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Dissecting Programmed Cell Death with Small Molecules

Yingjie Bai, Hiu C. Lam, and Xiaoguang Lei*

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CONSPECTUS: Programmed cell death (PCD) is fundamentally an

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indispensable process in all cellular activities, including cell develop-

ment, wound healing, and immune surveillance of tumors (Galluzzi, L.
et al. Cell Death Differ. 2018, 25, 486−541). Malfunctioning of PCD has
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been shown to be closely related to human diseases such as acute

pancreatitis, neurodegenerative diseases, and diverse types of cancers.
To date, multiple PCD processes have been discovered and the
corresponding regulatory pathways have been elucidated. For example,
apoptosis and autophagy are two PCD mechanisms that have been well
studied by sophisticated models and probe toolkits. However, limited
genetic and chemical tools for other types of PCD hamper the
elucidation of their molecular mechanisms. Our group has been
studying PCD using both function-oriented synthesis and chemical
biology strategies, including the development of diverse chemical
probes based on novel PCD modulators. For instance, in the
development of downstream programmed necrosis (or necroptosis)
inhibitor necrosulfonamide, we used a chemical probe to unveil a functional protein that was not previously implicated in
necroptosis, mixed lineage kinase domain-like protein (MLKL). In addition, high throughput screening and medicinal chemistry
enabled the discovery of bioymifi, a small molecule agonist which selectively causes oligomerization of the death receptor 5 (DR5),
to induce extrinsic apoptosis. Furthermore, we developed a biomimetic synthetic strategy based on diverse Diels−Alder reactions in
the total syntheses of ainsliadimers A and B, ainsliatrimers A and B, and gonchnatiolides A−C, which are natural product inhibitors
or activators for PCD. Using synthetic ainsliadimer A probe, we elucidated that ainsliadimer A inhibits the NF-κB pathway by
covalently binding to Cys46 of IKKβ and triggers apoptosis of cancer cells. We have also revealed that IKKβ is allosterically inhibited
by ainsliadimer A. In addition to total synthesis, we have developed a bioorthogonal click hetero-Diels−Alder cycloaddition of vinyl
thioether and o-quinolinone quinone methide (TQ-ligation) to facilitate small molecule target identification. The combination of
total synthesis and TQ-ligation enables subcellular imaging and identification of the cellular target of ainsliatrimer A to be PPARγ. In
addition, TQ-ligation has been applied in the discovery of heat shock protein 90 (HSP90) as one of the functional target proteins for
kongensin A. We also confirmed that kongensin A covalently attaches to Cys420 within HSP90 and demonstrated that kongensin A
blocks the interaction between HSP90 and CDC37 and subsequently inhibits necroptosis. Our development of these diverse PCD
modulators provides not only effective chemical tools for fundamental biomedical research, but also the foundation for drug
discovery targeting important human diseases such as cancers and inflammation caused by malfunction of PCD.

■ INTRODUCTION
Programmed cell death (PCD) can be classified into apoptosis
parthanatos, entotic cell death, and mitotic catastrophe as RCD
pathways.9 Since the characterization of novel PCD-related
and nonapoptotic PCDs based on the nature of ubiquitous signaling pathways has not yet been completed, space remains
causes and outcome of cell death. Apoptosis, the prototype
for component characterizations and development of small
PCD, was first described by Kerr et al. in 1972,1 characterized
by its morphological changes, unique biochemistry, and lack of molecule modulators for PCDs.
inflammation.1−4 Besides apoptosis, typical nonapoptotic
PCDs including autophagy,5 necroptosis,6 ferroptosis,7 and Received: November 29, 2019
pyroptosis,8 have been discovered recently.9,10 In addition to
PCDs, regulated cell death (RCD) has emerged to extend the
definition of PCDs to include the promiscuous cell death
pathways.9 In 2018, the Nomenclature Committee on Cell
Death accepted MPT-driven necrosis, NETotic cell deaths,11

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A Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 1. Small molecules modulating programmed cell death pathways including necroptosis, apoptosis, and the NF-κB pathway.
Indeed, recent discoveries of PCDs (e.g., ferroptosis9 and
oncosis12) were initiated from their corresponding inhibitors
or PCD pathway components via chemical screenings using
cellular phenotypes as readouts. However, the target
identification of these small molecule inhibitors posed a
challenge. Bioorthogonal reactions have been broadly applied
in bioimaging and target identifications.13,14 This methodology
might be applied in the study of PCDs with the corresponding
modulators by attaching chemical handles for various purposes
(e.g., attachment of biotin for affinity purification or
fluorescence for visualization). The rapid reaction time, high
spatiotemporal resolution, unique reactivity, and high specific-
ity features of these chemical biological tools are comple-
mentary to conventional studies in biochemistry and genetics.
Therefore, despite the fact that the reagents might be
somewhat toxic,15 bioorthogonal reactions have been currently
applied in many research areas including microbiology,
oncology, developmental biology, and neurobiology.14,16−19
Herein, we summarize our efforts in revealing the
mechanisms of PCDs using chemical biology approaches
with various small molecule probes and bioorthogonal
reactions which were developed in our laboratory (Figure 1).
First, we will discuss our chemical biology studies which led to
the discovery of PCD modulators necrosulfonamide and
bioymifi. Next, we present our endeavors using natural product
total synthesis and subsequent target identification of PCD-
inducers ainsliadimer A and ainsliatrimer A. Moreover, we
introduce the combination of biomimetic synthesis and TQ-
ligation that facilitates the target identifications of these PCD
modulators. Ultimately, the research summarized in this
Account alongside other studies of PCDs from different
research groups5,7,20 has collectively contributed to stimulate
fundamental biological discoveries and develop potential
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therapeutics for PCD-related human diseases such as cancers
and autoimmune diseases.
■ DISSECTING NECROPTOSIS WITH
NECROSULFONAMIDE
Necroptosis was first introduced by Yuan and co-workers in
2006, along with an antinecroptosis compound named as
necrostatin-1 (Nec-1) which targets receptor-interacting
protein 1 (RIPK1).6 Subsequently, multiple antinecroptosis
compounds targeting RIPK1 or RIPK3 have been identified.21
It has been shown that RIPK3 can phosphorylate itself and its
partner RIPK1, within the necrosome, an alternative name for
the RIPK1/RIPK3 complex.21 However, the biological
consequences of the phosphorylation related to necroptosis
and the necroptotic downstream substrate(s) of RIPK1/
RIPK3 complex remained elusive. Therefore, we initiated a
program in further deciphering the molecular pathway of
necroptosis. We used a cell-based assay of necroptosis for high-
throughput screening to discover antinecroptosis small
molecules. Two previously established cell lines, Jurkat and
HT-29 cells, were each treated with a combination of TNF-α
to initiate programmed cell death, a cell-permeable small
molecule that mimics Smac function (Smac mimetic) to
enhance the cell death signal, and the pan-caspase inhibitor z-
VAD-fmk to block apoptosis and initiate necroposis.22,23 After
screening ∼200 000 compounds using this cell-based necrop-
tosis assay, we identified a sulfonylaminopyrimidine hit as a
necroptosis inhibitor with an IC50 of less than 1 μM. Shortly
after structure−activity relationship (SAR) study, we discov-
ered a more potent derivative, with an IC50 of 124 nM, which
we named as necrosulfonamide. Subsequent cell-based bio-
logical evaluations of how necrosulfonamide inhibits necrop-
tosis were performed, and the results showed that the
formation of RIPK3 punctae in the necrosulfonamide treated
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Figure 2. Necrosulfonamide targets MLKL for antinecroptosis activity. (A) Live cell imaging upon the treatment of necrosulfonamide (NSA) and
necrostatin-1 (Nec-1). Expressing the mCherry-RIPK3 fusion protein in HeLa cells rendered them necrosis responsive. T, TNF-α; S, Smac
mimetic; Z, z-VAD-fmk. (B) Pull-down of RIPK3 in necrosulfonamide treated cells revealed the interacting protein MLKL. (C) Structure of the
chemical probe for target identification. (D) Graphical abstract of the mode of action on how necrosulfonamide blocks necroptosis.

cells was significantly different from the one of Nec-1 treated
cells. These results suggested that the cellular target(s) and
mode of action for necrosulfonamide might be different from
Nec-1 (Figure 2A).24
We then conducted co-immunoprecipitation experiments
with necrosulfonamide to examine the possible RIPK3-
interacting proteins. Indeed, pull down experiments and
subsequent mass spectrometry analysis revealed that the
mixed lineage kinase-domain like protein (MLKL) is an
interacting partner of RIPK3 (Figure 2B). MLKL was later
confirmed to be a crucial new component of necrosome, the
RIPK1/RIPK3-containing complex that promotes necroptosis.
The inhibition of necroptosis mediated by siRNA knockdown
of MLKL further demonstrated that MLKL is indispensable for
the necroptosis pathway. Therefore, our search for RIPK3
downstream inhibitors and the subsequent discovery of
necrosulfonamide have revealed that MLKL as one of the
central and essential components in the necroptosis process
(Figure 2D).24
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After the functional study of MLKL, we then proposed a
hypothesis that MLKL might be the direct cellular target of
necrosulfonamide. Therefore, we designed and synthesized a
necrosulfonamide probe for the pull-down assay. From our
SAR studies, we have gained crucial information about the
heterocyclic moiety of necrosulfonamide could tolerate
modifications without compromising its biological activity.
Then a small molecule probe was prepared which contained a
biotin tag, and a rigid polyproline linker between biotin and
necrosulfonamide to enhance the binding activity of the probe
to the potential target proteins (Figure 2C). Upon the
treatment of RIP3-overexpressing HeLa cell lysates with this
probe, a subsequent pull-down assay by streptavidin yielded
endogenous MLKL, which could be competed off by the
addition of excess necrosulfonamide as a binding competitor.
Mutagenesis experiments also revealed that the C86S mutant
of MLKL still mediated necroptosis despite the addition of
necrosulfonamide, suggesting that the Cys86 residue of MLKL
is essential for the binding of necrosulfonamide. Moreover,
previous SAR studies of necrosulfonamide suggested that the
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Figure 3. Bioymifi is a selective death receptor 5 (DR5) agonist to induce extrinsic apoptosis. (A) Structures of lead compounds A1, A2, C2, and
bioymifi. (B) RNA inferencing indicated that A2C2 (a 1:9 mixture of compounds A2 and C2) and bioymifi selectively target DR5. Error bars
represent the SD of experimental duplicates. (C) Bioymifi induced FLAG-labeled DR5 aggregation (red color). (D) Graphical abstract of the
mechanism on how bioymifi induces extrinsic apoptosis of cancer cells by selectively activating DR5. B, bioymifi; DISC, death-inducing signaling
complex; FADD, Fas-associated with death domain protein; TRADD, TNF receptor-associated death domain.

α,β-unsaturated amide group might act as a Michael acceptor
that covalently attaches to MLKL. Collectively, these data
revealed that MLKL plays a significant role in initiating
necroptosis and necrosulfonamide is a potent and selective
small molecule inhibitor of necroptosis by covalently targeting
MLKL.24
was not induced when cells were treated with either A2 or C2
alone. Our attention then turned to de novo syntheses of many
other analogues to further explore the SAR. Ultimately, we
developed a bromine-substituted thiazolidin-4-one derivative,
namely, bioymifi, which showed an improved activity with an
EC50 at 2 μM for inducing apoptosis (Figure 3A).27

■ DISCOVERY OF THE FIRST DR5 AGONIST

BIOYMIFI AS A POTENTIAL ANTICANCER AGENT
Because selective activation of specific apoptotic pathway has
been proven to be an effective anticancer strategy, we were
interested in searching for a compound to activate extrinsic
apoptosis, which is an apoptotic pathway initiated by death
receptors. 25,26 By screening over 200 000 compounds
combined with Smac mimetic in T98G cells, we discovered
an apoptosis-stimulating hit called A1 (Figure 3A). However,
our synthetic A1 was unfortunately not biologically active.
After an extensive investigation, we revealed that the structure
of “A1” compound from the commercial chemical library was
wrongly assigned. The real composition of “A1” was a 1:9
mixture of A2 and C2 (Figure 3A). To our surprise, apoptosis
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We then determined which apoptotic pathway was targeted
by bioymifi.24 Our initial study showed that the pan-caspase
inhibitor z-VAD-FMK rescued bioymifi-treated cells; therefore,
we proposed that a caspase-dependent pathway would be
involved in apoptosis. We excluded the intrinsic apoptotic
pathway, as RNAi targeting caspase-9 did not improve cell
survival after treatment with bioymifi. Moreover, biochemical
studies showed that caspase-3, caspase-8, and PARP are
cleaved consecutively after the treatment of bioymifi,
suggesting bioymifi kills cancer cells via the extrinsic apoptotic
pathway. We then hypothesized the target of bioymifi could be
a death receptor. After screening all known members of death
receptors by RNAi, we found that death receptor 5 (DR5) is a
possible cellular target of bioymifi (Figure 3B). Further
isothermal titration calorimetry (ITC) experiments illustrated
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Figure 4. Collective and biomimetic syntheses of sesquiterpene lactone oligomers.
that bioymifi reversibly binds to the ECD domain of DR5 with
a Kd of 1.2 μM similar to its LC50 to cancer cells. From our
studies, DR5 was found to be a functional cellular target of
bioymifi to inducing extrinsic apoptosis.27
Since death receptors have been shown to oligomerize to
activate the downstream apoptosis executors by the activation
of TNFα,28 we wondered if bioymifi triggers a similar
apoptosis-inducing mechanism. Therefore, full-length Flag-
tagged DR5 from cells was treated with bioymifi, assayed with
semi-denaturing detergent agarose gel electrophoresis (SDD-
AGE), and followed by immunoblot analysis. The high
molecule weight of DR5-Flag polymers indicated the
aggregation of DR5 was induced by bioymifi or A2C2. In
addition, aggregation of the purified DR5-ECD was observed
after the treatment with bioymifi or A2C2, which concluded
that ECD is required for aggregation. Subsequent immuno-
fluorescence studies on DR5-overexpressed HCC115 tumor
cells treated with bioymifi supported that bioymifi induces
oligomerization of DR5 on the cellular level (Figure 3C). In
conclusion, we have developed bioymifi as the first small
molecule agonist for DR5 to trigger extrinsic apoptosis of
cancer cells (Figure 3D).27
■
COLLECTIVE AND BIOMIMETIC SYNTHESES OF
SESQUITERPENOID NATURAL PRODUCTS
A family of sesquiterpenoid oligomer natural products were
isolated from Gochnatia and Ainslia plants. They have shown
potent biological activity to induce apoptosis of various tumor
cells, but the modes of action were unknown (Figure 4).29−32
We aimed to develop an efficient approach to access these
complex natural products to facilitate the subsequent mode of
action studies. Retrosynthetic analysis suggested that ainslia-
dimer A might be derived from two molecules of guaianolide-
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type sesquiterpenoid dehydrozaluzanin C. Starting from the
commercially available santonin, dehydrozaluzanin C was
synthesized in 12 steps. After screening conditions for the
hetero-Diels−Alder dimerization of dehydrozaluzanin C,
BINOL was found to be the optimal catalyst in promoting
the biomimetic dimerization. Hydrolysis of the alkenyl ether of
the dimeric product, followed by cyclization via an intra-
molecular aldol reaction furnished the total synthesis of
ainsliadimer A.33
We then extended the synthesis and demonstrated that the
similar biomimetic strategy could be applied for the syntheses
of other sesquiterpenoid oligomers, including ainsliatrimers A
and B, gochnatiolides A−C, and ainsliadimer B (Figure 4).34,35
The collective total syntheses of gochnatiolides A−C and
ainsliadimer B were achieved in two steps and three steps,
respectively, from dehydrozaluzanin C used as the common
precursor. The biomimetic Diels−Alder reaction was applied
as a key step under our optimized conditions to construct the
polycyclic ring systems. The sesquiterpenoid trimers, ainslia-
trimers A and B, were constructed in four steps via two
sequential biomimetic Diels−Alder reactions from dehydroza-
luzanin C. The first enantioselective syntheses also allowed the
unambiguous confirmation of the chemical structure and
absolute and relative configurations of these sesquiterpenoid
oligomers such as gochnatiolide B and ainsliatrimers A and B.
More importantly, this biomimetic strategy has accelerated the
development of effective small molecule probes for further
target identification of ainsliadimer A and ainsliatrimer A.33−36
■
MODE OF ACTION STUDY FOR AINSLIADIMER A
The biomimetic synthesis of ainsliadimer A has laid the
foundation for our mechanistic study of the apoptosis-inducing
activity of this natural product.33 First, we investigated which
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Figure 5. Ainsliadimer A covalently targets IKKβ to activate the NF-κB pathway and trigger apoptosis. (A) Structures of ainsliadimer A derived
positive probe and negative probe. (B) Pull-down assays by ainsliadimer A probes indicated the binding of IKKβ and IKKα. (C) Site-directed
mutagenesis revealed that Cys46 is the binding site. (D) Graphical abstract of the mode of action for ainsliadimer A.

apoptotic pathways were triggered by ainsliadimer A. We
found that the pan-caspase inhibitor z-VAD-FMK can rescue
BCG-823 cells treated with ainsliadimer A, indicating the
apoptosis pathway is involved. In addition, through real-time
PCR analysis, we found that multiple apoptosis inhibitory
proteins including Bcl-xl, c-IAP, and FLIP were globally
downregulated, suggesting the regulatory proteins correspond-
ing to their transcription factor pathway could be targeted.37
Subsequent investigation of the components in the nuclear
factor-κB (NF-κB) signaling pathway showed that ainsliadimer
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A inhibits the phosphorylation of IκB to block the NF-κB
pathway, which shares a comparable mechanism to the known
IκB kinase (IKK) inhibitor BMS-345541 in both LPS and
poly(I:C) induced models, suggesting ainsliadimer A might
directly block the NF-κB signaling pathway.37
We further designed and synthesized an ainsliadimer A-
derived chemical probe based on our SAR studies. This probe
contains an ainsliadimer A warhead, a PEG linker, and a biotin
tag. Given the fact that saturation of the α,β unsaturated
lactones led to the loss of function for ainsliadimer A, we also
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constructed a negative control (NC) probe through selective
reduction of the two conjugated alkene moieties for pull-down
assay (Figure 5A). We then used these two probes in the pull-
down experiments with cell lysates which were previously
activated by TNFα treatments. Two specific protein bands,
IKKα and IKKβ, were identified by mass spectrometry analysis
(Figure 5B). To reveal the binding site of ainsliadimer A (1),
we individually mutated the nine conserved cysteine residues
of IKKβ into alanine residues (Figure 5C). Among these
mutants, C46A was the sole mutant that lost the interaction
with ainsliadimer A (1). Further LC-MS/MS analysis also
confirmed that Cys46 residue of IKKβ is covalently attached
by ainsliadimer A.37
Taken together, we have elucidated the detailed mode of
action for ainsliadimer A. This remarkable natural product
selectively and covalently targets IKKβ to activate the NF-κB
pathway and trigger apoptosis of cancer cells.37
■
TQ-LIGATION FACILITATES NATURAL PRODUCT
TARGET IDENTIFICATION
In order to facilitate natural product target identification, we
have developed an efficient and robust bioorthogonal ligation
(TQ-ligation) by “click” hetero-Diels−Alder cycloaddition of
o-quinolinone quinone methide and vinyl thioether under
physiological conditions (Figure 6A).38 The compatibility of
Figure 6. New bioorthogonal ligation (TQ-ligation). (A) First
generation TQ-ligation. (B) Second generation TQ-ligation. (C)
Target identification of natural products using TQ-ligation.
TQ-ligation has been demonstrated in the imaging of taxol
derivative in live cells.38 The second generation TQ-ligation
was subsequently developed to improve the kinetic rate up to
18-fold compared to the prototype (Figure 6B).39 Moreover,
the second generation TQ ligation is mutually orthogonal to
the traditional strain-promoted azide−alkyne cyclization
(SPAAC) both in vitro and in vivo.39,40 We also found that
the second generation TQ ligation has a comparable rate
constant to that of the widely used SPAAC (k2 = 7.6 × 10−2
M−1 s−1 in CH3CN).39 We further demonstrated that the TQ-
ligation is an effective means to facilitate our target
identifications of the apoptosis inducers such as ainsliatrimer
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A and kongensin A (Figure 6C).41,42 We will discuss these
endeavors in details in the following sections.
■
AINSLIATRIMER A INDUCES APOPTOSIS BY
ACTIVATING PPARγ
In the mode of action study of the apoptosis inducer
ainsliatrimer A,41 we first synthesized a bioactive natural
product probe in which a vinyl thioether was attached onto the
3″-O position of ainsliatrimer A (TV-ainsliatrimer A) (Figure
7A). The HeLa cells were treated with TV-ainsliatrimer A,
followed by incubation with fluorescein-modified oQQM
precursor which would undergo “click” hetero-Diels−Alder
reaction with the vinyl thioether of TV-ainsliatrimer A,
resulting a distribution of the fluorescent small molecule
probes in cells. Pretarget imaging showed that fluorescence was
visualized in the nucleus exclusively, suggesting the cellular
targets of ainsliatrimer A should be located in the nucleus
(Figure 7A, B). Next, we investigated which nuclear protein(s)
might bind to ainsliatrimer A. We therefore synthesized the
biotinylated ainsliatrimer A probe using the TQ-ligation
strategy as well as the negative control probe from the
saturated TV-ainsliatrimer A derivative. Treatment of HeLa
cell lysates with these two probes, followed by pull-down assay
using the extracted cell nuclear proteins and LC-MS/MS
analysis, revealed that histone deacetylase 2 (HDAC2) and
peroxisome proliferator activated receptor γ (PPARγ) as two
candidate cellular targets (Figure 7D). siRNA knockdown at
the protein level demonstrated that PPARγ knockdown could
rescue cells treated with ainsliatrimer A while HDAC2
knockdown could not. Further validation experiments
elucidated that ainsliatrimer A is a selective PPARγ agonist
to trigger apoptosis.41 Along with our discovery, other
independent studies also demonstrated that the activation of
PPARγ could induce apoptosis.43,44 Therefore, the PPARγ
agonists such as ainsliatrimer A could serve as a promising lead
for anticancer therapy.
■ KONGENSIN A IS AN EFFECTIVE INHIBITOR OF
NECROPTOSIS BY TARGETING HSP90
To continue to search for novel small molecules modulating
programmed cell death such as necroptosis, we screened our
in-house ∼300 000 small molecule library and discovered
kongensin A (KA), a diterpenoid natural product isolated from
Croton kongensis as a hit to block necroptosis.45 Initial
validation assays showed that KA blocks necroptosis efficiently
at 2 μM. To identify the cellular target of kongensin A, we first
prepared a positive probe by attaching vinyl thioether onto KA,
and a negative control probe from KA derivative (Figure 8A).
HT-29 cells were incubated with the KA probe and NC probe,
followed by the treatment of biotin-modified oQQM precursor
for TQ-ligation. Pull-down assay followed by LC-MS/MS
analysis revealed that one of the cellular targets for KA is heat
shock protein 90 (HSP90). In addition, Cys420 residue was
confirmed to be the binding site by LC-MS/MS analysis and
site-directed mutagenesis (Figure 8B−D).42
Moreover, coimmunoprecipitation of HSP90 in kongensin
A-treated HeLa lysates revealed that KA inhibits the protein−
protein interaction between HSP90 and its co-chaperone cell
division cycle protein 37 (CDC37), a protein which can
stabilize multiple kinases such as CDK4.46 In addition, we
further demonstrated that the HSP90−CDC37 complex is
crucial for RIPK3-dependent necroptosis.47 In summary, we
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Figure 7. TQ-ligation facilitated the elucidation of mode of action for ainsliatrimer A. (A) Designed ainsliatrimer A TV-probe and fluorescein-
oQQM for imaging cellular distribution of ainsliatrimer A. (B) Fluorescent microscopy indicates nuclear localization of ainsliatrimer A. (C)
Structures of ainsliatrimer A probe and negative control probe. (D) Pull-down assays by ainsliatrimer A probe revealed potential binding proteins.

have elucidated the mode of action of KA: the covalent
interaction of KA to Cys420 of HSP90 disrupts the formation
of the HSP90−CDC37 complex and subsequently inhibits
RIPK3-dependent necroptosis.42 This study provided a new
means to develop a HSP90 inhibitor for the treatment of
necroptosis-related diseases such as inflammation and
atherosclerosis.48
immune disorders, which are caused by dysfunction of the
regulated cell death machinery.
Forward chemical genetics has been proven to be a very
powerful approach to the discovery of new targets. However,
the most significant challenge associated with this approach is
identification of the functional targets of small-molecule
probes. The great potential of chemical genetics cannot be

■
CONCLUSION
Our laboratory conducts research at the interface between
chemistry and biology. We systematically use bioactive small
molecules including complex natural products to dissect
fundamental biological process and to develop novel
therapeutic agents for the currently intractable human diseases.
Over the past decade, we have extensively utilized various small
molecule probes to dissect the extrinsic programmed cell death
pathways, including both apoptosis and programmed necrosis
(necroptosis), and illuminated a number of new molecular
mechanisms underlying these fundamental cellular processes.
These discoveries not only significantly enriched our current
understanding of programmed cell death mechanisms, but also
led to the discovery of a number of potential drug leads for the
treatment of cancers, ischemia-reperfusion injury, and auto-
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realized without the establishment of efficient means for target
identification. In this regard, synthetic chemistry plays an
important role in the development of effective chemical
probes, especially for the complex natural product derived
probes. Many issues such as thorough SAR studies, the choice
of labeling, and target ID methods should be carefully
considered for the chemical probe design and development.
Several leading chemical biologists have recently published a
commentary describing the key properties of successful
chemical probes.49 These include high potency, good
selectivity, and the availability of appropriate control
compounds. In our studies, we have inevitably applied many
different approaches, including direct labeling with biotin tags,
utilization of bioorthogonal ligation methods, or application of
pretarget imaging. In our opinion, no unified method could be
successfully applied in all cases. Extensive evaluations of
different approaches should be performed in order to obtain
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Figure 8. Kongensin A blocks necroptosis by inhibiting the protein−protein interaction of HSP90 and its co-chaperone CDC37. (A) Structures of
kongensin A and kongensin A thiovinyl ether probe. (B) Target identification revealed covalent binding of kongensin A on HSP90. (C) List of
possible interacting proteins for kongensin A. (D) Site-directed mutagenesis indicated the binding site of Hsp90 is C420. (E) Graphical abstract of
the mode of action of kongensin A.

the most conclusive results. Overall, our work to date provides In conclusion, we can certainly envision that the combined
a model workflow for the transformation of bioactive small efforts of function-oriented synthesis and forward chemical
molecules including complex natural products into effective genetics will continue to uncover the unknown molecular
chemical probes and the identification of their functional mechanisms of fundamental cellular process such as pro-
protein targets. grammed cell death to advance our understanding of human
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biology and provide new meaningful approaches to combat
human diseases.
■ AUTHOR INFORMATION
Corresponding Author
Xiaoguang Lei − Beijing National Laboratory for Molecular
Sciences, Key Laboratory of Bioorganic Chemistry and
Molecular Engineering of Ministry of Education, Synthetic and
Functional Biomolecules Center, College of Chemistry and
Molecular Engineering and Peking-Tsinghua Center for Life
Science, Peking University, Beijing 100871, China;
orcid.org/0000-0002-0380-8035; Email: xglei@
pku.edu.cn
Authors
Yingjie Bai − Beijing National Laboratory for Molecular
Sciences, Key Laboratory of Bioorganic Chemistry and
Molecular Engineering of Ministry of Education, Synthetic and
Functional Biomolecules Center, College of Chemistry and
Molecular Engineering, Peking University, Beijing 100871,
China
Hiu C. Lam − Beijing National Laboratory for Molecular
Sciences, Key Laboratory of Bioorganic Chemistry and
Molecular Engineering of Ministry of Education, Synthetic and
Functional Biomolecules Center, College of Chemistry and
Molecular Engineering, Peking University, Beijing 100871,
China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.accounts.9b00600
Notes
The authors declare no competing financial interest.
Biographies
Yingjie Bai received his B.Sc. degree at Peking University in 2017. He
is currently conducting his Ph.D. research under the supervision of
Prof. Xiaoguang Lei at Peking University to elucidate biological
functions of human endogenous lipids.
Hiu Chun Lam was born and raised in Hong Kong. In 2013, he
received his Bachelor degree in Chemistry with 1st class Honours at
the University of Adelaide, Australia. In 2018, Hiu completed his
Ph.D. in Chemistry under the supervision of Associate Professor
Jonathan George at the University of Adelaide. Hiu is currently a
postdoctoral fellow in Prof. Xiaoguang Lei’s group at Peking
University. His research interest focuses on the total synthesis and
mode of action study of complex natural products.
Xiaoguang Lei was born and raised in Beijing, China. He received
BSc degree in chemistry at Peking University in 2001 and then
received his Ph.D. in 2006 under the supervision of Prof. John. A.
Porco at Boston University. After 2 years as a postdoctoral fellow in
Prof. Samuel J. Danishefsky’s laboratory at Columbia University, Prof.
Lei joined the faculty at the National Institute of Biological Sciences
(NIBS) in Beijing as a Principal Investigator and Director of
Chemistry Center. In 2014, Prof. Lei moved to Peking University and
joined the faculty in the College of Chemistry and Molecular
Engineering. He is currently a Full Professor of Chemistry and
Chemical Biology at Peking University. His research program focuses
on natural product synthesis, chemical biology, synthetic biology, and
drug discovery.
J https://dx.doi.org/10.1021/acs.accounts.9b00600
■
pubs.acs.org/accounts Article
ACKNOWLEDGMENTS
We are grateful to our students, postdocs, and collaborators
who have been involved in these studies over the past decade.
Financial support from the National Natural Science
Foundation of China Grant (21625201, 21961142010,
21661140001, 91853202, and 21521003), the National Key
Research and Development Program of China
(2017YFA0505200), and the Beijing Outstanding Young
Scientist Program (BJJWZYJH01201910001001) is gratefully
acknowledged.
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