Letter
In Situ Proteome Profiling of C75, a Covalent Bioactive Compound
with Potential Anticancer Activities
Xiamin Cheng,† Lin Li,† Mahesh Uttamchandani,†,‡ and Shao Q. Yao*,†
†Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore, 117543
‡Defence Medical and Environmental Research Institute, DSO National Laboratories, 27 Medical Drive, Singapore 117510, Singapore
S
* Supporting Information
ABSTRACT: A library of cell-permeable, minimally tagged C75 analogues was synthesized and used to uncover biological
targets in human liver cancer cells. Known targets of C75, namely FASN and CPT1A, together with other unknown targets,
including PDIA3, TFRC, and GAPDH, were thus identified.
decreased fatty acid production through FASN inhibition is
espite recent success with the use of covalent drugs, for
Dexample, orlistat (weight loss), clopidogrel (antiplatelet), seen as a viable anticancer strategy.6 Prior work using isotopic
labeling and immuno precipitation validated FASN as the
cellular target of C75.6b In a separate study, a very similar
butyrolactone scaffold was shown to bind histone acetyltrans-
ferase GCN5, with a weak IC50 of approximately 100 μM,
highlighting that other proteins may have affinity to C75 as
well.8
and esomeprazole (peptic ulcer), there remain significant safety
concerns over the large-scale development of covalent-based
therapeutics.1 Such drugs could not only modify proteins but
also trigger an idiosyncratic immune response. Such concerns
are in part attributed to the difficulty in establishing the
spectrum of “off-target” binding events which can cause many
undesirable side effects.2
In the current study, we set out to identify unknown cellular
targets (on and off) of C75 at its early stages of development as
a potential therapeutic agent. A small library of eight activity-
based probes (ABPs) was first designed from the C75 scaffold.
The 5-membered-ring (4-methylene-2-octyl-5-oxotetrahydro-
furan-3-carboxylic acid) nucleophilic acceptor was retained as
the pharmacophore for binding to its respective targets. The
linker lengths were varied between 2 and 10 carbon atoms, with
both the cis and trans versions synthesized for probe 1f. The
terminal alkyne was introduced to the linker side chain. Several
azide-containing reporters were also synthesized, with a
fluorescent group (rhodamine B) and/or biotin (Figure 1b).
Briefly, by using 1g as an example, bromocarboxylic acid 6g
was treated with lithium (trimethylsilyl)acetylide, which was
made from trimethylsilylacetylene and butyllithium, to give
compound 5g (56% yield). Following reduction with lithium
aluminum hydride and deprotection of the trimethylsilyl
(TMS) group, 4g was obtained. After oxidation with PCC
and sodium acetate,9 the aldehyde 3g was eluted by diethyl
ether through a pad of silica gel and carefully concentrated. By
treatment with freshly prepared LDA and 6 N H2SO4, the
desired aldehyde 3g then was cyclized with intermediate 7 to
give compound 2g, which was deprotected under mild
conditions to give final product 1g.8 Compound 2g was not
To address this problem, we have recently developed a
convenient chemical proteomic method to unravel the global
target spectrum for any given covalent drug, using a click-based
in situ profiling approach.3,4 This method enables the repertoire
of drug-target interactions to be probed within living systems.
By introducing a very conservative modification onto the lead
molecule (namely, an alkyne tag) at solvent-accessible sites (to
retain pharmacodynamic properties), observations can be
reliably made to determine molecular behavior and interactions
within complex and dynamic cellular environments (Figure
1a).2 We had previously applied this strategy to successfully
profile the on- and off-targets of orlistat3 as well as those of
various kinase inhibitors.5 Here we extend the strategy by
building a library of a covalent bioactive compound, C75, which
has been particularly promising as a weight-loss and anticancer
agent through its ability to modulate fatty acid oxidation.6,7 C75
is a more stable synthetic mimic of cerulenin (a fungal
metabolite) (see the Supporting Information) and belongs to a
class of α-methylene-γ-butyrolactones.6 Various pathways have
been found to be modulated by C75, including inhibition of
fatty acid synthase (FASN) and activation of carnitine O-
palmitoyltransferase-1 (CPT1).7 The compound is also known
to alter the level of neuropeptide Y and AMP-activated protein
kinase (AMPK) activity in the hypothalamus, reducing food
intake.7b Many common human cancers, including breast,
ovarian, prostate, and colon, express high levels of FASN. Thus,
Received: January 21, 2014
Published: February 19, 2014
© 2014 American Chemical Society
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dx.doi.org/10.1021/ol500206w | Org. Lett. 2014, 16, 1414−1417