Bisubstrate-Type Chemical Probes Identify GRP94 as a Potential Target of Cytosine-Containing Adenosine Analogs

Article abstract of DOI:10.1021/acschembio.9b00965

We synthesized affinity-based chemical probes of cytosine-adenosine bisubstrate analogs and identified several potential targets by proteomic analysis. The validation of the proteomic analysis identified the chemical probe as a specific inhibitor of gluco

Full text of DOI:10.1021/acschembio.9b00965

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Article
Bisubstrate-type chemical probes identify GRP94 as a
potential target of cytosine-containing adenosine analogs
Dany Pechalrieu, Fanny Assemat, Ludovic Halby, Marlene Marcellin, Pengrong Yan, Karima
Chaoui, Sahil Sharma, Gabriela Chiosis, Odile Burlet-Schiltz, Paola B. Arimondo, and Marie Lopez
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00965 • Publication Date (Web): 19 Mar 2020
Downloaded from pubs.acs.org on March 20, 2020
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4 (10 µM)
KG-1
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5 (10 µM)
MOLM-13
K-562
MOLM-13
WM-266-4
WM-266-4
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Bisubstrate-type chemical probes identify GRP94 as a potential target of
cytosine-containing adenosine analogs.
a
a
a,b
c
d
Dany Pechalrieu , Fanny Assemat , Ludovic Halby , Marlene Marcellin , Pengrong Yan , Karima
c
d
d
c
a,b
Chaoui , Sahil Sharma , Gabriela Chiosis , Odile Burlet-Schiltz , Paola B. Arimondo * and Marie
a,e
Lopez *
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a
ETaC, CNRS FRE3600, Centre de Recherche et Développement Pierre Fabre, Toulouse, France
EpiCBio, Epigenetic Chemical Biology, Department Structural Biology and Chemistry, Institut Pasteur, CNRS
UMR n°3523, 28 rue du Dr Roux, 75015 Paris, France
Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, Toulouse,
b
c
France
d
Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, New York
Institut des Biomolécules Max Mousseron (IBMM), CNRS, Univ Montpellier, ENSCM UMR 5247, 240
avenue du Prof. E. Jeanbrau, 34296 Montpellier cedex 5, France
e
*
correspondence should be addressed to: Dr. Marie Lopez, IBMM CNRS, email: marie.lopez@cnrs.fr and Dr.
Paola B. Arimondo, EpiCBio Institut Pasteur - CNRS, email: paola.arimondo@pasteur.fr
GRAPHICAL ABSTRACT
Adenosine
moiety
Adenosine
moiety
GRP94
O
OH
TAMRA
Cytosine
moiety
Cytosine
moiety
O
O
N
N
HN
N
H
O
O
N
N
H
N
O
N
H
N
Bio
n
H
Bisubstrate-type
analogues
O
Chemical probes
In-cell target iden fica on
ABSTRACT
We synthesized affinity-based chemical probes of cytosine-adenosine bisubstrate analogues and
identified several potential targets by proteomic analysis. The validation of the proteomic analysis
identified the chemical probe as a specific inhibitor of glucose-regulated protein 94 (GRP94), a
potential drug target for several types of cancers. Therefore, as a result of the use of bisubstrate-type
chemical probes and a chemical-biology methodology, this work opens the way to the development of
a new family of GRP94 inhibitors that could potentially be of therapeutic interest.
KEY WORDS
Adenosine-cytosine analogues; chemical probes; affinity-based protein profiling (ABPP); heat shock
protein 90 (HSP90); GRP94 inhibitors
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INTRODUCTION
Bisubstrate-type enzyme inhibitors consist in the association of two distinct substrate derivatives of an
enzyme within the same molecule. In a previous work, we synthesized a library of molecules
containing the adenosine motif, present in the SAM (S-adenosyl-L-methionine) cofactor of
methyltransferases, bound to cytosine analogues to target DNA methyltransferases (DNMTs).
Surprisingly, this class of compounds appeared to target protein (i.e. histone) arginine
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methyltransferases (PRMTs). Here, in order to better understand the mode of action of these
bisubstrate-type enzyme inhibitors we used both conjugates 1 and 2 (FIG.1), inactive against DNMT,
PRMT and histone-lysine N-methyltransferases (KHMT), as a starting point to synthesize chemical
probes in order to identify their cellular target using affinity-based protein profiling (ABPP)
3
, 4
approach. In this strategy, appropriate chemical probes are used to pull-down the protein targets
they bind. We chose to incorporate a photoreactive moiety to ensure a covalent crosslink between the
chemical probe and protein target. The chemical probes consist of (i) an affinity moiety (e.g. inhibitor)
to bind to the target protein, (ii) a photoreactive group to trap the complexes in the cells upon
irradiation and (iii) a reactive entity to label and purify the complexes. The key feature consists of
using “click chemistry” to label the chemical probe with a fluorescent or affinity tag. We used copper
(I)-catalyzed azide-alkyne cycloaddition (CuAAC) as “click reaction” to tag our probes with biotin
5
and purify the associated proteins.
To this aim, we designed a chemical probe derived from the adenosine-cytosine scaffold common to
compounds 1 and 2 bearing a benzophenone and an alkyne moiety (compound 4, FIG.1) and an
inactive probe containing the probe reactive scaffold (in green) coupled to a Boc protecting group
(
compound 5, FIG .1). Five cancer cell lines were treated with the chemical probe, followed by UV
irradiation of the cells to initiate crosslinking and a click reaction was then carried out on the cellular
extracts to visualize and pull-down the proteins and analyze them by mass spectrometry. A clear
protein pattern was observed and the proteomic analysis results identified glucose-regulated protein 94
(
GRP94), a chaperone protein, as a potential target. This is not surprising since the adenosine motif is
present in the ATP (adenosine triphosphate) cofactor of GRP94. We then validated GRP94 as a target,
thus identifying the adenosine-substituted cytosine scaffold as a starting point for novel inhibitors of
this family of molecular chaperones.
RESULTS
In our quest for transition state analogues of DNMTs, we synthesized a chemical library of adenosine-
cytosine conjugates. Two conjugates (compounds 1 and 2), inactive against DNMT, KHMTs and
PRMTs (supporting information GRAPH SI-1) are depicted in FIGURE 1 together with the intermediate
3
, the corresponding chemical probe 4 and the probe 5 as control.
-
INSERT Figure 1-
Figure 1. Structure of the compounds 1-3 with the adenosine motif (red) linked to the 5-position of the cytosine
analogue (blue). Structure of the chemical probes 4 and 5 containing the probe reactive scaffold (green).
Chemical synthesis
Synthesis of the adenosine-cytosine analogues 1-3 (SCHEME 1) started from the ethyl cytosine-5-
carboxylate prepared according to the reported literature. The ethyl cytosine-5-carboxylate was then
N-alkylated with MOM, BOM or Boc-aminoethyl groups to afford compounds 6-8, respectively. The
6, 7
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ester group was then hydrolyzed under basic conditions to afford the carboxycytosine derivatives 9-11.
The carboxylic acids 9-11 were coupled to 2’,3’-O-isopropylidene-5’-deoxy-5’-aminomethyldenosine
to provide the amide derivatives 12-14 and the target compounds 1-3 after deprotection.
8
-INSERT Scheme 1-
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Scheme 1. Synthesis scheme of the adenosine-cytosine derivatives 1-3.
a) MOMCl or BOMBr, Boc-aminoethyl bromide, K
2
1
2
CO
’,3’-O-isopropylidene-5’-deoxy-5’-aminomethyladenosine, HATU, DIPEA, DMF, RT, 2h; d) TFA, H
h.
3
, DMF, RT, 18h; b) NaOH, MeOH/H
2
O, RT, 12h; c)
2
O, RT,
-INSERT Scheme 2-
Scheme 2. Synthetic scheme for chemical probe 4 (upper box) and control probe 5 (bottom box).
a) 5-Hexynoic acid, HATU, DIPEA, DMA, RT, 2h; b) Succinic anhydride, dioxane, 80 °C, 4h; c) HATU,
DIPEA, DMF, RT, 16h; d) Di-tert-butyl dicarbonate, NaOH, H O, dioxane, RT, 16h; e) HATU, DIPEA, DMA,
2
RT, 16h.
The synthesis of chemical probe 4 is presented in SCHEME 2. First, mono-functionalization of the
diamino-benzophenone was achieved using HATU as a coupling reagent and 5-hexynoic acid to yield
compound 15. A small linker was then added on the second primary amine of the benzophenone
following lactone ring opening with succinic anhydride, which afforded disubstituted benzophenone
16. The last step involved the coupling of the adenosine-based compound 3 with the benzophenone
derivative 16 using HATU as a coupling reagent.
The control probe 5 was synthesized using the alkynylated benzophenone 15 and the Boc-protected 5-
aminopentanoic acid 17 (SCHEME 2). The Boc group in control probe 5 was kept for in-cell
experiments in order to enable cell permeability.
ABPP experiments
For the gel-based experiments, breast cancer MCF-7 cells were first treated with increasing
concentrations of the probe 4. This was followed by UV irradiation to allow covalent binding of the
benzophenone moiety of 4 to the surrounding proteins, leading to an irreversible binding of the probe
to the potential protein targets. After cell lysis, the probe was then reacted with TAMRA-biotin-azide
through CuAAC, separated by SDS-PAGE, and visualized by in-gel fluorescence scanning to observe
the proteins bound to the probe (FIG. 2A AND 2B). We observed an increase in the labeled protein level
from 1 to 50 M of 4 with a highly intense band around 100 kDa. Total protein staining with SYPRO
Ruby was used as loading control (FIG. 2B AND 2C lower panel).
-INSERT Figure 2-
Figure 2. (A) Representation of the general strategy for target identification by using a chemical probe. (B-C)
TAMRA fluorescence (upper gels) and SYPRO Ruby stained total protein (bottom gels) profiles of MCF-7 cells
treated with a concentration range of probe 4 (B) or with 10 M of 4 with and without UV irradiation (left) or
after competition experiments with 100 M of competitors 1 and 2 (right) (C).
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A 10 M probe concentration was chosen for the following experiments because the best ratio
between fluorescence intensity of the band at 100 kDa over the background was obtained at this
concentration when compared to 25 or 50 M. As expected, in the absence of irradiation, only a weak
non-specific fluorescence was detected with no intense band at 100 kDa (FIG. 2C LEFT). To confirm
that the labeling is a consequence of recognition of the bisubstrate moiety, we performed competition
experiments by adding 100 M of compound 1 or 2 to the MCF-7 cell culture prior to treatment with
the chemical probe 4. Under these conditions, labeling of probe 4 was completely abolished by
competition with the free compounds 1 and 2 (FIG. 2C RIGHT), which confirm that labeling is due to
the affinity of the bisubstrate moiety for this target.
1
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Considering these promising results in MCF-7 cells, other cancer cell lines, i.e. leukemia cell lines K-
562, KG-1 and MOLM-13, and melanoma cell line WM-266-4, were likewise treated in triplicate with
probe 4 (FIG. 3). Similar to that observed in MCF-7 cell line, a band at 100 kDa was strongly labeled
in each of these cell lines. Additionally, other significant bands were observed at about 75 and 50 kDa
for K-562, KG-1 and WM-266-4 and about 50 and 37 kDa for K-562, KG-1 and MOLM-13. DMSO
and probe 5 controls were also analyzed by SDS-PAGE and the absence of fluorescent labeling was
confirmed under these conditions (two examples are shown in FIG. 3 right panels). As previously, total
protein staining with SYPRO Ruby was used as loading control (FIG. 3 lower panel).
-
INSERT Figure 3-
Figure 3. TAMRA fluorescence (upper gels) and total protein staining with SYPRO Ruby stain (bottom gels)
after affinity-based profiling in K-562, KG-1, MOLM-13 and WM-266-4 cells upon 1h treatment by 10 M of
probe 4, control probe 5 or 1% DMSO in triplicate.
Encouraged by these gel-based experiments, we next performed affinity-based pull-down experiments
in order to identify the protein targets of the transition state analogue probe 4. The experiments were
performed in three biological replicates in MCF-7, K-562, KG-1, MOLM-13 and WM-266-4 cell
lines. The active chemical probe 4 was compared to the inactive Boc probe 5.
After probe treatment, UV irradiation and CuAAC functionalization of the probe using the
trifunctional compound TAMRA-biotin-azide, probe-bound proteins were enriched with avidin beads.
After several washings, proteins were trypsin-digested and peptides subjected to LC-MS/MS analysis.
Proteomic analysis
To identify the potential protein targets, quantitative proteomic analysis was carried out on ABPP
samples obtained using chemical probe 4 and control probe 5. The Volcano plot reporting the
comparison results is presented in FIGURE 4 for KG-1 cell line and in supplementary information for
the other cell lines, i.e. MCF-7, K-562, MOLM-13 and WM-266-4 cell lines (GRAPH SI-2 to 5). For
KG-1 cell line, about 540 proteins were identified. However as expected, only 24 proteins were found
statistically over-represented (i.e. Log difference >1 or ratio >2 and -Log 10 p-value >1.33) when the
active probe 4 was compared to the inactive probe 5 containing only the Boc moiety (FIG. 4 and
TABLE SI-1).
-
INSERT Figure 4-
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Figure 4. Volcano plot of the proteomic data obtained following pull-down ABPP experiments with active
chemical probe 4 and inactive probe 5 in KG-1 cells. The statistical significance expression level change (p-
value) is represented as a function of the protein ratio between control and treated samples. Orange dots
represent the proteins with significantly over-represented in the treated sample.
For the other cell lines 165, 112, 401 and 213 proteins were identified with 26, 6, 40 and 19
statistically over-represented in MCF-7, K-562, MOLM-13 and WM-266-4 cell lines, respectively
0
1
2
3
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0
(
GRAPH SI-2 to 5 and TABLE SI-2 to 5)
The different data sets from the five cell lines analyzed were then compared. A Venn diagram analysis
revealed that, several proteins were significantly enriched in at least 3 cell lines (FIG. 5A and B, TABLE
SI-1 to 5).
-INSERT Figure 5-
Figure 5. Significantly over-represented proteins with chemical probe 4. (A) Venn diagram showing the
common and unique proteins statistically over-represented with the active probe 4 compared to the inactive
probe 5. (B) Table of commonly enriched proteins represented by green highlight in different cell lines. (C)
MS/MS counts and ratio values for GRP94 in KG-1, MCF-7, K-562, MOLM-13 and WM-266-4 cell lines.
*Ratio values are reported as a mean of the three replicates.
Among these proteins, HSP90B1, also known as GRP94 (M = 92 kDa), is the only protein found as
w
significantly enriched in all cell lines with high ratios and abundances in probe 4 treated samples
compare to the inactive Boc-containing probe 5 (FIGURE 5 A and C). Therefore, GRP94 represents a
promising target of our active chemical probe.
Target validation
To assess the specificity and the binding property of our probe with GRP94, fluorescence polarization
competition assays with recombinant GRP94 and HSP90α were performed under conditions of
9
equilibrium binding (FIG. 6). As controls, PU-H71, which is a pan-HSP90 inhibitor under conditions
1
0-15
of equilibrium binding but kinetically selects HSP90 residing in epichaperome networks,
and PU-
16-18
WS13, which is selective for GRP94 over other isoforms of HSP90,
were used and showed sub-
1
8
micromolar EC50s, as previously described. Interestingly, the EC50 binding of probe 4 is
approximately 10 M for GRP94 and shows no significant binding to HSP90α up to 50 M, the
highest concentration tested. This assay allows us to confirm that our probe binds to GRP94
specifically compared to HSP90α, confirming the proteomic data and gels. We also assessed
compounds 1 and 2 from which the chemical probe was derived. Although both compounds 1 and 2
showed minor binding to GRP94 at 50 M, no significant binding to HSP90α was observed at this
concentration. Therefore, the absence of the large benzophenone-containing moiety led to a lower
inhibition potency but the GRP94 selectivity is maintained for compounds 1 and 2.
-
INSERT Figure 6-
Figure 6. Fluorescence polarization competition assay for GRP94 (left) and HSP90α (right) performed under
conditions of equilibrium binding. Binding properties of 4, 1 and 2 were assessed and PU-H71 and PU-WS13
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were included as controls. Data are the mean value of two independent experiments run in triplicates (SD are
shown as error bars).
DISCUSSION
Here we applied the ABPP strategy using a chemical probe derived from adenosine-cytosine
conjugates to identify potential binding proteins. Since compounds 1 and 2 have no activity on
DNMT, HMTs and PRMTs, we were interested in synthesizing a chemical probe from them to use as
bait to trap proteins that bind by the APBB strategy. Chemical probe 4 was synthesized and after
incubation with five cancer cell lines, the in-cell UV-crosslinked proteins were labeled following click
chemistry with the fluorescent TAMRA for visualization and biotin for affinity purification. The
TAMRA label revealed, by SDS PAGE, a clear protein-labeling profile, with a common band around
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
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7
8
9
0
1
2
3
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7
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9
0
100 kDa. The proteins crosslinked by probe 4 in the five cancer cell lines were analyzed by
quantitative proteomics in comparison to the control probe 5 lacking the affinity moiety and with
DMSO. The proteomic data analysis showed that, in all tested cancer cell lines, the ATP-binding
molecular chaperone GRP94, of the HSP90 family, was selectively labeled by the chemical probe 4
over the DMSO control and control probe 5. Its detection level was significant and its molecular
weight is consistent with the 100-kDa band observed in SDS-PAGE. Additionally, equilibrative
nucleoside transporter SLC29A1 (ENT1), hydroxyacyl-CoA dehydrogenase trifunctional multienzyme
complex subunit alpha (HADHA), pyruvate carboxylase (PC) and propionyl-CoA carboxylase
(
PCCA) were also identified by proteomic analysis across at least three cell lines (Figure 5B).
SLC29A1 mediates cellular uptake of nucleosides and possess a high affinity for adenosine, chemical
19
moiety present in chemical probe 4. Its activity is inhibited by anticancer agents (dipyridamole and
2
0
dilazep) and in leukemia, a down regulation of SLC29A1 expression, due to a decrease in H3K27
2
1
acetylation, was observed in AraC-resistant cell lines. HADHA catalyzes mitochondrial oxidations
22
producing acetyl-CoA from fatty acids and is a component of RNA silencing machinery. HADHA
2
3
expression level is involved in risk of breast cancer and HADHA has a prognostic value in renal cell
2
4
carcinoma . PC and PCCA are carboxylases of therapeutic interest since the PC is up-regulated in
2
5
breast cancer and associated with growth and invasion
and PCCA dysfunction leads to severe
26
metabolic disorders . These carboxylases possess ATP and nucleotide binding sites. Their
identification is then compatible with the adenosine-cytosine chemical structure of the probe 4.
Despite the interest of these additional targets, GRP94 was selected since its molecular weight is
consistent with 100 kDa in-gel observed band. Therefore, further validation experiments were focused
on GRP94 and fluorescence polarization experiments confirmed selective binding of probe 4 for
GRP94 compared to HSP90α.
-
INSERT Figure 7-
Figure 7. Structures of PU-H71, a pan-HSP90 inhibitor under conditions of equilibrium binding, PU-WS13 and
NECA, GRP94-selective inhibitors over HSP90α and HSP90β and compounds 1, 2 and 4, new GRP94-selective
inhibitors.
GRP94 (encoded by the HSP90β1 gene), belonging to the heat shock protein 90 (HSP90) family, is an
essential chaperone that has ATPase activity enabling the folding of target proteins to their active
1
7, 27
conformation. HSP90s are very promising clinical targets and several clinical trials are ongoing.
Contrary to other HSP90 members that are located in the cytoplasm (HSP90α and HSP90β) or the
mitochondria (Trap-1), GRP94 resides in the endoplasmic reticulum (ER). GRP94 was shown to be
overexpressed in different types of cancers, including breast, colorectal, lung, pancreatic,
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gastrointestinal cancers and multiple myeloma. The expression of GRP94 is associated with
29-32
advanced stage, poor survival and cancer growth and metastasis
and GRP94 was identified as a
33
34, 35
biomarker of gastrointestinal cancer . GRP94 was also shown to be linked with chemoresistance.
All these data provide rationale for the high interest in GRP94 as a therapeutic target for the
development of anticancer therapies. However, the selective inhibition of GRP94 is highly complex
due to the high conservation of the ATP-binding site. Currently, only a few selective GRP94 inhibitors
and none have yet reached clinical evaluation. Thus there is a great interest in
identifying new chemical scaffolds as starting points for new inhibitors.
0
1
2
3
4
5
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4
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0
17, 36
have been reported,
In this work we used an innovative in cell ABPP strategy never applied in an HSP90 context.
Compared to the other chemical biology methods previously reported to study HSP90 binding
3
7
38, 39
properties that used co-immunoprecipitation
or on-beads immobilized affinity scaffold,
the
method reported here is carried out directly in live cells. Thereby, we identified compound 4 as a
GRP94-selective inhibitor over HSP90α. For the GRP94 inhibitor PU-WS13, its selectivity is due to
the occupation by the substituted phenyl ring of an allosteric pocket (FIG. 7 Site 2), which is not
present in HSP90α/β. On the other hand, PU-H71, a pan-HSP90 inhibitor under conditions of
equilibrium binding but kinetically selective for HSP90 residing in epichaperome networks, binds to
18
1
0
Site 1 present in both HSP90 α/β and GRP94. Our compound 4 bears an adenosine moiety (FIG. 7),
40
shared by GRP94-selective inhibitor NECA, that binds to the ATP binding site. Additionally, NECA
4
0, 41
was also shown to interact with Site 3 of GRP94.
From our results, we showed that the addition of
a cytosine substituted with MOM (compound 1) or BOM (compound 2) group to the adenosine moiety
results in a loss of activity. The bulky substituted cytosine might prevent from interacting within Site
. However, the cytosine substitution by a large benzophenone-containing linker leads to active and
selective GRP94 inhibitor. Thus, the affinity of compound 4 might be due, in addition to the presence
of the adenosine moiety, to the flexible and large benzophenone-containing scaffold that could reach
to the similar allosteric pocket as for PU-WS13 (Site 2). However, the reasons for the selectivity of 4
are still not clear and will require further investigation.
3
Therefore, in this study using an innovative in-cell ABPP strategy we identified the adenosine-
substituted cytosine analogues as an interesting starting point for further improvement.
Crystallographic data could give highly valuable information to improve the potency of our
compounds and afford potent and selective GRP94 inhibitors to be used potentially as therapeutic
anticancer agents.
ACKNOWLEDGMENT
This work was supported by PlanCancer2014 France (no. EPIG201401), the Centre national pour la
recherche scientifique (CNRS), the research center Pierre Fabre, the Région Occitanie, Toulouse
Métropole, FEDER (Fonds Européens de Développement Régional) and the French Ministry of
Research (Programme Investissement d’Avenir, Infrastructures Nationales en Biologie et Santé,
Proteomics French Infrastructure project, ANR 10-INBS-08). This work was also funded in part by
P01 CA186866 and P30 CA008748 (NCI Core Facility Grant). The authors thank Dr. T. Taldone
(
Sloan Kettering Institute) for discussion and proofreading of the manuscript.
CONFLICT OF INTEREST
G.C. has partial ownership in Samus Therapeutics Inc.
METHODS
Chemical Synthesis
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All chemicals were purchased from Sigma-Aldrich or Alfa Aesar. PU-H71, PU-WS13 and GM-cy3B
were synthesized and characterized as previously reported.
10, 17, 18, 42, 43
1
3
The NMR spectra were recorded on a Bruker Avance II spectrometer equipped with a C cryoprobe at
1
13
5
00 MHz for H and 125 MHz for C; 2D experiments were performed using standard Bruker
programs and atoms attribution was performed thanks to 2D correlations. Chemical shifts are given in
ppm. Coupling constants J are measured in Hz. Splitting patterns are designed as follows: s, singlet; bs
broad singlet; d, doublet; bd, broad doublet; t, triplet; brt, broad triplet; dd, doublet of a doublet; m,
multiplet; ddd, doublet of a doublet of a doublet; q, quartet; quint, quintet, sext, sextet.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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0
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1
2
3
4
5
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7
8
9
0
1
2
3
4
5
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8
9
0
HRMS-ESI were obtained on a Bruker MicroTOF.
Column chromatography was carried out on a puriflash 430 apparatus (Interchim) equipped with 30
m porated silica column.
Semi preparative HPLC was performed on an apparatus equipped with a VWR International LaPrep
pump P110, a VWR LaPrep P314 Dual l absorbance detector and EZChrom software. C18 reversed-
phase column (Waters x-bridge, RP-18, 25 x 250 mm, 5 m) were used for semi preparative HPLC
with a binary gradient elution (solvent A: H
flow rate of 20 mL.min and the chromatogram was monitored at 250 and 320 nm.
2
O/0.01% TEA and solvent B: CH CN/0.01% TEA), a
3
-1
General procedure for synthesis of compounds 1, 2 and 3
Water (100 l) was added to a solution of 12 (102 mg; 0.20 mmol), 13 (126 mg; 0.22 mmol) or 14
121 mg; 0.20 mmol) in TFA (1 mL). The reaction mixtures were stirred at room temperature for 1h.
(
The solvent was removed and the residues were co-evaporated three times with 1N ammonia in
methanol under reduce pressure. The residues were purified by reversed phase HPLC using a linear
gradient H O/acetonitrile:100/0→20/80 with 0.2% of TEA to give the desired products.
2
1
-(methoxymethyl)-N-((5’-deoxyadenosin-5’-yl)methyl)cytosine-5-carboxamide (1)
Compound 1 was obtained as a white amorphous solid (74 mg; 0.16 mmol; 80%).
NH
a3 a2
2
N
N
N
a5
a1
a4
N
H
a10
O
c1
c2
N
O
a6
a7
a9
a8
a11
H
2
N
c5
c3
O
HO OH
N
N
c4
c6
c7
O
1H NMR (500 MHz, DMSO-d
NH), 7.79 (s, 1H, HNH), 7.30 (s, 2H, HNH), 5.88 (d, J= 5.0 Hz, 1H, Ha6), 5.49 (d, J= 5.8 Hz, 1H, HOH),
.22 (d, J= 5.5 Hz, 1H, HOH), 5.04 (s, 2H, Hc6), 4.66 (q, J= 5.3 Hz, 1H, Ha7), 4.10 (q, J= 5.4 Hz, 1H,
6
) δ 8.43-8.27 (m, 3H, 1HNH, Ha5, Hc3), 8.16 (s, 1H, Ha1), 7.79 (brs, 1H,
H
5
Ha8), 3.93 (dt, J= 4.4, 9.0 Hz, 1H, Ha9), 3.33-3.29 (m, 1H, Ha11), 3.28 (s, 3H, Hc7), 3.24-3.16 (m, 1H,
Ha11), 2.04-1.80 (m, 2H, Ha10).
13C NMR (125 MHz, DMSO-d
149.8 (Ca4), 148.4 (Cc1), 140.3 (Ca5), 119.6 (Ca3), 99.1 (Cc2), 88.1 (Ca6), 81.0 (Ca9), 79.6 (Cc6), 73.8
a7), 73.5 (Ca8), 56.7 (Cc7), 36.4 (Ca11), 33.4 (Ca10).
) δ 165.3 (Cc5), 164.5 (Cc1), 156.5 (Ca2), 154.5 (Cc4), 153.1 (Ca1),
6
(
C
+
HRMS-ESI (m/z) calculated for C18
H
24
N
9
O [M+H] : 462.1844; found: 462.1942.
6
1
-(benzoxymethyl)-N-(5’-deoxyadenosin-5’-yl)methyl)cytosine-5-carboxamide (2)
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5
5
5
6
Compound 2 was obtained as a white amorphous solid (96 mg; 0.18 mmol; 81%).
NH
a3 a2
2
N
N
N
a5
a1
a4
N
H
a10
O
c1
c2
N
O
a6
a7
a9
a8
a11
H
2
N
c5
c3
O
HO OH
N
N
c4
c6
O
c7
c8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c9
c10
c11
1H NMR (500 MHz, DMSO-d
a1), 7.81 (brs, 1H, HNH), 7.39-7.21 (m, 7H, Hc9, Hc10, Hc11, 2HNH), 5.88 (d, J= 5.0 Hz, 1H, Ha6), 5.49
d, J= 5.8 Hz, 1H, HOH), 5.25-5.16 (m, 3H, HOH, Hc6), 4.66 (ddd, J= 5.3, 10.9 Hz, 1H, Ha7), 4.60 (s,
6
) δ 8.42 (s, 1H, Hc3), 8.39-8.26 (m, 3H, 2HNH and Ha5), 8.16 (s, 1H,
H
(
2H, Hc7), 4.14-4,08 (m, 1H, Ha8), 3.94 (dt, J= 4.5, 9.1 Hz, 1H, Ha9), 3.34-3.26 (m, 1H, Ha11), 3.23-3.18
(m, 1H, Ha11), 2.01-1.82 (m, 2H, Ha10).
13C NMR (125 MHz, DMSO-d
148.9 (Ca4), 147.3 (Cc3), 142.5 (Ca5), 137.6 (Cc8), 128.7 (Cc10), 128.3 (Cc11), 128.2 (Cc9), 119.4 (Ca3),
6
) δ 163.6 (Cc5), 160.3 (Cc1), 152.1 (Ca4), 150.3 (Cc2), 149.0 (Ca1),
9
3
8.7 (Cc2), 88.4 (Ca6), 82.2 (Ca9), 78.6 (Cc6), 73.9 (Ca7), 73.8 (Ca8), 71.4 (Cc7), 55.5 (Cc7), 36.5 (Ca11),
3.2 (Ca10).
+
HRMS-ESI (m/z) calculated for C24
H
28
N
9
O
7
[M+H] : 538.2757; found: 538.2161.
1
-(2-aminoethyl)-N-(5’-deoxyadenosin-5’-yl)methyl)cytosine-5-carboxamide (3)
Compound 3 was obtained as a white amorphous solid (69 mg; 0.15 mmol; 75%).
NH
a3 a2
2
N
N
N
a5
a1
a4
N
H
a10
O
c1
c2
N
O
a6
a7
a9
a8
a11
c3
H
2
N
c5
HO OH
N
N
c6
c4
O
c7
NH
2
1H NMR (500 MHz, DMSO-d
.18-8.06 (m, 3H, Ha1 and 2HNH), 7.57 (d, J= 23.6 Hz, 1H, HNH), 7.30 (s, 2H, HNH), 5.87 (d, J= 4.9 Hz,
1H, Ha6), 5,65-5,04 (m, 2H, HNH), 4.66 (t, J= 5.2 Hz, 1H, Ha7), 4.11 (t, J= 5.0 Hz, 1H, Ha6), 3.94 (tt, J=
6
) δ 8.35 (s, 1H, Hc3), 8.28 (s, 1H, Ha5), 8.24 (t, J= 5.1 Hz, 1H, HNH),
8
9
2
.0, 4.3 Hz, 1H, Ha9), 3.68 (t, J= 6.2 Hz, 2H, Hc6), 3.24-3.14 (m, 2H, H, Ha11), 2.88-2.70 (m, 2H, Hc7),
.03-1.79 (m, 2H, Ha10),
13C NMR (125 MHz, DMSO-d
49.8 (Ca4), 149.7 (Ca1), 140.3 (Cc3), 119.6 (Ca3), 97.8 (Cc2), 88.1 (Ca6), 81.8 (Ca9), 73.8 (Ca6), 73.5
a7), 52.5 (Cc6), 40.5 (Cc7), 36.3 (Ca11), 33.5 (Ca10).
) δ 165.7 (Cc5), 164.4 (Cc1), 156.5 (Ca2), 154.7 (Cc4), 153.1 (Ca1),
6
1
(
C
+
HRMS-ESI (m/z) calculated for C H N O [M+H] : 461.2004; found: 461.2011.
1
8 25 10 5
Synthesis of chemical probe 4
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To a solution of 4-((4-(4-(hex-5-ynamido)benzoyl)phenyl)amino)-4-oxobutanoic acid 16 (6.7 mg; 16
mol), HATU (8 mg; 20 mol) and DIPEA (7 l; 38 mol) in DMF (0.4 mL) was added 3 (6.3 mg; 4
mol). The mixture was stirred at room temperature for 16 hours. The residue was then directly
purified by reversed phase HPLC using a linear gradient H O/acetonitrile:100/0→20/80 with 0.01% of
2
TEA to afford 4 as a amorphous white solid (8.3 mg; 9.8 mol; 72%).
1H NMR (500 MHz, DMSO) δ 10.33 (s, 1H, HNH), 10.30 (s, 1H, HNH), 8.32 (s, 1H, Ha5), 8.28 (s, 1H,
1
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H
(
5
c3), 8.24 (brt, 1H, HNH), 8.14 (s, 1H, Ha1), 8.08 (brt, 1H, HNH), 7.77-7.71 (m, 4H, Hp9, Hp13), 7.71-7.66
m, 4H, Hp8, Hp14), 7.28 (brs, 2H, HNH2), 5.86 (d, J= 5.1 Hz, 1H, Ha6), 5.51-5.46 (m, 1H, HOH), 5.24-
.18 (m, 1H, HOH), 4.64 (t, J= 4.9 Hz, 1H, Ha7), 4.08 (t, J= 5.2 Hz, 1H, Ha8), 3.98-3.91 (m, 1H, Ha9),
3.72 (t, J= 5.9 Hz, 2H, Ha11), 3.32-3.26 (m, 3H, Hc6 and Hc7), 3.21-3.12 (m, 1H, Hc7), 2.83 (t, J= 2.7
Hz, 1H, Hp1), 2.61 (t, J= 7.0 Hz, 2H, Hp17), 2.48 (t, J= 7.4 Hz, 2H, Hp5), 2.41 (t, J= 7.1 Hz, 2H, Hp18),
2
.24 (dt, J= 2.8, 7.3 Hz, 2H, Hp3), 1.96-1.81 (m, 2H, Ha10), 1.78 (quint, J= 7.4Hz, 2H, Hp4).
13C NMR (125 MHz, DMSO) δ 193.4 (Cp11), 171.6 (Cp19), 171.2 (Cp6, Cp16), 165.2 (Cc1), 164.1 (Cc4),
56.07 (Cc5), 154.1 (Ca2), 152.7 (Ca1), 149.4 (Ca4), 149.0 (Cc3), 143.0 (Cp15, Cp7), 139.8 (Ca5), 131.7
1
(
(
C
p10 and Cp12), 130.9 (Cp9 and Cp13), 119.2 (Ca3), 118.2 (Cp8 and Cp14), 97.6 (Cc2), 87.7 (Ca6), 83.9
Cp2), 81.3 (Ca9), 73.4 (Ca7), 73.1 (Ca8), 71.8 (Cp1), 49.2 (Ca11), 37.8 (Cc6), 35.8 (Cc7), 35.2 (Cp5), 33.1
(Ca10), 31.6 (Cp17), 30.0 (Cp18), 23.8 (Cp4), 17.3 (Cp3).
+
HRMS-ESI (m/z) calculated for C41
H
45
N
12
O [M+H] : 849.3427; found: 849.3436.
9
N-(4-(4-(5-(Boc-amino)pentanamido)benzoyl)phenyl)hex-5-ynamide (5)
HATU (396.0 mg; 1.04 mmol) was solubilized in DMA (2 mL) at RT under argon. The carboxylic
acid 17 (215.0 mg; 0.99 mmol) and DIPEA (0.19 mL; 1.39 mmol) was added to the solution. After 20
min of stirring at room temperature, the compound 15 (198.9 g; 0.65 mmol) was then added. The
reaction mixture was stirred at room temperature for 16 h. The residue was diluted with ethyl acetate
and washed with water and brine, and dried over sodium sulfate. The solvent was removed and the
residue was purified by silica gel flash chromatography using a linear gradient of cyclohexane/ethyl
acetate:0/100→80/20 to afford the title compound 5 as an amorphous white solid (309.8 mg; 0.57
mmol; 88% yield).
1H NMR (500 MHz, DMSO) δ 10.31 (s, 1H, HNH), 10.25 (s, 1H, HNH), 7.78-7.67 (m, 8H, Hp8, Hp9,
Hp13 and Hp14), 6.82 (t, J= 5.5 Hz, 1H, HNH), 2.93 (q, J= 6.5 Hz, 2H, Hp20), 2.83 (t, J= 2.7 Hz, 1H, Hp1),
2.48 (t, J= 7.7 Hz, 2H, Hp5), 2.35 (t, J= 7.5 Hz, 2H, Hp17), 2.24 (dt, J= 2.5, 7.0 Hz, 2H, Hp3), 1.78
(
(
quint, J= 7.2 Hz, 2H, Hp4), 1.58 (quint, J= 7.7 Hz, 2H, Hp18), 1.41 (quint, J= 7.2 Hz, 2H, Hp19), 1.37
s, 9H, HBoc).
13C NMR (125 MHz, DMSO) δ 193.4 (C
p11), 171.8 (Cp6), 171.2 (Cp16), 155.6 (CBoc), 143.1 (Cp7), 143
p15), 131.7 (Cp10, Cp12), 130.9 (Cp13, Cp9), 118.2 (Cp8, Cp14), 84 (Cp2), 77.4 (CBoc), 71.7 (Cp1), 39.4
p20), 36.2 (Cp17), 35.2 (Cp5), 29.1 (Cp19), 28.2 (CBoc), 23.8 (Cp4), 22.4 (Cp18), 17.3 (Cp3).
(
(
C
C
+
HRMS-ESI (m/z) calculated for C29
H
35
N
3
O
5
Na [M+Na] : 528.2469; found: 528.2471.
Cell culture
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KG-1, MOLM-13 and K-562 human leukemia cell lines, WM-266-4 human melanoma cancer cells
and MCF-7 human breast cancer cells were obtained from ATCC (USA). Cells were grown in RPMI
1
640 GlutaMAX™ (ThermoFisher Scientific) medium containing 10% FCS (Lonza) for K-562 and
MCF-7 cells and 20% for KG-1 and MOLM-13 cells, WM-266-4 cell line was grown in EMEM
containing 10% FCS at 37 °C and under 5% CO
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In situ labeling of cancer cells for in-gel visualization
For probe labeling assays, suspension cells were counted and adherent cells were grown until
approximately 90-80% confluent before treatment. They were first washed twice with PBS and
resuspended in their medium without FCS for the incubation with probes. Probe was added at desired
concentration and incubated for 1 h at 37 °C. Chemical probe 4 was used for measurement sample and
chemical probe 5 was used as control experiments for proteomic analysis. DMSO (1% v/v) was added
in control samples. Competitor (1 or 2) was added 30 min after the test compound to chase. Following
treatment, cells were washed once with PBS and resuspended in fresh medium for the UV-irradiation
step. After 1 h of exposition at 365 nm, cells were lysed in lysis buffer (RIPA buffer (Sigma-Aldrich)
+
cOmplete™, EDTA-free Protease Inhibitor Cocktail (PIC) (Roche Diagnostics)) on ice for 15 min
followed by 3 min of sonication (30 s / 30 s on/off, medium power, Diagenode Bioruptor®). Protein
concentrations were determined and proteome samples were diluted in PBS at a final concentration of
-1
1
1
mg.mL for the CuAAC ligation. The premix reagents (60 M TAMRA-biotin-azide, 1 mM CuSO
4
,
mM TCEP and 100 M TBTA, final concentrations) were added to the lysate. The samples were
agitated for 1h at room temperature. The proteins were next precipitated by addition of
methanol/chloroform/water (2/0.5/1) and pelleted by centrifugation at 15,000 × g for 5 min. After
removal of supernatant, protein pellet was further washed in methanol and resuspended in PBS buffer
containing 2% SDS and 10 mM DTT. Läemmli sample buffer was then added and the samples were
heated for 10 min at 95 °C, separated by 1D SDS-PAGE and visualized using Typhoon 9410 (GE
Healthcare).
In situ labeling of cancer cells for gel and MS analysis
In parallel to SDS-PAGE visualization, samples were enriched on avidin-coupled agarose beads
(
Pierce™ NeutrAvidin™ Agarose, ThermoFisher Scientific) (50 L, pre-washed three times in 0.2%
SDS in PBS) by incubation with gentle shaking for 2 h at room temperature to bind and enrich biotin-
labeled proteins. The supernatant was then removed and the beads were washed with 1% SDS in PBS
(
3 x 1 mL), 4 M urea in 50 mM ammonium bicarbonate (AMBIC) (2 x 1 mL) and 50 mM AMBIC (5
x 1 mL).
On-bead proteins were then resuspended in 50 L of 50 mM AMBIC and reduced with 10 mM
dithiothreitol at 55 °C for 30 min. After on-bead reduction, samples were washed twice with 50 mM
AMBIC and again resuspended in 50 L of 50 mM AMBIC for alkylation of cysteines by 10 mM
iodoacetamide in the dark for 30 min. Beads were then washed twice with 50 mM AMBIC and
resuspended in 50 l of 50 mM AMBIC for protein digestion with trypsin (1 g, Promega), overnight
at 37 °C. Supernatant was kept in clean Eppendorf and beads were washed with 50 mM AMBIC
followed by 0.1% TFA in H O. Supernatants of each washing step were combined, evaporated to
2
dryness and resuspended for desalting step before LC-MS/MS analysis.
Peptides were analyzed by nanoLC-MS/MS using an UltiMate 3000 RSLCnano system coupled to an
Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). 5 l of each sample
were loaded on a C-18 precolumn (300 m ID x 5 mm, Thermo Fisher) in water containing 5%
-1
acetonitrile and 0.05% TFA and at a flow rate of 20 L.min . After 5 min of desalting, the precolumn
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was switched online with the analytical C-18 column (75 m ID x 50 cm, Reprosil C18) equilibrated
in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2%
formic acid). Peptides were eluted using a 5 to 50% gradient of solvent B over 105 min at a flow rate
of 300 nL.min . The orbitrap Velos was operated in a data-dependent acquisition mode with the
XCalibur software. Survey scan MS were acquired in the Orbitrap on the 350-1800 m/z range with the
resolution set to a value of 60000. The 20 most intense ions per survey scan were selected for CID
fragmentation. Dynamic exclusion was employed within 60s to prevent repetitive selection of the
same peptide.
-1
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Raw mass spectrometry files were processed with the MaxQuant software (version 1.5.2.8) for
database search with the Andromeda search engine and quantitative analysis. Data were searched
against human entries of the Swissprot protein database. Carbamidomethylation of cysteines was set as
a fixed modification whereas oxidation of methionine and protein N-terminal acetylation were set as
variable modifications. Specificity of trypsin digestion was set for cleavage after K or R, and two
missed trypsin cleavage sites were allowed. The precursor mass tolerance was set to 20 ppm for the
first search and 4.5 ppm for the main Andromeda database search. The mass tolerances MS/MS mode
was set to 0.5 Da. Andromeda results were validated by the target-decoy approach using a reverse
database at both a peptide and a protein FDR of 1%. For label-free relative quantification of the
samples, the “match between runs” option of MaxQuant was enabled with a time window of 0.7 min,
to allow cross-assignment of MS features detected in the different runs.
Specifically labeled proteins were identified by comparison to the alkyne probe-treated samples with
the controls. The “LFQ” metric from the MaxQuant “protein group.txt” output was used and ratio
between alkyne probe and controls were calculated. According to different thresholds/filters, proteins
were considered specific if ratios are > 2 or Log difference is > 1, Log10 p-value > 1.33, the sum of
MS2 count of probe/control > 5 and then if there is a statistically significant difference (Student’s t–
test, p = 0.05).
FP competition assay under conditions of equilibrium binding
The GRP94 and HSP90α FP competition assays were performed on an Analyst GT instrument
(
Molecular Devices, Sunnyvale, CA) and carried out in black 96-well microplates (Corning, no. 3650)
9
, 44
in a total volume of 100 L in each well as previously described
. A stock of 10 M Cy3B-GM was
prepared in DMSO and diluted with Felts buffer [20 mM HEPES (K) pH 7.3, 50 mM KCl, 2 mM
-1
DTT, 5 mM MgCl
2
, 20 mM Na
2
MoO
4
, and 0.01% NP40 with 0.1 mg.mL BGG]. To each well was
added the fluorescent dye labeled ligand (6 nM Cy3B-GM for GRP94 and HSP90α), recombinant
protein (10 nM GRP94 or HSP90α) and the tested inhibitor (initial stock in DMSO) in a final volume
of 100 L of Felts buffer. Compounds were added in duplicate wells. For each assay, background
wells (buffer only), tracer controls (free, fluorescent dye labeled ligand only), and bound controls
(
fluorescent dye labeled ligand in the presence of protein) were included on each assay plate. The
assay plate was incubated on a shaker at 4 °C for 24 h and the FP values (in mP) were measured. The
fraction of fluorescent dye labeled ligand bound to GRP94 or HSP90α was correlated to the mP value
and plotted against values of competitor concentrations. The inhibitor concentration at which 50% of
bound fluorescent dye labeled ligand got displaced was obtained by fitting the data. For Cy3B-GM, an
excitation filter at 530 nm and an emission filter at 580 nm were used with a dichroic mirror of 561
nm. All experimental data were analyzed using SoftMax Pro 6.3 and plotted using Prism 6.0
(
Graphpad Software Inc., San Diego, CA) and binding affinity values are given as relative binding
affinity values (EC50, concentration at which 50% of fluorescent ligand was competed off by
compound). PU-H71 and PU-WS13 were included as controls.
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6
SUPPORTING INFORMATION AVAILABLE
Synthesis and characterization of compounds 6-17. H and 13C NMR spectra of compounds 1-5.
Inhibition percentages of various DNMT, KHMTs and PRMTs for compounds 1-4. Table of
proteomic analysis result tables (Table SI-1 to SI-5) and volcano plot (Graph SI-1 to SI-5). This
material is available free of charge via the Internet.
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Guianvarc'h, D., Ferroud, C., and Arimondo, P. B. (2017) Rational design of bisubstrate-type
analogs as inhibitors of DNA methyltransferases in cancer cells, J. Med. Chem. 60, 4665-4679.
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Cerchietti, L., Levine, R., Melnick, A., Larson, S. M., Lewis, J. S., Guzman, M. L., and Chiosis,
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Wang, T., Lecomte, N., Janjigian, Y., Younes, A., Batlevi, C. W., Guzman, M. L., Roboz, G. J.,
Koziorowski, J., Zanzonico, P., Alpaugh, M. L., Corben, A., Modi, S., Norton, L., Larson, S. M.,
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