Design and synthesis of tag-free photoprobes for the identification of the molecular target for CCG-1423, a novel inhibitor of the Rho/MKL1/SRF signaling pathway

Article abstract of DOI:10.3762/bjoc.9.111

CCG-1423 and related analogues represent a new class of inhibitors of Rho/MKL1/SRF-mediated gene transcription, a pathway that has been implicated in both cancer and fibrosis. The molecular target for these compounds is unknown. To facilitate its identification, a series of tag-free photoaffinity probes was designed and synthesized, each one containing a photoactivatable group and an acetylenic end group for subsequent attachment to a fluorescent tag using click chemistry. All were confirmed to maintain biological activity in a cell-based assay for inhibition of SRE-Luc expression. The functional activity of the most potent probe 24 was further confirmed in an assay for PC-3 cell migration. Photolysis of 24 in intact PC-3 cells followed by cell lysis, click ligation of a fluorescent dye, and gel electrophoresis revealed specific labeling of a single 24 kDa band that could be blocked with an active competitor. Future work will focus on identifying the labeled protein(s).

Full text of DOI:10.3762/bjoc.9.111

Design and synthesis of tag-free photoprobes for the
identification of the molecular target for CCG-1423,
a novel inhibitor of the Rho/MKL1/SRF
signaling pathway
Jessica L. Bell1, Andrew J. Haak2, Susan M. Wade2, Yihan Sun3,
Richard R. Neubig2 and Scott D. Larsen*1
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 966–973.
1Vahlteich Medicinal Chemistry Core, Department of Medicinal
Chemistry, College of Pharmacy, University of Michigan, Ann Arbor,
MI 48109, USA, 2Department of Pharmacology, University of
Michigan Medical School, University of Michigan, Ann Arbor, MI
48109, USA and 3College of Pharmacy, University of Michigan, Ann
Arbor, MI 48109, USA
doi:10.3762/bjoc.9.111
Received: 13 February 2013
Accepted: 26 April 2013
Published: 21 May 2013
This article is part of the Thematic Series "Synthetic probes for the study
of biological function".
Email:
Scott D. Larsen* - sdlarsen@umich.edu
Guest Editor: J. Aubé
* Corresponding author
© 2013 Bell et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
CCG-1423; click ligation; photoaffinity labeling; Rho pathway inhibitor;
tag-free photoprobe; target identification
Abstract
CCG-1423 and related analogues represent a new class of inhibitors of Rho/MKL1/SRF-mediated gene transcription, a pathway
that has been implicated in both cancer and fibrosis. The molecular target for these compounds is unknown. To facilitate its identifi-
cation, a series of tag-free photoaffinity probes was designed and synthesized, each one containing a photoactivatable group and an
acetylenic end group for subsequent attachment to a fluorescent tag using click chemistry. All were confirmed to maintain bio-
logical activity in a cell-based assay for inhibition of SRE-Luc expression. The functional activity of the most potent probe 24 was
further confirmed in an assay for PC-3 cell migration. Photolysis of 24 in intact PC-3 cells followed by cell lysis, click ligation of a
fluorescent dye, and gel electrophoresis revealed specific labeling of a single 24 kDa band that could be blocked with an active
competitor. Future work will focus on identifying the labeled protein(s).
Findings
Serum-induced signaling through Rho leads to gene-transcrip- early and muscle-specific genes. These signals involve release
tional effects, which are mediated by serum response factor from cytosolic actin of the transcriptional coactivator MKL1,
(SRF), a MADS box transcription factor that binds to the serum first identified in megakaryocytic leukemia (also known as
response element (SRE) in the promoters of various immediate- myocardin-related transcription factor MRTF-A). Upon acti-
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Beilstein J. Org. Chem. 2013, 9, 966–973.
vation, MKL1 translocates to the nucleus where it binds to SRF would no longer retain its affinity for 2. There are in fact reports
to collaborate in the activation of the transcription of a number of target ID studies that were only successful when performed
of genes including c-fos and SRF itself [1-4]. This pathway has using intact cells [14-16]. We considered this an ideal approach
been implicated in cancer metastasis [5] and, more recently, in in light of our current hypothesis regarding the mechanism of
fibrosis [6,7], making it an intriguing drug target.
action of 1 (redistribution of MKL primarily into the cytosol),
which may require an intact actin cytoskeleton or nucleus. We
We recently reported a series of compounds that inhibit gene thus elected to design photoaffinity probes that were tag-free,
expression mediated by this Rho/MKL1/SRF signaling pathway i.e., lacking either a biotin or fluorescent tag [17]. There are a
[8]. The lead CCG-1423 (1, Figure 1) was identified in a cell- number of distinct advantages to this approach: (1) the probes
based high-throughput screen as an inhibitor of expression of a would retain low molecular weights and polar surface areas, and
luciferase reporter gene driven by the serum response element therefore good cell permeability, (2) minimal structural change
promoter (SRE-Luc) [9]. Analysis of the mechanism of 1 would maximize the likelihood of maintaining affinity for the
showed that it acts at or downstream of MKL1 but upstream of target; and (3) affinity for the target could be confirmed in cell-
SRF. Recent observations from our lab and others [10] have based phenotypic assays prior to any photolabeling studies.
shown that 1 blocks MKL1 nuclear localization. The specific Tag-free photoprobes have also been reported to result in much
molecular target leading to inhibition of MKL1 function by 1, less nonspecific binding relative to corresponding biotinylated
however, is not known.
photoprobes [18].
Identification of the macromolecular target(s) of 1 would allow The design of our tag-free photoprobes followed the model
us to employ rational and structure-based drug design to create pioneered by the Cravatt group in performing activity-based
more potent and selective therapeutics for the treatment of protein profiling (ABPP) [19,20]. This entails the incorporation
RhoA-related disorders. Early efforts at optimization in our of a ligand for recognition by the target, a reactive functionality
laboratory led to nipecotic (bis)amide analogue 2 (CCG- for covalently bonding to the target, and either an azide or
100602, Figure 1), which was less potent (IC50 9.8 µM versus acetylene moiety as a latent linker for subsequent ligation of a
1.5 µM), but maintained similar maximal efficacy to that of 1 tag for isolation and/or visualization (e.g., biotin or fluorophore)
with significantly less cytotoxicity [8]. We therefore selected 2 via click chemistry. This technology has been highly successful
as a template for the design of affinity probes.
in profiling enzyme activity in living cells and even in whole
organisms [21]. In ABPP, covalent linkage by the reactive func-
The successful isolation/identification of molecular targets for tionality is usually dependent upon a particular enzymatic reac-
low-affinity ligands and/or low-abundance targets requires tion, but photoactivatable groups (PGs) have also been used
photolabeling [11]. This permits extensive isolation and purifi- when this is not possible [22].
cation without premature dissociation from the target. Based on
the structure–activity relationship (SAR) of 2, we hypothesized We envisioned adapting this technology to intracellular target
that we could integrate photoactivatable functional groups identification as depicted in Figure 2. Whole cells would be in-
without loss of activity. A variety of such groups that differ in cubated with a photoprobe (A) after confirmation of its bio-
their reactivities and chemoselectivities are available [12,13].
logical activity. Following exposure to UV light, the cells
would be lysed, releasing the labeled proteins bound covalently
Photolabeling of cellular targets is typically done after cell lysis. to the probe (B). Click chemistry would then be applied to
However, if the molecular target for 2 is a multiprotein com- covalently attach a biotin or fluorescent tag for visualization
plex, membrane-bound, or integral to the nucleus, the process of and/or isolation (C). Any isolated proteins would be digested
cell lysis could compromise its integrity to such an extent that it and identified by high-resolution mass-spectral analysis.
Figure 1: Structures of lead Rho/MKL1/SRF inhibitor 1 and conformationally restricted analogue 2.
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Beilstein J. Org. Chem. 2013, 9, 966–973.
We selected benzophenone and azide as the PGs for incorpor-
ation into 2 based on synthetic ease and their complementary
photoreactivities (benzophenone inserts into C–H bonds, azide
into heteroatom–H bonds) [12]. Furthermore, we had previ-
ously established that benzophenone is tolerated on the benz-
amide ring with retention of the biological activity (3, Table 1)
[23]. In addition to a PG, we needed a suitable linking function-
ality for attachment of a clickable acetylene group. Preliminary
work (data not shown) established that an ether (versus amide)
linker was superior with regard to maintenance of biological
activity. A small set of model compounds was thus prepared to
evaluate the impact of ether linker and PG placement on bio-
logical activity (see below in Table 1).
Scheme 1 depicts the general synthesis of model benzophenone-
probes 8. Boc-protected nipecotic acid (4) was reacted with
substituted anilines 5 under standard EDC-mediated amidation
conditions, followed by TFA-catalyzed deprotection to afford
amides 6. A subsequent second amidation with benzoic acids 7
afforded final bis(amide) analogues 8.
We also synthesized a model azide (Scheme 2). Synthesis began
with the acetylation of 4-chloro-3-nitroaniline (9) followed by
reduction of the nitro group using iron and hydrochloric acid to
Figure 2: Strategy for tag-free photolabeling in whole cells (PG =
photoactivatable group, TAG = fluorescent group or biotin).
Scheme 1: General synthesis of model benzophenone probes.
Scheme 2: Synthesis of aryl azide model probe 14.
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Beilstein J. Org. Chem. 2013, 9, 966–973.
generate aniline 10. The azido group was introduced by diazoti- Preparation of the photoaffinity probe 19 is shown in Scheme 3.
zation/azidation to provide 11. Deacetylation with potassium Aniline 15 was Boc-protected [24], and then alkylated using
hydroxide revealed aniline 12, which was then coupled with 4 propargyl bromide to produce ether 16. Following deprotection,
and deprotected to give amine 13. Final amidation with aniline 17 was coupled with Boc-protected nipecotic acid (4),
3-methoxy-5-trifluoromethylbenzoic acid provided model probe giving amide 18 after deprotection. Final coupling with
14.
3-benzoylbenzoic acid afforded the final probe 19.
Table 1 presents biological activity data for all of the new Synthesis of a photoprobe based on the most active model 8c is
model probes. These include: potency at inhibiting the Rho/ shown in Scheme 4. Acid 20 was esterified prior to alkylation
MKL1/SRF-driven expression of a luciferase reporter gene with propargyl bromide, affording ester 22. Saponification, fol-
(IC50 SRE.L) and a measure of maximal efficacy, as indicated lowed by amidation with the previously prepared piperidine 6c,
by percent inhibition of SRE.L at the maximum dose tested provided the final probe 24.
(100 μM) [8,9]. We have noted in our previous work that both
of these parameters are important for predicting activity in Finally, two azide photoprobes were prepared. The first one, 25
inhibiting cell migration [8]. As shown in Table 1, all of the (Scheme 5), based on model 14, was synthesized in a straight-
model probes retained good activity relative to the lead com- forward manner by simply amidating acid 23 with previously
pound 2 with little to no cytotoxicity. Based on these results we prepared azide-containing piperdine 13 (Scheme 2). The second
decided to install acetylenes in our final probes through ether probe, 28, reversing the placement of the azide and acetylene,
linkages at the positions of the methoxy groups of the most was prepared from aniline 26. Introduction of the azide was
active models 8a, 8c and 14.
accomplished via diazotization/azidation, followed by standard
Table 1: Effects of model photoprobes on transcription and cytotoxicity in PC-3 cellsa.
compound
structure
IC50 SRE.L
(µM)b
% inhibition SRE.L
(100 µM)b
% inhibition WST-1
(100 µM)c
3
9.9
8.3
11
75
84
64
0
0
0
8a
8b
8c
14
5.3
7.0
60
77
0
13
aFor detailed assay descriptions, see Evelyn et al. [8,9]; bInhibition of Rho-pathway selective serum response element-luciferase reporter gene
expression; cInhibition of mitochondrial metabolism of WST-1.
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Beilstein J. Org. Chem. 2013, 9, 966–973.
Scheme 3: Synthesis of benzophenone photoaffinity probe 19.
Scheme 4: Synthesis of benzophenone photoaffinity probe 24.
Scheme 5: Synthesis of aryl azide photoaffinity probes.
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Beilstein J. Org. Chem. 2013, 9, 966–973.
amide coupling of 27 and 18 to afford final aryl azide photo- tion into the scratch wound with an IC50 (4.7 μM) comparable
probe 28.
to its potency at inhibiting SRE.L (2.7 μM).
Table 2 summaries the biological data for the four final candi- Preliminary photolabeling studies with 24 were undertaken in
date photoprobes. Fortuitously, all of the probes remained PC-3 prostate cancer cells. Intact cells were treated with 0.3 µM
active in our assay, although the benzophenone analogues 19 24 for 30 min. To facilitate the identification of specifically
and 24 were more potent than the azide probes 25 and 28. labeled proteins, a parallel competition experiment was also
Based on its superior potency, we selected benzophenone 24 performed by treating cells with 0.3 µM 24 and a large excess
(IC50 = 2.7 μM) for further studies.
(10 µM) of 29 (Figure 4), i.e., an analogue of 2 with slightly
greater potency (IC50 = 6.4 µM) that was identified in later
Prior to photolabeling, we wanted to confirm retention of func- SAR studies [23]. UV irradiation was applied for 30 min at
tional activity in a PC-3 prostate cancer cell model of migration. room temperature by using the long-wavelength setting of a
Cells (5.0 × 105) were plated in DMEM containing 10% FBS hand-held illuminator (366 nm, UVL-56 lamp, UltraViolet
and grown to confluence in a 12-well plate. After 24 h, a scratch Products, San Gabriel CA). Cells were then lysed and subse-
was made using a 200 µL pipette tip. Medium was replaced quently fluorescently tagged by using an InvitrogenTM Click-
with DMEM containing 0.5% FBS and varying concentrations iT® Reaction Buffer Kit to attach azido-Cy5.5 dye for visualiza-
of 24 or 0.1% DMSO control. Images of the wound were taken tion.
at the beginning of the experiment by using a bright-field
inverted microscope (Leica DM IRB) at 2.5× magnification. Electrophoresis of the tagged lysates was performed, using
After 24 h the cells were fixed (10% formalin) and stained either 20 or 10 µg of protein, and is visualized in Figure 5. Lane
(0.5% crystal violet) to obtain high-contrast images. Area quan- 1 contains 0.3 µM of compound 24 alone, and lane 2 includes a
tification of the wound was determined computationally using much higher concentration of the competitor 29. Lane 3
the ImageJ® software (NIH). The results are summarized in contains 0.3 µM of compound 24 alone without UV treatment.
Figure 3. Probe 24 was clearly able to inhibit PC-3 cell migra- Although there is obviously a high degree of nonspecific
Table 2: Effects of tag-free photoprobes on transcription and cytotoxicity in PC-3 cellsa.
compound
structure
IC50 SRE.L % inhibition SRE.L
% inhibition WST-1
(100 µM)
(µM)
(100 µM)
19
6.6
57
0
0
24
25
2.7
11
17
60
75
54
4
2
28
aSee Table 1 for assay descriptions.
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Beilstein J. Org. Chem. 2013, 9, 966–973.
Figure 3: Photoprobe 24 (CCG-206559) retains biological activity to block prostate cancer migration. A. Cellular migration determined by wound
assay. PC-3 prostate cancer cells were grown to confluence in 12-well plates and a scratch was made with a 200 µL pipette. Images were taken at
the beginning of the experiment and after 24 h in DMEM containing 0.5% FBS and the indicated concentration of 24 or 0.1% DMSO. After 24 h the
cells were stained with crystal violet to produce high-contrast images. Shown are examples of 10 μM 24 and DMSO after 24 h, from experiments with
similar initial wound areas as determined by using image analysis software. B. Quantification of wound assay migration. The change in migration over
24 h was determined by the difference between the area of the wound after 24 h and the initial area of the wound. The percent inhibition was plotted
by normalizing the compound-treated cells to the DMSO control. Results are expressed as the mean (±SEM) of triplicate experiments. A nonlinear
least-squares regression curve was fit for CCG-206559 inhibition of migration, IC50 = 4.7 μM.
binding under these conditions, a single band appears at approx-
imately 24 kDa (as indicated by the white box) that was
competed off by 29 and does not appear without UV treatment,
suggesting that it is a specific binding protein that has been
successfully photolabeled.
Although the level of fluorescent labeling in the non-UV-irradi-
Figure 4: Structure of the competitor used in the photolabeling experi-
ment.
ated control lanes 3 is clearly diminished relative to lanes 1 and
2, it is nevertheless much higher than expected. Unfortunately,
we do not yet have a satisfactory explanation for this. Future
experiments will include using an even lower concentration of
photoprobe (which is reported to minimize nonspecific binding
[11]) and rigorous exclusion of light during processing of the
cell lysates.
In conclusion, we have designed and synthesized tag-free
benzophenone and aryl azide photoaffinity probes that retain the
biological activity of the lead Rho/MKL1/SRF transcription
inhibitor 2 in whole cells. Significantly, a preliminary photola-
beling study with the most potent photoprobe 24 in whole
prostate cancer cells was successful at detecting specific
binding to one or more proteins at 24 kDa. Future work will
focus on identifying the labeled protein(s). We first plan to
repeat the photolabeling study with 24 and tag the labeled
proteins by clicking with azido-biotin, thereby enabling isola-
tions with streptavidin. Following purification, the 24 kDa
region of the lanes (± competitor 29) will be trypsin-digested
Figure 5: SDS-PAGE gel of photolabeling experiment in whole PC-3
cells. Lane 1 contains 0.3 µM 24 after 30 min UV treatment; lane 2
and analyzed for differences by high resolution LC/MS/MS. If
contains 0.3 µM 24 and 10 µM 29 after 30 min UV treatment; and lane
3 contains 0.3 µM 24 without UV treatment.
this is not successful, future work will entail using probe 24 and
competitor 29 in SILAC experiments [25].
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