Multiprotein Dynamic Combinatorial Chemistry: A Strategy for the Simultaneous Discovery of Subfamily-Selective Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3

Article abstract of DOI:10.1002/asia.201800729

Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically templated DCC systems employ only a single biomolecule to direct the self-assembly process. To expand the scope of DCC, herein, a novel multiprotein DCC strategy has been developed that combines the discriminatory power of a zwitterionic “thermal tag” with the sensitivity of differential scanning fluorimetry. This strategy is highly sensitive and could differentiate the binding of ligands to structurally similar subfamily members. Through this strategy, it was possible to simultaneously identify subfamily-selective probes against two clinically important epigenetic enzymes: FTO (7; IC₅₀=2.6 μm) and ALKBH3 (8; IC₅₀=3.7 μm). To date, this is the first report of a subfamily-selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins; thus it is of broad scientific interest.

Full text of DOI:10.1002/asia.201800729

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Multi-Protein Dynamic Combinatorial Chemistry: A Novel Strategy
that Leads to Simultaneous Discovery of Subfamily-Selective
Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3
Authors: Mohua Das, Yang Tianming, Dong Jinghua, Fransisca
Prasetya, Xie Yiming, Kendra Wong, Adeline Cheong, and
Esther C. Y. Woon
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Multi-Protein Dynamic Combinatorial Chemistry: A Novel Strategy
that Leads to Simultaneous Discovery of Subfamily-Selective
Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3
Mohua Das, Tianming Yang, Jinghua Dong, Fransisca Prasetya, Yiming Xie, Kendra H. Q. Wong,
Adeline Cheong, and Esther C. Y. Woon*[a]
Abstract: Dynamic combinatorial chemistry (DCC) is a powerful
supramolecular approach for discovering ligands for biomolecules. To
date, most, if not all biologically-templated DCC employ only a single
biomolecule in directing the self-assembly process. To expand the
scope of DCC, herein, we developed a novel multi-protein DCC
strategy which combines the discriminatory power of zwitterionic
‘thermal-tag’ with the sensitivity of differential scanning fluorimetry.
This strategy is highly sensitive and could differentiate the binding of
ligands to structurally-similar subfamily members. Through this
strategy, we were able to simultaneously identify subfamily-selective
probes against two clinically-important epigenetic enzymes, FTO (7;
IC50 = 2.6 µM) and ALKBH3 (8; IC50 = 3.7 µM). To our knowledge, this
is the first report of a subfamily-selective ALKBH3 inhibitor. The
developed strategy could, in principle, be adapted to a broad range of
proteins, thus it shall be of broad scientific interest.
approaches employ only a single template in directing the self-
assembly process; this severely limits the applications of DCC
approaches.
To expand the scope and potential of DCC, in this study, we
explored the concept of multi-protein DCC, where two or more
protein templates were used concurrently in the same dynamic
system. We envisaged that such a strategy would enable the
discovery of ligands against several proteins of interest
simultaneously, thus greatly multiply the power and efficiency of
DCC. Another distinct advantage of the multi-protein DCC
approach is that it permits the use of several structurally and/or
functionally related protein isoforms in concert. This ensures that
only ligands that are highly selective for a particular protein
isoform or target will be assembled and identified. Although it is
also possible to achieve highly selective inhibition from
conventional DCC approaches (for instance, Greaney et al.[18]
successfully
identified
isozyme-selective
glutathione
Introduction
S-transferases inhibitors using an acyl hydrazone-based DCC),
single-templated DCC only generate hits against one target at a
time.
Dynamic combinatorial chemistry (DCC) is
a
powerful
supramolecular approach for discovering ligands for biological
targets. The idea was first independently conceived and
developed by the Sanders and Lehn groups (for excellent reviews,
see Refs 1-10).[1-10] In the DCC method, simple building blocks
are linked together by reversible covalent chemistry to generate
dynamic libraries of structures. Because of the reversible nature
of these libraries, they are highly responsive to external influence,
such that the introduction of a template triggers rapid structural
adaptation of the library members, resulting in the assembly of
structures that are highly complementary to the template. To date,
DCC has been successfully applied to a range of biological
templates, including enzymes, receptors, transmembrane
transporters, nucleotides, and polymer-supported targets.[11-17]
However, most, if not all, such biologically-templated DCC
The development of the proposed multi-protein DCC system
poses a number of analytical challenges. Not only is there a
requirement to analyse several protein-ligand interactions
simultaneously, it is also necessary to assign binding of ligands to
specific proteins in
a complex, multi-component mixture.
Furthermore, structures generated from reversible self-assembly
system are constantly exchanging, and this further complicates
their analysis.
Currently, only few methods have been reported for the analysis
of single-templated DCC,[8,19] this include HPLC analysis,[20,21]
NMR studies,[22-24] X-ray crystallography,[25] size-exclusion
chromatography (SEC)[26,27] and, more recently, the use of
polymer-scaffolded (PS-DCL),[28] DNA[29-32] or peptide nucleic
acids (PNA)-encoded[33,34] DCC libraries. However these methods
only detect amplification of the best binders in the presence of the
template, and could not distinguish which template is responsible
for the observed amplification, hence they are unsuitable for the
proposed multi-protein DCC system. Although it is possible to
directly observe different protein-ligand complexes using native
protein mass spectrometric (MS) techniques, the results of MS
analyses are not always representative of what exists in solution,
since different non-covalent complexes survive the transition from
solution to gas phase differently.[35] Indeed, our group[14] and that
[a]
M. Das, Dr. T. Yang, J. Dong, F. Prasetya, Y. Xie, K. H. Q. Wong, A.
Cheong, Asst. Prof. E. C. Y. Woon*
Department of Pharmacy
National University of Singapore
18 Science Drive 4, Singapore 117543
E-mail: esther.woon@nus.edu.sg
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. The multi-protein DSF detection strategy and its application on dynamic self-assembly system. (A) In this approach, ‘thermal tags’ which, by design, are
capable of exclusively modulating the melting temperature of fusion partner proteins without compromising their structures and functions are used to fine-tune the
melting temperature of the target proteins, such that their individual melting profiles could be simultaneously monitored in a single DSF melting analysis. When used
in combination with DCC, formation of stable protein-ligand complexes can be easily detected by shifts in Tm of proteins engaged in ligand binding. (B) Structures
of thermal tags investigated in this study. Both the KE and RD tags are zwitterionic peptides with alternating positively (blue) and negatively (red) charged amino
acids. (C) Schematic diagram of the fusion constructs. The KE or RD tag was genetically appended to the C-terminus of FTO, ALKBH3 and ALKBH5, which are
clinically-important proteins belonging to the iron- and 2-oxoglutarate (2OG)-dependent AlkB oxygenases. The starting and terminating residue numbers of the
proteins are given in parentheses. The fusion constructs also contain N-terminus His6-TEV tag to facilitate protein purification (for details of expression and
purification of the fusion proteins, see Supporting Information)
of Poulsen[36] have previously observed fragmentation of protein-
ligand complexes under certain MS ionisation conditions. To date,
no method exists which allow the analysis of multiple protein-
ligand interactions in a dynamic system. This severely limits the
potential applications of DCC-based approaches.
This approach is conceptually similar to DSF-based ‘thermal shift
assay’ which has been widely used for the detection of protein-
ligand interactions with stable ligands.[43] However, prior to this
work, it is not known whether DSF could be applied to the analysis
of dynamic library. We are also not aware of the combined use of
‘thermal tag’ with DSF to facilitate the study of multiple protein-
ligand interactions. As we shall demonstrate, multi-protein DSF is
a simple, yet powerful strategy for monitoring protein-templated
self-assembly. Through this method, one is able to assess Tm shift
and thermodynamic parameters of an equilibrating system in a
single, rapid DSF experiment. We further provide proof of
principle that the combined use of multi-protein DSF strategy and
DCC can enable simultaneous templating of several target
proteins. This is demonstrated by the concurrent discovery of
subfamily-selective inhibitors against two clinically-important
epigenetic enzymes, FTO and ALKBH3.
Recent studies demonstrated that zwitterionic polymers, in
general, are able to confer a wide range of biophysical properties
when conjugated to proteins, such as increased water solubility,
pH resistance, and antifouling property.[37-40] More recently, Jiang
et al. showed that poly(carboxybetaine) polymers and
poly(Glu-Lys) polypeptides were able to improve the thermal
stability of fusion partner proteins, rendering them more resistant
to heat inactivation.[41,42]
Inspired by these interesting observations, herein, we present a
novel detection strategy, which enables the simultaneous
analysis of several protein-ligand complexes in a multi-protein
DCC system (Figure 1A). It combines the discriminatory power of
‘thermal tag’ with the sensitivity of differential scanning fluorimetry
(DSF) technique. In this approach, the proteins of interest are
To the best of our knowledge, the present study represents not
only the first report of multi-protein DCC method, but also a new
strategy for probing dynamic chemical systems, which hopefully
will further our understanding of protein-directed self-assembly,
genetically labelled with a zwitterionic peptide-based thermal tags, and inspire new applications for DCC-based approaches.
which by design are capable of modifying the thermal stability of
the host proteins without disrupting their structural and functional
properties. The expectation is that, by appending appropriate
thermal tags, one could specifically fine-tune the melting
Results and Discussion
temperatures (Tm) of the proteins such that their individual melting
profiles could be simultaneously monitored in a single DSF
melting analysis. When used in combination with DCC, formation
of protein-ligand complexes can be easily detected by an increase
in Tm of the proteins that are engaged in ligand binding.
Thermal tag design and protein engineering
In this study, we selected three members of the iron- and
2-oxoglutarate (2OG)-dependent AlkB oxygenases, namely FTO,
ALKBH3 and ALKBH5, as our protein templates.[44] These AlkB
proteins are currently of intense biological and medical interest
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of all recombinant fusion proteins was confirmed by SDS-PAGE
and ESI-MS analyses (Figure S1).
Table 1. Thermal stability data for ALKBH5, FTO, ALKBH3, and their
corresponding KE- and RD-tagged fusion proteins[a]
ALKBH5
FTO
Tm (oC)a
ALKBH3
Introduction of a single thermal tag triggered dramatic
increase in the thermal stability and melting temperature of
the AlkB subfamilies
Thermal
tag
Tm
Tm
Tm
Tm (oC)a
Tm (oC)a
(oC)a
(oC)a
(oC)a
We initially investigated the impact of thermal tags on protein
melting characteristics. DSF-based melting analyses showed that
all KE-tagged and RD-tagged ALKBH5 fusion proteins unfold
cooperatively to produce monophasic melting transitions similar
to that observed with unmodified ALKBH5, implying that the
proteins remained stably folded when conjugated with KE or RD
tag (Figures 2A and S2A). The melting temperatures of the
ALKBH5 fusion proteins are however significantly higher than that
of unmodified ALKBH5 (Tm = 40.3 ± 0.1 °C; Table 1), suggesting
a marked improvement in their thermal stability. The extent of
thermal stabilisation appears to be dependent on the length of the
thermal tag used, as demonstrated by observation that relatively
short KE6 tag increases the Tm of ALKBH5 by 2.1 °C, whilst
longer KE12 and KE18 tags triggered a more dramatic Tm
increase of 4.9 °C and 9.2 °C, respectively. Similar trend (i.e.
longer tag leading to larger Tm increase) was also observed with
RD tags, although correspondingly smaller Tm increases of 1.8 °C,
3.4 °C and 8.2 °C were produced by RD6, RD12 and RD18 tags,
respectively. Thus the RD tags are slightly less effective in
improving protein thermal stability than KE tags of the same
length.
40.3 ± 0.1
42.4 ± 0.2
45.2 ± 0.2
49.5 ± 0.3
42.1 ± 0.2
43.7 ± 0.1
48.5 ± 0.2
--
45.1 ± 0.2
48.3 ± 0.1
52.6 ± 0.2
55.4 ± 0.3
47.8 ± 0.1
49.7 ± 0.1
53.3 ± 0.2
--
49.4 ± 0.3
53.6 ± 0.1
57.4 ± 0.1
61.7 ± 0.1
52.5 ± 0.3
53.2 ± 0.1
57.9 ± 0.2
--
Untagged
KE6
2.1
4.9
9.2
1.8
3.4
8.2
3.2
7.5
10.3
2.7
4.6
8.2
4.2
8.0
12.3
3.1
3.8
8.5
KE12
KE18
RD6
RD12
RD18
[a]The Tm values of the fusion proteins were measured at a protein concentration
of 2 μM (in 50 mM HEPES buffer, pH 6.0) using DSF melting analysis.
because of their critical roles in several key cellular processes,
such as epigenetic gene regulation and RNA metabolism.[45-47] It
is also increasingly clear that dysregulation of these enzymes may
underlie the pathogenesis of a range of human diseases,
including metabolic diseases, neurodegenerative diseases and
cancers.[48-51] However, despite their clinical significance, to date,
there have only been few reports of inhibitors that selectively
target these enzymes, in part due to their close structural similarity,
which renders the development of subfamily-selective inhibitors
particularly challenging. In this regard, the selection of AlkB
subfamilies for the present study not only allows us to 1) evaluate
the power of multi-protein DCC strategy to simultaneously
discover probes against several clinically-important targets, but
also 2) its potential in directing the self-assembly of probes with
subfamily-selectivity.
Interestingly, the thermal stabilisation effect of KE and RD tags
also extends to other AlkB subfamilies, such as FTO and ALKBH3,
where modification of both proteins with either KE or RD tag again
triggered remarkable increase in their Tms (Figures 2 and S2). As
observed for ALKBH5, FTO and ALKBH3 containing longer tags
exhibited a larger Tm increase than their counterparts with shorter
tags (Table 1). However, KE tag of the same length appears to
have a greater impact on ALKBH3 than on FTO and ALKBH5. For
instance, KE18 tag produced a Tm increase of 12.3 °C in ALKBH3,
but only 10.3 °C in FTO, and 9.2 °C in ALKBH5 (Table 1). In
contrast, RD tag of the same length triggered a similar Tm
increase in all three proteins.
To implement the proposed strategy (Figure 1A), we first
designed a series of thermal tags that are capable of conferring
different melting temperatures to the AlkB proteins. As potential
candidates, we considered short zwitterionic peptides, that are 6,
12 or 18 residues in length, with either alternating Lys/Glu (KE)
sequences (abbreviated as KE6-, KE12-, and KE18-tags) or
alternating Arg/Asp (RD) sequences (abbreviated as RD6-,
RD12-, and RD18-tags) (Figure 1B). Recent studies suggested
that zwitterionic polymers such as poly(carboxybetaine) polymers
and poly(Glu-Lys) polypeptides can increase the thermal
resistance of fusion partner proteins.[41,42] However, we are not
aware of the use of zwitterionic peptides to specifically modulate
the melting temperature of proteins. The effect of zwitterionic
peptides on the melting behaviour of proteins has also not been
systematically studied.
These findings are notable because it suggests that the
attachment of a single KE or RD tag as short as six residues in
length is sufficient to induce a considerable change in the thermal
stability and melting temperature of the fusion protein. It further
suggests that one can, in principle, fine-tune the melting
temperature of AlkB subfamilies by incorporating KE or RD tag of
appropriate length. The mechanism by which the KE/RD-tags
enhances the melting temperatures of their partner proteins is
unclear at present, nevertheless, it has been suggested that
zwitterionic peptide, being strongly hydrophilic in nature, has the
propensity to pull water away from the hydrophobic regions of the
protein.[41,42] Presumably this stabilises the protein folded
structures and thus increases its thermal stability and melting
temperature. Alternatively, it could also result from ionic
interaction between the protein and the zwitterionic tag or a
To investigate the suitability of zwitterionic peptides as thermal
tags, the KE and RD sequences were genetically appended to the
C-terminus of FTO, ALKBH3 and ALKBH5 using protein
engineering techniques; modelling analysis suggests that fusion
of a relatively short peptide to the C-terminus of AlkB subfamilies
is likely to be minimally disruptive on the structure and function of
the protein (Figure 1C, for details of gene constructs and protein
expression, see Supporting Information). Successful expression
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change in the oligomerisation state of the fusion protein. Further
studies are needed to understand the underlying mechanisms.
natively folded conformations (Figures 2E,F and S2; PDB ID:
4CXW[53] and 2IUW[54]).
The thermal tags have little impact on the overall
conformation of the AlkB subfamilies
The thermal tags do not alter the catalytic activity of the AlkB
subfamilies
Intriguingly, despite having a profound impact on thermal stability,
the thermal tags do not significantly alter the overall secondary
structures of the AlkB subfamilies, as clearly demonstrated by
far-UV circular dichroism (CD) spectroscopy. In particular, all KE-
and RD-tagged ALKBH5 share well-ordered secondary structure
elements, with a positive maximum at 194 nm and a negative
band at 215 nm, which are similar to that observed for
unmodified ALKBH5 (Figures 2D and S2D). Spectral fitting further
indicates approximately equal α-helical (24-27%) and β‐sheet
contents (26-28%) in all ALKBH5 fusion proteins, in agreement
with the reported X‐ray crystal structure of native ALKBH5 (PDB
ID: 4NJ4).[52] Similarly, the CD characteristics and secondary
structure contents of FTO and ALKBH3 fusion proteins are also
highly consistent with that of their unmodified equivalents,
suggesting that all thermally-tagged proteins continued to adopt a
We were also able to demonstrate that the thermal tags have little
impact on the catalytic activities of AlkB demethylases. Notably,
ALKBH5
and
FTO
preferentially
demethylate
N6-methyladenosine (m6A) substrates, whilst ALKBH3 favours
N1-methyladenosine (m1A) substrates.[55,56] Consistent with their
inherent substrate specificity, detailed kinetic analyses revealed
that ALKBH5 and FTO modified with either KE or RD tag
displayed similar affinities (Km) and catalytic efficiencies (kcat/Km)
for m6A substrate as their unmodified proteins (Figures 2G,H and
S2), whilst ALKBH3 conjugated with either KE or RD tag retained
> 90% of its canonical demethylase activity against m1A substrate
(Figures 2I and S2I). This finding is interesting because, contrary
to our result, previous study showed that the attachment of larger
zwitterionic peptides, such as poly(Glu-Lys) 10KDa and 30KDa in
length, in fact led to significant increase in the substrate affinities
of β-lactamases.[42] Apparently the greater number of charged
Figure 2. Effects of KE thermal tags on the thermal stability, structural conformation, and catalytic activity of AlkB demethylases (for the effects of RD thermal tags,
see Figure. S2). Representative first derivative plots showing the melting profiles of (A) ALKBH5, (B) FTO, and (C) ALKBH3 and their corresponding KE-tagged
fusion proteins (colour-coded) at a protein concentration of 2 μM. In all cases, introduction of a single KE tag triggered a significant increase in the melting
temperature of the proteins. The extent of thermal stabilisation increases with the length of the thermal tag used. (D-F) Superimposition of representative far UV-CD
spectra for KE-tagged proteins and native proteins reveals negligible conformational change in the presence of the KE tags. The inset shows the secondary structure
contents of the fusion proteins derived from CD spectra fit, which are highly consistent with that of native proteins (obtained from X‐ray crystal structures), suggesting
that the fusion proteins remained natively folded when conjugated with KE tags. (G-I) Steady-state kinetic analyses indicate that modification with KE tag does not
significantly alter the binding affinities and the demethylase activities of AlkB enzymes. The Km and kcat values were determined by keeping a constant enzyme
concentration of 0.5 μM. Error bars represent SD of three replicates.
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ALKBH5-KE6 (Tm = 42.4 °C), FTO-KE12 (Tm = 52.6 °C) and
ALKBH3-KE18 (Tm = 61.7 °C); these proteins were selected as
they have reasonably large difference in Tm between them. We
appreciate that most proteins undergo aggregation during thermal
unfolding, we were therefore concerned that protein aggregates
derived from the melting of one protein might interfere with the
melting behaviour of other proteins within the same mixture.
Nevertheless, in our multi-protein DSF analysis, three distinct
denaturation peaks were observed which resembled the melting
profile of ALKBH5-KE6 superimposed with that of FTO-KE12 and
ALKBH3-KE18, suggesting that mixing of fusion proteins do not
significantly alter their individual melting characteristics
(Figures 3A,B). Consistent with our detection strategy, there was
also a marked separation of individual melting peaks (Tm > 9 °C),
which enables any ligand-induced Tm shift to be clearly detected
without merging of peaks, as demonstrated by ligand binding
experiments with 2,4-pyridine dicarboxylic acid 1 (a known
‘generic’ inhibitor of AlkB subfamilies),[57] and FTO-selective
inhibitor 2 (PubChem ID : 126970771; Figure 3C,D).[53] By
comparison, DSF analysis of a mixture of untagged ALKBH5,
FTO, and ALKBH3 produced an overlapping melting profile which
is difficult to deconvolute (Figure 3E). Thus the combined use of
thermal tags with DSF enables the simultaneous analysis of
different fusion proteins in a mixture, even structurally-similar
subfamily members which is otherwise challenging to differentiate.
Figure 3. The thermal tags provide a basis for differential analysis of several
proteins simultaneously. The DSF-based melting profiles of ALKBH5-KE6, FTO-
KE12 and ALKBH3-KE18, when analysed (A) individually and (B) as a mixture,
revealed that mixing of fusion proteins do not affect their individual melting
behaviour. Ligand binding experiments with (C) ‘generic’ inhibitor 1 (100 µM)[57]
and (D) FTO-selective inhibitor 2 (100 µM) [53] demonstrated the capability of
DSF detection strategy to monitor several protein-ligand interactions in a single
DSF screen. (E) DSF analysis of a mixture of unmodified ALKBH5, FTO, and
ALKBH3 produced an overlapping melting profile, demonstrating the power of
the thermal tag approach in discriminating structurally-similar subfamily
members.
Notably, in our multi-protein DSF strategy, different thermal tags
were applied to different AlkB subfamilies. Hence one possible
issue is that fusion proteins bearing longer, more stabilising tags
might be less sensitive to further stabilisation by ligands than
those with shorter tags. However, titration of each fusion protein
against increasing concentrations of 1 revealed that proteins with
KE18 tag exhibited similar concentration-dependent increase in
Tm as those with KE12 and KE6 tags (Figure S3). Thus the length
of thermal tag used does not significantly impact the magnitude of
ligand-induced Tm shift.
residues in longer peptides confer stronger hydrophilic properties
which promotes hydrophobic interactions between the substrate
and its binding site. Our result therefore suggests that the effects
of zwitterionic tags on protein activity could be reduced through
the use of relatively short 6-18mer peptide sequences. Moreover
the apparent lack of influence on protein conformations and
catalytic activities further suggests that both KE and RD tags likely
exist as separate entities, structurally and functionally
independent from the proteins to which they were attached.
Analysis of self-assembly dynamics with multi-protein DSF
detection strategy
On the basis of these promising results, we next investigated
whether the developed detection strategy could be used in
combination with DCC to achieve multi-protein targeting. As our
model dynamic reaction, we chose the reversible acyl hydrazone
reaction between scaffold ligand 3 and aldehydes (Figure 4A).
The acyl hydrazone exchange is one of the most well-established
reversible reactions, first introduced by the Sanders group[58] and
subsequently by others[18,59,60] for the preparation of dynamic
library. By design, scaffold 3 contains a hydrazide moiety that is
free to participate in acyl hydrazone exchange with a set of
aldehydes. It also contains a pyridyl function which, based on
modelling analysis, is expected to bind to the AlkB subfamilies via
chelation with the active site Fe(II). Another notable feature is that
3 exhibits similar binding affinity for ALKBH5-KE6 (KD,app = 37.9
Taken together, our results clearly demonstrated that both KE-
and RD-tags are highly versatile thermal tags that are able to
exclusively fine-tune the thermal stability and melting temperature
of AlkB proteins, without compromising their structural
conformation and catalytic activity. To our knowledge, this is the
first report demonstrating that zwitterionic peptides could provide
a general strategy for bioengineering proteins with the desired
melting characteristics.
The thermal tags provide a basis for recognition and analysis
of multiple proteins in a mixture
To establish whether thermal tag-induced Tm change could
facilitate the discrimination of different fusion proteins in the same
mixture, we performed one-pot DSF analysis of a mixture of
± 4 μM; Tm = 1.5 °C), FTO-KE12 (KD,app = 40.4 ± 4 μM; Tm
=
0.7 °C) and ALKBH3-KE18 (KD,app = 31.0 ± 3 μM ; Tm = 1.4 °C),
thus it does not inherently favour any particular protein template
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KE12 and ALKBH3-KE18 increased steadily over time, giving Tm
shifts of 6.1 °C and 4.0 °C, respectively after 5 h incubation
(Figure 5D,H). This was accompanied with an increase in
amplification of 5e and 5h to 35% and 32% of total adduct
concentration, respectively (Figure 5I). Detailed analysis revealed
an excellent linear relationship between Tm shift and adduct
concentration, hence the magnitude of Tm shift provides a direct
indication of the extent of adducts amplification (Figure 5J).
Although some degree of fluorescence quenching occurred in the
presence of the library components, the observed Tm shifts were
highly reproducible (standard deviations < 1 °C). To determine if
heating of the DCL during DSF analysis could affect DCL
equilibration, we performed HPLC analysis of the DCL at various
temperatures, ranging from 37 °C to 70 °C (Figure S5). The
results revealed no significant change in DCL equilibration even
when heated to a temperature of 70 °C, although increasing DCL
temperature did cause a decrease in retention time, which led to
the merging of some adduct peaks.
The identity of the preferentially binding hydrazones was
subsequently confirmed by separate DSF experiments in which
the ten aldehydes 4a-j were individually mixed with 3, and then
analysed for Tm shift with the fusion proteins. This established
adducts 5e and 5h to be the specific binder for FTO-KE12 and
ALKBH3-KE18, respectively. Consistent with DCC result, no Tm
shifts were observed for ALKBH5-KE6 for all combinations of 3
and aldehydes.
Figure 4. Formation of acyl hydrazone dynamic library. (A) The scaffold ligand
3 contains a pyridyl function which is expected to bind to the AlkB subfamilies
via chelation with the active site Fe(II), leaving the hydrazide side chain (blue)
free to participate in acyl hydrazone exchange with aldehydes. To facilitate
rapid acyl hydrazone exchange, the reaction was performed under slightly
acidic condition (pH 6.0) in the presence of a nucleophilic catalyst aniline.[48] (B)
Structures of aldehydes used in this study.
The combined use of multi-protein DSF detection with DCC
enables multi-protein targeting
We appreciate that, to date, DSF analysis has primarily been used
to study the binding of stable ligands to protein. Therefore Tm shift
data derived from labile and interchanging protein-ligand
complexes may not reflect actual ligand binding affinity. To verify
our multi-protein DCC result, we determined the binding affinities
of the identified hits 5e and 5h using NMR-based water relaxation
(Figures S6-S8). The weak potency of 3 against all three proteins
also enables the effect of hydrazone substitution to be monitored.
In a typical multi-protein DCC system, the dynamic library was set
up by mixing all three fusion proteins i.e. ALKBH5-KE6 (2 μM),
FTO-KE12 (2 μM), ALKBH3-KE18 (2 μM) with MnCl2 (50 μM;
substitute for Fe(II)) and scaffold 3 (40 μM), in the presence of ten
aldehydes 4a-j, each aldehyde at 20 μM (library A; Figure 4B).
The use of a fairly low concentration of aldehydes (10-fold relative
to individual protein concentration) was a deliberate strategy to
only detect high affinity ligand. To facilitate rapid acyl hydrazone
exchange, the reaction was performed under slightly acidic
condition (pH 6.0) in the presence of aniline (5 mM), which serves
as a nucleophilic catalyst.[18,61] The dynamic exchange was
monitored using both DSF and HPLC analyses (Figure 5 and S4).
assay.[14,62] In this method, the apparent binding constants (KD,app
)
were determined by monitoring the bulk water relaxation rate,
which decreased when water access to paramagnetic Mn(II) in
the active site was hindered through binding of ligands to the
metal.
Our NMR assays support results from DCC, where a substantial
increase in the binding affinity of 3 for FTO-KE12 (KD,app = 40.4
± 4μM) was observed only in the presence of aldehyde 4e
(KD,app (5e) = 0.46 ± 0.1 μM; Figure 5K); the presence of other
aldehydes has negligible effect on the binding affinity of 3 (Figure
S6). As anticipated, the affinity of
3 for ALKBH3-KE18
Initially at t0, the reaction mixture consisted predominantly of
scaffold 3 and free aldehydes, with no apparent hydrazone adduct
formation or Tm shift (Figure 5A,E). After a 1-hour incubation, a
distinct Tm shift could be detected for FTO-KE12 (Tm = 3.9 °C;
Figure 5B). This was likely induced by specific binding of 5e to
FTO-KE12, as supported by the concomitant amplification of
hydrazone adduct 5e in HPLC analysis (Figure 5F). As the
reaction progressed, amplification of a second hydrazone adduct
5h became apparent at 2 h, which resulted in a slight Tm shift for
ALKBH3-KE18 (Tm = 2.7 °C; Figure 5C,G). The Tms of FTO-
(KD,app = 31.0 ± 3 μM) was improved only when combined with
aldehyde 4h (KD,app (5h) = 1.0 ± 0.2 μM; Figure 5L), and not with
other aldehydes (Figure S7). Finally, the binding of 3 to ALKBH5-
KE6 (KD,app = 37.9 ± 4 μM) was also not significantly altered by
any of the aldehydes investigated (Figure S8). Thus there is
qualitative agreement between Tm shift data derived from DCC
screen and ligand binding affinities. Our results therefore clearly
demonstrated that DSF technique can indeed be used to monitor
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and analyse self-assembly dynamics in multi-component DCC
system.
5k to each protein was reduced to near zero (Tms 0.1-0.3 °C;
Figure S9D). DSF-based titration experiments further revealed
that when all three proteins were present, a considerably higher
concentration of 5k (31 µM) was required to produce a significant
Tm shift of 1.0 °C in FTO-KE12, compared to just 3 µM when only
FTO-KE12 was present (Figure S10). In sharp contrast, the
binding of selective ligands to their protein targets was not
affected by the presence or absence of other proteins. For
instance, the binding of 5e (10 µM) to FTO-KE12 elicits a similar
Tm shift of 5 °C whether in the presence of a single or multiple
protein templates (Figure S9E,F). Importantly, in dynamic library
containing both selective and non-selective adducts 5e and 5k
(set up by adding aldehyde 20 µM 4e to the above dynamic
library), only the specific binding of 5e to FTO-KE12 could be
detected (Figure S9G). Taken together, the results suggest that
the extent of Tm shifts in multi-templating DCC experiment is
primarily dependent on the affinity of the ligands for the target
proteins. Presumably, strong binders, such as 5e, dominates the
DCL equilibrium at the expense of weak binders, such as 5k,
whilst weak binders dominate the DCL equilibrium over the non-
binders.
Importantly, the successful identification of adducts 5e and 5h
validates the capability of our multi-protein DCC approach to
identify highly selective ligands against several targets
simultaneously. Notably, adducts 5e differ from 5h by only a
single isosteric replacement of para-CF3 with ortho-Me group,
thus this approach has the sensitivity to detect subtle active site
selectivity between structurally-related subfamily members.
Comparing the effects of single-templating and multi-
templating on ligand selection
We next compared the impact of single-templating and
multi-templating on ligand selection. We performed another set of
DCC experiments in which the dynamic library was constituted by
mixing scaffold 3 and aldehydes 4k-t (library B, Figure 4B). When
the dynamic library was screened against each protein
individually (single-templating), we observed substantial binding
of adduct 5k to ALKBH5-KE6 (Tm = 4.8 °C), FTO-KE12 (Tm
= 3.0 °C), and ALKBH3-KE18 (Tm = 3.4 °C; Figure S9A-C).
However, when the same dynamic library was screened against
all three proteins concurrently (multiple-templating), the binding of
Figure 5. The multi-protein DSF detection strategy enables the analysis of self-assembly dynamics. The dynamic library was constituted by mixing ALKBH5-KE6
(2 μM), FTO-KE12 (2 μM), ALKBH3-KE18 (2 μM), scaffold ligand 3 (40 μM), aldehydes 4a-j (library A, each aldehyde at 20 μM), MnCl2 (50 μM) and aniline (5 mM)
in HEPES buffer (50 mM, pH 6.0). (A-D) DSF melting analyses showed time-dependent increase in Tm shift for FTO-KE12 and ALKBH3-KE18, suggesting the
simultaneous assembly of hydrazone adducts capable of substantial binding to FTO-KE12 and ALKBH3-KE18. (E-H) HPLC analyses of the dynamic library revealed
concomitant amplification of adducts 5e and 5h, at the expense of other hydrazone adducts. The peaks were assigned based on retention times of pure aldehydes
and adducts. (I) The concentrations of the adducts were determined based on their respective HPLC peak areas, using calibration plots obtained from pure adducts.
The percentages of adduct formation were calculated relative to the starting concentration of scaffold ligand 3 (40 μM). (J) There is an excellent linear relationship
between Tm shift and ln (adduct concentration), thus the magnitude of Tm shift provides a direct indication of the extent of adducts amplification. (K-L) NMR-based
water relaxation assay confirms adducts 5e and 5h to be the specific binders for FTO-KE12 and ALKBH3-KE18, respectively. Error bars represent SD of three
replicates.
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Scheme 1. Synthesis of stable sulphonamide analogues 6-15. aCompounds 6-9 are stable analogues of identified hydrazone hits 5c, 5e, 5h and 5k, respectively,
wherein the relatively labile acyl hydrazone moiety was replaced with a stable sulphonamide moiety. Reagents and conditions: (i) substituted benzenesulfonamides,
Et3N, 1-hydroxybenzotriazole hydrate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, CH2Cl2, 25 °C; (ii) LiOH, tetrahydrofuran-water 4:1, 25 °C. See
Experimental Section and Supporting Information for full synthesis, characterisation and NMR spectra of compounds.
Figure 6. Activity profiling of the sulphonamide analogues against representatives AlkB subfamilies. (A) Consistent with multi-protein DCC results, 7 demonstrates
distinct selectivity for FTO, with substantially reduced inhibition and Tm shift against other AlkB subfamilies, whilst 8 exhibited > 100-fold selectivity for ALKBH3 over
FTO and ALKBH5. 7 and 8 also discriminates against other human 2OG oxygenases, as demonstrated by their poor inhibitory activities against JMJD2A (IC50
s
>100 µM). The reported values are average of three replicates. Enzyme inhibition curves of (B) 7, (C) 8, and (D) 9, as determined using HPLC-based assay. Error
bars represent SD of three replicates. For enzyme inhibition curves of compounds 3 and 6, see Figure S11.
Multi-protein DCC led to the identification of subfamily-
selective probes for FTO and ALKBH3
values in low micromolar range, which represents more than
10-fold improvement in activity compared with scaffold ligand 3.
Notably, compound 9 (analogue of 5k), which was identified by
the single-templated DCC, was also found to be a relatively potent,
non-selective inhibitor, and control compound 6 (analogue of 5c)
To establish whether the hits identified from our multi-protein DCC
screen were in fact active inhibitors of AlkB demethylases, we
synthesised stable analogues of 5c, 5e, 5h and 5k, wherein the
relatively labile acyl hydrazone moiety was replaced with a stable
sulphonamide moiety (Scheme 1). It should be emphasised that
the key result of any DCC experiment is the information about the
most effective structural combinations of building blocks. Hence
labile groups like hydrazones are often replaced to form stable
ligands.[63]
a
poor inhibitor against all AlkB enzymes investigated
(IC50 > 25 µM).
The selectivity profiles of compounds 7 and 8 are also strikingly
consistent with multi-protein DCC results (Figure 6). In particular,
we observed remarkable selectivity of
7 for FTO, with
substantially reduced inhibition and Tm shift against ALKBH3
(IC50 = 69.1 µM, Tm = 1.8 °C) and ALKBH5 (IC50 = 201.3 µM,
Tm = 0.3 °C), whilst 8 exhibited > 100-fold greater activity for
ALKBH3 over FTO (IC50 = 105.6 µM, Tm = 0.9 °C) and ALKBH5
(IC50 = 128.2 µM, Tm = 0.8 °C). Further profiling studies revealed
that 7 and 8 also discriminate against other human 2OG
oxygenases, as demonstrated by their poor inhibitory activities
against ALKBH2 (IC50s > 40 µM) and JMJD2A (IC50s > 100 µM; a
histone demethylase critically involved in chromatin
remodelling).[64] Our results therefore validate the multi-protein
The resulting stable analogues, 6-9, were evaluated for activity
against a panel of untagged AlkB subfamilies using both HPLC-
based demethylase assay and Tm shift assay (for assay
conditions, see Supporting Information).
As shown in Figure 6, the Tm shift and inhibition data are in close
agreement with multi-protein DCC results. In particular,
compounds 7 (analogue of 5e) and 8 (analogue of 5h) were
indeed potent inhibitors of FTO (IC50 = 2.6 µM, Tm = 8.1 °C) and
ALKBH3 (IC50 = 3.7 µM, Tm = 7.4 °C), respectively, with IC50
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Figure 7. Selectivity studies of analogues of (A) ALKBH3-selective inhibitor 8, and (B) FTO-selective inhibitor 7. In all compounds investigated, the introduction of
a methyl or trifluoromethyl substituent at the ortho-position favours ALKBH3 selectivity, whereas a translocation of these groups to the meta- or para-positions
promotes FTO selectivity, as apparent from a comparison of the selectivity profile of 8 with 10 and 11, or that of 7 with 15. Likewise, the addition of either a m-methyl
or p-methyl group to 8 (i.e. 12-14) drives selectivity towards FTO. The reported values are average of three replicates.
DCC screen as
identification of subfamily-selective probes.
a
reliable method for the simultaneous
(i.e. 10) or para-positions (i.e. 11) drives selectivity towards FTO.
This change in selectivity was particularly apparent when
comparing the activity of 8 with that of analogues 12-14. In all
cases, the addition of either a m-methyl or p-methyl group caused
a substantial increase in selectivity towards FTO.
A similar trend was also observed for 7, whereby a translocation
of its trifluoromethyl substituent from the para- to ortho-position
(i.e. 15) resulted in a 14-fold reduction in potency against FTO,
coupled with an increase (3-fold) in potency towards ALKBH3
To rationalise the selectivity of 7 and 8, a series of mono-, di-, and
tri-methyl substituted analogues 10-15 was prepared and
evaluated for activities against the AlkB subfamilies. The assay
result was summarised in Figure 7. It suggests that the
introduction of a methyl substituent at the ortho-position (i.e. 8)
favours ALKBH3 selectivity, whereas shifting it to either the meta-
Figure 8. Rationalisation for the selectivity of 7 for FTO over ALKBH3. The structural models of the (A) FTO-7 complex, and (B) ALKBH3-7 complex built using
reported crystal structures of FTO (white surface and white sticks; PDB ID 4CXW[53]) and ALKBH3 (salmon surface and salmon sticks; PDB ID 2IUW[54]). The right
panels show the close-up views of interactions between 7 and active site residues in FTO and ALKBH3. Whereas 7 forms highly favourable interactions with FTO,
it is unable to fit into the binding pocket of ALKBH3 due to steric clash between its 4-trifluoromethylphenyl group and the side chain of Tyr143.
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(Figure 7B). Notably, these substitution changes have very little
or no effect on activities towards ALKBH2 and ALKBH5. The
structural models of FTO-7 and ALKBH3-7 complexes built using
the reported crystal structures of FTO (PDB ID 4CXW[53]) and
ALKBH3 (PDB ID 2IUW[54]; Figure 8) reveal that compound 7 is
able to coordinate with the active site Fe(II) in a bidentate manner
and participate in hydrogen-bonding interactions with the side
chains of Arg96 (2.4Å and 2.5Å). The pyridyl carboxylate group is
inhibitors would be of interest as functional probes and, possibly,
therapeutic leads.
One limitation of this approach is that it is only applicable to
proteins that are amenable to Tm modification by the thermal tags.
Conceivably the use of a larger number of proteins will produce
significant Tm signal overlap, and this limits the number of protein
templates that can be analysed concurrently. Moreover, it is
necessary to carry out separate experiments to determine the
identity of the best binders. Nevertheless, the method is relatively
simple, cheap and amenable to a high-throughput format.
Moreover unlike current techniques, such as HPLC and NMR
analyses, it detects ligands binding rather than amplification of the
best binders, which greatly simplifies spectral analysis, thus
potentially permits the use of larger, more complex dynamic
library. Although this study focuses on the development of multi-
protein DCC, the developed method is highly versatile and may,
in principle, be adapted to a wide range of studies, such as
parallel profiling of compound library, high-throughput mapping of

further stabilised through formation of salt bridges with both N of
Arg319 (2.5Å and 2.7Å), and hydrogen-bonds with the hydroxyl
groups of Tyr295 (2.7Å) and Ser318 (2.6Å). Modelling studies
further suggest that 7 might not be able to fit into the binding
pocket of ALKBH3 due to potential steric clash between its
4-trifluoromethylphenyl group and the side chain of Tyr143. This
may contribute to the poor activity of 7 against ALKBH3 (Figure
8B). However the proposed model is unable to account for the
activity of compounds 8 and 15, since similar steric clash is also
possible between these compounds and ALKBH3. Further
crystallographic studies are needed to determine the exact
structural basis for the selectivity of these inhibitors. Studies are
also currently underway to determine the potency and selectivity
of 7 and 8 in cells, which will provide insight on their usefulness
as functional probes and, possibly, therapeutic leads.
protein
substrate
specificity,
and
the
study
of
protein-protein/nucleic acid interactions. Thus, the approach
outlined here shall be of general scientific interest.
To the best of our knowledge, this study represents not only the
first example of multi-protein DCC, but also a new strategy for
probing dynamic chemical system, which hopefully will further our
understanding of protein-directed self-assembly, and inspire new
applications for DCC-based approaches.
Conclusions
Overall, we used
a combination of protein engineering,
spectroscopic, thermodynamic, kinetic, biochemical and thermal
denaturation studies to investigate the effects of zwitterionic tags
on protein structures and functions. Our results revealed that the Experimental Section
incorporation of a single KE or RD peptide, as short as 6 to 18
General procedures for compound synthesis
residues in length, can trigger dramatic increase in thermal
stability and melting temperature of the host proteins without
disrupting their structural conformations and catalytic activities.
We further showed that both KE and RD tags are highly versatile
thermal tags and are generally applicable to a range of proteins,
including AlkB subfamilies such as FTO, ALKBH3, and ALKBH5.
To our knowledge, this is the first report showing that zwitterionic
peptides can be used to exclusively fine-tune the melting
temperature of proteins. Importantly, by exploiting thermal tag-
induced Tm change, we developed a novel DSF-based detection
strategy which enables the analysis of several protein-ligand
interactions simultaneously. The assay is remarkably sensitive
and could effectively distinguish the binding of ligands to different
proteins, including structurally-similar subfamily members, which
is extremely challenging to achieve. Through the combine use of
multi-protein DSF and DCC, we were able to achieve
simultaneous discovery of highly selective probes against several
target proteins. This was demonstrated by the identification of
compounds 7 and 8, which not only exhibit exceptional selectivity
for FTO (IC50 = 2.6 µM, Tm = 8.1 °C) and ALKBH3 (IC50 = 3.7 µM,
Tm = 7.4 °C), respectively over other AlkB subfamilies, but also
discriminate against structurally-related human 2OG oxygenases,
such as JMJD2A. Notably, to date, there is no report of subfamily-
selective ALKBH3 inhibitor. In light of the biological and clinical
significance of FTO and ALKBH3, we envisaged that the identified
Starting materials, reagents, and solvents were obtained from commercial
sources and used as received. Progress of the reactions were monitored
by thin layer chromatography, which was performed on precoated
aluminum-backed plates (Merck, silica 60 F254). Purification of
intermediates and final products was carried out using flash column
chromatography conducted on silica gel (230-400 mesh). Melting points
were determined using a Gallenkamp melting point apparatus. Infrared
spectra
were
recorded
on
a
PerkinElmer Spectrum
100
FT-IR spectrometer using neat compound. 1H and 13C NMR spectra were
recorded using TMS as the internal standard with a Bruker Advance 400
Ultrashield NMR spectrometer at 400 MHz and 100 MHz, respectively.
Chemical shifts (δ) were reported in ppm downfield from the internal
standard. The signals were quoted as s (singlet), d (doublet), t (triplet), m
(multiplet), br (broad). Coupling constants J were given in Hz (± 0.5 Hz).
ESI-MS spectra were recorded on
a Shimadzu LC-MS2020 mass
spectrometer. High resolution mass spectra (HRMS) were recorded using
Bruker MicroTOF-QII mass spectrometer. The purity of synthesised
compounds was confirmed to be higher than 95% using analytical reverse-
phase HPLC (Ultimate 3000). Compound 1 was commercially available
and used without any further purification. Compound 2 was synthesised as
previously reported.[53] Synthesis and characterisation of selected
compounds 7, 8, 17, 21, 22 are described below. For synthesis of other
compounds investigated in this study, see Supporting Information.
4-(Methoxycarbonyl)picolinic acid (17). To a solution of dimethyl
pyridine-2,4-dicarboxylate 16 (1.4 g, 7.3 mmol) in methanol (20 mL),
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1.2 mL of aqueous sodium hydroxide (6 M) solution was added. The
mixture was then stirred at room temperature for 10 h, after which the
solvent was evaporated in vacuo. The resulting residue was washed with
ethyl acetate (20 mL), then dissolved in water (40 mL) and the aqueous
solution acidified to pH 2 with conc. HCl at 0 °C. The mixture was then
filtered to give 17 (368 mg, 28%) as a white solid. m.p. 230-231 °C; 1H
NMR (400 MHz, DMSO-d6): δ = 3.92 (s, 3H; CO2CH3), 8.05 (d, J = 3.6 Hz,
1H), 8.38 (s, 1H), 8.91 (d, J = 4.4 Hz, 1H), 13.95 (s, 1H; CO2H); 13C NMR
(100 MHz, DMSO-d6): δ = 52.5 (CO2CH3), 123.4, 126.0, 139.4, 148.4,
150.9, 164.5 (CO2CH3), 165.2 (CO2H); IR (neat) υ/cm-1 = 1707 (CO); MS
(ESI-) m/z: calcd. for C8H6NO4 [M-H]- : 180.0. Found 180.3.
dichloromethane, and washed by citric acid aqueous solution (10%). The
organic layer was neutralized by aqueous saturated NaHCO3 solution and
dried over anhydrous Na2SO4. Then the solvent was removed in vacuo
and the residue was purified by flash column chromatography
(dichloromethane / methanol 10 : 1). The fractions collected were further
purified by triturating with n-hexane-isopropanol and the precipitate was
separated by filtration to give 22 (70.9 mg, 13%) as a white solid. Rf = 0.60
(dichloromethane/methanol 4 : 1); m.p. 265-266 °C; 1H NMR (400 MHz,
DMSO-d6): δ = 2.53 (s, 3H; CH3), 3.89 (s, 3H; CO2CH3), 7.16-7.18 (m, 1H),
7.22-7.32 (m, 2H), 7.89-7.90 (m, 1H), 7.94 (dd, J = 4.8, 1.4 Hz, 1H), 8.45
(m, 1H), 8.87 (d, J = 4.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ = 20.1
(CH3), 52.3 (CO2CH3), 123.3, 124.8, 125.5, 128.4, 129.9, 130.9, 135.7,
143.9, 147.5, 147.9, 149.9, 165.2 (CONH), 166.5 (CO2CH3); IR (neat)
Methyl
2-(((4-(trifluoromethyl)phenyl)sulfonyl)carbamoyl)
υ/cm-1
=
1175 (SO), 1349, 1709 (CO); MS (ESI-) m/z: calcd. for
isonicotinate (21). To a suspension of 4-(methoxycarbonyl)picolinic acid
17 (154 mg, 0.8 mmol) in dichloromethane (3mL), triethylamine (301 mg,
2.9 mmol), 1-hydroxybenzotriazole hydrate (172 mg, 1.2 mmol), 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (245 mg,
1.2 mmol) were added sequentially at room temperature. The reaction was
C15H13N2O5S [M-H]-: 333.0. Found 333.1.
2-((o-Tolylsulfonyl)carbamoyl)isonicotinic acid (8). To a solution of
methyl 2-((o-tolylsulfonyl)carbamoyl)isonicotinate 22 (49 mg, 0.1 mmol) in
tetrahydrofuran : water (1 : 1, 8 mL), lithium hydroxide monohydrate (12.2
mg, 0.2 mmol) was added. The reaction mixture was stirred at room
temperature for 10 h. The aqueous layer was washed with
stirred
at
room
temperature
for
30
minutes.
Then
4-(trifluoromethyl)benzenesulfonamide (230 mg, 1 mmol) was added. The
reaction mixture was stirred at room temperature for 3 days. The reaction
mixture was then diluted with dichloromethane, and washed by citric acid
aqueous solution (10%). The organic layer was neutralized by aqueous
saturated NaHCO3 solution and dried over anhydrous Na2SO4. Then the
solvent was removed in vacuo and the residue was purified by flash
dichloromethane, and then was acidified to pH
1 using aqueous
hydrochloric acid solution (2N). The precipitate was separated by filtration
to give 8 (23.7 mg, 51%) as a white solid. m.p. 246-247 °C; 1H NMR (400
MHz, DMSO-d6): δ = 2.61 (s, 3H; CH3), 7.40-7.47 (m, 2H), 7.60 (m, 1H),
7.97 (m, 1H), 7.98 (m, 1H), 8.43 (m, 1H), 8.86 (m, 1H); 13C NMR (100 MHz,
DMSO-d6): δ = 19.6 (CH3), 122.6, 124.9, 126.2, 130.4, 132.4, 133.7, 137.1,
137.2, 140.1, 149.2, 150.5, 163.6 (CONH), 165.4 (CO2H); IR (neat) υ/cm-1
= 1149 (SO), 1342, 1638 (CO), 1651 (CO), 3006 (OH); MS (ESI-) m/z:
calcd. for C14H11N2O5S [M-H]-: 319.0. Found 319.1; HRMS (ESI) m/z: calcd.
for C14H11N2O5S [M-H]-: 319.0394. Found 319.0934.
column chromatography (dichloromethane
/ methanol 10 : 1). The
fractions collected were further purified by triturating with n-hexane-
isopropanol and the precipitate was separated by filtration to give 21 as a
white solid (268 mg, 81%). Rf = 0.60 (dichloromethane/methanol 4 : 1);
m.p. 230-231 °C; 1H NMR (400 MHz, DMSO-d6): δ = 3.89 (s, 3H; CO2CH3 ),
7.79 (m, 2H), 7.86 (m, 2H), 8.02 (m, 1H), 8.44 (s, 1H), 8.71 (m, 1H); 13C
1
NMR (100 MHz, DMSO-d6): δ = 52.3 (CO2CH3), 123.4, 123.9 (q, JC-F
= 270.8 Hz), 124.8 (q, 3JC-F = 3.7 Hz), 125.5, 127.8, 133.1 (q, 2JC-F = 31.7
Hz), 147.3, 147.5, 149.4, 150.0, 165.1 (CONH), 167.0 (CO2CH3); 19F NMR
(376.3 MHz, DMSO-d6): δ = 61.20; IR (neat) υ/cm-1 = 1170 (SO), 1263
(CF), 1353, 1730 (CO); MS (ESI-) m/z: calcd. for C15H10F3N2O5S [M-H]-:
387.0. Found 387.1.
Plasmid and thermal tag construction
DNA fragments encoding full length human FTO and N-terminally
truncated ALKBH5 (residues 66-292) were cloned into pNIC28-Bsa4 to
generate N-terminal His6-tagged FTO1-505 and N-terminal His6-tagged
ALKBH566-292 constructs, respectively, as previously described.[53,55] DNA
for full length human ALKBH3 was cloned into pET28a to generate an
N-terminal His6-tagged ALKBH31-286 construct, as previously described.[56]
The above constructs were then subjected to mutagenesis experiments
using the QuikChange mutagenesis kit (Agilent Technologies) whereby
DNA encoding various the KE and RD tag sequences (commercially
synthesised by GenScript USA) were fused to the C-terminus of the
respective genes. The sequences of all of fusion protein constructs were
confirmed by DNA sequencing.
2-(((4-(Trifluoromethyl)phenyl)sulfonyl)carbamoyl)isonicotinic acid
(7). To
a solution of methyl 2-(((4-(trifluoromethyl)phenyl)sulfonyl)
carbamoyl)isonicotinate 21 (59.5 mg, 0.1 mmol) in tetrahydrofuran : water
(1 : 1, 4 mL), lithium hydroxide monohydrate (15.3 mg, 0.3 mmol) was
added. The reaction mixture was stirred at room temperature for 18 h. The
aqueous layer was washed with dichloromethane, and then was acidified
to pH 1 using aqueous hydrochloric acid solution (2 N). The precipitate
was separated by filtration to give 7 (40.5 mg, 70%) as a white solid. m.p.
252-253 °C; 1H NMR (400 MHz, DMSO-d6): δ = 8.01 (m, 3H), 8.20 (m,
2H), 8.42 (s, 1H), 8.85 (m, 1H); 13C NMR (100MHz, DMSO-d6): δ = 122.0,
123.4 (q, 1JC-F = 271.3 Hz), 125.1, 126.3 (q, 3JC-F = 3.7 Hz), 127.4, 128.7,
133.0 (q, 2JC-F = 32.2 Hz), 140.9, 143.6, 149.0, 150.2, 164.3 (CONH), 165.3
(CO2H); 19F NMR (376.3 MHz, DMSO-d6): δ = 61.65; IR (neat) υ/cm-1
= 1274 (CF), 1167 (SO), 1362, 1657 (CO), 1716 (CO), 3007 (OH); MS
(ESI-) m/z: calcd. for C14H8F3N2O5S [M-H]- : 373.0. Found 373.1; HRMS
(ESI) m/z: calcd. for C14H8F3N2O5S [M-H]-: 373.0112. Found 373.0115.
Protein expression and purification
Full length human FTO,[47,53,55] human ALKBH3,[56] human
[53,55]
[46,53,56]
ALKBH566-292
JMJD2A1-359
,
human
ALKBH256-258
and
human
[53]
was expressed and purified as previously reported, with
modifications. In brief, all the constructs for the proteins and their KE/RD
tagged fusion proteins were transformed into E. coli BL21 (DE3) Rosetta
cells. The transformed cells were grown at 37 °C until an OD600 of 0.6
was reached. Protein expression was then induced with isopropyl
β-D-1-thiogalactopyranoside (IPTG, 0.5 mM, Gold Biotechnology). Cell
growth was continued at 16 °C for 16 h, after which the cells were
harvested by centrifugation and the resulting cell pellet was stored
at -80 °C. The frozen cell pellets were then thawed, resuspended in lysis
buffer and disrupted by French Press. Further purification of the protein
was achieved using Ni affinity chromatography and gel filtration, as
described below. Full length human FTO was sub-cloned into
pNIC28-Bsa4 to generate a His6-tagged FTO1–505 construct. FTO in lysis
Methyl 2-((o-tolylsulfonyl)carbamoyl)isonicotinate (22). To
a
suspension of 4-(methoxycarbonyl)picolinic acid 17 (280 mg, 1.5 mmol) in
dichloromethane (5 mL), triethylamine (549 mg, 5.4 mmol), 1-
hydroxybenzotriazole
hydrate
(314
mg,
2.3
mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (441 mg,
2.3 mmol) were added sequentially at room temperature. The reaction was
stirred at room temperature for 30 minutes. Then o-toluenesulfonamide
(292 mg, 1.7 mmol) was added. The reaction mixture was stirred at room
temperature for 48 h. The reaction mixture was then diluted with
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buffer (25 mM Tris, pH 7.5, 500 mM NaCl, 40 mM imidazole and 5 mM
β-mercaptoethanol (β-ME) was purified using Ni affinity chromatography
(GE healthcare), followed by gel filtration using HiLoad superdex 200
26/60 (GE healthcare) into the final buffer (25 mM Tris buffer, pH 7.5, 100
mM NaCl, 5% (v/v) glycerol and 5 mM β-ME). Full length ALKBH3 was
sub-cloned into pET28a to generate a His6-tagged ALKBH31–286 construct.
ALKBH3 in lysis buffer (50 mM Sodium Phosphate, pH 8.0, 300 mM NaCl,
10 mM imidazole and 5 mM β-ME) was purified using Ni affinity
chromatography (GE healthcare), followed by gel filtration using a HiLoad
superdex 75 16/60 (GE healthcare) into the final buffer (25 mM Sodium
Phosphate buffer, pH 8.0, 150 mM NaCl, 5% (v/v) glycerol and 5 mM
β-ME). For human ALKBH5, a His6-tagged ALKBH566–292 construct in
pNIC28-Bsa4 was used. ALKBH5 in lysis buffer (20 mM Tris, pH 8.0,
500 mM NaCl, 40 mM imidazole and 5 mM β-ME) was first purified using
Ni affinity chromatography (GE healthcare), followed by anion
chromatography using a 5 mL HiTrap Q HP column (GE healthcare) and
gel filtration using HiLoad superdex 75 16/60 (GE healthcare) into the final
buffer (20 mM Tris buffer, pH 8.0, 100 mM NaCl and 5 mM β-ME). For
human ALKBH2, a His6-tagged ALKBH256-258 construct in pET28b was
used. ALKBH2 in lysis buffer (50 mM Sodium Phosphate buffer, pH 8.0,
300 mM NaCl, 10% (v/v) glycerol, 5 mM β-ME) was purified by Ni affinity
chromatography (GE healthcare), followed by anion chromatography
using a 5 mL HiTrap Q HP column (GE healthcare) and gel filtration using
a HiLoad superdex 75 26/60 (GE healthcare) into a final buffer of 10 mM
Tris, pH 8.0, 100 mM NaCl, 5 mM β-ME. For human JMJD2A, a
His6-tagged JMJD2A1-359 construct in pNIC28-Bsa4 was used. The
transformed E.coli cells were grown in Terrific Broth (TB) supplemented
with 8 g/L of glycerol and appropriate antibiotics. JMJD2A in lysis buffer
containing 100 mM HEPES, 500 mM NaCl, 10 mM Imidazole, 10% glycerol,
0.5 mM TCEP, pH 8.0, Benzonase, Protease Inhibitor Cocktail Set III was
purified using Ni-NTA Superflow (Qiagen) column followed by HiLoad
16/60 Superdex-200 column (GE Healthcare) into a final buffer of 20 mM
HEPES, pH 7.5, 300 mM NaCl, 10% (v/v) glycerol, 0.5 mM TCEP. All
KE/RD tagged proteins were expressed using similar methods. For details,
see Supporting Information.
Circular dichroism (CD) spectroscopy
The proteins were exchanged into 10 mM sodium phosphate buffer (pH
6.0). Far UV-CD spectra were recorded at a protein concentration of 0.1
mg/mL at 25 °C on a JASCO J-810 spectropolarimeter over a wavelength
range of 190-250 nm with a scan rate of 20 nm/min. All the spectra were
subtracted with the buffer blank and smoothed using the Savitsky-Golay
algorithm (polynomial order 10). All measurements were performed in
triplicates.
Steady-state kinetic analyses of nucleic acid demethylation by
untagged ALKB proteins and their tagged counterparts
Steady-state kinetic analysis is done using HPLC-based demethylation
assays. The substrates used are 5’-GG(m6A)CU-3’ (for FTO);
5’-AAAGCAG(m1A)ATTCGAA-3’ (for ALKBH3) and 5’-GG(m6A)CU-3’ (for
ALKBH5). The Km and kcat values were determined by keeping a constant
enzyme concentration of 0.5µM and varying the substrate concentrations
(0.5, 1, 2, 3, 5 and 10μM), as previously reported.[53] The concentration of
demethylated product at different substrate concentrations was plotted as
a function of time. The initial velocity (V0) for each substrate concentration
was determined from the slope of the curve at the beginning of a reaction.
The Michaelis–Menten curve was fitted using non-linear regression, and
the kinetic constants (Vmax, Km) of the substrate was estimated using
GraphPad Prism 6.0. All reactions were performed at 4 ºC in triplicate and
were adjusted to ensure that less than 20% of the substrate was consumed.
Multi-protein DCC approach
The dynamic library was constituted by mixing ALKBH5-KE6 (2 μM), FTO-
KE12 (2 μM), ALKBH3-KE18 (2 μM), scaffold ligand 3 (40 μM), aldehydes
4a-j (library A, each aldehyde at 20 μM), MnCl2 (50 μM) and aniline (5 mM)
in HEPES buffer (50 mM, pH 6.0) in a final reaction volume of 50 µL. The
reaction was incubated at 25 ºC for the specified time points (see Figure
5), after which SYPRO orange (5x; Invitrogen) was added, and the reaction
mixture analysed using a MiniOpticon™ Real-Time PCR Detection System
(Bio-Rad). Melting curve was obtained by steady heating from 25 °C to
95 °C at a rate of 1 °C/min. Fluorescence were detected on FAM channel,
with readings taken every 0.5 °C. The fluorescence intensity data was then
fitted to Boltzmann Sigmoidal curve using GraphPad Prism 6.0 to
determine melting temperature (Tm) of the proteins. The assay was
performed in triplicate, with standard deviations typically < 1 °C.
Differential Scanning Fluorimetry (DSF) melting analysis
All DSF-based experiments were performed using a MiniOpticon Real-
Time PCR Detection System (Bio-Rad), monitoring protein unfolding using
SYPRO orange (Invitrogen) according to the reported method.[43,53]
To characterise the effects of thermal tags on the melting temperature (Tm)
of proteins:
The reaction mixtures contained proteins (2 μM), MnCl2 (50 μM; active site
metal) and 5x SYPRO orange (Invitrogen; fluorescent dye) in a final
volume of 50 μL. Reagents were prepared in 50 mM HEPES buffer, pH
6.0 except MnCl2, which was dissolved as 100 mM stocks in 20 mM HCl,
then further diluted in MilliQ water. Fluorescence were detected on FAM
channel, with readings taken every 0.5 °C in the range 25-95 °C, with the
temperature increased linearly by 1 °C/min. The fluorescence intensity
data was then fitted to Boltzmann Sigmoidal curve using GraphPad Prism
6.0 to determine melting temperature (Tm) of the proteins. The Tm shift
caused by the thermal tags was determined by subtraction of the Tm of
untagged protein from the Tm of fusion protein. The assay was performed
in triplicate for each protein, with standard deviations typically < 1 °C.
For thermal shift assay with inhibitors:
HPLC analysis of multi-protein DCC
The DCL was analysed using a Zorbax C18 column (4.6 mm x 250 mm)
at a flow rate of 1 mL/min at 25 °C. Gradient used: from 95% solvent A
(water + 0.1% TFA) to 15% solvent B (MeOH) over 20 min, then to 60%
solvent B over 30 min. The UV detection wavelength was set at 254 nm.
The concentration of adducts 5e and 5h were calculated based on their
peak areas, using calibration plots obtained from pure standards. The
percentages of adduct formation were calculated relative to the starting
concentration of scaffold ligand 3 (40 μM).
NMR water relaxation experiment
The reaction mixtures contained proteins (2 μM), MnCl2 (50 μM),
compounds (100 μM) and 5x SYPRO in a final volume of 50 μL. The
inhibitors tested were prepared in 100% DMSO and added such that the
final concentration of DMSO was not more than 1% v/v of assay mix. The
Tm was determined as described above. The Tm shift caused by the
addition of each inhibitor was determined by subtraction of the ‘reference’
Tm (derived from protein incubated with MnCl2 and 1% v/v DMSO) from
the Tm obtained in the presence of the inhibitor. The assay was performed
in triplicate for each inhibitor, with standard deviations typically < 1 °C.
NMR assay was performed as previously reported, with modifications.[14,65]
The experiments were conducted at 500 MHz using a Bruker DRX 500
spectrometer equipped with a standard 5 mm z-gradient TXI probe. Unless
otherwise stated, all experiments were conducted at 298 K in conventional
3 mm diameter NMR tube (Norell). A freshly prepared mixture of protein
(50 μM), Mn(II) (50 μM) and aldehyde (500 μM) buffered with 50 mM Tris
dissolved in 10% H2O and 90% D2O (pH 6.0) was titrated against scaffold
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ligand 3 at varying concentrations(10, 25, 50, 80, 100 μM). A 1:1 molar
ratio of protein to Mn(II) was used to minimise any contribution to relaxation
rate changes from binding of small molecules to free Mn(II). Inversion
recovery experiments were performed with two scans with a relaxation
delay of at least 5 times T1 (longitudinal relaxation time constants) between
transients. Pulse tip-angle calibration using the single-pulse nutation
method was undertaken for each sample ensuring accurate 90° and 180°
pulses. The data were plotted as the fractional change in relaxation rate
(1 - R1/R1(0)) against ligand concentration. KD curves were fitted using
GraphPad Prism 6.0.The assay was performed in triplicate.
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Entry for the Table of Contents
Layout 2:
FULL PAPER
Mohua Das, Tianming Yang, Jinghua
Dong, Fransisca Prasetya, Yiming Xie,
Kendra H. Q. Wong, Adeline Cheong,
and Esther C. Y. Woon*
Page No. – Page No.
Multi-Protein
Dynamic
Combinatorial Chemistry: A Novel
Strategy that Leads to Simultaneous
Discovery of Subfamily-Selective
Inhibitors
for
Nucleic
Acid
Demethylases FTO and ALKBH3
A novel multi-protein DSF strategy which uses thermal tag to exclusively fine-tune the melting temperature of the target proteins
such that their individual melting profiles could be monitored in a single DSF melting analysis. When combined with DCC, formation
of protein-ligand complexes can be detected by shifts in Tm of proteins engaged in ligand binding. This strategy led to the discovery
of selective ligands for FTO and ALKBH3 simultaneously.
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