DNA-Encoded Dynamic Chemical Library and Its Applications in Ligand Discovery

Article abstract of DOI:10.1021/jacs.8b09277

Dynamic combinatorial library (DCL) has emerged as an efficient tool for ligand discovery and become an important discovery modality in biomedical research. However, the applications of DCLs have been significantly hampered by low library diversity and limited analytical methods capable of processing large libraries. Here, we report a strategy that has addressed this limitation and can select cooperatively binding small-molecule pairs from large-scale dynamic libraries. Our approach is based on DNA-mediated dynamic hybridization, DNA-encoding, and a photo-cross-linking-based decoding scheme. To demonstrate the generality and performance of this approach, a 10 000-member DNA-encoded dynamic library has been prepared and selected against six protein targets. Specific binders have been identified for each target, and we have validated the biological activities of selected ligands for the targets that are implicated in important cellular functions including protein deacetylation and sumoylation. Notably, a series of novel and selective sirtuin-3 inhibitors have been developed. Our study has circumvented a major obstacle in DCL and may provide a broadly applicable method for ligand discovery against biological targets.

Full text of DOI:10.1021/jacs.8b09277

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Article
DNA-encoded Dynamic Chemical Library
and Its Applications in Ligand Discovery
Yu Zhou, Chen Li, Jianzhao Peng, Liangxu Xie, Ling Meng, Qingrong
Li, Jianfu Zhang, Xiang David Li, Xin Li, Xuhui Huang, and Xiaoyu Li
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09277 • Publication Date (Web): 01 Nov 2018
Downloaded from http://pubs.acs.org on November 1, 2018
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DNA-encoded Dynamic Chemical Library and Its Applications in
Ligand Discovery
Yu Zhou,†,‡ Chen Li,† Jianzhao Peng,§,‡ Liangxu Xie,¶ Ling Meng,‡ Qingrong Li, §,‡ Jianfu Zhang,‡
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Xiang David Li,‡ Xin Li,‡,* Xuhui Huang,*,¶ and Xiaoyu Li*,‡
† Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen
Graduate School, 2199 Lishui Road West, Shenzhen, China 518055
‡ Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
§ Department of Chemistry, Southern University of Science and Technology, 1088 Xueyuan Road, Shenzhen, China 518055
¶ Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
SAR, China
DNA-encoded chemical library; dynamic combinatorial library; high throughput screening; ligand discovery; drug target.
ABSTRACT: Dynamic combinatorial library (DCL) has emerged as an efficient tool for ligand discovery and become an important
discovery modality in biomedical research. However, the applications of DCLs have been significantly hampered by low library
diversity and limited analytical methods capable of processing large libraries. Here we report a strategy that has addressed this
limitation and can select cooperatively binding small molecule pairs from large-scale dynamic libraries. Our approach is based on
DNA-mediated dynamic hybridization, DNA-encoding, and a photo-crosslinking-based decoding scheme. To demonstrate the
generality and performance of this approach, a 10,000-member DNA-encoded dynamic library has been prepared and selected against
6 protein targets. Specific binders have been identified for each target, and we have validated the biological activities of selected
ligands for the targets that are implicated in important cellular functions including protein deacetylation and sumoylation. Notably, a
series of novel and selective sirtuin-3 inhibitors have been developed. Our study has circumvented a major obstacle in DCL and may
provide a broadly applicable method for ligand discovery against biological targets.
Introduction
procedures (e.g. separation of protein from bound ligands prior
to analysis).6, 33
Screening large-scale libraries against biological targets to
identify novel ligands is one of the most important discovery
modalities in biomedical research.1 In contrast to the classical
high throughput screening (HTS) where the protein target is
interrogated by individual compounds, with dynamic
combinatorial library (DCL), the target acts as the template to
direct the in situ synthesis of binders from a mixture of building
blocks (BBs) undergoing dynamic exchange through reversible
chemical reactions.2-16 Compared with static library, DCL
allows for spontaneous compound synthesis by mixing library
BBs without spatial separation, and it also has the benefit for
high-affinity binders to be amplified from a large population of
non-binding background and then identified by comparing the
equilibria with and without the target. DCL has shown great
potential and wide utilities in numerous studies;2-25 however,
the development and applications of DCL have been
significantly limited by low library diversity, and the lack of
In response, we report a strategy that circumvents these
limitations and enables the selection of large-scale dynamic
libraries based on DNA-mediated dynamic assembly and DNA
encoding. Nucleic acids have been widely used as scaffolds for
ligand display to interrogate biological targets,34-51 while DNA-
encoded chemical library (DEL), originally proposed by Brenner
and Lerner,52-53 has become an efficient screening technology in
drug discovery.54-78 Similar to DCL, DEL also employs mixed
compounds in library processing, but DELs can contain millions to
even billions of compounds due to the high encoding capacity of
DNA molecules. Recently, the Neri group has developed an elegant
method named Encoded Self-Assembling Chemical (ESAC)
library, capable of selecting large-scale DNA-encoded dual-
pharmacophore libraries;43, 79-80 however, ESAC libraries are static
and not suitable for DCL applications (e.g. for non-immobilized or
endogenous proteins). The Hamilton group used proteins to direct
dynamic DNA hybridization,34 a concept lately elaborated in more
complex systems by Zhang,81 Winssinger,45 and us.82 These studies
have demonstrated that the principle of DNA encoding could be
used for DCL; however, the preparation of DNA-encoded dynamic
library (DEDL) with actual chemical diversity and the selection for
de novo ligand discovery have yet to be realized. Previously, we
reported a proof-of-principle, single-display DEDL with no
chemical diversity.82 In this study, first, we designed a novel dual-
display dynamic DEDL method and performed methodology
development with model systems. Second, to demonstrate the
utilities of this approach in ligand discovery, a 10,000-member
DEDL was prepared and selected against 6 protein targets.
analytical methods suitable for large libraries remains as a
26
major problem.6,
Typically, chromatography, mass
spectrometry, NMR, crystallography, spectroscopic methods,
and bioassays are used for hit identification in DCL, but these
methods lack the capability of resolving large libraries. Otto,
Miller, Erlanson and the respective co-workers have developed
several elegant strategies for large DCLs;27-32 however, most
libraries only contain a few hundred compounds and new
approaches capable of processing large DCLs are highly
desired.6 In addition, DCL selections usually require high
protein consumption and involve tedious sample processing
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encoding capacity of DNA molecules,61 this approach allows
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for the preparation and selections of large-scale DCLs.
Moreover, DNA-encoding-based selections use very small
amount of proteins and is capable of detecting small
equilibrium shifts arising from low-affinity binders by counting
the copy numbers of individual DNA sequences.61
Initial validations: the dynamic system, p-stilbazole-
mediated crosslinking, and hit decoding strategy
First, we validated the dynamic nature of the system: two
partially complementary DNAs (FA-S1/FB-S1) were
conjugated with desthiobiotin (DB), a streptavidin (SA) ligand
with an affinity of ~330 nM.84-85 A fluorescein (FAM) and a
quencher were installed on FA-S1 and FB-S1 to detect duplex
formation; results show that FA-S1/FB-S1 formed dynamic
duplexes and SA promoted the formation of the bivalent DB-
DB duplexes in a thermodynamically controlled manner
(Figure S1). Next, a pair of p-stilbazoles were incorporated
(FA-1/FB-1; Figure 2a) and incubated at 0 °C to form stable
duplexes. After brief UV irradiation (10 s), ~45% crosslinking
yield was observed (i), while little crosslinking was observed in
negative controls (no UV, missing a B base, mismatched
hybridization regions, misaligned B-bases; ii-v). In contrast, at
30 °C when the duplex is dynamic, <10% crosslinking was
observed (i, Figure 2b), while SA addition increased the yield
to 50-60% (ii). In addition, only ~10% crosslinking was
observed with the non-specific protein BSA (bovine serum
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Figure 1. a) Scheme for library design and selection. Two sets of
DNA-encoded small molecules (FA/FB) with short
a
complementary region form the DNA-encoded dynamic library
(DEDL). Target addition shifts the equilibrium promoting the
formation of high-affinity duplexes. UV irradiation locks the
shifted equilibrium through p-stilbazole (B-base)-mediated photo-
crosslinking. Crosslinked duplexes are isolated and the sequences
at the encoding sites are decoded for hit identification (see Figure
2). Red dots: p-stilbazoles. b) Structures of the B base and the
crosslinked B-B pair in a DNA duplex.
albumin) or without the DB ligand (iii-iv). In
a
thermodynamically controlled system, the same equilibrium
can be reached from different starting points. To test this, FA-
1 and FB-1 were mixed either in different orders or at different
starting temperatures but with the same final equilibrating
conditions. Results show that similar crosslinking yields were
obtained, indicating that the same equilibrium was reached in
these experiments (Figure 2c). Collectively, these results have
validated the dynamic nature of the system and that
p-stilbazole-mediated crosslinking is an efficient and specific
stop mechanism for dynamic libraries.
Specific binders have been identified for each target and the
ligand binding affinities and selectivities have been validated
with biophysical methods. Finally, we have validated the
biological activities of selected ligands in a cellular context for
the targets that are implicated in important biological functions.
Results
Our strategy is shown in Figure 1, two sets of small molecules
(library BBs) are encoded with two DNA strands (FA and FB),
respectively. When the “dynamic hybridization region” is short
(6-7 bases), the FA/FB duplexes are dynamic and continuously
exchange DNA strands,82 thereby creating a DCL of small
molecule pairs. Adding the target protein shifts the equilibrium
and promotes the formation of high-affinity duplexes at the
expense of non-binders. Following the selection, “hit
compounds” can be identified by reading the enriched DNA
sequences at the encoding sites. In DCL selections, the dynamic
exchange needs be stopped to “freeze” the shifted equilibrium
prior to hit decoding.3-4 Here we chose photo-crosslinking as the
stop mechanism, as it is kinetically fast and can be
imposed/withdrawn conveniently with little perturbation to the
system.82 Previously, we used psoralen as the crosslinker;82
however, psoralen has been mostly used at the end of DNA as
a terminal crosslinker, and it may affect the properties of DNA
duplex (e.g. sequence specificity and duplex stability) when
incorporated internally. In addition, psoralen has shown only
modest crosslinking efficiency.82 Asanuma and co-workers
reported that p-stilbazoles are excellent interstrand DNA photo-
crosslinkers and are orthogonal to natural nucleobases with
little impact on the properties of DNA duplexes (Figure 1b).83
We embedded a pair of p-stilbazoles (B-bases) in the
hybridization region. After UV-irradiation, crosslinked FA/FB
duplexes are isolated for hit identification by PCR and
sequencing to read the DNA codes. Identified small molecule
pairs are then tethered with various linkers and evaluated for
binding affinities and biological activities. Considering the high
After crosslinking, it is necessary to read the encoding
sequences of both FA and FB, so that the selected small
molecule pairs, which may bind the target cooperatively, can be
identified.43 However, regular PCR cannot read through the
unnatural B-B linkage. To address this issue, we employed a
relay-primer-bypass strategy.86 As shown in Figure 2e, a relay
primer hybridizes at a non-coding region of the crosslinked
FA/FB duplex to mask the unnatural linkage. Along with a
terminal primer, enzyme-mediated extension and ligation forms
a PCR-compatible DNA template that contains both of the
encoding sequences. To test this strategy, two DNA strands
(FA-2/FB-2) were crosslinked and subjected to the relay-
primer-bypass experiment. As shown in Figure 2f, with the
relay and terminal primers, the crosslinked FA-2/FB-2 was
successfully PCR-amplified (lane 2), while no products were
observed when either of the primers was omitted (lane 3-4).
Furthermore, the PCR products were sequenced to confirm that
the sequences at both encoding sites have been correctly
decoded (Figure 2g).
Model selections with the streptavidin-desthiobiotin system.
After these preliminary studies, we prepared 10 model
libraries by mixing DB-conjugated DNAs with excess of non-
binding background DNAs. The model libraries were subjected
to the selection procedure shown in Figure 1 with and without
SA, and Sanger sequencing was used as a rapid and convenient
way to decode the results. In all cases, the DB-sequences have
been enriched after the selections with SA, while mixed
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sequences were observed after the selections without SA
(Figure S3-S4).
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Figure 2. a) (i) FA-1/FB-1 formed stable duplexes at 0 °C and were irradiated; (ii) no UV; (iii) no B-base in FA-1; (iv) mismatched
hybridization region; (v) misaligned B-bases. Reactions were analyzed with electrophoresis (15% PAGE). M: marker. b) FA-1/FB-1 formed
dynamic duplexes at 30 °C (incubated for 1 h) and irradiated: (i) no protein; (ii) with SA (0.2 µM); (ii*): with SA (0.1 µM). (iii) with BSA;
(iv) without DB in FA-1; (v), FA-1 only. Reactions were analyzed with 15% PAGE. c) FA-1 and FB-1 were mixed at different orders or
starting temperatures, irradiated, and analyzed with 15% PAGE. Lane 1-9: same as 1-9 in the column graph. d) Fluorescence polarization
(FP) analysis of the SA-binding affinity of DB-DB on a stable DNA duplex carrying a fluorescein (FAM) label.87 DNA duplex: 5 nM; SA:
varied; volume: 70 µL; buffer: 1x phosphate-buffer saline (PBS, pH 7.2). Error bars indicate standard deviations of three repeats. See the SI
for FP experiment details. e) Scheme for the relay primer bypass experiment. f) Crosslinked FA-2/FB-2 duplexes (Figure S2) were subjected
to the procedures in e); ligated DNA was PCR-amplified with long primers to afford a 176-bp DNA product, mimicking the DNA length in
next-generation sequencing (NGS). Gel image (3% agarose): M, ladder; lane 1, PCR of a 176-nt DNA standard; lane 2, PCR products from
e); lane 3, no relay primer; lane 4: no terminal primer. g) Sanger sequencing of the PCR products from lane 2 in e). DNA: 0.2 µM; protein:
0.2 µM; buffer: 1x PBS (pH 7.2); UV: 365 nm, 10 sec. Extension and ligation: T4 DNA polymerase, 10 units, T4 DNA ligase, 20 units, 16
°C, 60 min. Red dots: p-stilbazoles.
Moreover, to obtain a more quantitative measure of the
selection method, the SA-binding affinity of DB-DB on a stable
DNA duplex carrying a FAM group was determined as 21.7 nM
(Kd) using fluorescence polarization (FP), following a previous
reported method (Figure 2d).87 Therefore, these model
selections have shown that the prototypical high-affinity
binders may be enriched from large excess of background in the
library.
by a unique sequence (Figure 3a). The small molecules were
conjugated to the respective DNA strands, HPLC-purified,
characterized by mass spectrometry, and then mixed at equal
ratio to form the 10,000-member dynamic library. After the
library preparation, the library was first selected against SA and
next-generation sequencing (NGS) was used for hit
identification; a control selection without SA was also
performed. Raw sequencing data were processed with a custom
Python script to quantitatively tally the codons for each BB pair
and then calculate the enrichment fold (post-selection% with
the target/post-selection% without the target), which
eventually generates a scatter plot for each target (x-axis:
sequencing counts after selection with the target; y-axis:
enrichment fold after the selection).82 In the plot, BB pairs with
Preparation and selections of a 10,000-member DNA-
encoded dynamic library.
After model selections, a large-scale DEDL with actual
chemical diversity was prepared by mixing two sets of DNA-
conjugated BBs, each containing 100 small molecules encoded
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high enrichment folds and high post-selection sequencing Besides DB, the library also contains iminobiotin (IB),
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counts are considered as potential hits, while the ones with low
sequence counts are not considered due to statistical under-
sampling88 (see the SI for details on data processing and
calculation methods).
another SA ligand but with much lower affinity (Figure 3h).
Monovalent IB binds SA at >100 µM (Kd) and bivalent IB-IB
binders have affinities in the high nM to mid-µM range.87 We
r
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(Figure S5-S6), relay-primer-bypass (Figure S7), and next
generation sequencing (NGS) data analysis methods and script. b)-
g): Scatter plots of selection results after data processing with a
custom script. Selected BBs are labeled as “FA-FB”. x-axis:
sequencing counts after selection with the target; y-axis:
enrichment fold = (post-selection% with the target)/(post-
selection% without the target).88 h)-i): Structures of the selected
BBs and the linkers.
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that DB and IB would be good representatives for typical small-
molecule-protein interactions, and they may even be considered
as “affinity markers” in library selections. Indeed, DB-DB, DB-
IB, IB-DB, and IB-IB pairs have all been selected by SA
(Figure 3b). Moreover, as a quantitative measure, we
determined the SA-binding affinity of IB-IB DNA duplex as
20.4 µM by FP (Figure S5a). These results have suggested that
nM to mid-µM binders could be identified with this method,
which covers the range of interactions most common in small
molecule ligand discovery. In addition, several other DB-
containing duplexes were also enriched. In addition, the library
contains raloxifene (RA; Figure 3h), an estrogen receptor α
(ER-α) ligand89 and bidentate RAs are known to cooperatively
bind ER-α dimers.41, 90 As shown in Figure 3c, the RA-RA pair
has been enriched from the ER-α selection. Next, aiming for de
novo discovery, we selected the library against 4 additional
targets: a) Sirtuin 3 (SIRT3), a sirtuin family protein and an
important metabolism regulators in cells;91 b) SUMO-
activating enzyme E1 (SAE1), transcription intermediary factor
1β (TRIM28), and SUMO-conjugating enzyme 9 (UBC9): all
play important roles in sumoylation, a type of protein post-
translational modifications.92 These proteins are also implicated
in many human diseases. As shown in Figure 3d-g, for each
target, several small molecule pairs have been enriched and the
structures are shown in Figure 3h. To control for false positives
arising from protein-DNA interactions, a “blank library” with
the same DNA composition but without the small molecules
was also selected. Results show that none of the selected pairs
in Figure 3 were enriched from the blank library (Figure S8-
S9).
Hit preparation and validation.
Previously, Seitz, Winssinger, Neri, and the respective co-
workers have elegantly shown that small molecule fragment
pairs selected from DNA-encoded or DNA-display libraries
could be elaborated into high-affinity binders by screening
different linkers41, 43, 49, 80, 93 or using rigid scaffolds.87 Here we
selected 6 linkers with varied lengths, flexibility, and
lipophilicity (L1-L6; Figure 3i) to tether the selected small
molecules.93 Tethered compounds (“hits”) were synthesized
off-DNA and their binding affinities were determined with
surface plasmon resonance (SPR; Figure 4a). Due to the large
number of compounds if all 6 linkers were to be used, we only
prepared representative compounds with selected linkers for
each target.
Figure 3. a) A 10,000-member dynamic library was selected
against 6 targets. Library DNA, 0.2 µM; protein, 0.2 µM; volume:
200 µL; buffer: 1x PBS (pH 7.2); the solution was incubated at 30
ºC for 1 h before irradiation (365 nm; 10 s). Crosslinked library was
purified and subjected to the relay-primer bypass experiment
(Figure 2e). See the SI for details on library synthesis, BB
structures, DNA sequences, characterization data, gel-purification
SIRT3 enriched four BB pairs (Figure 3d). Linking 23 and 11
generated a series of ligands with micromolar affinities (Figure
4a). 23-L3-11 has the highest affinity (Kd: 14.7 µM), while
shorter or longer linkers lowered the affinity, suggesting that
L3 may be more suitable for placing 23 and 11 into the binding
pockets. The pair of 81 and 3 also gave similar results. 77/39
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and 16/39 compounds appeared to be much better binders. linker dependence is also observed for SAE1 binders (Figure
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Joining the pairs directly (77-39 and 16-39) significantly
improved the binding affinity. 77-39 has a Kd of 2.14 µM,
although still being micromolar, this has already placed 77-39
among the most potent SIRT3 binders reported.94 As a
complementary method to determine binding affinity, 77-39
and 77-L6-39 were labeled with fluorescein and the affinities
for SIRT3 were measured with FP. The results are consistent
with SPR (4.53 µM and 16.0 µM, respectively; Figure 4b). The
4a): 13-L1-65 has the highest affinity (Kd: 9.38 µM), while the
affinity decreases with increased linker length. Finally, for
UBC9 and TRIM28, hit compounds with shorter linkers
exhibited moderate affinities.
As an indication of binding selectivity, none of the individual
BBs or the truncated “single-BB-linker” conjugates show
appreciable binding in SPR (Table S1 and Figure S14-S17).
and FP, and the results show no detectable binding (Figure
S18). Collectively, these results have proven the selectivity of
the bivalent binders and the binding cooperativity of the two
BBs.
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In general, most ligands have shown affinities ranging from
low to mid-µM, demonstrating the selection affinity threshold
of this method. In addition, 77-39 and 16-39 DNA duplexes
have shown affinities of 25.5 µM and 38.6 µM for SIRT3,
respectively (Figure 4c), suggesting that the method may
identify
Figure 5. a) Scheme of the DPAL experiments. b) Structures of
BP-1 and BP-2. c) Western blot analysis of SIRT3 labeling. Lane
1: with BP-1/CP; lane 2: with 50x 77-39; lane 3: labeling with BP-
2/CP; lane 2: with 50x 77-L6-39. SIRT3: 1 µM; probe: 2 µM;
buffer: 1x PBS, 0.1 M NaCl, pH 7.2, volume: 50 µL; UV: 365 nm,
30 s. BP/CP may partially renature and form the SIRT3-CP/BP
complex under SDS-PAGE condition.95 d) BP/CP probes were
used for affinity pulldown of SIRT3 from HeLa cell lysates and the
captured proteins were analyzed with Western blot. Lane 1-4: same
as c). DNA probes: 5 µM; lysates: 1.4 µg/mL; 70 µg used.
Figure 4. a) Hit compounds were synthesized “off-DNA” by
tethering BBs with various linkers and the binding affinities were
determined with SPR. The results (in disassociation constants; Kd)
are shown in tables. Sensorgrams and fitting curves are shown in
Figure S10-S13. b) FP analysis of SIRT3-binding affinities of
FAM-labeled 77-39, 77-L6-39, 77, and 39 (off-DNA). c) FP
analysis of 77-39 and 16-39 on stable DNA duplexes carrying a
FAM label and the negative controls (-NH2: amino-modified
DNA). Error bars indicate standard deviations of three repeats. n.d.:
not detectable. In FP, ligand or DNA duplex: 5 nM; protein: varied;
volume: 70 µL; buffer: 1x PBS (pH 7.2).
modest affinity binders that could be further elaborated into
more potent ligands with proper linker choice. Moreover, the
enrichment folds of 77-39 and 77-L6-39 are similar to the IB-
IB pair in the SA selection, and their binding affinities are also
comparable, which suggests that enrichment fold may be used
to estimate the binding affinity of the selected binders when
compared with the ones of known ligands.
Previously we developed a protein labeling method named
DPAL (DNA-programmed affinity labeling; Figure 5a).95 Here
we used DPAL to further validate the SIRT3-ligand interaction.
Two DNA-conjugated binding probes based on 77-39 were
prepared (BP-1 and BP-2; Figure 5b). We reason that DNA
conjugation between the two BBs may not interfere with SIRT3
binding; indeed, both probes gave similar affinities as the
original ligands (Figure S10e). Then, the BPs were mixed with
a biotinylated, photo-reactive capture probe (CP) and incubated
with SIRT3, respectively. After irradiation, the mixture was
analyzed with Western blot. Results show that SIRT3 was
In corroboration, FP data also show that individual BBs (39, 77,
and 16) have weak affinities for SIRT3 (Figure 4b and S14).
The BB 39 has been selected by multiple targets; however, 39
alone and all the 39-linker conjugates showed very weak or
undetectable affinities (Table S1), suggesting that, although 39
by itself may be non-selective, the cooperativity of 39 with the
other binding partner has led to the enhanced affinity and
selectivity of these ligands.26 To further validate the target-
binding selectivity, the affinities of 77-39 and 77-L6-39 for
SIRT5, another sirtuin protein, were measured with both SPR
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specifically labelled by BP-1 and BP-2, while little labeling
typical fragment-based libraries.96-99 As a proof-of-principle
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was observed in the presence of the free ligand competition
(Figure 5c). Furthermore, the probes were used to affinity-
purify SIRT3 protein from cell lysates with streptavidin beads.
As shown in Figure 5d, the probes were able to capture and
isolate SIRT3 from cell lysates and ligand competition
abolished the capture, validating the SIRT3-ligand binding in a
cellular context.
study, we only tested a few representative linkers, further
elaborations on the linker structure should be able to improve
the binding affinity.100 Moreover, a larger library with higher
chemical diversity would certainly be beneficial for identifying
more potent compounds. Collectively, these selections have
demonstrated the generality of our method and that it may be
used for de novo discovery of cooperative binding partners that
can be developed into selective binders.
In general, the affinities of the identified ligands are modest
but are comparable to the ones from the primary screening of
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Figure 6. a)-c) SIRT3 inhibition assay results. d)-g) SIRT1, 2, 3, 5 inhibition assay results. n.d.: not detectable. h) Structures of the
compounds. i) The effect of 77-39 (100 µM, 24 h) on mitochondrial protein acetylation in HeLa cells. Left: Western blot; COX IV:
cytochrome c oxidase (input control); Kac: acetylated lysine. Right: Coomassie blue (CB) staining. j) Cytotoxicity assay (HeLa cells) of 77-
39. k) The effect of 77-39 on the ATP level in cells (100 µM, 24 h); antimycin (100 µM, 24 h) was used as the positive control. See the SI
for experimental details.
Biological validation of SIRT3 ligands.
39 are more potent than nicotinamide (Figure 6a-c), and 77-39
exhibited excellent inhibition activity (IC50: 4.50 µM).
Importantly, individual BBs (16, 77, 39) showed very weak
inhibition activities (Figure 5c), indicating that both BBs are
required for inhibition, presumably due to the cooperative
binding. Next, using the same assay, the compounds were
assayed against SIRT1 and SIRT2, two Class-I sirtuins also
having robust deacetylation activities. As shown in Figure 6d-
g, all compounds appeared to be SIRT3-selective. The most
potent inhibitor, 77-39, is 14-fold more selective against
SIRT2; 16-L6-39, although less potent, exhibited >26-fold
selectivity. Both compounds showed little inhibition on SIRT1.
Moreover, SIRT5 is a Class-III sirtuin with weak deacetylation
activity but is an efficient desuccinylase;102 therefore, using the
same assay but with a succinylated peptide substrate, we tested
SIRT3 is a Class-I sirtuin and it regulates many important
cellular functions such as metabolic homeostasis and ATP
regulation.101 Small molecule modulators are valuable tools to
study SIRT3. However, only a few SIRT3 inhibitors have been
developed and selective ones are even more rare.94 Comparing
with known SIRT3 ligands, the 77/39 and 16/39 compounds
exhibited good affinities and 77-39 is one of the most potent
binders.94 Therefore, we investigated whether the ligands are
also selective SIRT3 inhibitors. First, the compounds were
tested with a SIRT3 inhibitor assay, which measures the
deacetylation of an acetylated p53 peptide substrate by SIRT3.
Nicotinamide, a widely used pan-sirtuin inhibitor, was used as
the positive control. Results show that 16-L6-39, 16-39, and 77-
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Journal of the American Chemical Society
the compounds against SIRT5 and indeed observed little more biologically complex systems, such as endogenous
proteins and live cells.111 However, it is important to point out
that, since the two BBs are displayed on a DNA duplex with
short linkers, this method is limited to the targets where the
binding pockets are fairly adjacent. Varying the linker length
may further expand the target scope for this method. Finally, at
present, this method is limited to identifying small molecule
pairs, and a tethering process is still required for ligand
maturation. As demonstrated in many reports,43, 87, 100, 112 finding
a suitable linker for optimal binding is a highly challenging
task. Further methodology advancement to simultaneously
select both the library building blocks and the linker structure
is highly desired,49 and we will report our progress on this front
in due course.
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inhibition. In addition, we assayed the 23/11 compounds and
they also appeared to be selective SIRT3 inhibitors (Figure
S19). Collectively, these assay results have demonstrated the
selectivity of the SIRT3 inhibitors. Moreover, we used
computational methods to study the modes of ligand-SIRT3
binding. The results indicate that the ligands prefer to bind the
co-factor-binding site on SIRT3 and also provide explanations
for the linker effect observed among the ligands (Figure S20-
S24).
9
Furthermore, we tested the cellular activities of 77-39. As a
mitochondrial deacetylase, SIRT3 inhibition increases the
acetylation level of mitochondrial proteins.103 We treated HeLa
cells with 77-39 (100 µM), prepared the mitochondrial extracts,
and probed the protein acetylation with an anti-acetylated-Lys
antibody. Results show that 77-39 increased the acetylation of
mitochondrial proteins in general, while the overall protein
expression was not significantly altered (Figure 6i) and 77-39
showed little cytotoxicity (Figure 6j). SIRT3 is also a key ATP
regulator and cells lacking SIRT3 exhibited decrease of ATP
levels.104 Using antimycin (an ATP production inhibitor) as the
positive control, we measured the overall ATP level in HeLa
cells treated with 77-39, and significant reduction of ATP was
observed (Figure 6k). Collectively, these results have
demonstrated the biological activities of 77-39 in a cellular
context.
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ASSOCIATED CONTENT
Supporting Information. Synthetic procedures, characterization
data, library information, selection protocols and data analysis
methods, protocols of protein expression, protocols of library
synthesis, and other experimental details. This material is available
free of charge via the Internet at http://pubs.acs.org.”
AUTHOR INFORMATION
Corresponding Author
* xiaoyuli@hku.hk; xuhuihuang@ust.hk; lx418@hku.hk;
Author Contributions
Preliminary validation of SAE1, UBC9, TRIM28 ligands.
The manuscript was written through contributions of all authors.
Sumoylation is a type of post-translational modifications
where proteins are conjugated with small ubiquitin-related
modifiers (SUMOs).92 The sumoylation cascade comprises E1,
E2, and E3 enzymes.105 SAE1 is an E1 enzyme and UBC9 is an
E2 enzyme; ligands of these enzymes have been shown to be
sumoylation inhibitors.106-107 We tested 40-L6-57 (an SAE1
ligand) and 21-L1-73 (a UBC9 ligand) with a biochemical
assay measuring the sumoylation efficiency of RanGAP1, a
known sumoylation substrate.108 As shown in Figure S25a, both
compounds inhibited RanGAP1 sumoylation in a dose-
dependent manner, while unrelated compounds have little
effect (Figure S26). Although this assay is not target-specific,
the results suggested that the compounds may act via their
respective target. Finally, we preliminarily investigated the
TRIM28 ligand 95-L6-39. The bromodomain of TRIM28 is a
well-known “reader” for lysine acetylation (Kac).109
Previously, we have developed a screening assay for Kac-
reader inhibitors using an H3K9 peptide affinity probe (Figure
S25b).110 In this assay, 95-L6-39 exhibited moderate inhibition
Notes
The authors declare no competing financial interests
ACKNOWLEDGMENT
This work was supported by grants from the Research Grants
Council of the Hong Kong Special Administrative Region, China
(Project No.: AoE/P-705/16, 17321916, 17302817, 16305817,
Collaborative Research Fund C7029-15G), seed funding for basic
research (201601159018), grants from National Natural Science
Foundation of China (21572014, 21521003, 21572191 and
91753130), and Shenzhen Science and Technology Innovation
Committee (JCYJ20170413173837121). We thank Drs. Xiucong
Bao, Zheng Liu, Gaofei Tian, Yiwen Cui at HKU for the
recombinant proteins used in this work. We thank Prof. Jing Zhao,
Dr. Wei Wei, and Tongyang Zhu at Nanjing University and Prof.
Yan Cao at Shanghai Second Military Medical Officer for their
assistance with the SPR experiments. We thank Center for
Genomic Science at the University of Hong Kong for MALDI
analysis support.
against TRIM28 (Figure S25c), whereas 57-L6-39,
a
structurally similar compound but not a TRIM28 ligand, did not
show any activity.
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