Identification of Targets of the HIF-1 Inhibitor IDF-11774 Using Alkyne-Conjugated Photoaffinity Probes

Article abstract of DOI:10.1021/acs.bioconjchem.6b00305

We developed a hypoxia-inducible factor-1 (HIF-1) inhibitor, IDF-11774, as a clinical candidate for cancer therapy. To understand the mechanism of action of IDF-11774, we attempted to isolate target proteins of IDF-11774 using bioconjugated probes. Multifunctional chemical probes containing sites for click conjugation and photoaffinity labeling were designed and synthesized. After fluorescence and photoaffinity labeling of proteins, two-dimensional electrophoresis (2DE) was performed to isolate specific molecular targets of IDF-11774. Heat shock protein (HSP) 70 was identified as a target protein of IDF-11774. We revealed that IDF-11774 inhibited HSP70 chaperone activity by binding to its allosteric pocket, rather than the ATP-binding site in its nucleotide-binding domain (NBD). Moreover, IDF-11774 reduced the oxygen consumption rate (OCR) and ATP production, thereby increasing intracellular oxygen tension. This result suggests that the inhibition of HSP70 chaperone activity by IDF-11774 suppresses HIF-1α refolding and stimulates HIF-1α degradation. Taken together, these findings indicate that IDF-11774-derived chemical probes successfully identified IDF-11774's target molecule, HSP70, and elucidated the mode of action of IDF-11774 in inhibiting HSP70 chaperone activity and stimulating HIF-1α degradation in cancer cells.

Full text of DOI:10.1021/acs.bioconjchem.6b00305

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Article
Identification of targets of the HIF-1 inhibitor IDF-11774
using alkyne-conjugated photoaffinity probes
Hyun Seung Ban, Ravi Naik, Hwan Mook Kim, Bo-Kyung Kim, Hongsub Lee, Inhyub Kim, Hee-
Chul Ahn, Yerin Jang, Kyusik Jang, Yumi Eo, Kyung Bin Song, Kyeong Lee, and Misun Won
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00305 • Publication Date (Web): 07 Jul 2016
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Identification of targets of the HIF-1 inhibitor IDF-11774 using alkyne-conjugated photoaffinity
probes
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Hyun Seung Ban1,2‡, Ravi Naik3‡, Hwan Mook Kim4, BoꢀKyung Kim5, Hongsub Lee6, Inhyub Kim5,7,
Heechul Ahn3, Yerin Jang3, Kyusik Jang3, Yumi Eo3, Kyung Bin Song8, Kyeong Lee3*, and Misun
Won5,7*
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1Metabolic Regulation Research Center, KRIBB, Daejeon 305ꢀ806, Korea
2Biomolecular Science, University of Science and Technology, Daejeon 305ꢀ350, Korea
3College of Pharmacy, Dongguk UniversityꢀSeoul, Goyang 410ꢀ820, Korea
4Gachon University, College of Pharmacy, Incheon 406ꢀ840, Korea
5Personalized Genomic Medicine Research Center, KRIBB, Daejeon 305ꢀ806, Korea
6ILDONG Pharmaceutical Co. Ltd., Hwaseong, Kyungiꢀdo 445ꢀ811, Korea
7Functional Genomics, University of Science and Technology, Daejeon 305ꢀ350, Korea
8Department of Food Science and Technology, Chungnam National University, Daejeon 305ꢀ764,
Korea
Correspondence to:
Misun Won, Personalized Genomic Medicine Research Center, KRIBB, 125 Gwahangro, Ounꢀdong,
Yusongꢀgu, Daejeon 305ꢀ806; Korea, Tel: 82ꢀ42ꢀ860ꢀ4178; e-mail: misun@kribb.re.kr
Kyeong Lee, College of Pharmacy, Dongguk UniversityꢀSeoul, 32 Donggukro, Ilsandongꢀgu, Goyang
410ꢀ820, Korea, Tel: 82ꢀ31ꢀ961ꢀ5214; e-mail: kaylee@dongguk.edu.
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ABSTRACT: We developed a hypoxiaꢀinducible factorꢀ1 (HIFꢀ1) inhibitor, IDFꢀ11774, as a clinical
candidate for cancer therapy. To understand the mechanism of action of IDFꢀ11774, we attempted to
isolate target proteins of IDFꢀ11774 using bioconjugated probes. Multifunctional chemical probes
containing sites for click conjugation and photoaffinity labeling were designed and synthesized. After
fluorescence and photoaffinity labeling of proteins, twoꢀdimensional electrophoresis (2DE) was
performed to isolate specific molecular targets of IDFꢀ11774. Heat shock protein (HSP) 70 was
identified as a target protein of IDFꢀ11774. We revealed that IDFꢀ11774 inhibited HSP70 chaperone
activity by binding to its allosteric pocket, rather than the ATPꢀbinding site in its nucleotideꢀbinding
domain (NBD). Moreover, IDFꢀ11774 reduced the oxygen consumption rate (OCR) and ATP
production, thereby increasing intracellular oxygen tension. This result suggests that the inhibition of
HSP70 chaperone activity by IDFꢀ11774 suppresses HIFꢀ1α refolding and stimulates HIFꢀ1α
degradation. Taken together, these findings indicate that IDFꢀ11774ꢀderived chemical probes
successfully identified IDFꢀ11774’s target molecule, HSP70, and elucidated the mode of action of
IDFꢀ11774 in inhibiting HSP70 chaperone activity and stimulating HIFꢀ1α degradation in cancer cells.
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■ INTRODUCTION
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Hypoxiaꢀinducible factorꢀ1 (HIFꢀ1) is associated with poor cancer prognosis and resistance to
various therapies. Under normoxia, HIFꢀ1α is rapidly degraded by the VHLꢀmediated ubiquitinꢀ
proteasomal degradation pathway. However, under hypoxic conditions, HIFꢀ1α translocates to the
nucleus and dimerizes with the HIFꢀ1β subunit.1, 2 The HIFꢀ1α/β dimer binds to hypoxiaꢀresponse
elements (HRE) of the sequence 5’ꢀRCGTGꢀ3’ (where R is A or G) in the promoter and activates the
transcription of target genes involved in angiogenesis, glycolysis, metastasis, proliferation, and
resistance to apoptosis.3 Importantly, HIFꢀ1 promotes angiogenesis via vascular endothelial growth
factor (VEGF), thereby regulating tumor growth and metastasis.4, 5 Moreover, HIFꢀ1 reprograms cell
metabolism by coordinating the transcription of its target genes, such as glucose transporter (GLUT),
hexokinase, pyruvate kinase M2, lactate dehydrogenase A, and pyruvate dehydrogenase kinase.6
Therefore, the inhibitors of HIFꢀ1 or metabolic enzymes may impair the metabolic adaptability of
cancer cells, making them more sensitive to anticancer therapy.7
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A growing number of HIFꢀ1 inhibitors have been reported.8 Candidate HIFꢀ1 inhibitors include
mTOR C1/C2 inhibitors, HIFꢀ1 degradation inhibitors, and topoisomerase inhibitors. Clinical studies
of the potential candidates PXꢀ478 (Phase I) and BAYꢀ87ꢀ2243 (Phase I) have been performed.9, 10
HIF inhibitory mechanism has been understood that PXꢀ478 inhibited HIFꢀ1α translation and BAYꢀ
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87ꢀ2243 inhibited mitochondrial oxygen consumption.10,
Some topoisomerase inhibitors are
currently in clinical studies for use in various combination therapeutics, and other small molecules are
still in the discovery stage.12 As part of the ongoing effort to develop HIF inhibitors, we have
developed IDFꢀ11774, an orally administered HIFꢀ1 inhibitor derived from the aryloxyacetylamino
benzoic acid scaffold.13ꢀ15 Recently, IDFꢀ11774 was approved as a clinical candidate for a phase I
study of cancer therapy by the Korean Food and Drug Administration (KFDA).16
The identification of molecular targets of drugs is a bottleneck in phenotypeꢀbased drug discovery,
and various methodologies have been developed. Currently, biochemical methods, such as affinity
chromatography and drug affinity responsive target stability (DART), genetic interactions using
model organisms, and chemical biology approaches using activityꢀbased probes (ABP) are being
exploited.17 Recently, other chemical biology techniques, such as photoaffinity labeling, biotinylation,
and click conjugation, have been considered very useful and reliable tools for detecting biological
target proteins.18ꢀ20 In this study, we report the target and mechanism of action of IDFꢀ11774 using
chemical probes.
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■ RESULTS AND DISCUSSION
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Generation of chemical probes for target identification of IDF-11774. Adamantyl
phenoxyacetic acid is a unique component of bioactive chemicals with a wide range of activities.13, 15,
21ꢀ23 We constructed focused chemical library using this scaffold, and compound IDFꢀ11774 exhibited
the most clear activity and most desirable profile (Figure 1).16
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IDFꢀ11774 reduced the HREꢀluciferase activity of HIFꢀ1α (IC50=3.65 µM) and blocked HIFꢀ1α
accumulation under hypoxia in HCT116 human colon cancer cells (Figures S1A and B). In addition,
we found that IDFꢀ11774 suppressed the mRNA expression of HIFꢀ1 target genes VEGF and EPO but
not HIFꢀ1α (Figure S1C).
Figure 1. Target identification scheme of IDFꢀ11774 using chemical probes.
To understand the mechanism of action of IDFꢀ11774 in inhibiting HIFꢀ1α accumulation, we
investigated its molecular targets using activityꢀbased probes. We designed and synthesized IDFꢀ
11774ꢀderived multifunctional chemical probes (Figure 1). These multifunctional probes contained
two functional moieties: trifluoromethyl diazirine and acetylene. Trifluoromethyl diazirine, a
precursor of carbene, has valuable properties, such as photoactivity at a long wavelength, good
chemical stability, and rapid photolysis.24 The acetylene click reaction for the conjugation of azideꢀ
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linked fluorescent dyes is highly specific and bioorthogonal.19 The synthesis of IDFꢀ11774 and its
chemical probes 1-4 is shown in Schemes S1ꢀS3.
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A structureꢀactivity relationship (SAR) study suggested that adamantane constitutes a major part
of the pharmacophore for HIFꢀ1 inhibition and that the Nꢀmethyl piperazine moiety is crucial for
solubility and activity.25 Therefore, we used chemical probes with or without the adamantane group as
a control and clickable acetylene group at the piperazine end (Figure 2A). The results showed that the
adamantylꢀcontaining probe 2 retained the inhibitory effect on hypoxiaꢀinduced accumulation and
transcriptional activation of HIFꢀ1α, whereas the adamantylꢀfree probe 1 did not (Figures 2A, 2B and
S2A).
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Using these chemical probes, we then performed lightꢀinduced covalent photocrosslinking and
click conjugation of the fluorescent dyes Cy5 and Cy3 to probes 1 and 2, respectively, in HCT116
cells. Next, the proteins conjugated to each probe were separated using SDS–PAGE, and a protein of
approximately 70 kDa specifically bound to probe 2 was visualized using inꢀgel fluorescence
scanning (Figure S2B). We then performed twoꢀdimensional electrophoresis (2DE) to select the
protein that binds specifically to chemical probe 2 and not to probe 1. When the proteins conjugated to
probe 1ꢀCy5 or probe 2ꢀCy3 were separated, a green fluorescent spot of approximately 70 kDa was
determined to be specific to probe 2 (Figure 2C). After inꢀgel digestion, mass spectrometry analysis
revealed that the protein bound to probe 2 was heat shock protein (HSP) 70 (Table S1).
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Figure 2. Identification of target proteins of IDFꢀ11774. (A) Chemical structures of probes and
their HIF inhibitory activity. (B) Effects of chemical probes on HIFꢀ1α accumulation. (C) Imaging of
the proteinꢀconjugated probe in 2ꢀDE. Photoaffinityꢀlabeled proteins conjugated with a fluorescent
dye (controlꢀCy5 and IDFꢀ11774 probeꢀCy3) were separated using 2ꢀDE and visualized using inꢀgel
fluorescence scanning.
Binding of the recombinant HSP70 protein to biotin-conjugated IDF-11774 probes. To
determine whether IDFꢀ11774 binds to HSP70, an affinity pullꢀdown assay was performed using
biotinylated probes (Figure 3A). The adamantylꢀfree, biotinylated probe of IDFꢀ11774 (probe 3) was
used as a negative control. As shown in Figure 3B, binding of HSP70 to the biotinylated IDFꢀ11774
probe (probe 4) was detected. In the competition assay, binding of HSP70 with probe 4 was almost
abolished in the presence of excess IDFꢀ11774 (Figure 3C), implicating the interaction of probe 4
with HSP70.
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Figure 3. Binding of chemical probes to recombinant HSP70. (A) Chemical structures of
biotinylated probes. (B) Binding of probe 4 to HSP70. A pullꢀdown assay of cell lysates was
performed using probe 4 (5 ꢀM) and streptavidin resin. (C) Competitive binding of probe 4 and IDFꢀ
11774 (50 ꢀM) to HSP70.
IDF-11774 binds to the allosteric domain of HSP70 NBD. Next, we investigated the binding
site of IDFꢀ11774 on HSP70, which consists of the NBD and SBD. To evaluate the direct binding of
IDFꢀ11774 to HSP70, recombinant HSP70 proteins were prepared as shown in Figures 4A and S3. It
was revealed that probe 2, not probe 1, directly bound to fullꢀlength HSP70 (HSP70ꢀFL) in an in vitro
binding assay using photoaffinity labeling and click conjugation of Cy3. In addition, the binding of
probe 2 to HSP70ꢀFL was almost abrogated in the presence of IDFꢀ11774 as a competitor (Figure 4B).
In the domainꢀbinding assay, probe 2 selectively bound to the HSP70ꢀNBD (Figure 4C), and the
binding was attenuated by addition of IDFꢀ11774 (Figure S4). These results suggest that probe 2
directly binds to the NBD and not the SBD of HSP70.
To understand the possible binding modes of IDFꢀ11774 in the NBD region, a docking study of
the crystal structure of HSP70 (PDB 1S3X) was performed. As expected, a known HSP70 ATPase
inhibitor, VERꢀ155008 bound to the ATPꢀbinding pocket. Interestingly, IDFꢀ11774 was found to bind
to an allosteric pocket according to our computational analysis (Figures 4D and 4E).26 In this
allosteric pocket, IDFꢀ11774 has hydrophobic interactions with Trp90, Phe68, and Val59, whereas the
oxygen of the carbonyl group interacts with Arg261 by hydrogen bonding. The adamantyl ring moiety
of IDFꢀ11774 is neatly located within the hydrophobic area of the NBD allosteric pocket, and
piperazine fits well in the hydrophilic area.
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Figure 4. Binding model of IDFꢀ11774 to HSP70. (A) Binding of IDFꢀ11774 probes to
recombinant HSP70. FL: full length, NBD: nucleotideꢀbinding domain, SBD: substrateꢀbinding
domain. (B) Binding of probes to recombinant HSP70 (FL) and a competition assay in the presence of
IDFꢀ11774 was performed. (C) Binding of probes to the NBD region of HSP70. (D) Docking model
of IDFꢀ11774. IDFꢀ11774 sits within the allosteric site of HSP70 NBD (magenta), whereas VERꢀ
155008 sits within the ATPꢀbinding site of HSP70 NBD (green). (E) Closeꢀup view of IDFꢀ11774ꢀ
HSP70. The NBD region of HSP70 is displayed in pale gray or as colored ribbons.
Inhibition of the chaperone activity of HSP70 by IDF-11774. The molecular chaperone HSP70,
facilitates protein folding to prevent protein aggregation.27 During HSP70 activation, ATP hydrolysis
at the NBD of HSP70 stimulates the binding of a substrate to the SBD of HSP70, leading to refolding
of the substrate.28 To clarify whether IDFꢀ11774 regulates HSP70 molecular functions, we examined
the effects of IDFꢀ11774 on the ATPase activity of recombinant HSP70 protein. Consistent with the
findings of a previous report,29 VERꢀ155008 (Figure S6A)30 inhibited the ATPase activity of HSP70
(Figure 5A). However, IDFꢀ11774 did not inhibit the ATPase activity of HSP70. Therefore, we next
determined whether IDFꢀ11774 affects the chaperone activity of HSP70. An assay of chaperone
activity was performed by determining the degree of refolding of a denatured firefly luciferase in the
presence of recombinant HSP70. We found that IDFꢀ11774 inhibited the refolding of denatured
luciferase with an IC50 of 13.4 µM (Figure 5B). Similarly, the chemical probe 2, but not 1, also
suppressed the chaperone activity of recombinant HSP70 without affecting the ATPase activity of
HSP70 (Figure S5A and B). As expected, VERꢀ155008 inhibited luciferase refolding in the assay
using recombinant HSP70 with an IC50 value of 23.5 µM (Figure 5B), presumably inhibiting the
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ATPase activity of HSP70. Of note, treatment of cells with IDFꢀ11774 or VERꢀ155008 reduced levels
of the HSP70 target proteins HER2 and Rafꢀ1 (Figure 5C).29 In addition, VERꢀ155008 inhibited
hypoxiaꢀinduced HIFꢀ1α protein accumulation, HREꢀdependent luciferase activity, and expression of
HIFꢀ1 target genes (Figure 5D, S6B and S6C), indicating that HSP70 is involved in the regulation of
HIFꢀ1α accumulation.
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Figure 5. Inhibition of HSP70 chaperone activity by IDFꢀ11774. The effect of IDFꢀ11774 on the
ATPase activity (A) and chaperone activity (B) of HSP70. VERꢀ155008, an ATPase inhibitor of
HSP70, was used as a control. (C) The effect of IDFꢀ11774 on the degradation of HSP70 target
proteins. The expression of HER2 and Rafꢀ1 was detected in the presence of IDFꢀ11774 or VERꢀ
155008 using Western blot analysis. (D) Inhibition of HIFꢀ1 accumulation and HREꢀluciferase
activity by VERꢀ155008.
Inhibition of mitochondrial respiration and ATP production by IDF-11774. Because HIFꢀ1α
regulates oxygen homeostasis and energy metabolism,31 we further investigated the effects of IDFꢀ
11774 on mitochondrial respiration by determining the oxygen consumption rate (OCR) using the
XF24 Extracellular Flux Analyzer. During mitochondrial respiration, ATP production was inhibited by
oligomycin after basal respiration, then maximum respiration was induced by
trifluorocarbonylcyanide phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation. Under
these conditions, IDFꢀ11774 lowered the OCR during respiration (Figure 6A) and reduced the
intracellular ATP level (Figure 6B). Similarly, the HSP70 inhibitor VERꢀ155008 also lowered the
OCR and inhibited ATP production (Figures S7A and S7B). Next, using a hypoxiaꢀdetecting probe,
MAR, it was shown that both IDFꢀ1174 and VERꢀ155008 increased the intracellular oxygen tension
due to the reduced OCR, suggesting stimulation of HIFꢀ1α degradation under hypoxia (Figure 6C).
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Taken together, IDFꢀ11774 significantly reduced mitochondrial respiration and ATP production in
HCT116 cells.
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Figure 6. Effect of IDFꢀ11774 on mitochondrial metabolism. (A) The effect of IDFꢀ11774 on
mitochondrial respiration. The OCR was measured by adding oligomycin (1 µM), FCCP (0.5 µM),
and rotenone (1 µM)/antimycin A (1 µM) to HCT116 cells. (B) Effect of IDFꢀ11774 on intracellular
ATP content. (C) The intracellular oxygen tension was detected by the hypoxiaꢀsensitive probe MAR
(0.5 µM). The scale bar indicates 100 µM.
Although many efforts have been made to develop HIFꢀ1 inhibitors, few HIFꢀ1 inhibitors are in
clinical trials. We developed HIFꢀ1α inhibitors by screening a focused library of aryloxyacetylamino
benzoic acids followed by lead optimization. To understand the mode of action of IDFꢀ11774, we
designed and synthesized multifunctional chemical probes for photoaffinity labeling and click
conjugation.25, 32 After fluorescence labeling and isolation of the target molecule using 2DE, HSP70
was identified as a target protein binding directly to IDFꢀ11774.
The HSP70 family are multifunctional molecular chaperones that aid in the folding of newly
synthesized proteins, the solubilization of protein aggregates, the degradation of target proteins and
the assembly of protein complexes.33 In mammalian cells, the HSP70 family includes heatꢀinducible
HSP70 (HSPA1A, HSPA1B), constitutively expressed HSC70 (HSPA8), endoplasmic reticulum
resident BiP (HSPA5), and mitochondrial mortalin (HSPA9). HSP70 chaperone activity correlates
with the malignancy, clinical cancer stage, and poor prognosis of various cancers, suggesting the
HSP70 family proteins may be targets for anticancer therapy.34ꢀ36
Various mechanisms employed by HSP70 inhibitors have been reported:37 i) inhibition of ATPase
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activity;29, 38 ii) allosteric control of HSP70;39, 40 and iii) targeting HSP70ꢀinteracting proteins, such as
the Jꢀdomain protein family and nucleotide exchange factors.41 VERꢀ155008 binds to the ATPꢀbinding
pocket of the NBD of HSP70,29 whereas 2ꢀphenylethynesulfonamide (PES) interacts with the SBD of
HSP70 to disrupt the HSP70 complex of coꢀchaperones.42 We revealed that IDFꢀ11774 interfered with
HSP70 chaperone activity without affecting its ATPase activity by binding to the allosteric pocket in
the NBD, thereby reducing the refolding of HSP70 substrates HIFꢀ1α, Her2 and Raf1. The binding of
IDFꢀ11774 to the allosteric pocket of the HSP70 NBD may be advantageous due to its increased
specificity compared with the ATPꢀbinding pocket.
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It has been reported that simultaneous inhibition of HSP70 and HSC70 is necessary to reduce cell
viability.43 Interestingly, a pullꢀdown assay using a biotin probe demonstrated that IDFꢀ11774 also
bound HSC70 but not GRP78 (Figure S8A). Moreover, both IDFꢀ11774 and chemical probe 2
inhibited refolding activity of HSC70, while probe 1 did not (Figures S8B and S8C). Therefore, IDFꢀ
11774 exerts antiꢀcancer activity by binding to both HSP70 and HSC70 in cancer cells.
■ CONCLUSIONS
To elucidate the mechanism of action of HIFꢀ1 inhibitor IDFꢀ11774, we designed and synthesized
the multifunctional chemical probe 2 directed against the HIFꢀ1 inhibitor IDFꢀ11774. Using the probe
and chemical biology techniques, HSP70 was identified as a target protein of IDFꢀ11774. IDFꢀ11774
inhibited the chaperone activity of HSP70, presumably by binding to its allosteric pocket without
affecting ATPase activity. Furthermore, IDFꢀ11774 inhibited mitochondrial respiration and ATP
production, thereby increasing intracellular oxygen tension. This result suggests that the inhibition of
HSP70 chaperone activity by IDFꢀ11774 suppresses HIFꢀ1α refolding and stimulates HIFꢀ1α
degradation. Taken together, we report multifunctional chemical probes of IDFꢀ11774 identified
molecular target HSP70 and elucidated the mode of action of a novel HIFꢀ1 inhibitor.
■ EXPERIMENTAL PROCEDURES
General Synthesis Methods and Materials. All commercial chemicals were of reagent grade and
were used without further purification. Solvents were dried using standard procedures. All reactions
were performed under an atmosphere of dry argon in flameꢀdried glassware. The proton nuclear
magnetic resonance (1HꢀNMR) spectra were collected using a Varian (300, 400, or 500 MHz)
spectrometer (Varian Medical Systems, Inc., Palo Alto, CA, USA). 13CꢀNMR spectra were recorded
on a Varian (100 MHz) spectrometer. The chemical shifts are provided in parts per million (ppm)
downfield with coupling constants in hertz (Hz). The mass spectra were recorded using highꢀ
resolution mass spectrometry (HRMS) in electron ionization mode on a JMSꢀ700 mass spectrometer
(Jeol, Japan) or on a G2 QTOF mass spectrometer. The products from all reactions were purified by
flash column chromatography using silica gel 60 (230–400 mesh Kieselgel 60). Furthermore, thinꢀ
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layer chromatography on 0.25ꢀmm silica plates (E. Merck; silica gel 60 F254) was used to monitor
reactions. The purities of the final products were checked by reversedꢀphase highꢀpressure liquid
7
8
chromatography (RP-HPLC) performed on a Waters Corp. HPLC system equipped with an ultraviolet
(UV) detector set at 254 nm. The mobile phases were (A) H2O containing 0.05% trifluoroacetic acid
and (B) CH3CN. HPLC employed an YMC Hydrosphere C18 (HSꢀ302) column (5ꢀµm particle size,
12ꢀnm pore size) with a diameter of 4.6 mm and a length of 150 mm. The flow rate was 1.0 ml/min.
The IDFꢀ11774 and its probe compounds purity was assessed using either a gradient of 90% B to 100%
B in 35 min (Method A) or a gradient of 95% B to 100% B in 35 min (Method B). The IDFꢀ11774
derivatives purity was assessed using either a gradient of 25% B to 100% B in 35 min (Method C) or
a gradient of 80% B to 100% B in 35 min (Method D). The purities of all biologically evaluated
compounds were >95% for both methods A and B.
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Synthesis Procedures. The compound IDFꢀ11774 and chemical probes 1-4 were synthesized as
shown in Schemes S1-S3.
2-(4-Adamantan-1-yl-phenoxy)-1-(4-methylpiperazin-1-yl)ethanone (IDF-11774): To a solution of
2ꢀ(4ꢀ adamantanꢀ1ꢀylꢀphenoxy)acetic acid (7) (0.3 g, 1.04 mmol) and 1ꢀmethyl piperazine (0.10 g,
1.04 mmol) in DMF (5.0 ml) were added EDC.HCl (0.24 g, 1.25 mmol), HOBT (0.17 g, 1.25 mmol),
and DIPEA (0.37 ml, 2.08 mmol). The reaction mixture was stirred overnight at room temperature
and then partitioned between EtOAc and brine. The organic layer was separated, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column
chromatography (CH2Cl2: MeOH = 9:1) to give 2ꢀ(4ꢀadamantanꢀ1ꢀylꢀphenoxy)ꢀ1ꢀ(4ꢀmethylpiperazinꢀ
1
1ꢀyl)ethanone as a white solid (0.31 g, 80.3% yield). H NMR (400 MHz, CDCl3)
δ 7.27 (d, J = 8.0
Hz, 2H), 6.89 (d,
J
= 8.0 Hz, 2H), 4.65 (s, 2H), 3.65–3.57 (m, 4H), 2.40–2.36 (m, 4H), 2.29 (s, 3H),
166.6, 155.6,
2.08 (brs, 3H), 1.88–1.87 (m, 6H), 1.76–1.74 (m, 6H); 13C NMR (100 MHz, CDCl3)
δ
144.7, 126.0, 114.1, 67.8, 55.1, 54.6, 46.0, 45.3, 43.3, 42.0, 36.8, 35.6, 28.9; HRMS [M+H] calcd.
[C23H33N2O2]: 369.2542, found: 369.2542; purity 100.0% (as determined by RPꢀHPLC: method A, tR
= 14.06 min; method B, tR = 15.02 min).
2-(2-(3-(Trifluoromethyl)-3H-diazirin-3-yl)phenoxy)acetic acid (9a): A solution of ethyl 2ꢀ(2ꢀ (3ꢀ
(trifluoromethyl)ꢀ3Hꢀdiazirinꢀ3ꢀyl)phenoxy)acetate (0.12 g, 0.41 mmol) in THF/H2O (3:1 2 ml) was
treated with lithium hydroxide monohydrate (0.07 g, 1.66 mmol) and stirred at room temperature for
12 hr. The reaction mixture was then acidified with 10% HCl to pH 4 and partitioned between EtOAc
and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo to obtain 2ꢀ(2ꢀ(3ꢀ(trifluoromethyl)ꢀ3Hꢀdiazirinꢀ3ꢀyl)phenoxy)acetic acid as a white solid
(0.08 g, 74.0% yield). 1H NMR (400 MHz, CDCl3)
7.12 (t, = 8.0 Hz, 1H), 6.96 (d, = 8.0 Hz, 1H), 4.66 (s, 2H); MS (ESI) m/z 261 (M+H).
δ 7.72 (d, J = 8.0 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H),
J
J
2-(4-Adamantan-1-yl-2-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenoxy)acetic acid (9b): A solution
of ethyl 2ꢀ(4ꢀadamantanꢀ1ꢀylꢀ2ꢀ(3ꢀ(trifluoromethyl)ꢀ3Hꢀdiazirinꢀ3ꢀyl)phenoxy)acetate) (0.2 g, 0.47
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mmol) in THF/H2O (3:1 2 ml) was treated with lithium hydroxide monohydrate (0.07 g, 1.89 mmol)
and stirred at room temperature for 12 hr. The reaction mixture was then acidified with 10% HCl to
pH 4 and then partitioned between EtOAc and brine. The organic layer was separated, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo to obtain 2ꢀ(4ꢀadamantanꢀ1ꢀylꢀ2ꢀ(3ꢀ
1
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(trifluoromethyl)ꢀ3Hꢀdiazirinꢀ3ꢀyl) phenoxy) acetic acid as a white solid (0.1 g, 55.5% yield). H
NMR (400 MHz, CDCl3)
δ 7.81 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 4.75 (s,
2H), 2.12 (brs, 3H), 1.91–1.88 (m, 6H), 1.79–1.76 (m, 6H); MS (ESI) m/z 395 (M+H).
1-(4-(Prop-2-ynyl)piperazin-1-yl)-2-(2-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenoxy)ethanone
(Probe 1): To a solution of 2ꢀ(2ꢀ(3ꢀ(trifluoromethyl)ꢀ3Hꢀdiazirinꢀ3ꢀyl)phenoxy)acetic acid (9a) (0.1 g,
0.27 mmol) and 1ꢀ(propꢀ2ꢀynyl)piperazine (0.033 g, 0.27 mmol) in DMF (4.0 ml) were added
EDC.HCl (0.062 g, 0.32 mmol), HOBt (0.044 g, 0.32 mmol), and DIPEA (0.12 ml, 0.68 mmol). The
reaction mixture was stirred at room temperature overnight and then partitioned between EtOAc and
brine. The organic layer was separated, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The resulting residue was purified by silica gel column chromatography (CH2Cl2: MeOH
9:1) to give 1ꢀ(4ꢀ(propꢀ2ꢀynyl)piperazinꢀ1ꢀyl)ꢀ2ꢀ(2ꢀ(3ꢀ(trifluoromethyl)ꢀ3Hꢀdiazirinꢀ3ꢀ
yl)phenoxy)ethanone as a white solid (0.04 g, 35.1% yield). 1H NMR (400 MHz, CDCl3)
7.31–7.29
= 8.0 Hz, 1H), 4.69 (s, 2H), 3.69–3.63 (m, 4H), 3.31 (s,
=
δ
(m, 2H), 7.00 (t,
J
= 8.0 Hz, 1H), 6.95 (d,
J
2H), 2.57–2.53 (m, 4H), 2.24 (t,
J
= 4.0 Hz, 1H); 13C NMR (100 MHz, CDCl3)
δ 165.2, 164.5, 134.2,
130.6, 130.3, 126.4, 123.2, 120.5, 77.9, 73.8, 62.0, 51.5, 51.2, 46.8, 44.2, 41.8: HRMS [M+H] calcd.
[C17H18F3N4O2]: 367.1382, found: 367.1382; purity 100.0% (as determined by RPꢀHPLC: method A,
t
R = 12.07 min; method B,
tR = 13.30 min).
2-(4-Adamantan-1-yl-2-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenoxy)-1-(4-(prop-2-
ynyl)piperazin-1-yl)ethanone (Probe 2): To a solution of 2ꢀ(4ꢀadamantanꢀ1ꢀylꢀ2ꢀ(3ꢀ(trifluoromethyl)ꢀ
3Hꢀdiazirinꢀ3ꢀyl)phenoxy)acetic acid (9b) (0.08 g, 0.20 mmol) and 1ꢀ(propꢀ2ꢀynyl)piperazine (0.025
g, 0.20 mmol) in DMF (2.0 ml) were added EDC.HCl (0.046 g, 0.24 mmol), HOBt (0.032 g, 0.24
mmol), and DIPEA (0.09 ml, 0.5 mmol). The reaction mixture was stirred overnight at room
temperature and then partitioned between EtOAc and brine. The organic layer was separated, dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by
silica gel column chromatography (hexane:EtOAc = 2:8) to give 2ꢀ(4ꢀadamantanꢀ1ꢀylꢀ2ꢀ(3ꢀ
(trifluoromethyl)ꢀ3Hꢀdiazirinꢀ3ꢀyl)phenoxy)ꢀ1ꢀ(4ꢀ(propꢀ2ꢀynyl)piperazinꢀ1ꢀyl)ethanone as a white
solid (0.03 g, 30.0% yield). 1H NMR (400 MHz, CDCl3)
(d, = 8.0 Hz, 1H), 4.89 (s, 2H), 4.89 (s, 2H), 3.67–3.66 (m, 2H), 3.44–3.43 (m, 2H), 3.38 (s, 2H),
2.60–2.55 (m, 4H), 2.28 (t,
NMR (100 MHz, CDCl3)
δ 7.72 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 6.89
J
J
= 4.0 Hz, 1H), 2.09 (brs, 3H), 1.88–1.87 (m, 6H), 1.74–1.71 (m, 6H);13C
165.8, 156.0, 144.9, 132.9, 128.0, 127.9, 121.2, 113.2, 78.0, 73.7, 68.7,
δ
51.9, 51.4, 46.8, 45.3, 43.1, 42.0, 36.6, 35.8, 31.6, 28.8; HRMS [M+H] calcd. [C27H32F3N4O2]:
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501.2477, found: 501.2477; purity 100.0% (as determined by RPꢀHPLC: method A, tR = 17.67 min;
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method B, tR = 18.60 min).
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8
9
tert-Butyl 4-(2-phenoxyacetyl)piperazine-1-carboxylate (11a): To a solution of 2ꢀphenoxyacetic
acid (10) (0.5 g, 3.28 mmol) and tertꢀbutyl piperazineꢀ1ꢀcarboxylate (0.61 g, 3.28 mmol) in DMF (5.0
ml) were added EDC.HCl (0.75 g, 3.94 mmol), HOBt (0.53 g, 3.94 mmol), and DIPEA (1.46 ml, 8.21
mmol). The reaction mixture was stirred overnight at room temperature and then partitioned between
EtOAc and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The resulting residue was purified by silica gel column chromatography
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(hexane:EtOAc = 3:7) to give tertꢀbutyl 4ꢀ(2ꢀphenoxyacetyl)piperazineꢀ1ꢀcarboxylate as a white solid
1
(0.56 g, 65.1% yield). HꢀNMR (CDCl3, 400 MHz)
δ 7.31 (t, J = 12.0 Hz, 2H), 6.99 (t, J = 8.0 Hz,
1H), 6.95 (d,
J = 8.0 Hz, 2H), 4.70 (s, 2H), 3.58–3.56 (m, 4H), 3.44–3.40 (m, 4H), 1.46 (s, 9H); MS
(ESI) m/z 321 (M+H).
tert-Butyl 4-(2-(4-Adamantan-1-ylphenoxy) acetyl) piperazine-1-carboxylate (11b): To a solution
of (4ꢀadamantanꢀ1ꢀylꢀphenoxy) acetic acid (7) (0.5 g, 1.74 mmol) and tertꢀbutyl piperazineꢀ1ꢀ
carboxylate (0.32 g, 1.74 mmol) in DMF (5.0 ml) were added EDC.HCl (0.4 g, 2.09 mmol), HOBt
(0.28 g, 2.09 mmol), and DIPEA (0.78 ml, 4.36 mmol). The reaction mixture was stirred overnight at
room temperature and then partitioned between EtOAc and brine. The organic layer was separated,
dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified
by silica gel column chromatography (hexane:EtOAc = 2:8) to give tertꢀbutyl 4ꢀ(2ꢀ(4ꢀadamantanꢀ1ꢀ
1
ylphenoxy) acetyl) piperazineꢀ1ꢀcarboxylate as a white solid (0.6 g, 75.6% yield). HꢀNMR (400
MHz, CDCl3)
3.43–3.40 (m, 4H), 2.08 (brs, 3H), 1.87–1.86 (m, 6H), 1.76–1.71 (m, 6H), 1.46 (s, 9H); 13C NMR
(100 MHz, CDCl3) 166.9, 155.4, 154.5, 144.9, 126.0, 114.0, 80.3, 67.9, 45.4, 43.3, 42.0, 36.7, 35.6,
28.9, 28.4; HRMS [M+H] calcd. [C27H39N2O4]: 455.2910, found: 455.2910; purity 100.0% (as
determined by RPꢀHPLC: method C, R = 26.4 min; method D, R = 27.2 min).
δ 7.27 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 8.0 Hz, 2H), 4.67 (s, 2H), 3.59–3.56 (m, 4H),
δ
t
t
2-Phenoxy-1-(piperazin-1-yl)ethanone (12a): Trifluoroacetic acid (0.48 ml, 6.24 mmol) was added
to a suspension of tertꢀbutyl 4ꢀ(2ꢀphenoxyacetyl)piperazineꢀ1ꢀcarboxylate (11a) (0.5 g, 1.56 mmol) in
dichloromethane (5.0 ml) and stirred overnight at room temperature. The solvent was evaporated
under reduced pressure to afford a crude solid, which was purified by silica gel column
chromatography (CH2Cl2: MeOH = 9:1) to obtain 2ꢀphenoxyꢀ1ꢀ(piperazinꢀ1ꢀyl)ethanone as a white
1
solid (0.31g, 91.2% yield). HꢀNMR (CDCl3, 400 MHz)
δ
9.93 (brs, 1H), 7.31 (t,
J = 12.0 Hz, 2H),
7.03 (t,
J
= 8.0 Hz, 1H), 6.90 (d,
J
= 8.0 Hz, 2H), 4.70 (s, 2H), 3.89–3.88 (m, 4H), 3.12–3.10 (m, 4H);
MS (ESI) m/z 221 (M+H).
2-(4-Adamantan-1-ylphenoxy)-1-(piperazin-1-yl) ethanone (12b): Trifluoroacetic acid (0.3 ml, 3.95
mmol) in dichloromethane (4.5 ml) was added to a suspension of tertꢀbutyl 4ꢀ(2ꢀ(4ꢀadamantanꢀ1ꢀ
ylphenoxy) acetyl) piperazineꢀ1ꢀcarboxylate (11b) (0.45 g, 0.98 mmol) and stirred overnight at room
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temperature. The solvent was evaporated under reduced pressure to afford a crude solid, which was
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purified by silica gel column chromatography (CH2Cl2: MeOH = 9:1) to obtain 2ꢀ(4ꢀadamantanꢀ1ꢀ
1
ylphenoxy)ꢀ1ꢀ(piperazinꢀ1ꢀyl) ethanone as a white solid (0.31g, 88.6% yield). HꢀNMR (400 MHz,
CDCl3)
4H), 3.20–3.13 (m, 4H), 2.07 (brs, 3H), 1.85–1.84 (m, 6H), 1.78–1.70 (m, 6H); 13C NMR (100 MHz,
CDCl3) 167.2, 155.0, 145.4, 126.2, 114.0, 67.5, 43.4, 43.3, 43.2, 42.2, 38.8, 36.7, 35.6, 28.9; HRMS
[M+H] calcd. [C22H31N2O2]: 355.2386, found: 355.2386; purity 100.0% (as determined by RPꢀHPLC:
method C, R = 9.7 min; method D, R = 11.3 min).
δ 9.75 (s, 1H), 7.27 (d, J = 8.0 Hz, 2H), 6.83 (d, J = 8.0 Hz, 2H), 4.65 (s, 2H), 3.86–3.84 (m,
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δ
t
t
(2-{2-[2-(2-Chloro-acetylamino)-ethoxy]-ethoxy}-ethyl)-carbamic acid tert-butyl ester: To a
solution of 1,2ꢀbis(2ꢀaminoethoxy)ethane (0.3 g, 2.03 mmol) and triethylamine (0.56 g, 4.06 mmol) in
methanol (4.0 ml) were added diꢀtertꢀbutyl dicarbonate (0.55 ml, 2.43 mmol). The reaction mixture
was stirred at room temperature overnight, and the methanol and TEA were removed in vacuo to yield
an oily residue. This residue was dissolved in CH2Cl2 and washed with a solution of sodium
carbonate. The combined extracts were dried on anhydrous MgSO4, filtered, and concentrated in
vacuo. The resulting residue was purified by silica gel column chromatography (nꢀHexane:EtOAc =
2:8) to form monoꢀprotected diamine as yellow oil (0.1 g, 66.7% yield). The TLC control showed
almost no bisꢀprotected diamine. The yellow oil was used without further purification. A solution of
chloroacetyl chloride (0.17 g, 1.58 mmol) in CH2Cl2 (3 ml) was added dropwise over 20 min to a
solution of monoꢀprotected diamine (0.32 g, 1.32 mmol) and TEA in CH2Cl2 (3 ml) at −20°C. The
resulting brown solution was left to stir at room temperature for 24 h. The reaction solution was
removed in vacuo, and the residue was dissolved in CH2Cl2 and washed with a solution of sodium
carbonate. The combined extracts were dried on anhydrous MgSO4, filtered, and concentrated to give
1
as brown oil (0.3 g, 70.4% yield). H NMR (300 MHz, CDCl3)
δ 7.03 (s, 1H), 5.06 (s, 1H), 3.95 (s,
2H), 3.38–3.51 (m, 10H), 3.20 (m, 2H), 1.33 (s, 9H); MS (ESI) m/z 325 (M+H).
tert-Butyl 2-(2-(2-(2-(4-(2-phenoxyacetyl)piperazin-1-yl)acetamido)ethoxy)ethoxy)ethylcarbamate
(13a): To the solution of (2ꢀ{2ꢀ[2ꢀ(2ꢀchloroꢀacetylamino)ꢀethoxy]ꢀethoxy}ꢀethyl)ꢀcarbamic acid tert
ꢀ
butyl ester (0.29 g, 0.90 mmol) in anhydrous acetone (10 ml) were added compound 12a (0.1 g, 0.45
mmol), K2CO3 (0.069 g, 0.90 mmol), Cs2CO3 (0.07 g, 0.22 mmol), and KI (0.04 g, 0.22 mmol). The
reaction mixture was heated at 60°C for 36 h and cooled to room temperature. The mixture was
evaporated under reduced pressure, and the residue was washed with water. The solution was
extracted with ethyl acetate, and the combined organic layers were dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The crude product was purified by silica gel column
chromatography (CH2Cl2:MeOH = 9:1) to obtain tertꢀbutyl 2ꢀ(2ꢀ(2ꢀ(2ꢀ(4ꢀ(2ꢀphenoxyacetyl)piperazinꢀ
1
1ꢀyl)acetamido)ethoxy)ethoxy)ethylcarbamate as a yellow oil (0.14 g, yield 60.8%). H NMR (400
MHz, CDCl3)
δ 7.35 (t, J = 12.0 Hz, 1H), 7.31 (t, J = 8.0 Hz, 2H), 7.00 (t, J = 8.0 Hz, 1H), 6.94 (d, J
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= 8.0 Hz, 1H), 4.98 (t,
J
= 8.0 Hz, 1H), 4.69 (s, 2H), 3.64ꢀ3.62 (m, 4H), 3.60–3.47 (m, 10H), 3.31–
5
6
3.29 (m, 2H), 3.02 (s, 2H), 2.53–2.49 (m, 4H), 1.44 (s, 9H); MS (ESI) m/z 509 (M+H).
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8
tert-Butyl
2-(2-(2-(2-(4-(2-(4-adamantan-1-yl-phenoxy)acetyl)piperazin-1-
yl)acetamido)ethoxy)ethoxy)ethylcarbamate (13b): To the solution of (2ꢀ{2ꢀ[2ꢀ(2ꢀchloroꢀ
acetylamino)ꢀethoxy]ꢀethoxy}ꢀethyl)ꢀcarbamic acid tertꢀbutyl ester (0.18 g, 0.56 mmol) in anhydrous
acetone (10 ml) and DMF (4 ml) were added compound 12b (0.1 g, 0.28 mmol), K2CO3 (0.078 g,
0.56 mmol), Cs2CO3 (0.045 g, 0.14 mmol), and KI (0.023 g, 0.14 mmol). The reaction mixture was
heated at 60°C for 36 h and cooled to room temperature. The mixture was evaporated under reduced
pressure, and the residue was washed with water. The solution was extracted with ethyl acetate, and
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the combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo
.
The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH = 9:1) to obtain
tertꢀbutyl
2ꢀ(2ꢀ(2ꢀ(2ꢀ(4ꢀ(2ꢀ(4ꢀadamantanꢀ1ꢀylꢀphenoxy)acetyl)piperazinꢀ1ꢀ
1
yl)acetamido)ethoxy)ethoxy)ethylcarbamate as a yellow oil (0.12 g, yield 66.6%). H NMR (400
MHz, CDCl3)
= 8.0 Hz, 1H), 4.66 (s, 2H), 3.65ꢀ3.63 (m, 4H), 3.60–3.48 (m, 10H), 3.32
2.53 2.50 (m, 4H), 2.08 (brs, 3H), 1.88–1.86 (m, 6H), 1.76 1.74 (m, 6H), 1.44 (s, 9H); MS (ESI) m/z
643 (M+H).
δ
7.36 (t,
J
= 12.0 Hz, 1H), 7.28 (d,
J
= 8.0 Hz, 2H), 6.86 (d,
J = 8.0 Hz, 2H), 5.07 (t, J
–3.30 (m, 2H), 3.03 (s, 2H),
–
–
5-((3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-N-(2-(2-(2-(2-(4-(2-
phenoxyacetyl)piperazin-1-yl)acetamido)ethoxy)ethoxy)ethyl)pentanamide (Probe 3): Compound 13a
(0.1 g, 0.19 mmol) was dissolved in the 4ꢀml mixture of TFA and CH2Cl2 (1:3), and the solution was
stirred at room temperature for 3 h. The solvents were then removed and coꢀevaporated three times
with toluene to obtain crude free amine product 14a. Without further purification, the amine product
14a was dissolved in 2 ml DMF, to which 0.5 ml of TEA was added. Subsequently, (+)ꢀbiotin
Nꢀ
hydroxysuccinimide ester (0.08 g, 0.23 mmol) was added to the solution, and it was stirred overnight
at room temperature. The reaction was quenched by water and extracted with 10% MeOH/DCM. The
organic layers were dried over anhydrous MgSO4, filtered, and then concentrated under reduced
pressure. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH
8.5:1.5) to give the compound 5ꢀ((3a ,4 ,6a )ꢀ2ꢀoxohexahydroꢀ1Hꢀthieno[3,4ꢀd]imidazolꢀ4ꢀyl)ꢀ ꢀ(2ꢀ
(2ꢀ(2ꢀ(2ꢀ(4ꢀ(2ꢀphenoxyacetyl)piperazinꢀ1ꢀyl)acetamido)ethoxy)ethoxy)ethyl)pentanamide as a pale
yellow solid (0.06 g, 48.4% yield). 1H NMR (400 MHz, CDCl3)
7.41 (t, = 12.0 Hz, 1H), 7.30(t,
= 8.0 Hz, 2H), 7.01 (t, = 8.0 Hz, 1H), 6.94 (d, = 8.0 Hz, 1H), 6.63 (t, = 8.0 Hz, 1H), 6.42 (s, 1H),
5.42 (s, 1H), 4.70 (s, 2H), 4.50–4.47 (m, 1H), 4.31–4.28 (m, 1H), 3.65–3.53 (m, 12H), 3.51–3.47 (m,
2H), 3.43–3.39 (m, 2H), 3.15–3.12 (m, 1H), 3.04 (s, 2H), 2.91–2.88 (m, 1H), 2.73 (d, = 12.0 Hz,
1H), 2.54–2.49 (m, 4H), 2.23 (t,
= 8.0 Hz, 2H), 1.74–1.62 (m, 4H), 1.47–1.43 (m, 2H); 13C NMR
(100 MHz, CDCl3) 173.4, 169.9, 166.7, 163.8, 157.7, 129.7, 121.8, 114.6, 70.2, 69.9, 69.8, 67.7,
=
S
S
R
N
δ
J
J
J
J
J
J
J
δ
61.8, 61.4, 60.1, 55.6, 53.5, 53.0, 45.3, 42.1, 40.5, 39.1, 38.7, 35.9, 31.6, 28.2, 28.1, 25.6; HRMS
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[M+H] calcd. [C30H47N6O7S]: 635.3227, found: 635.3227; purity 100.0% (as determined by RPꢀ
HPLC: method A, tR = 7.31 min; method B, tR = 22.76 min).
7
8
N-(2-(2-(2-(2-(4-(2-(4-Adamantan-1-yl-phenoxy)acetyl)piperazin-1-
yl)acetamido)ethoxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanamide (Probe 4): The compound 13b (0.1 g, 0.15 mmol) was dissolved in the 4ꢀml mixture
of TFA and CH2Cl2 (1:3), and the solution was stirred at room temperature for 3 h. Then the solvents
were removed and coꢀevaporated three times with toluene to obtain crude free amine product 14b.
Without further purification, the amine product 14b was dissolved in 2 ml DMF, to which 0.5 ml of
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TEA was added. Subsequently, (+)ꢀbiotin Nꢀhydroxysuccinimide ester (0.06 g, 0.18 mmol) was added
to the solution, and it was stirred overnight at room temperature. The reaction was quenched by water
and extracted with 10% MeOH/DCM. The organic layers were dried over anhydrous MgSO4, filtered,
and then concentrated under reduced pressure. The crude product was purified by silica gel column
chromatography (CH2Cl2: MeOH = 8.5:1.5) to give the compound
1ꢀylꢀphenoxy)acetyl)piperazinꢀ1ꢀyl)acetamido)ethoxy)ethoxy)ethyl)ꢀ5ꢀ((3a
oxohexahydroꢀ1Hꢀthieno[3,4ꢀd]imidazolꢀ4ꢀyl)pentanamide as a white solid (0.07 g, 58.8% yield). H
NMR (400 MHz, CDCl3) 7.40 (t, = 12.0 Hz, 1H), 7.28 (d, = 8.0 Hz, 2H), 6.88 (d, = 8.0 Hz,
2H), 6.57 (t, = 12.0 Hz, 1H), 6.33 (s, 1H), 5.34 (s, 1H), 4.67 (s, 2H), 4.50–4.47 (m, 1H), 4.31–4.28
(m, 1H), 3.73–3.57 (m, 12H), 3.53–3.49 (m, 2H), 3.44–3.40 (m, 2H), 3.16–3.13 (m, 1H), 3.04 (s, 2H),
2.91–2.87 (m, 1H), 2.73 (d, = 12.0 Hz, 1H), 2.54–2.51 (m, 4H), 2.23 (t, = 8.0 Hz, 2H), 2.08 (brs,
3H), 1.87–1.85 (m, 6H), 1.75–1.70 (m, 6H), 1.68–1.64 (m, 4H), 1.45–1.41 (m, 2H); 13C NMR (100
MHz, CDCl3) 173.3, 169.8, 166.8, 163.7, 155.5, 144.9, 126.0, 114.1, 70.2, 70.1, 69.9, 69.8, 67.8,
N
ꢀ(2ꢀ(2ꢀ(2ꢀ(2ꢀ(4ꢀ(2ꢀ(4ꢀadamantanꢀ
S
,4 ,6a )ꢀ2ꢀ
S
R
1
δ
J
J
J
J
J
J
δ
61.7, 61.4, 60.1, 55.5, 53.5, 53.0, 45.4, 43.3, 42.1, 40.5, 39.1, 38.7, 36.7, 35.9, 35.6, 28.9, 28.2, 28.1,
25.6; HRMS [M+H] calcd. [C40H61N6O7S]: 769.4322, found: 769.4322; purity 100.0% (as determined
by RPꢀHPLC: method A, tR = 13.69 min; method B, tR = 15.22 min).
Molecular Modeling. All docking studies were performed using the SurflexꢀDock program
(Tripos, Princeton, NJ) with the SYBYL module by employing the following protocol. Proteins were
prepared by repairing side chain, randomly adding hydrogen atoms, adding charge to the proteins with
Amber7 FF99 for biopolymers and GasteigerꢀMarsili for ligands, and fixing the amide side chains.
Furthermore, staged minimization was set up with 1000 steps/stage at maximum iteration, none at
initial optimization, 0.5 kcal/(mol*Å) gradient at termination, and tripos at force field. After these
steps, we obtained a total energy of −15667.875 kcals/mol. Second, ligands were prepared via energy
minimization with 1000000 steps at maximum iteration, simplex at initial optimization, 0.05
kcal/(mol*Å) gradient at termination, tripos at force field, and GasteigerꢀMarsili at charge. Using this
method, we acquired a total energy of 19.1594 kcals/mol. Subsequently, the binding mode was
calculated using the Surflex Dock score (Total_score, Crash, and Polar).
Cell culture. Human colorectal carcinoma HCT116 cells were cultured in a 5% CO2 atmosphere
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at 37°C in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) supplemented with 5%
fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco). Cells were
seeded in a tissue culture plate and incubated at 37°C for 20 h for subsequent experiments. Hypoxic
conditions were achieved by replacing cells with 1% O2, 94% N2, and 5% CO2 in a multigas incubator
(Sanyo, Osaka, Japan).
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Immunoblot analysis. Cells lysates subjected to SDSꢀpolyacrylamide gel electrophoresis (PAGE),
transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and
immunoblotted with primary antibody. After further incubation with horseradish peroxidase (HRP)ꢀ
conjugated secondary antibody, the blot was subject to enhanced chemiluminescence. antiꢀHIFꢀ1α
(BD Transduction Laboratories, San Diego, CA, USA), HSP70 (Santa Cruz), HER2 (Cell Signaling),
Rafꢀ1 (Santa Cruz), and antiꢀβꢀactin (Abcam, Cambridge, UK).
Reporter gene assay. HCT116 cells expressing a hypoxia response element (HRE)ꢀdependent
firefly luciferase reporter construct (HREꢀLuc) and a cytomegalovirusꢀRenilla luciferase reporter
construct were established with the Cignal™ Lenti Reporter assay system (SABiosciences, Frederick,
MD, USA) according to the manufacturer’s instructions. Cells stably expressing the reporter gene
were selected with puromycin. Cells were incubated for 12 h with or without drugs in normoxic or
hypoxic conditions. After removing the supernatant, luciferase activity was measured with a Dualꢀ
Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions
with a Victor™ X Light luminescence reader (Perkin Elmer, Boston, MA, USA).
Identification of IDF-11774-binding protein. Photoaffinity labelling using 360ꢀnm UV radiation
(UVP, Upland, CA, USA) and Click reactions of the probe and Cy3 azide or Cy5 azide (Click
Chemistry Tools, Scottsdale, AZ, USA) were performed as described.25 Proteins conjugated to the
probe were separated by SDSꢀPAGE and fluorescence was detected with a Typhoon 9410 imaging
system (GE Healthcare, Piscataway, NJ, USA). Then, 2ꢀdimensional electrophoresis (2ꢀDE) was
performed as described.25 Isoelectric focusing was carried out using immobilized pH gradient (IPG)
strips (Immobiline DryStrip; pH 3ꢀ11; 7 cm; GE Healthcare) in the Ettan IPGphor system (GE
Healthcare). The strip was transferred to a 12% polyacrylamide gel, and electrophoresis was
performed. The fluorescenceꢀvisualized and CBBꢀstained spots were excised from the gel and
trypsinized for proteasomal analysis using LC/ESIꢀQꢀTOF MS. Peptide identification was performed
with the database on the Mascot website (www.matrixscience.com).
Pull-down assay with biotinylated probe. NeutrAvidin UltraLink resin (200 µl, Thermo,
Waltam, MA) and biotinylated probe (125 nmol) were incubated for 1 h in 400 µl of binding buffer
(50 mM Tris, pH 7.5, and 150 mM NaCl) in Pierce Spin Columns (Thermo). After incubation with
cell lysate (200 µg) for 1 h, the columns were washed three times with wash buffer (50 mM Tris, pH
7.5, 150 mM NaCl, and 2 M urea). Bound proteins were eluted in an elution buffer (1.2% SDS in PBS)
followed by boiling for 5 min in a sample buffer (50 mM Tris, pH 7.4, 4% SDS, 10% glycerol, 4% 2ꢀ
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thioethanol, and 0.05 mg/ml bromophenol blue) at a ratio of 4:1 and then separated by SDS/PAGE.
Protein level was detected by immunoblot analysis.
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Preparation of recombinant HSP70 protein. HSP70 gene was obtained from the Korean Human
Gene Bank (KUGI) at KRIBB. The DNA fragment encoding the HSP70ꢀFL(aa 1ꢀ613), nucleotideꢀ
binding domain HSP70ꢀNBD (aa 1ꢀ388) and substrateꢀbinding domain HSP70ꢀSBD (aa 386ꢀ613)
were subcloned into the pHis vector (Merck, Germany). The recombinant HSP70 protein was
expressed in E. coli Rosetta 2 (DE3) and purified by NiꢀNTA affinity chromatography, TEV cleavage
and sizeꢀexclusion chromatography (Figure S1).
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In vitro binding of probe to recombinant HSP70. The recombinant human HSP70 (2 µg) was
incubated for 10 min with probe 2 (10 µM) in the presence or absence of IDFꢀ11774 (100 µM)
followed by photoaffinity labeling and the click reaction. After the click reaction, proteins were
precipitated with methanol/chloroform/water (60/15/40, v/v) and denatured by boiling for 5 min in a
5× sample buffer. Proteins were separated by SDSꢀPAGE, and fluorescence was detected with a
Typhoon 9410 imaging system.
HSP70 ATPase activity assay. A mixture of HSP70 (1 µg) or HSC70 (1 µg, StressMarq, Victoria,
Canada) and HSP40 (0.14 µg, StressMarq) was incubated in reaction buffer (20 mM Tris, pH 7.5, 50
mM KCl, 5 mM MgCl2, and 1 µM ATP). After incubation for 30 or 60 min, ATPase activity was
determined by measuring ATP content using an ENLITEN rLuciferase/Luciferin reagent (Promega).
HSP70 chaperone activity assay. The chaperone activity of HSP70 and HSC70 was determined
by a luciferase refolding assay.44 Luciferase (500 µg/ml) was denatured in denature buffer (30 mM
TrisꢀHCl, pH 7.4, 6 M guanidineꢀHCl, and 5 mM DTT) for 60 min at room temperature. The
refolding reaction was performed in 50 µl of refolding buffer (20 mM Tris, pH 7.5, 50 mM KCl, 5
mM MgCl2, 10 mM DTT, and 1 mM ATP) containing HSP70 (0.5 µg), HSP40 (0.3 µg) and denatured
luciferase (0.125 µg). After incubation for 30 min, luciferase activity was determined with a
Luciferase Assay System (Promega).
Determination of ATP content. Intracellular ATP content was determined by an ENLITEN ATP
Assay System (Promega) according to the manufacturer’s instructions.
Measurement of mitochondrial respiration. The oxygen consumption rate (OCR) in cells was
measured in 24ꢀwell plates using the XF24 extracellular flux analyzer. HCT116 cells were incubated
for 24 h on XF24 cell culture plates (Seahorse Biosciences) and further incubated for 30 min at 37°C
without CO2 in XF assay media (pH 7.4; Seahorse Biosciences) containing various concentrations of
drugs. After determining basal OCR, oligomycin (1 µM), carbonylcyanide p-
trifluoromethoxyphenylhydrazone (0.5 µM), and rotenone (1 µM)/antimycin A (1 µM) were added
sequentially, and OCR was determined following each addition.
Detection of oxygen tension. The intracellular oxygen tension was detected using a hypoxiaꢀ
detecting probe mono azo rhodamine (MAR, Goryo Chemical, Japan) as described.45
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■ AUTHOR INFORMATION
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Corresponding Author
*misun@kribb.re.kr
*kaylee@dongguk.edu
Author Contributions
‡Contributed equally to this work.
Notes
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The authors declare no competing financial interest.
■ ACKNOWLEDGMENT
We thank Mi Young Lee, Sung Wook Chae, and AꢀRang Im at KMꢀBased Herbal Drug Research
Group, Korea Institute of Oriental Medicine, Korea, for technical assistance. This work was supported
by the National Research Foundation (NRF) grants (2014R1A2A2A01005455 and
2015M3A9A8032460), Health Technology R&D grant (HI13C2162) and KRIBB Initiative
program.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX.
Schemes of probe synthesis, NMR charts, and additional biological data (mass spectrometric analysis,
cell basedꢀHIF assay, in vitro HSP70 assay, recombinant HSP70 purification, and mitochondrial
respiration) (PDF)
Abbreviations
2DE, twoꢀdimensional electrophoresis; EPO, erythropoietin; GLUT, glucose transporter; HIF,
hypoxiaꢀinducible factor; HRE, hypoxiaꢀresponse element; HSP, heat shock protein; NBD,
nucleotideꢀbinding domain; OCR, oxygen consumption rate; SBD, substrateꢀbinding domain; VEGF,
vascular endothelial growth factor
■ Table of Contents
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Table of Contents
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IDF-11774
Biotin-conjugated probe
Pull-down
Target Identification
Mechanism of Action
Photoaffinity
UV
N3-FL
Click
Multifunctional imaging probe
Inhibition of HSP70 chaperone activity
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Figure 1
Bioconjugate Chemistry
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Aryloxyacetylamino
benzoic acid analogues
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HIF-1 inhibition
in cancer cells
SAR
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IDF-11774
Photoaffinity
UV
N3-FL
Click
Pull-down
Biotin-conjugated probe
Multifunctional imaging probe
Target Identification
Mechanism of Action
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Figure 2
(A)
(B)
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O2 (%) 20
1
-
-
-
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-
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Probe 1
Probe 2
+
-
+
HRE-Luc
IC50 (µM)
HIF-1α
β-Actin
Cmpd
R
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Probe 1
Probe 2
H
>20
Adamantyl
6.20 0.57
±
(C)
pI
3
11
100
75
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MW
(kDa)
Probe 2-Cy3
Probe 1-Cy5
100
75
*
*
50
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MW
(kDa)
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Figure 3
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(A)
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Probe 3: R = H
Probe 4: R = Adamantyl
(C)
Probe 4
Unbound
Bound
Unbound Bound
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+
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Probe 3
Probe 4
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IDF-11774
+ (10x)
HSP70
HSP70
b-Actin
b-Actin
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