A Light-Controllable Chemical Modulation of m6A RNA Methylation

Article abstract of DOI:10.1002/anie.202103854

Bioactive small molecules with photo-removable protecting groups have provided spatial and temporal control of corresponding biological effects. We present the design, synthesis, computational and experimental evaluation of the first photo-activatable sma

Full text of DOI:10.1002/anie.202103854

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: A Light-controllable Chemical Modulation of m6A RNA
Methylation
Authors: Ling Lan, Ying-Jie Sun, Xiao-Yang Jin, Li-Jun Xie, Li Liu, and
Liang Cheng
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10.1002/anie.202103854
Angewandte Chemie International Edition
RESEARCH ARTICLE
A Light-controllable Chemical Modulation of m6A RNA
Methylation
Ling Lan,[a][c] Ying-Jie Sun,[a][c] Xiao-Yang Jin,[a][c] Li-Jun Xie,[a] Li Liu[a][c] and Liang Cheng*[a][b][c]
Dedicate to Prof. emeritus Dong Wang on the occasion of his 80th birthday.
[a]
L. Lan, Y.-J. Sun, X.-Y. Jin, Dr. L.-J. Xie, Prof. L. Liu and Prof. L. Cheng
Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Laboratory of Molecular Recognition and Function, CAS Research/Education
Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences
Beijing 100190, China
E-mail: chengl@iccas.ac.cn
[b]
[c]
Prof. L. Cheng
Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences
Hangzhou 310024, China
L. Lan, Y.-J. Sun, X.-Y. Jin, Prof. L. Liu and Prof. L. Cheng
University of Chinese Academy of Sciences
Beijing 100049, China.
Supporting information for this article is given via a link at the end of the document.
Abstract: Bioactive small molecules with photo-removable
protecting groups have provided spatial and temporal control of
corresponding biological effects. Herein we presented the design,
synthesis, computational and experimental evaluation of the first
photo-activatable small-molecule methyltransferase agonist. By
blocking the functional N-H group on MPCH with a photo-removable
ortho-nitrobenzyl moiety, we have developed a promising photo-
caged compound that had completely concealed its biological
activity. Short UV light exposure of cells treated with that caged
N6-Methyladensoine m6A is one the most abundant internal
modifications in mRNAs, presenting in 1~4‰ of all adenosines
in global cellular RNAs.[6] Dysregulated m6A methylation levels
has been proven to affect RNA metabolism and hence lead to
various diseases.[7] For example, a decrease of m6A caused by
amplified demethylase activity contributed to the onset of
obesity,[8] while increased levels of m6A methylation were
associated with the progression of human abdominal aortic
molecule in
a
few minutes resulted in
a
considerable
aneurysm[9] and human brain developing.[10]
Therefore,
hypermethylation of m6A modification in transcriptome RNAs,
implicating a rapid release of the parent active compound. This
study validates for the first time the photo-activatable small organic
molecular concept in the field of RNA epigenetic research, which
represents a novel tool in spatiotemporal and cellular modulation
approaches.
considerable progress has been made in the discovery and
development of methyltransferase/demethylase-based organic
molecules for cancer therapy.[11] Despite these advances, our
understanding of intracellular functionalities of m6A is inadequate
by the lack of approaches allowing for a selective modulation of
this methylation process with high spatial and temporal precision.
Non-invasive manipulation of cellular processes by light-
activated biomacromolecules and chemical probes has recently
developed as an emerging scientific field.[12] However, being
able to control RNA epigenetics with spatiotemporal control still
represented a challenge. More recently, we have demonstrated
an in vitro artificial demethylation of m6A through flavin
mononucleotide facilitated biocompatible photo-oxidation[13] and
a small molecular triggered deprenylation of cytokinins in live
plant Arabidopsis thaliana.[14] In this report, we demonstrated
that a photo-responsive chemical method is feasible for a
precise spatiotemporal modulation of particular RNA
modification, m6A for example, in live cells (Scheme 1A).
Introduction
Till now, over 170 post-transcriptional RNA epigenetic
modifications have been identified on a variety of RNAs,
including mRNA, tRNA, rRNA, lncRNA, miRNA, snoRNA and
other non-coding RNAs.[1]
Similar to that highly diverse
distribution, the functions of those epigenetics varied
significantly in diverse physiological processes, such as genetic
information translation and regulation processes.[2]
Their
abnormal change have been shown in the association with a
plethora of human disease.[3] Thus, a precise regulation of a
particular RNA variant is highly desirable. Indeed, classical
genetic approaches such as knockouts/downs, mutations, and
overexpression of corresponding RNA-modifying enzymes
(RMEs) have led to the discovery of several significant
4]
molecular mechanisms behind those epigenetic marks.[1a,
Furthermore, the development of chemical tools targeting those
RMEs have attracted considerable attention in the last decade.[5]
However, an efficient approach to manipulate a particular
modification in cellular environment with high a spatiotemporal
precision is still a challenge.
1
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10.1002/anie.202103854
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RESEARCH ARTICLE
The agonism was believed to arise from multiple cooperative
interactions between piperidine ring with surrounding residues in
METTL3/14 complex, as established in previous studies. In
contrast to traditional approaches by randomly grafting photo-
caging groups onto chemical functionalities, we used the
computational docking to precisely guide and evaluate the
caging strategy. The program AutoDock 4.2 was used to
characterize the binding of METTL3/14 complex (PDB: 5K7W)
with small compounds (Schemes 1B-D, Tables 1 and S2).[16] It
showed that the agonist 1 could preferably occupy a cavity of
SAM binding channel in a similar manner with a high affinity by
interacting mainly with the Glu532 (with N-H), His538 and Lys513
(both with C=O) residues. The binding energy (G) was
calculated as -6.86 kcal·mol-1, while the binding sites were
deduced with Lys513 and His538 (both with the carboxyl group),
and Glu532 (with the amino group), respectively. These binding
results were highly consistent with previous report (G = -6.94
kcal·mol-1, binding at Lys513 and Glu532).[15] However, we also
observed some other binding modes other than at the SAM
channel (for other inferior binding modes and their interacting
sites, see Supporting Information for details). These allosteric
binding effects should be taken into consideration when high
concentration of small organic molecules are used.[15] We
anticipated that a simple and yet appropriate N-caging group
would not only obstruct the key interaction with Glu532, but would
also impede other favorable bonding by inducing a large
conformational change of the chair-like structure of the
piperidine. We thus designed two masked compounds bearing
2-nitrophenyl methyl ethyl (2) and [4,5]dioxole substituted (3)
photo-caging substituents at the key N-H (Scheme 1E) and
calculated their binding affinities with METTL3/14 complex
(Schemes 1C-D, Table 1). We did observe significant changes
in their structures, which caused the disappearance of all crucial
interactions embedding in the METTL3/14-SAM-binding
channel.[17] On the other hand, some trivial contacts with
surrounding residues were detected (Table 1), which suggested
that the entire large size molecules produced differential
interactions, which were consistent with their much lower binding
Scheme 1. (A) Concept of a light-triggered RNA methylation of adenosine
promoted by the methyltransferase METTL3.
A METTL3 agonist was
energies (G
=
-5.56 and -6.24 kcal·mol-1, respectively),
conjugated with a photo-caging group in the active site that blocked its binding.
The methylation underwent without interruption, yielding a normal m6A level.
Irradiation with light (hv) removes the caging group, releasing the compound
and activating METTL3, resulting in higher m6A methylation level. (B) The
docking model and the interaction diagram with the lowest energy of
METTL3/14 complex with compound 1 (as shown in (E)). The yellow dashed
lines were used to indicate the key hydrogen bonds between the two
molecules. (C) The docking model and the interaction diagram with the lowest
energy of METTL3/14 complex with compound 2 (as shown in (E)). (D) The
docking model and the interaction diagram with the lowest energy of
METTL3/14 complex with compound 3 (as shown in (E)). (E) Structures of the
METTL3 agonist 1 and the caged compounds 2-3. Blue part represents the
photo-caging groups.
dissociation constants (Ki = 83.95 and 26.79 μM, respectively)
and ligand efficiencies (LE = 0.26) comparing with compound 1
(Ki = 9.43 μM and LE = -0.69). Thus, the temporal concealing of
N-H and the relatively large substituents would make it possible
to be a brake on the compound binding with METTL3 and thus
totally block its agonism. Transcriptome RNAs would then be
methylated without interruption and yielding a normal m6A
amount. If the caging group were removed upon light exposure
to release their precursor that would then subsequently activate
METTL3, a higher m6A methylation level would be accomplished.
Therefore, the molecular docking suggested it provide an
efficient
means
to
modulate
the
corresponding
methyltransferase activity and regulate the m6A RNA level
without RMEs engineering.
Results and Discussion
Given the essential role of methyltransferases in the transferring
methyl group from S-adenosylmethionine (SAM) to designated
RNAs, we sought to achieve a light-regulated control over its
function with photo-stimulated small molecules. We exemplify
the feasibility of this opto-modulation approach by utilizing a
piperidine-3-carboxylate 1 (MPCH, Schemes 1B, E), an organic
molecule that was recently identified to activate the triprotein
complex METTL3/14/WTAP pertaining to m6A installation.[15]
Table 1. Thermodynamic parameters for the binding interactions between
METTL3/14 and three small molecules 1-3 by molecular docking calculations[a]
G
Ki
Ligand efficiency
(LE)
H-bond(s)[b]
(kcalmol-1)
(μM, 298K)
Lys513 (-C=O)
His538 (-C=O)
1
-6.86
9.43
-0.69
2
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10.1002/anie.202103854
Angewandte Chemie International Edition
RESEARCH ARTICLE
+
Glu532 (-NH2
)
(A)
Gln550 (-NO2)
Asn549 (-NO2)
Gly407 (-C=O)
2
3
-5.56
-6.24
83.95
26.79
-0.26
-0.26
Asn549 (-NO2)
[a]
Full calculations and predictions performed with AutoDock 4.2 was listed in
Table S2. [b] Main bindings between the METTL3/14 complexes with the small
molecules 1-3. Surrounding amino acid residues and their corresponding
interacting chemical functionalities within the small molecules (in parentheses)
were listed.
(B) 2.0
1.5
(C)
150
2 (+hv)
3 (+hv)
1
2
2 (+hv)
3
3 (+hv)
100
50
0
-0.3
-0.4
-0.5
1.0
0.5
Based on our model, we synthesized two masked agonists 2-3
by introducing 2-nitrophenyl methyl ethyl and [4,5]dioxole
substituted photo-caging substituents onto the key N-H
0 102030
Time (s)
0.0
0
0
0
0
0
0
0
0
0
0
0
5
0
5
0
5
0
0
0
0
0
2
3
3
4
4
5
2
4
6
8
(Schemes 1E).
This was achieved by employing
a
Wavelength [nm]
Irradiation time (s)
regioselective and efficient alkylation with appropriate 2-
nitrobenzyl bromides (See Supporting Information for details).
Simpler 2-nitrobenzyl protecting groups (R’ = H) were not utilized
(Scheme 2A).[18] Although they have been proven to be good
photo-removable groups for aliphatic amines, the generated 2-
nitroso benzaldehydes 4 (R’ = H) upon photolysis may condense
with the release amine to form imine byproducts and thus
hamper the biological activity restoration. In contrast, 2-nitroso
acetophenones like 5 (R’ = Me) would reduce the risk of those
side effects (Scheme 2A). To determine the profiles and kinetics
of the photo-responsive release, we measured the photolytic
characterization of these compounds (Schemes 2B-C). UV
spectra of compounds 1-3 (Scheme 2B) revealed that precursor
1 showed no absorption at 230-500 nm range, whereas the
photo-caged 2 possessed a characteristic absorption around
262 nm and the dioxole moiety in 3 shifted the characteristic
absorption to around 356 nm. In order to examine the photo-
dissociation speediness, we used a light-emitting diode (LED)
reactor at a wavelength of 365 nm (100 mW·cm-2) to irradiate
compounds 2-3 (200 μM). Mass spectra and UV analysis of
samples collected over a period of 5 minutes confirmed that
agonist 1 was smoothly released under this condition, with the
generation of corresponding 2-nitroso acetophenones 5[19] as
revealed by UV absorbance (Schemes 2C-D, Figures S1-S2).
Furthermore, after 2 minutes of irradiation, approximately 80% of
the bioactive antagonist 1 was regenerated from compound 2,
and the maximum restoration was reached within 5 minutes of
irradiation (kobs = 6.45 × 10-3 s-1, t1/2 = 106.6 s). Previous reports
have indicated that electron-rich substituted 2-nitrobenzyl groups
like [4,5]dioxolyl 3 usually photolyzed more efficiently than its
unsubstituted counterpart.[20] Conversely, a lower uncaging
efficiency of photolysis of 3 was observed in our case, with the
(D)
(E)
4×106
4×106
1
1
3
0
0
4×106
5×106
2
0
0
3×106
2×106
2+hv
3+hv
0
0
0
2
4
6
8
0
2
4
6
8
Retention time (min)
Retention time (min)
Scheme 2. (A) Photo-induced de-caging reaction of compounds 2-3. (B) UV-
vis absorption spectra of compounds 1-2 at 200 μM and compound 3 at 150
μM in DMSO/H2O (v/v 1/1000) before and after light irradiation (λ = 365 nm,
100 mW·cm-2). Black line: 1 without hv irradiation. Ice blue: compound 2
before light irradiation. Navy blue: compound 2 under light irradiation after 5
minutes. Salmon: compound 3 before light irradiation. Maroon: compound 3
under light irradiation after 10 minutes. (C) Time course for photo-induced
uncaging of 2 (navy blue) and 3 (maroon) at λ = 365 nm. The Y axial
corresponds to the absorbance change of generated nitrosoketones. The
calculation is detailed in the SI materials. (Data collected at λ = 262 nm for 2
and 356 nm for 3, respectively). Inset: Determination of the initial rate
constants (kobs) for the photo-deprotection of compounds 2 (navy blue) and 3
(maroon). (D) Liquid chromatography mass spectrometry (LC-MS) total ion
current (TIC) diagram of the photo-deprotection reaction of caged compounds
2. Black: compound 1. Ice blue: compound 2 before light irradiation. Navy
blue: compound 2 under light irradiation after 5 minutes. The new peak was
identical to compound 1 according to the retention time. (E) LC-MS (TIC
diagram) of the photo-deprotection reaction of caged compounds 3. Black:
compound 1. Salmon: compound
compound 3 under light irradiation after 10 minutes. The new peak was
identical to compound 1 according to the retention time.
3 before light irradiation. Maroon:
To further assess the inactivity of caged compound 2, as well as
its reactivation ability upon light exposure, we performed a
bioluminescence-based assay to measure the activities of
methyltransferases METTL3/14 under the modulation of small
molecules 1 and 2 (Scheme 3). During the transmethylation
reaction of adenosine to m6A, the methyl donor SAM was
converted to a more stable and yet uncharged byproduct S-
adenosyl homocysteine (SAH) accompanied by the release of
methylated RNA from METTL3/14-RNA complex. In this assay,
we incubated the METTL3/14 complex with appropriate RNA
substrates and compounds 1 or 2 and then hydrolyzed the key
by-product SAH to homocysteine and adenine. The latter was
phosphorylated to afford adenosine triphosphate (ATP), and was
reaction progress curve reached plateau after 10 minutes (kobs
=
2.61 × 10-3 s-1, t1/2 = 265.5 s) (Scheme 2C). The reason for the
decreased performance of dioxole substituent was not clear. In
the photo-caging concept, it is essential that the parent molecule
be speedily released by light exposure. Otherwise, a long-time
irradiation would cause DNA damage along with the photo-
caging group cleavage. Thus, those data confirmed that caged
compound 2 possessed excellent uncaging kinetics and was
then chosen for enzymatic and in vitro investigations.
3
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10.1002/anie.202103854
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RESEARCH ARTICLE
then quantified using the classical luciferase reaction. Thus, the
methyltransferases activity (m6A generation) was quantified by
the luminescence output (Scheme 3A). Two ssRNAs 5’-
performance of compound 2 upon light irradiation could be used
to differentially modulate the m6A methylation in live cells, and
thus allow us to spatially and temporally investigate the
epigenetic effects in biological events (Scheme 4). We tested
three different cell lines: A549, MCF-7 and HeLa. As expected,
no change was observed after exposure to compounds 1 or 2
(100 nM) in HeLa cell line with or without light irradiation,
confirming that the light-activation manipulation strategy together
with the developed small organic molecules had no statically
significant effect on the cell viability (Figure S7). With a
concentration of both molecules 1 and 2 at 1 nM, which matched
to the amount used in previous enzymatic experiments with
greatest activity (Figure S8), RNA dot blot analysis[21]
demonstrated that cells incubated with caged compound 2
without light irradiation failed to exhibit significant m6A
methylation increase compared with the control groups,
suggesting that the installation of photo-caging groups
completely detached its activity and also the caged compound
has negligible side-effects to the live cells (Scheme 4B, Figure
S9). Consistently, 5 minutes of light irradiation revealed that
UACACUCGAUCUGGACUAAAGCUGCUC-3’
and
5’-
CUGGACUGGACUGG’-3’ containing one and two m6A
consensus motifs (GGACU) respectively, were used in this
assay (Underlined letter A indicated the methylation position).
We first validated the luminescence signal by utilizing the
agonist 1 (Figure S4). In that case of ssRNA with two motifs, an
increase of luminescence signal (ΔΔSAH%
= 46.5%) was
observed at 1 nM concentration of 1, clearly indicating that the
enzymes were fully and functionally activated (Figures S5-S6).
As expected, the caged compound 2 did not cause any
significant change in dark conditions, which was consistent with
its relatively weak binding affinity as determined before (Table 1)
and again indicated that the N-photocaging sufficiently
suppressed the agonism effect. However, its activation was
efficiently reinstated after exposure to the 365 nm light (100
mW·cm-2) within as short as 5 minutes (ΔΔSAH% = 43.7%). The
renovated activity of 2 was approximately comparable to the
original compound 1, therefore confirming its photo-responsive
activity (Scheme 3B). Thus, the rapid uncaging of 2 by light
irradiation has enabled the modulation of corresponding
methyltransferase METTL3/14 complex activity for m6A
methylation with the same efficacy as the unmodified 1.
mRNAs within cells incubated with compound
2
were
considerably hyper-methylated when compared to other
intervention groups (control and without irradiation). Of note,
comparable levels of m6A with 2 under light irradiation have
been observed for all three cell lines compared to those treated
with unmodified 1 (Scheme 4B, Figure S9). In order to exclude
other possible effect factors of regulating m6A level, we identified
the target METTL3 and METTL14 proteins with western blot
from HeLa cells incubated with compounds 1 and 2, with or
(A)
without light irradiation (Figure S10).
The expression of
METTL3/14 was determined before and after chemical treatment
and irradiation treatments, and the results clearly proved that
there is no side-effect on the corresponding m6A
methyltransferases’ expression. Taken together, these results
not only supported previous enzymatic analysis showing the
agonist effects could be predominately removed by introducing a
photo-caging group, but also suggested that the caged
compound 2 is particularly stable for potential therapeutic
utilization in living organisms, in which it can be promptly
initiated by light irradiation in the needing time and place without
genetic approaches.
(B)
20
*
15
10
5
*
*
0
_
_
_
1
2
hv
+
_
_
_
+
_
+
+
_
Scheme 3. (A) Bioluminescence-based assay used for the determination of
METTL3/14 complex activity on adenosine methylation of two ssRNAs 5’-
UACACUCGAUCUGGACUAAAGCUGCUC-3’
CUGGACUGGACUGG’-3’ upon treatment with small compounds 1 and 2 with
or without light irradiation (Underlined letter indicated the methylation
and
5’-
A
position). The by-product SAH was used as the luminescence trigger for
detection. (B) The effects of photo-caged small molecule for the METTL3/14
complex on the RNA substrate methylation using enzymatic assay in (A). The
Y axial corresponds to the ratio of SAH generated from the original SAM (1
μM), and the calculation is detailed in the Supporting Information. Error bars
indicate mean ± SEM (n = 3~6), normalized to DMSO-treated samples for
each sample. Unpaired students’ t test. *p < 0.05.
Having established such an effective approach to disrupt the
METTL3 agonism, we next investigated whether the adjustable
4
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10.1002/anie.202103854
Angewandte Chemie International Edition
RESEARCH ARTICLE
compound. In addition, since the decaging/activation process
occurred with fast dynamics (< 5 minutes), ultraviolet radiation
caused cytotoxicity is reduced to minimal. On the other hand,
the opto-control system that we used is based on the chemical
agonist of methyltransferases, which is principally suitable for
utilization in living systems, as it can be swiftly initiated by light
irradiation, without the requirement of addition/deletion of
endogenous enzymes. This may be useful in the case of
dealing with other epigenetic marks. Since the caging groups
can be easily changed, it would be a useful starting point to
expand to other caged ligands with miscellaneous chemical
properties to be installed during the preparation step followed by
stimuli-activation. For example, by combining two or more
chemical components (agonist and/or inhibitors) into an
assembly that is covalently linked by a photo-caging group, one
can achieve an efficient dual modulation of multiple RMEs
simultaneously with appropriate light-irradiation. It fuses the
gains of photo-responsive chemical tools in terms of flexibility
without protein engineering[22] and the distinguish activities (i.e.,
both activation and inhibition of function) can be evoked
respectively. This approach might be, therefore, an excellent
technique to precisely modulate RNA modification for the control
of cell activities.
Scheme 4. (A) Schemic demontrstation of this chemical approach in
controlling METTL3/14 complex function with light to modulate the m6A RNA
methylation status in live cells. Red circles represent m6A modification. Gray
ball (within the left) represents compound 2, which upon light irradiation was
Acknowledgements
converted to
1
(blue ball, within the right). (B) Determination of m6A
abundance in the three human cancer cell lines upon 1 or 2 (1 nM for A549
and HeLa, 100 nM for MCF-7) treatment for 4 hours by dot blot assay. The
RNA loading amount is from 0.8 μg to 0.4 μg. Cells upon treatment with 2
after 2 hours incubation were irradiated for 5 minuts (365 nm, 100 mW·cm-2). A
methylene blue staining (MB) was used as loading control.
This work was supported by the National Key R&D Program of
China
(2016YFA0602900,
2017YFA0208100,
and
2020YFA0707901), National Natural Science Foundation of
China (22022704, 91853124, 21977097, and 21778057) and
Chinese Academy of Sciences. Dr. L.-J. Xie is supported by the
Postdoctoral Innovative Talents Support Program (BX20200337).
We thank Prof. Cai-Guang Yang, Dr. Yue Huang at Shanghai
Institute of Materia Medica, CAS and Prof. Xia Dong from
Institute of Chemistry, CAS for insightful discussions and help
with dot-blot experiments.
Conclusion
Stimuli-responsive chemical probes that target RMEs involved in
RNA epigenetic modification are powerful techniques for
controlling their activities. Their application to modulate RNA
modification processes in vitro or in vivo, especially during
embryonic development, might provide an effective tool to
understand the complication of this epitranscriptomic regulatory
system. Additionally, these methods can also be cherished as
excellent starting points for developing novel drugs against
several diseases such as cancers and viral infections pertaining
to RNA modification dysregulation. However, to the best of our
knowledge, such stimuli-gated chemical molecules for
epitranscriptomic modulation of intracellular processes like m6A
Keywords: RNA modification • Epigenetics • Small organic
agonist • Photo-decaging • Spatiotemporal control
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methylation has not been realized yet.
Here, we have
developed a novel chemical modular for m6A based on a photo-
caging strategy with the agonist compound MPCH 1. Molecular
docking of this molecule helped propose binding modes within
the methyl donor SAM-binding sites of METTL3 and helped
guide the photo-caging substituents identified in this work, which
is one of the main perspectives of this work. Our synthesized
compound 2 showed little binding affinity and activation effects
against the m6A methyltransferase complex METTL3/14 in dark.
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Importantly, we observed
a clear gain-of-function in the
biochemical assay and live human cancer cells upon light
exposure, which significantly altered the cellular m6A levels. It is
expected that the observed effects are produced by the
extremely rapid photo-decaging to release the precursor
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Entry for the Table of Contents
This work demonstrated a novel route for the spatiotemporal control of intracellular RNA epigenetic modifications. The agonism effect
of a small compound can be concealed through appropriate photo-responsive caging groups, and the synthesized compounds
exhibited a high sensitivity of external light irradiation and rapidly restored their effects in live cells.
7
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