Affinity-Driven Covalent Modulator of the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Cascade

Article abstract of DOI:10.1002/anie.201801618

Traditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)-(+)-isochaihulactone (1) exhibited significant inhibition against multiple-drug-resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off-target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,β-unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI-oriented activity-based proteome profiling. By MS/MS and computer-guided molecular biology approaches, an affinity-driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the “ON/OFF” switch of apoptosis through non-canonically nuclear GAPDH translocation, which bypasses the common apoptosis-resistant route of MDR cancers.

Full text of DOI:10.1002/anie.201801618

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201801618
German Edition: DOI: 10.1002/ange.201801618
Medicinal Chemistry
Affinity-Driven Covalent Modulator of the Glyceraldehyde-3-Phos-
phate Dehydrogenase (GAPDH) Cascade
Jeffy Chern, Chun-Ping Lu, Zhanxiong Fang, Ching-Ming Chang, Kuo-Feng Hua, Yi-Ting Chen,
Cheng Yang Ng, Yi-Lin Sophia Chen, Yulin Lam,* and Shih-Hsiung Wu*
Abstract: Traditional medicines provide a fertile ground to
explore potent lead compounds, yet their transformation into
modern drugs is fraught with challenges in deciphering the
target that is mechanistically valid for its biological activity.
Herein we reveal that (Z)-(+)-isochaihulactone (1) exhibited
significant inhibition against multiple-drug-resistant (MDR)
cancer cell lines and mice xenografts. NMR spectroscopy
showed that 1 resisted an off-target thiolate, thus indicating that
1 was a target covalent inhibitor (TCI). By identifying the
pharmacophore of 1 (a,b-unsaturated moiety), a probe derived
from 1 was designed and synthesized for TCI-oriented activity-
based proteome profiling. By MS/MS and computer-guided
molecular biology approaches, an affinity-driven Michael
addition of the noncatalytic C247 residue of GAPDH was
found to control the “ON/OFF” switch of apoptosis through
non-canonically nuclear GAPDH translocation, which
bypasses the common apoptosis-resistant route of MDR
cancers.
Bupleurum scorzonerifolium). Despite being an active inhib-
itor of several model cancer cell lines,[1,2] its drug-efficacy
target remains unclear. Moreover, owing to the low extraction
yields, in previous studies a racemic mixture of (Z)-isochai-
hulactone was used for characterization,[2] leaving the enan-
tioselective cytotoxicity of each enantiomer ambiguous.
Multiple-drug resistance (MDR) in prostate cancer has
become a severe problem owing to limited available targeted
agents. The majority of the resistance is due to resurgence of
the androgen receptor (AR) signaling, which is a primary
culprit for therapeutic failure in incurable prostate cancers
(PCa), especially in castration-resistant PCa (CRPC).[3] First-
line AR-directed therapies (ADTs; e.g., enzalutamide
(Xtandiꢀ) and abiraterone (Zytigaꢀ)) depend on the drugg-
ability of the AR ligand binding domain (LBD); however,
relapses emerge when affinity-related mutations (e.g.,
ART877A in LNCaP) or complete loss of LBD (known as AR
variants; e.g., ARV7 in 22Rv1) occur (models used in this
study are listed in Table 1), which leads to MDR. The
uncontrollable signaling of AR variants results in the
constitutive expression of many anti-apoptosis-associated
proteins, which thereby fuels the creation of a vicious cycle
of MDR to clinical-setting combination therapies.[4–7]
T
raditional medicines are versatile compounds with a variety
of biological effects and a structurally diverse set of scaffolds
for lead discovery. Nonetheless, a lack of knowledge of the
mechanistic targets of a compound often impedes further
structure-based drug design or therapeutic development. In
this study, we aimed to identify the mechanistic target of the
active lignin 1 (a natural product isolated from the root of
Targeted covalent inhibitors [TCIs; Eq. (1)] are a valuable
tool for drug discovery. Compounds containing a weak
electrophilic warhead initially bind to the receptor protein
so that it is positioned to subsequently form a covalent adduct
with a noncatalytic nucleophile, thus providing an extended
duration of action, increased selectivity, and lower off-target
reactivity.[8,9] The lower off-target reactivity of a TCI prevents
[*] J. Chern, S.-H. Wu
Institute of Biological Chemistry, Academia Sinica (Taiwan)
E-mail: Shwu@gate.sinica.edu.tw
J. Chern, C.-M. Chang, S.-H. Wu
Chemical Biology and Molecular Biophysics Program
Taiwan International Graduate Program, Academia Sinica
Taipei (Taiwan)
Table 1: IC50 [mm][a] of 1–4 and derivatives 22a and 22b with the C C
bond reduced in drug-resistant prostate cancers.
=
(Z)-Isochaihulactone
(E)-Anhydropodorizol
J. Chern, Y.-T. Chen, S.-H. Wu
compound
1
2
3
4
Department of Chemistry, National Taiwan University
Taipei (Taiwan)
PC3[b]
1.10Æ0.03
0.96Æ0.02
1.02Æ0.06
7.20Æ0.03
6.50Æ0.02
10.1Æ0.04
13.50Æ0.03
12.50Æ0.03
15.80Æ0.04
8.70Æ0.03
13.80Æ0.06
20.10Æ0.03
LNCaP[c]
22Rv1[d]
C.-P. Lu
Department of Food Science, Fu Jen Catholic University
Taipei (Taiwan)
=
FDA-approved drug
C C reduction
22a 22b
Z. Fang, C. Y. Ng, Y. Lam
Department of Chemistry
National University of Singapore (Singapore)
E-mail: chmlamyl@nus.edu.sg
compound
enzalutamide[e]
LNCaP[c]
22Rv1[d]
6.20Æ0.04
52.58Æ0.05
59.16Æ0.03
85.86Æ0.05
61.43Æ0.05
130.30Æ0.01
K.-F. Hua, Y.-L. S. Chen
Department of Biotechnology and Animal Science
National Ilan University, Ilan (Taiwan)
[a] Values are the mean Æstandard deviation of at least triplicate results.
[b] Human AR-(À) PCa, resistant to all ADTs (bone metastasis).
[c] Human ARFL-(+) PCa, resistant to first-generation ADTs (lymph-node
metastasis). [d] Human ARFL- and ARV7-(+) CRPC, resistant to all ADTs
(from CWR22). [e] Next-generation ADT (Xtandiꢀ): most effective drug
for advanced CRPC treatment.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201801618.
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alkylation-derived drug resistance and detrimental side
effects.[10]
All four stereoisomers 1–4 of isochaihulactone were
obtained by asymmetric synthesis (see Scheme S1 and Fig-
ure S1 in the Supporting Information). With an efficient
strategy for 6b preparation (6b is a solid which could be
readily purified by recrystallization), expedient synthesis of
the compound was achieved in large quantity. To study their
structure–activity relationships (SARs), we evaluated some
MDR prostate cancers in the presence of each isomer
(Table 1). From the cytotoxicity assay, 1 (IC50 ꢀ 1 mm) was
found to be the most potent inhibitor of the four isomers (IC50
ꢀ 6.50–20.10 mm). Each SAR of 1–4 was in agreement with
their corresponding sub-G1 accumulation (see Figure S2a).
The difference in potency between 1 and 2 (6.5–10-fold)
suggested that enantioselective cytotoxicity existed. To dem-
onstrate the cancer selectivity of 1, we examined whether the
effective concentration of 1 would affect normal prostate cells
(PZ-HPV-7). In a trypan blue exclusion assay, PZ-HPV-7 cells
were found to be less susceptible to 1 than 22rv1 cells (73 vs.
44%; see Figure S3a). Notably, some populations within PZ-
HPV-7 even grew in the presence of 1 (see Figure S3b,c).
Meanwhile, to investigate the preclinical potency of 1, nude
mice carrying a tumor (ca. 120 mm3) were subjected to
subcutaneous injection of 1. The data showed that 1 reduced
the tumor volume of mouse LNCaP xenografts by activating
caspase-dependent apoptosis in a caspase-dependent manner
(Figure 1b,c; no histological changes in the brain, liver,
kidney), which was in agreement with cell-based findings
(i.e., PS externalization, caspase-3 activity, Z-VAD-fmk
treatment, and changes in hallmark proteins; see Fig-
ure S2b–e). Collectively, these findings exhibited a promising
safety profile and potency of 1 for application of the
treatment in vivo.
Figure 1. a) Structures of isochaihulactone isomers 1–4, derivatives
=
22a and 22b of 1 with the C C bond reduced, and TCI-based probes 5
and 6. b) LNCaP xenograft mice administered subcutaneous injection
on days 0–4 for 5 days. Data points designate the tumor volume
(mm3) after inoculation at the indicated time. c) Immunohistochemical
staining of activated caspase-3 from tumor tissues (on day 31 after
inoculation). Scale bars: 100 mm.
Compound 1 consists of a a,b-unsaturated moiety in
conjugation with an a-benzylidene moiety substituted with
three electron-donating methoxy groups. On the basis of
previous reports, these substituents would make the Michael
acceptor less electrophilic,[11] which might serve as a good
TCI-like property that we could utilize to facilitate our target
identification. We evaluated the reactivity of 1 to thia-
Michael addition in the presence of 7 mm[12] cysteamine (2-
aminoethanethiol) to mimic a biologically relevant base-
deprotonated thiolate system (Figure 2a).[13,14] The treatment
of 1 with cysteamine did not result in any signal difference in
the NMR spectrum (Figure 2c; 10–48 h), whereas a rapid
Michael addition reaction with benzylidene aldehyde was
observed by monitoring the aldehyde proton signal, which
shifted from dH = 9.69 to 10.21 ppm in a time-dependent
manner (Figure 2b; see also Figure S4). Nevertheless, the
Figure 2. a) Thia-Michael addition by cysteamine. b,c) NMR spectra
after the reaction of benzylidene acetaldehyde (b) or 1 (c) with
cysteamine at the indicated concentrations (+: 7 mm; + + +:
200 mm). Signature 1H NMR shifts are highlighted.
reaction with 1 proceeded when excess cysteamine was added,
which yielded a signal at dH = 7.44 ppm accompanied by the
loss of the aromatic proton signal at dH = 7.36 ppm (Fig-
ure 2c; see also Figure S5). The 13C NMR spectrum of this
product showed a carbonyl signal (dC = 172.2 ppm; see Fig-
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ure S6), whereas mass spectroscopy measurements provided
Subsequently, by using a relative quantitative proteomic
monoisotopic masses are consistent with cysteamine–1 and
cysteine–1 adducts (see Figure S7). These findings confirmed
that the a,b-unsaturated moiety of 1 is essential for reaction
with the sulfhydryl group of the thiol species, even though the
reaction occurs sluggishly. To demonstrate whether the
Michael acceptor of 1 was the prerequisite for potency, we
approach, we calculated the label-free quantification (LFQ)
ratio of streptavidin-enriched 5 to vehicle-labeled proteomes
(LFQ5:vehicle). From LFQ analysis (see Table S1 in the
Supporting Information; LFQ5/LFQvehicle > 20;), after ruling
out mismatched molecular weights, GAPDH, MDH2, and
ETFA were believed to be the potential targets. Validation
through RNA interference and cDNA overexpression of
GAPDH, MDH2, and ETFA revealed that only the knock-
down of GAPDH showed a survival benefit in response to
1 (ca. sixfold; see Figures S3a,d and S8; GAPDH-depleted
cells were continuously cultured in pyruvate-containing
media to avoid toxicity[15]).
=
reduced the C C bond in 1 to give 22a and 22b (Figure 1a)
for cytotoxicity assays. The results showed that both 22a and
22b were significantly less potent than 1 (Table 1). Taken
together, these results indicated that 1 contains an off-target
Michael acceptor as the pharmacophore.
To identify the target of 1, we modified 1 by incorporating
a biorthogonal azido group to facilitate target visualization or
enrichment to give probes 5 and 6 for TCI-oriented activity-
based proteome profiling (TCI-ABPP; see Figure S9a and
Scheme S1b,c). Ideally, these TCI-based probes would bind
to the target, remain for a residence time, and reorient
themselves to a suitable position for Michael addition with
neighboring nucleophilic side chains (usually cysteine). After
in situ labeling, the given proteome should be covalently
modified by the probe to allow subsequent Staudinger
ligation by a phosphine–biotin reporter tag (p-biotin; see
Figure S9a). By streptavidin blot analysis, one clear band
ranging from 35 to 40 kDa was visualized (Figure 3a), while
the labeling efficiency of each probe revealed a high corre-
lation with their corresponding IC50 value (5: 5.12 mm; 6:
12.20 mm). Since positive staining in a streptavidin blot reflects
the number of covalent adducts present, these results again
demonstrated that the formation of a Michael adduct plays
a critical role in the inhibitory activity of 1.
The exact modification site of 1 in GAPDH was identified
by incubating 1 with purified GAPDH at the physiological
pH value. After trypsin digestion, the peptides were analyzed
by MS/MS, which revealed that peptides with monoisotopic
masses that were consistent (< 5 ppm error) with the non-
catalytic C247 modification were observed (Figure 3c). A
parallel experiment performed using wild-type (WT) and
C247A GAPDH for 5-mediated phosphine–Cy5 ligation (p-
Cy5; see Figure S9b) provided the same conclusion that C247
was the only modification site (see Figure S9c). Interestingly,
human GAPDH contains three cysteine residues (Figure 3b),
yet the intramolecularly competing C152 residue, as the most
reactive thiolate in GAPDH, formed no adduct with 1, as
neither enzymatic activity nor a glycolytic network change
was observed (see Figure S10a,b), in close agreement with
thiolate-tolerating nature of 1 (Figure 2c). Hence, based on
the concept of TCI, the affinity for GAPDH should be the key
to lower the reaction barrier to give the final GAPDH–
1 adduct. By isothermal titration calorimetry (ITC), we
determined the affinity of 1 for GAPDHC247A (avoiding bond
formation heat). By ITC, this binding event was characterized
as an entropy-driven reaction with nanomolar affinity (KD =
0.26 mm; see Figure S9d). To date, all covalent inhibitors of
GAPDH, such as koningic acid, 3-bromopyruvate, and
orlistat (see Figure S10c–e),[16–18] rely on the alkylation of
C152 to modulate the function of GAPDH with their highly
reactive electrophiles. Collectively, these results suggested the
Michael adduct of GAPDH–1 was modulated by an affinity-
driven alkylation on C247 to overcome the energy barrier of
the thiolate-tolerating warhead in 1.
GAPDH was initially believed to be solely a glycolytic
enzyme in most cells. However, subsequent studies have
shown that GAPDH participates in many unexpected pro-
cesses, such as vesicle trafficking, cytoskeletal dynamics,
RPL13a protection, telomerase inhibition, and cell
death.[19,20] Based on GAPDH–1 adduct formation, we
covalently docked 1 with C247 of GAPDH. Consistent with
entropy-driven binding (ÀTDS dominance;[21] see Fig-
ure S9e), 1 was buried in a hydrophobic pocket located in
the middle of the a/b two-layer sandwich (Figure 4a), where it
revealed hydrophobic contacts to two functional motifs. For
clarity, we divided the pocket into 1,3-benzodioxole (BzD;
Figure 4b) and 3,4,5-trimethoxybenzyl subpockets (MOBz;
Figure 4c) related to SIAH1-dependent apoptosis (see Fig-
ure S11a) and the cytosol-restricted trans activation of AR
through interaction with a polyQ tract,[22,23] respectively.
Figure 3. a) Streptavidin blot of lysate treated with p-biotin-ligated 1, 5,
or 6, or a vehicle (see Figure S9a for a TCI-ABPP flowchart). a) pKa
values of each cysteine residue in human GAPDH. H179 deprotonates
C152 (blue dashed line; active site). c) Representative MS2 spectrum
of 1-derived peptide; Dm1 +398 Da (+C22H24O7).
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tion was further demonstrated by nuclear fractionation and
confocal imaging. Both results showed that GAPDH entered
the nucleus in response to 1 (Figure 5c; see also Figure S11b),
whereas no entrance was observed when SIAH1-depleted
cells were used (Figure 5d).
Since C152 nitrosylation or other oligomerization of
GAPDH could result in a misinterpretation of our findings,
we verified each circumstance carefully to rule out possible
biases. Based on the experimental results (see Figure S12),
none of the above-mentioned biases were found. Importantly,
when exogenously FLAG-fused GAPDH (WT and C247S)
were used for nuclear fractionation, only C247S interfered
with 1-dependent nuclear GAPDH translocation (Figure 5e).
Likewise, complementation studies of C247S showed a more
significant survival benefit than WT in shGAP-infected 22rv1,
whereas no significant changes in the case of shSIAH1-
infected 22rv1 were observed (Figure 5 f). Taken together,
these results demonstrated the ON/OFF switch of cytotoxicity
through GAPDH–1 adduct formation.
Turning now to the AR-relevant motif near MOBz, helix-
11 interacts with a polyQ tract of AR located in the N-
terminal transactivation domain (NTD; Figure 6a; see also
Figure S13c). To examine whether GAPDH plays a role in
RPL13a-like stabilization (depending on the nitrosylated
state of C247[19]) to increase the half-life of AR (since
GAPDH increases AR transactivation in the cytosol[23]), we
assayed the abundance of AR upon treatment with 1. From
western blots, 1 was found to promote endogenous ARFL and
ARV7 degradation in a dose-dependent manner, whereas the
Figure 4. a) Covalent docking of the GAPDH–1 adduct. The relative
positions of each cysteine are given. Subpockets b) BzD and c) MOBz
occupied by the ligand are indicated. Important interacting motifs (b-
hairpin in BzD and Helix-11 in MOBz) are highlighted.
In the first scenario, SIAH1 with a nuclear localization
signal (NLS), as a “shuttle”, facilitates the translocation of
GAPDH from the cytosol to the nucleus, where GAPDH
initiates apoptosis (see Figure S11a).[24] To validate whether
the cancer-selective cytotox-
icity of 1 was the result of this
pathway, we depleted SIAH1
using
lentivirus-mediated
shRNA (see Figure S3d) to
determine whether it would
prevent 1-mediated cell
death. From cytotoxicity
assays, a significant efficacy
difference
between
the
SIAH1-depleted and non-
depleted cells was found
(Figure 5a). Next, since
hydrophobic contact with
L288 located at the hinge of
the b-11 sheet could cause
binding to the BzD moiety to
result in alteration of the
relative position of the apop-
tosis-relevant motif at K227
of the b-hairpin (Figure 4b),
immunoprecipitation (IP) of
endogenous GAPDH to
monitor its interaction with
SIAH1
was
performed.
Upon treatment with 1,
SIAH1 was found to increase
in a dose-dependent manner
(Figure 5b). The 1-mediated
Figure 5. a) Dose–response curves of control (pLKO)- and shSIA-infected 22rv1. b) Enhanced interaction
between SIAH1 and GAPDH. c,d) Nuclear GAPDH accumulation in 22rv1 (c) or shSIA-infected 22rv1 (d).
e) Illustration and western blot showing ON/OFF switching of nuclear GAPDH accumulation with
exogeneous FLAG-GAPDHs; N.R.: no reaction. f) Complementation studies of 22rv1 with indicated
nuclear GAPDH transloca- genotypic background.
4
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Acknowledgements
RNAi reagents were obtained from RNAi Core Facility, AS,
Taiwan. Grants from the Ministry of Education (Singapore;
MoE Tier 2: R-143-000-478-112) and the Ministry of Science
and Technology (Taiwan; MOST 103-2113M-001-029-MY3
and NSC 103-2923-M-001-006-MY3) are acknowledged. We
thank Dr. Feng-Ling Yang (IBC, AS, Taiwan) for critically
reading the manuscript.
Conflict of interest
The authors declare no conflict of interest.
Figure 6. a) Western blots showing that both WT and C247S GAPDH
bind to AR, and illustration of the hypothesized GAPDH-mediated AR
stabilization. b) ARDLBD degradation in AR-null cells (quantified by
triplicate western blots of ARDLBD in response to 1; see also Fig-
ure S13d). c) Loss of AR binding in ubiquitin-incompetent ARDLBD/K311R
Keywords: asymmetric synthesis ·
castration-resistant prostate cancer · covalent inhibitors ·
enzymes · multiple-drug resistance
.
addition of epoxomicin (EPO, a proteasome inhibitor)
prevented further 1-mediated 26S proteasome degradation
(Figure S13a,b), thus indicating the occurrence of “LBD-
independent” AR degradation (Figure S13c). To understand
whether this effect was due to the GAPDH–1 adduct, we set
up an AR-null model, which expressed AR lacking LBD
(ARDLBD), and coinfected it with WT or C247S GAPDH.
First, there was no observable difference in endogenous ARV7
binding between WT and C247S (Figure 6a). A complemen-
tation study of WT and C247S in 1-mediated V5-ARDLBD
degradation revealed that the degradation rate was slower for
C247S than WT (Figure 6b; see also Figure S13d). Moreover,
to show the unequivocal involvement of GAPDH-1 in
ARDLBD association, we constructed the mutant V5-ARDLBD/
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noncatalytic C247 residue of GAPDH can be selectively
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Manuscript received: February 6, 2018
Accepted manuscript online: April 17, 2018
Version of record online: && &&, &&&&
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ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2018, 57, 1 – 7
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Medicinal Chemistry
Overcoming resistance: A new class of
inhibitors that target the noncatalytic
C247 residue of GAPDH in an affinity-
driven manner to initiate SIAH1-depen-
dent apoptosis and androgen-receptor
degradation has been discovered (see
picture). The affinity-driven Michael
addition of isochaihulactone was found
to control the “ON/OFF” switch of
apoptosis by a mechanism that bypasses
the common apoptosis-resistant route of
multiple-drug-resistant cancers.
J. Chern, C.-P. Lu, Z. Fang, C.-M. Chang,
K.-F. Hua, Y.-T. Chen, C. Y. Ng,
Y.-L. S. Chen, Y. Lam,*
S.-H. Wu*
&&&& — &&&&
Affinity-Driven Covalent Modulator of the
Glyceraldehyde-3-Phosphate
Dehydrogenase (GAPDH) Cascade
Angew. Chem. Int. Ed. 2018, 57, 1 – 7
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!