BMAL1 and CLOCK proteins were prepared and their
binding to an E-box DNA fragment evaluated. Screening of a
chemical library led to identification of 5,8-quinoxialinedione
(1), which significantly inhibited binding of BMAL1 and
CLOCK to E-box DNA at low micromolar concentrations. The
results of a structure–activity relationship study are also reported.
The 69-kDa BMAL1 and 95-kDa CLOCK proteins contain
basic helix-loop-helix (bHLH) domains, Per-ARNT-Sim (PAS)
domains A and B, and transactivation domains (TADs). The
bHLH is an N-terminal DNA-binding domain,31 whereas the
PAS domains serve as a versatile sensor in signalling pathways.32
Previous studies reported that more than 30% of BMAL1 and
nearly 60% of CLOCK are predicted to be disordered26 by
computational simulation of disordered probability33 and that the
intrinsically flexible PAS domains adopt various conformations
depending on bound ligands or interacting partners.32,34 Recent
X-ray structural analysis of the heterodimer of mouse
BMAL1/CLOCK revealed that the dimerisation interface
involves not only the leucine zippers in bHLH, but also both the
PAS A and B of each protein, engaging contact areas of ~1,950
and 700 Å2, respectively, through electrostatic and hydrophobic
interactions including aromatic residues such as Trp427 of
BMAL1 and Trp284 of CLOCK.34 These structural features
clearly indicate that the PAS domains play an essential role in
dimer formation, thus in the DNA binding. Therefore, we
prepared a model protein of BMAL1 and CLOCK comprising
both bHLH and PAS domains, with (His)6 and thioredoxin, a
soluble tag, attached at each N terminus (Fig. 1a).35
Recombinant human BMAL1 (68 kDa) and CLOCK (62 kDa)
were expressed in Escherichia coli according to a standard
method (see ESI†). Each protein was obtained as an inclusion
body, which was dissolved in urea, purified by an Ni-NTA
column, and dialysed to refold. The protein electrophoresis
analysis showed that the resulting BMAL1 was fairly pure,
whereas CLOCK contained two unknown protein bands at
around 30 kDa (Fig. S1). Further purification attempts were
unsuccessful. Thus, we decided to proceed with evaluation of the
DNA-binding properties of these proteins.
An in vitro FP-based binding assay was performed by
incubating protein (500 nM) with a 5'-fluorescein (FAM)-
labelled double-stranded oligo DNA fragment (10 nM)
possessing either a Per2 E-box sequence or a mutated sequence
as a control.34 To mimic the crowded environment of the nucleus,
10% v/v PEG200 was added (Fig. S2).36 While BMAL1,
CLCOK, or a 1:1 mixture of the proteins failed to show apparent
binding to the mutated dsDNA (Fig. 1b, white bars), an
equimolar mixture of the proteins showed a significantly
increased FP value compared with each protein alone (Fig. 1b,
red bars).37 These results suggest that the recombinant proteins
retain the ability to form the heterodimer, thereby binding to E-
box DNA. Furthermore, the titration experiments confirmed that
1:1 binding of an equimolar mixture of BMAL1/CLOCK to E-
box DNA had a Kd value of 36 ± 10 nM (Fig. 1b, inset: red circle),
which is consistent with a previously reported value (59 ± 10
nM).34 No binding was observed with the control DNA (Fig. 1b,
2 | J. Name., 2012, 00, 1-3
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Because quinones undergo two-electron reductVioiewn AtroticlaefOfnolirnde
39
DOI: 10.1039/D0CC04861E
the corresponding hydroquinones under biological conditions,
we suspected that 1 would convert to 16 in solution. Evaluation
of 16–18 demonstrated that hydroquinones 16 and 17 were as
potent as 1, whereas the dimethylether derivative 18 was inactive
(Fig. 4). Notably, HPLC analysis showed that 16 was readily
converted to 1 in an aqueous solution at room temperature (Fig.
S5). These results indicate that 1 and 16 exist in equilibrium and
suggest that either 1 or 16, or both, may act as the active species.
The PAS A and B domains of BMAL1 and CLOCK are
cysteine-rich domains (13 and 8 residues, respectively).
Considerable effort has recently focused on the development of
PAS-directed inhibitors for treating cancer40 and neurological
disorders,41 and an allosteric inhibitor for hypoxia-inducible
factors 2 (HIF 2) is currently being tested in a clinical trial.42
Studies have revealed that this aromatic compound binds to a
hydrophobic cleft in the PAS B domain of HIF 2, inducing a
structural alteration and thereby disrupting dimerisation with
ARNT protein.43 The three-dimensional structures of the PAS B
domains of HIF 2 and BMAL1 are highly preserved and
BMAL1 notably possesses Cys373 near the hydrophobic cleft
(Fig. S6). Because nature uses covalently bound quinone
cofactors, such as cysteine tryptophylquinone,44 and the covalent
attachment of quinones to cysteine residues has been well
studied,43 it is reasonable to hypothesise that hydrophobic
electrophiles may react with these cysteine residues.
We attempted MALDI-TOF analysis of the chemically treated
proteins, and the spectra observed for BMAL1 were significantly
broadened compared with a non-treated control and CLOCK,
suggesting that BMAL1 may be more vulnerable to chemical
reaction with 1 than CLOCK (Fig. S7). We then tested whether
masking of cysteine residues affects the inhibitory activity of 1.
When BMAL1 and CLOCK were separately treated with
iodoacetamide, dialysed, and then combined, the same level of
DNA binding as the control was observed, but the inhibitory
activity of 1 was significantly reduced (Fig. S8). Furthermore,
electrophoresis analysis demonstrated that treatment of 1 causes
smearing in the case of BMAL1, but this was less evident for
CLOCK (Fig. S9a). This smearing clearly disappeared when
proteins were pre-treated with iodoacetamide (Fig. S9b). These
results indicate that 1 preferably reacts with nucleophilic
residues such as cysteines in BMAL1. Cell-based evaluation of
1 by luciferase reporter assay45 did not show significant activity
at 10 M against BMAL1/CLOCK interaction, presumably due
in part to its low stability in the cellular environment.
Fig.
4 Inhibitory activities of compounds 1–18 (50 M) against binding of
BMAL1/CLOCK (500 nM) to 5'-FAM-labelled E-box DNA fragment (10 nM). n = 3. The
mean value ±SEM are presented.
inset: white triangle), clearly demonstrating the selective binding
of the resulting heterodimer to the E-box fragment.
To explore synthetic compounds that are able to disrupt the
binding of BMAL1/CLOCK to DNA, a chemical library of
heterocycles (1,785 compounds) was screened in a 96-well
format (ESI†). The results identified 5,8-quinoxalinedione 1 (Fig.
2) as a hit compound that significantly reduced the FP value (Fig.
S3). Compound
1 demonstrated concentration-dependent
inhibitory activity against the binding of BMAL1/CLOCK to E-
box DNA with an IC50 value of about 1 M (Fig. 3, red bars).
Importantly, structurally similar and bulkier benzoquinoxaline-
5,10-dione 4 was inactive (Fig. 3, white bars). These results
validate the inhibitory activity of 1 for DNA binding.
Next, we wanted to assess the specificity of the inhibitory
activity of 1. To this end, we tested 1 for binding of specificity
protein 1-zinc finger transcription factor (Sp1ZF) to a GC box
DNA fragment by conducting an electrophoretic mobility-shift
(EMSA) assay.38 The results indicated that 1 did not interfere
with the binding of Sp1ZF to the GC box DNA at the
concentration range of 1–10 M (Fig. S4). Thus, the activity of
1 is selective for BMAL1/CLOCK and E-box over Sp1ZF and
GC box, suggesting that the activity is likely not caused by
nonspecific binding of 1 to a protein or DNA fragment.
The distinct activities observed for 1 and 4 prompted us to
perform structure–activity relationship studies of 2–15 (Fig. 2).
Interestingly, the dimethyl derivative 2 was less active, whereas
dibromo-substituted 3 was as potent as 1, suggesting that the
electrophilicity of the quinone moiety is important (Fig. 4). All
benzo/pyrazino derivatives 4–12 were less active, indicating that
addition of the fused ring to the enedione of 1 hampers inhibition.
Furthermore, the results showing that the thiadiazolopyrazine
derivative 13 was less active than 1 while the dibromo-quinone
derivative 14 was equally potent as 3 clearly indicated that the
quinone moiety is required for activity. As seen in 15,
replacement of the dibromo groups in 14 by amino groups
reduced activity, which is consistent with the trend observed for
1–3, in which electron-deficient compounds with
addition/substitution reactivity at the enedione unit (R = H or Br)
were more potent.
In conclusion, a fluorescent-based chemical library screening
using recombinant BMAL1 and CLOCK permitted
identification of 1 as a potent inhibitor of binding to E-box DNA
at low micromolar concentrations. Our results suggest that 1
covalently reacts with protein(s) and may modulate the dimer
formation. Although further study is required in order to
elucidate the details of the mechanism of action and improve
cellular activity, to our knowledge, this is the first example of a
synthetic compound that disrupts binding of BMAL1/CLOCK to
an E-box DNA fragment. This simple low-molecular-weight
heterocycle may serve as a useful scaffold for the development
of BMAL1/CLCOK inhibitors.
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20 L. I. Iconaru, D. Ban, K. Bharatham, A. Ramanathan, W.
We acknowledge support from the Japanese Ministry of
Education, Culture, Sports, Science and Technology (18H04376
to T.S., 18K04394 to J.O.), Japan Society for the Promotion of
Science (20H02719 to T.S., 18H02106 and 20H04769 to J.O.),
Astellas Foundation for Research on Metabolic Disorders, Naito
Foundation, and Uehara Foundation (J.O.). We thank Prof.
Takeyuki Suzuki of the Comprehensive Analysis Center of ISIR,
Osaka University, for assistance with mass analysis.
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DOI: 10.1039/D0CC04861E
Identification of synthetic inhibitors for the DNA binding of intrinsically
disordered circadian clock transcription factors
Table of Contents
Fluorescent-based library screening for the DNA-binding of disordered BMAL1 and
CLOCK identified heterocyclic inhibitors which possess low micromolar activity.