Selective Targeting of AF9 YEATS Domain by Cyclopeptide Inhibitors with Preorganized Conformation

Article abstract of DOI:10.1021/jacs.0c10324

YEATS domains are newly identified epigenetic "readers"of histone lysine acetylation (Kac) and crotonylation (Kcr). The malfunction of YEATS-Kac/Kcr interactions has been found to be involved in the pathogenesis of human diseases, such as cancer. These di

Full text of DOI:10.1021/jacs.0c10324

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Article
Selective Targeting of AF9 YEATS Domain by Cyclopeptide
Inhibitors with Preorganized Conformation
Yixiang Jiang,∥ Guochao Chen,∥ Xiao-Meng Li,∥ Sha Liu, Gaofei Tian, Yuanyuan Li,* Xin Li,*
Haitao Li,* and Xiang David Li*
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ABSTRACT: YEATS domains are newly identified epigenetic
“readers” of histone lysine acetylation (Kac) and crotonylation
(Kcr). The malfunction of YEATS−Kac/Kcr interactions has been
found to be involved in the pathogenesis of human diseases, such
as cancer. These discoveries suggest that the YEATS domains are
promising novel drug targets. We and others recently reported the
development of YEATS domain inhibitors. Although these
inhibitors have a general preference toward the AF9 and ENL
YEATS domains, selective inhibitors targeting either YEATS
domain are challenging to develop as these two proteins share a
high structural similarity. In this study, we identified a proximal site outside the acyllysine-binding pocket that can differentiate AF9
YEATS from ENL YEATS. Combinatorial targeting of both the acyllysine pocket and this additional site by conformationally
preorganized cyclopeptides enabled the selective inhibition of the AF9 YEATS domain. The most selective inhibitor, JYX-3, showed
a 38-fold higher binding affinity toward AF9 YEATS over ENL YEATS. Further investigations indicated that JYX-3 could engage
with AF9 in living cells, disrupt the YEATS-dependent chromatin recruitment of AF9, and suppress the transcription of AF9 target
genes.
We recently developed the first class of peptide-based
YEATS domain inhibitors with submicromolar activity by
targeting a unique π−π−π stacking that underlies the YEATS−
Kcr recognition (Figure S1).25 The high affinity between the
YEATS domains and our inhibitors was achieved by replacing
the original crotonyl group with expanded π systems to
enhance the π−π−π stacking interactions (e.g., furan in XL-07i
and oxazole in XL-13a, Figure 1A). Research by others has also
led to the identification of a set of small molecule YEATS
inhibitors.26−29
The reported inhibitors showed a general preference toward
the YEATS domains of AF9 and its paralog ENL, which are
known to participate in the regulation of gene transcription by
serving as a member of transcriptional complexes, for example,
the super elongation complex (SEC).30 While it is clear that
the presence of these two paralogs in one complex is mutually
exclusive;31 the different roles played by either AF9- or ENL-
containing complexes in specific gene regulation events have
been poorly understood. The development of selective
INTRODUCTION
■
Chemical inhibitors are valuable tools to probe the biological
functions of target proteins. Target selectivity is a key issue
when developing a chemical inhibitor. This is especially true
when the target protein shares high structural homology with
other family members, such as protein kinase.1,2 Canonical
kinase inhibitors that target structurally conserved adenosine
triphosphate (ATP)-binding pockets frequently lead to
polypharmacology because of poor selectivity.3−10 One
strategy to improve the selectivity of a kinase inhibitor involves
combinatorial targeting of the ATP pocket and a second site
that is more structurally diverse, for example, cavities proximal
to the ATP cleft, the substrate-binding region, or regulatory
domains of the catalytic pocket.11−14
YEATS domains are newly identified epigenetic “readers”
that recognize histone lysine acetylation (Kac) and crotony-
lation (Kcr) via conserved aromatic “sandwich” cages.15−19
The human genome encodes four YEATS domain-containing
proteins, AF9, ENL, GAS41, and YEATS2, which exist in
multiple chromatin-remodeling and histone-modifying com-
plexes.20 Given the critical roles played by these complexes in
gene regulation and chromatin biology, aberrant YEATS-
dependent readouts of histone modifications have been
associated with the pathogenesis and progression of human
diseases, such as cancer.21−24 These discoveries suggest that
YEATS domains are promising drug targets.
Received: October 5, 2020
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Figure 1. Development of cyclopeptide inhibitors selective for AF9 YEATS domain. (A) Structures and inhibitory activities of previously reported
YEATS inhibitors XL-07i and XL-13a. (B) Superimposition of structures of AF9 YEATS−XL-07i (PDB: 5YYF) and ENL YEATS−H3K27ac
(PDB: 5J9S) complexes in their acyllysine-binding pockets. (C) Steric clash between the Cbz group of XL-07i and the Loop 8 surface of ENL
YEATS. Small to large and green to red disks indicate slight to significant steric interactions. (D) XL-07i forms different contacts with AF9 YEATS.
Cbz is involved in different π stackings with H107 and H111 of AF9 but not with N107 and N111 of ENL. Red dash lines indicate hydrogen
bonding. (E) Design strategy of the cyclopeptide inhibitors.
inhibitors should provide useful tools to functionally
distinguish isoforms of transcriptional complexes containing
either AF9 or ENL. However, it is extremely challenging to
pursue the selectivity between AF9 and ENL YEATS, as the
residues that shape the acyllysine-binding pockets in these two
domains are identical (Figure 1B).
We reasoned that targeting an additional site outside the
acyllysine-binding pocket would be a solution to increase the
inhibitor selectivity. Here, we describe the identification of a
site proximal to the acyllysine-binding pocket that distin-
guishes AF9 YEATS from ENL YEATS. By developing
cyclopeptide inhibitors with preorganized conformation that
target both the acyllysine pocket and the proximal site, we
achieved selective inhibition of the AF9 YEATS domain. The
most selective inhibitor, JYX-3, showed a 38-fold higher
binding affinity toward AF9 YEATS over ENL YEATS.
YEATS as it formed different π stackings with the imidazole
rings of AF9 H107 and H111 residues (Figure 1D).25 Notably,
these two positions in ENL are Asn instead of His, indicating
the absence of the π stackings in this region of ENL. Taken
together, inhibitors with proper functional groups approaching
the Loop 8 region in YEATS domains should show selectivity
between AF9 and ENL.
We noticed that, to place its Cbz group in the Loop 8 region
of AF9 YEATS, XL-07i peptide adopted a constrained
conformation by forming an intramolecular hydrogen bond
between the amide carbonyl group on the side-chain of Gln
and the guanidine proton of Arg (Figure 1D). We reasoned
that cyclization of the original linear peptides by replacing the
hydrogen bond with covalent linkages could rigidify the
inhibitors into the AF9 YEATS-favored conformation (Figure
1E). On the basis of the structures of XL-07i and XL-13a, we
first developed four cyclopeptide inhibitors (JYX-1−JYX-4,
Table 1) carrying either a furan or an oxazole ring at the lysine
side chain. The original amide of Gln was removed, and the
alkyl chain was covalently linked to the guanido group. The
guanidine was retained as it contributed to the YEATS−
inhibitor interaction by forming a charge stabilized hydrogen
bond with AF9 D103 (Figure 1D). An inhibitor lacking the
conjugated π system (JYX-0) at the lysine side-chain was also
prepared as an inactive control.
We adopted a fluorescence-based competitive photo-cross-
linking assay25,32,33 to estimate the inhibitory activity of these
four cyclopeptides on AF9 YEATS (Figure S3A). As shown in
Figure 2A, all of the four inhibitors could efficiently block the
interaction between AF9 YEATS and a crotonylated peptide
probe 1 (Figure S3B). Specifically, JYX-3 (IC50 = 0.41 μM)
and JYX-4 (IC50 = 0.56 μM) exhibited higher potencies than
JYX-1 (IC50 = 1.78 μM) and JYX-2 (IC50 = 2.23 μM), which
was consistent with our previous result that the oxazole ring
was preferred by AF9 YEATS compared with the furan ring.25
RESULTS AND DISCUSSION
■
Development of AF9 YEATS-Selective Cyclopeptide
Inhibitors. In our previous study, we developed two potent
pentapeptide inhibitors, XL-07i and XL-13a, which showed
slightly higher selectivity for AF9 YEATS over ENL YEATS
(3−5 folds, Figure 1A).25 To uncover the different binding
behaviors between AF9 and ENL YEATS on interacting with
XL-07i and XL-13a, we superposed the crystal structure of
ENL YEATS bound to a histone H3K27ac peptide over the
structure of the AF9 YEATS−XL-07i complex. Although the
acyllysine-binding pockets of these two proteins are well-
aligned (Figure 1B), the surface at the Loop 8 region of ENL
YEATS, mainly the L108 and N111 side-chains, clashed with
the XL-07i N-terminal carboxybenzyl (Cbz) group (Figure
1C). Similar incompatibles were also observed when the apo
ENL YEATS or ENL YEATS in complex with an acetylated
lysine were superposed with AF9 YEATS−XL-07i (Figure S2).
On the contrary, the Cbz group of XL-07i was favored by AF9
B
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Table 1. Structures of JYX-0 to JYX-8 and Their Inhibitory Activity toward AF9 and ENL YEATS Domains
More importantly, JYX-3 and JYX-4 showed 31-fold and 19-
fold decreased inhibitory activity on ENL YEATS (JYX-3, IC50
= 12.80 μM; JYX-4, IC50 = 10.84 μM) than that on AF9
YEATS, respectively (Figure 2B), indicating a substantially
increased selectivity toward AF9 over ENL compared with the
linear precursor XL-13a (only 3 folds). As expected, the
negative control inhibitor JYX-0 showed no affinity to either
AF9 or ENL YEATS as it failed to compete the probe-1-
induced labeling toward these two domains (Figure S4).
We next examined the selectivity of JYX-3 against a panel of
epigenetic regulators (Figures S5−S8), including another two
human YEATS domains (GAS41 and YEATS2),25 canonical
Kac “readers” (bromodomains of BRD2(2), BRD3(2),
BRD4(1), BRD4(2), BRD9, TAF1(2), and CECR2),34 lysine
acylation “erasers” (Sirt2, Sirt3, Sirt5, and Sirt6),35,36 and
histone methylation “readers” (ING2 and SPIN1).35 JYX-3
showed little inhibitory effects toward all the tested proteins.
Further trials to diversify the covalent linkages (JYX-5−JYX-8)
that cyclized the inhibitors revealed that a simple 4-carbon
chain (as in JYX-3) was the best linker among all the tested
ones (Table 1 and Figure S9). The introduction of any moiety
into the carbon chain would diminish the inhibitor selectivity
for AF9 YEATS.
Structural Features in the Loop 8 Region of AF9
YEATS Underlie the Inhibitor Selectivity. To study the
molecular basis of how the cyclopeptide engages with AF9
YEATS, we determined the crystal structure of AF9 YEATS in
complex with JYX-3 at 3.05 Å resolution (Figure 3A and Table
S1). In the complex, JYX-3 exhibits an almost identical binding
pose to that of its linear counterpart XL-07i. Similar to XL-
07i/13a, JYX-3 also keeps all the key features critical for AF9
YEATS binding, including Koxa-accommodating hydrogen
bonding and hydrophobic interactions, guanido group-D103
hydrogen bonding, and π stacking interactions between Cbz
and H107 and H111 side-chains (Figure 3A,B). Compared
with XL-07i, there is a highly coordinated hydrogen bonding
network involving AF9 D103, the Gln side-chain amide of XL-
07i, and a structural water molecule not seen for JYX-3 (Figure
S10), which might account for the slightly reduced inhibitory
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Figure 3. Crystal structure of AF9 YEATS domain in complex with
JYX-3. (A) Overall structure of AF9 YEATS (gray) bound to JYX-3
(salmon). Key AF9 residues (yellow) accommodating the inhibitor
are highlighted. (B) Two-dimensional interaction diagram of JYX-3
with AF9 YEATS. Numbers show the distances of the indicated
hydrogen bonds in Å.
assessment of the inhibitory activities of these cyclopeptides
(Table 2 and Figure S11) revealed that the inhibitors’
selectivity was indeed sensitive to structural changes of the
moieties in contact with the Loop 8. Specifically, AF9 YEATS
tolerated a broad spectrum of changes at this site, while ENL
YEATS disfavored almost any substitutions larger than acetyl
in size. By casting a careful glance at the Loop 8 of ENL
YEATS, the overall structure is firmly stabilized by multiple
intramolecular hydrogen bonds (Figure 4A), which make the
loop too rigid to host any foreign ligands requiring conforma-
tional changes. In contrast, the Loop 8 of AF9 YEATS is likely
to be more flexible to accommodate guests as it only involves
one hydrogen bond. When the linear peptide inhibitor, XL-
13a, approaches ENL YEATS, it is easy for the peptide to
adopt conformational changes to avoid direct clashes between
the Cbz group and the Loop 8 of the protein. After cyclization,
the conformation of the inhibitor has been fixed, the rigidity of
both the cyclopeptide and the Loop 8 of ENL YEATS
Figure 2. Determination of inhibitory activity of cyclopeptide
inhibitors toward AF9 and ENL YEATS domains. (A and B)
Competitive photo-cross-linking assay to determine the inhibitory
activities of the cyclopeptides JYX-1 to JYX-4 toward AF9 YEATS
(A) and JYX-3 and JYX-4 toward AF9 and ENL YEATS, respectively
(B). Protein concentration was 5 μg/mL. After photo-cross-linking,
the probe-1-labeled proteins were conjugated to rhodamine-N3 and
visualized by in-gel fluorescence scanning. All curves were normalized
between 100% and 0% at the highest and lowest fluorescence
intensities, respectively. Data are reported as mean s.d., n = 2.
activity of JYX-3 (IC50 = 0.41 μM) than XL-13a (IC50 = 0.24
μM) toward AF9 YEATS.
To gain more insight into how interactions between the Cbz
group and the Loop 8 of AF9 YEATS contributed to the
selectivity of the cyclopeptide inhibitors, we further synthe-
sized a collection of JYX-3 analogues (JYX-9−JYX-33) with
the Cbz substituted by different functional groups. The
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Table 2. Structures of JYX-9 to JYX-33 and Their Inhibitory Activity toward AF9 and ENL YEATS Domains
eventually lead to a substantial decrease in their binding
affinity, and therefore, increase the selectivity of the cyclo-
peptide toward AF9 YEATS.
On the contrary, the Cbz group in JYX-3 seemed not to be
simply accommodated by the Loop 8 of AF9 YEATS but
provided key interactions with the protein by it forming
additional π stackings with H107 and H111, respectively
(Figure 3A,B). Indeed, the two corresponding residues in ENL
YEATS turned out to be asparagine (N107 and N111), which
also are the only two residues that differ AF9 YEATS from
ENL YEATS in their Loop 8 regions (Figure 4A). To further
investigate the contribution of the Cbz-mediated π interactions
to the selectivity, we generated AF9 and ENL YEATS mutants
with their 107 and 111 residues swapped, i.e., the H107N and
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Figure 5. JYX-3 is selective for endogenous AF9. (A) Photo-cross-
linking pull-down in nuclear extracts (1 mg/mL) with probe 1 in the
presence or absence of JYX-3. After photo-cross-linking, the probe-1-
labeled proteins were conjugated to biotin-N3 and enriched by
streptavidin. The eluted protein mixtures were analyzed by
immunoblotting. (B) Quantification of the inhibitory effects of JYX-
3 on the enrichment of AF9 and ENL in (A). Data are reported as
mean s.d. (n = 3). (C) CETSA to show the engagement of JYX-3
with endogenous AF9, but not ENL, in living cells. Hela cells treated
with or without JYX-3 were heated at indicated temperatures. The
soluble proteins were analyzed by immunoblotting. β-Actin was used
as a loading control. Each blotting in (A) and (C) is representative of
three independent experiments.
Figure 4. Structure features of AF9 YEATS Loop 8 underlie the
inhibitor selectivity. (A) Loop 8 region of ENL YEATS is more
rigidified than that of AF9. (B and C) ITC measurements for the
binding affinities of JYX-3 with AF9 and ENL YEATS (B) and AF9NN
and ENLHH mutants (C).
artificial membrane permeability assay (PAMPA),38 we first
demonstrated that the cyclization indeed increased the
lipophilicity and passive transport rate of the inhibitor (Figures
S12 and S13). Although there would still be a large space to
improve, JYX-3 behaved comparably to theophylline, a model
drug known to have relatively low cell membrane perme-
ability.39 In addition, JYX-3 exhibited a higher stability than its
linear precursor XL-13a (Figure S14). We next applied the
cellular thermal shift assay (CETSA)40,41 to determine whether
JYX-3 could target AF9 in living cells. HeLa cells treated with
or without 20 μM JYX-3 were heated at different temperatures.
Soluble proteins were then extracted and analyzed by
immunoblotting. As expected, JYX-3 helped AF9 resist the
high-temperature-induced denaturation, while the thermal
stability of ENL remained unchanged upon the inhibitor
treatment (Figure 5C). The CETSA results indicated that JYX-
3 could traverse the cell membrane and selectively engage with
the endogenous AF9, but not ENL, under the tested
conditions.
H111N dual mutations in AF9 (AF9NN) and N107H and
N111H dual mutations in ENL (ENLHH). Isothermal titration
calorimetry (ITC) measurements showed that JYX-3 had a 38-
fold binding preference for AF9 YEATS (Kd = 0.37 μM) over
ENL YEATS (Kd = 14.1 μM), while the Kd values of the same
inhibitor toward AF9NN and ENLHH were 6.04 and 1.52 μM,
respectively (Figure 4B,C and Table S2). Compared with their
wild-type counterparts, the 16-fold affinity drop of AF9NN and
9-fold binding enhancement of ENLHH to JYX-3 completely
reversed their preference toward the cyclopeptide inhibitor.
Taken together, investigations by changing the structure
features of either the cyclopeptides or the YEATS domains
strongly suggested that the Cbz-His-His unit in the Loop 8
region should be a main contributor to the selectivity of JYX-3
toward AF9 YEATS.
JYX-3 Is Cell Permeable and Selectively Engages with
Endogenous AF9. We next monitored the engagement of
JYX-3 with endogenous AF9. To this end, HeLa S3 nuclear
extracts were photo-cross-linked with the probe 1 in the
presence of JYX-3. The probe-1-labeled and thus enriched
proteins were subjected to immunoblotting analysis against
anti-AF9 and anti-ENL antibodies. In the presence of 2 μM
JYX-3, 60% enrichment of endogenous AF9 was blocked while
the enrichment of ENL left unaffected (Figure 5A,B). At 20
μM, JYX-3 inhibited more than 90% AF9 enrichment, while
the enrichment of ENL was still as much as 60%. This result
revealed that, at the endogenous protein level, JYX-3 also
showed satisfactory AF9 selectivity.
JYX-3 Disrupts the Chromatin Association of AF9 and
Downregulates the Expression of AF9 Target Genes. To
assess the ability of JYX-3 to inhibit the chromatin association
of AF9, we performed a fluorescence recovery after photo-
bleaching (FRAP) assay42 using U2OS cells transfected with a
full-length AF9 fused with GFP. A significant increase in the
fluorescence recovery rate was observed when the cells were
treated with JYX-3, which phenocopied the loss-of-Kac-
binding AF9 mutant (F59A) (Figure 6A−C). The increased
mobility of AF9−GFP was, therefore, likely to arise from the
displacement of AF9 YEATS from the acetylated chromatin by
JYX-3. FRAP using ENL−GFP, however, showed no differ-
ences in the recovery rates acquired from samples in the
presence and absence of JYX-3 (Figure 6D−F), further
supporting the AF9 selectivity of the inhibitor in cellular
environment.
Encouraged by the in vitro results, we proceeded to examine
the performance of JYX-3 in living cells. Using the immobilized
artificial membrane (IAM) chromatography37 and the parallel
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Figure 6. JYX-3 disrupts the YEATS-dependent chromatin association of AF9. (A) FRAP assay using AF9−GFP. (B and C) FRAP curves (B) and
recovery t1/2 (C) of each group in (A). (D) FRAP assay using ENL−GFP. (E and F) FRAP curves (E) and recovery t1/2 (F) of each group in (D).
In (A) and (D), shown are the representative nuclei of transfected U2OS cells pre- or postbleaching at indicated time points. Red circles indicate
the bleached regions. The pan-HDAC inhibitor SAHA was used to increase the global Kac level.42 Scale bar = 4 μm. Data are reported as mean
s.e.m., n ≥ 20. P values are based on the two-tailed Student’s t-test. ****P < 0.0001, n.s.: not significant.
We next sought out to explore the effects of JYX-3 on the
AF9 YEATS-dependent regulation of gene expression. The
YEATS domain is known to be essential for the enrichment of
AF9 at its target genes, for example, MYC and PABPC1.43 AF9
may further recruit histone H3K79 methyltransferase DOT1L,
leading to elevated H3K79 dimethylation (Kme2) or
trimethylation (Kme3) levels and transcription activation of
the target genes. By performing chromatin immunoprecipita-
tion and quantitative PCR (ChIP-qPCR) in HeLa cells, we
detected reduced abundances of AF9 and H3K79me3, but not
ENL, on MYC and PABPC1 upon JYX-3 treatment (Figure
7A), suggesting that the cyclopeptide could disrupt the
YEATS-dependent enrichment of AF9 on its target genes
and could further prevent the recruitment of DOT1L. In
addition, the exposure of HeLa cells to JYX-3 induced
significant losses in the transcription levels of MYC and
PABPC1 (Figure 7B), which was in good accordance with the
unloading of AF9 from these two genes. Our observations
agree with the previous discoveries that AF9 downregulation or
Kac-binding-impairing mutations in AF9 YEATS led to
transcription defects of its target genes.43
CONCLUSION
In summary, we developed the first AF9 YEATS domain-
■
specific inhibitor with 38-fold or higher selectivity over ENL
Figure 7. JYX-3 perturbs the YEATS-dependent gene regulation
and other human YEATS domains. The selectivity of the
activity of AF9. (A) ChIP-qPCR analysis of AF9 target genes in HeLa
inhibitor is achieved by preorganizing the conformation of a
cells treated with inhibitors. (B) RT-qPCR analysis shows mRNA
previously reported peptide-based inhibitor via structure-
levels of AF9 target genes in HeLa cells treated with inhibitors (20
guided cyclization, which places a Cbz group to the Loop 8
region of YEATS domains. Such conformation is only favored
by the Loop 8 of AF9 YEATS, in which the H107 and H111
μM). Data are presented as mean s.e.m., n ≥ 3. P values are based
on the two-tailed Student’s t-test. *P < 0.05, **P < 0.01, n.s.: not
significant.
G
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residues form extensive π stacking interactions with the Cbz.
These interactions are absent in other YEATS domains as their
corresponding histidine positions are substituted with non-
aromatic amino acids. The development of selective YEATS
domain inhibitors has been challenging as the acyllysine-
binding pockets of all YEATS domain are highly conserved.
Our study provides a novel strategy to realize the selectivity by
targeting an additional site outside the acyllysine-binding
pocket that distinguishes one YEATS from another. We
envision that similar methods can also be used to develop
inhibitors selective for YEATS domains of ENL, YEATS2, and
GAS41, as well as other structurally conserved epigenetic
“readers”, such as bromodomain and chromodomain.44,45
Xiang David Li − Departments of Chemistry, The University
of Hong Kong, Hong Kong, China; orcid.org/0000-0002-
2797-4134; Email: xiangli@hku.hk
Authors
Yixiang Jiang − Departments of Chemistry, The University of
Hong Kong, Hong Kong, China
Guochao Chen − MOE Key Laboratory of Protein Sciences,
Beijing Advanced Innovation Center for Structural Biology,
Beijing Frontier Research Center for Biological Structure,
Department of Basic Medical Sciences, School of Medicine
and Tsinghua-Peking Center for Life Sciences, Tsinghua
University, Beijing 100084, China
Xiao-Meng Li − Departments of Chemistry, The University of
Hong Kong, Hong Kong, China
Sha Liu − Departments of Chemistry, The University of Hong
Kong, Hong Kong, China
ASSOCIATED CONTENT
■
sı
* Supporting Information
Gaofei Tian − Departments of Chemistry, The University of
Hong Kong, Hong Kong, China
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c10324.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c10324
Figures of complex structures, superimposition of
structures, schematic diagram illustrating the compet-
itive photo-cross-linking assay, in-gel fluorescence
scanning, in-gel fluorescence and competition curves,
IAM chromatography analysis, PAMPA analysis of the
inhibitors, and stability analysis of the inhibitors, tables
of data collection and refinement statistics and ITC
thermodynamic parameters of different YEATS do-
mains, discussions of reagents, antibodies, and instru-
mentation used, molecular cloning, protein expression,
and purification, cell culture, preparation of nuclear
extracts, photo-cross-linking, Cu(I)-catalyzed azide-
alkyne cycloaddition/click chemistry, in-gel fluorescence
scanning, isothermal titration calorimetry measurements,
immunoblotting, chromatin immunoprecipitation and
quantitative PCR, quantitative PCR analysis, cellular
thermal shift assay, fluorescence recovery after photo-
bleaching, immobilized artificial membrane chromatog-
raphy, parallel artificial membrane permeability assay,
stability test of inhibitors, crystallization, design, syn-
thesis, and characterization of photoaffinity probes,
synthesis methods used, and data strings, schemes of
synthetic pathways and LC−MS spectra (PDF)
Author Contributions
∥Y.J., G.C., and X.-M.L. contributed equally to this work.
Notes
The authors declare the following competing financial
interest(s): X.L. and X.D.L. are co-inventors on a patent
(Publication No. WO 2019/101195) related to the cyclo-
peptide inhibitors reported in this manuscript.
ACKNOWLEDGMENTS
■
The authors would like to acknowledge the support from
National Natural Science Foundation of China (91753203 to
X.D.L. and H.L., 91753130 to X.D.L., 31725014 to H.L., and
31871283 and 31922016 to Y.L.), Excellent Young Scientists
Fund of China (Hong Kong and Macau) (21922708 to
X.D.L.), National Key R&D Program of China
(2020YFA0803300 to H.L.), Beijing Natural Science Founda-
tion (5182014 to Y.L.), Beijing Metropolis for the Beijing
Nova Program (Z181100006218068 to Y.L.), and China
Association for Science and Technology for the Young Elite
Scientists Sponsorship Program (YESS20170075 to Y.L.). The
authors would like to acknowledge the support from the Hong
Kong Research Grants Council (RGC) Collaborative Research
Fund (CRF C7029-15G and C7017-18G to X.D.L.), the Areas
of Excellence Scheme (AoE/P-705/16 to X.D.L.), the General
Research Fund (GRF 17121120, 17126618, and 17125917 to
X.D.L.), and the RGC Postdoctoral Fellowship (Scheme
2020/21 to X.L.). Y.L. is a Tsinghua Advanced Innovation
Young Scientist. The authors would like to thank the staff
members at beamline BL17U1 of Shanghai Synchrotron
Radiation Facility and Dr. S.F. at Tsinghua Center for
Structural Biology for their assistance in data collection and
the China National Center for Protein Sciences Beijing for
providing facility support.
AUTHOR INFORMATION
■
Corresponding Authors
Yuanyuan Li − MOE Key Laboratory of Protein Sciences,
Beijing Advanced Innovation Center for Structural Biology,
Beijing Frontier Research Center for Biological Structure,
Department of Basic Medical Sciences, School of Medicine
and Tsinghua-Peking Center for Life Sciences, Tsinghua
University, Beijing 100084, China; Email: liyuanyuan@
tsinghua.edu.cn
Xin Li − Departments of Chemistry, The University of Hong
Kong, Hong Kong, China; orcid.org/0000-0001-9698-
1883; Email: lx418@hku.hk
Haitao Li − MOE Key Laboratory of Protein Sciences, Beijing
Advanced Innovation Center for Structural Biology, Beijing
Frontier Research Center for Biological Structure, Department
of Basic Medical Sciences, School of Medicine and Tsinghua-
Peking Center for Life Sciences, Tsinghua University, Beijing
100084, China; Email: lht@tsinghua.edu.cn
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