Remodeling natural products: Chemistry and serine hydrolase activity of a rocaglate-derived β-lactone

Article abstract of DOI:10.1021/ja412431g

Flavaglines are a class of natural products with potent insecticidal and anticancer activities. β-Lactones are a privileged structural motif found in both therapeutic agents and chemical probes. Herein, we report the synthesis, unexpected light-driven di-epimerization, and activity-based protein profiling of a novel rocaglate-derived β-lactone. In addition to in vitro inhibition of the serine hydrolases ABHD10 and ACOT1/2, the most potent β-lactone enantiomer was also found to inhibit these enzymes, as well as the serine peptidases CTSA and SCPEP1, in PC3 cells.

Full text of DOI:10.1021/ja412431g

Article
pubs.acs.org/JACS
Remodeling Natural Products: Chemistry and Serine Hydrolase
Activity of a Rocaglate-Derived β‑Lactone
Neil J. Lajkiewicz,† Armand B. Cognetta, III,‡ Micah J. Niphakis,‡ Benjamin F. Cravatt,*,‡
and John A. Porco, Jr.*,†
†Department of Chemistry, Center for Chemical Methodology and Library Development (CMLD-BU), Boston University, 590
Commonwealth Avenue Boston, Massachusetts 02215, United States
‡Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,
United States
S
* Supporting Information
ABSTRACT: Flavaglines are a class of natural products with
potent insecticidal and anticancer activities. β-Lactones are a
privileged structural motif found in both therapeutic agents
and chemical probes. Herein, we report the synthesis,
unexpected light-driven di-epimerization, and activity-based
protein profiling of a novel rocaglate-derived β-lactone. In
addition to in vitro inhibition of the serine hydrolases ABHD10
and ACOT1/2, the most potent β-lactone enantiomer was also found to inhibit these enzymes, as well as the serine peptidases
CTSA and SCPEP1, in PC3 cells.
INTRODUCTION
■
Flavaglines, specifically rocaglate derivatives, are cyclopenta[b]-
benzofuran natural products isolated from the genus Aglaia that
have been shown to be potent anticancer agents.1 Figure 1
Figure 2. (A) β-Lactone 5 possessing a rocaglate scaffold. (B) β-
Figure 1. Rocaglates that exhibit potent biological activity.
Lactone-containing natural products.
shows three natural products: silvestrol (1),2 methyl rocaglate
(2),3a,b and rocaglaol (3)3b as well as the fully synthetic protein
(or lipostatin, 7, Figure 2). Furthermore, these chemotypes
translation inhibitor hydroxamate 4.4 Rocaglates have also been
have appeared in the clinical agents salinosporamide A
(marizomib or NPI-0052, 8), a phase I clinical candidate for
shown to have other pharmacological activities including anti-
cancer,10 and tetrahydrolipstatin (THL or orlistat), which is
currently an approved treatment for obesity.11 The mechanism
of biological action of β-lactones is typically through covalent
acylation of a functional nucleophilic residue in the active sites
of enzymes to produce stable and inactive acyl-enzyme
adducts.12 Thus, while β-lactones can target a wide array of
protein families,13 enzymes possessing catalytically essential
nucleophiles are particularly susceptible to inactivation. Because
these chemotypes show a wide range of activities across serine
inflammatory, neuroprotective, and cardioprotective effects.1j
As methyl rocaglate (2) has a secondary hydroxyl syn-facial and
β-substituted to its ester moiety, we considered that a β-lactone
structure 5 may be installed on the rocaglate core (Figure 2)
and the resulting “remodeled”5 natural product may possess
distinct pharmacological activity from other rocaglates and β-
lactones.6
β-Lactones are an important class of enzyme inhibitors that
have found broad utility as both chemical probes7 and drugs.8
There are numerous bioactive β-lactones found in nature,
several of which are widely used in biomedical research,
including omuralide (lactacystin-β-lactone, 6)6b and lipstatin9
Received: December 6, 2013
Published: January 21, 2014
© 2014 American Chemical Society
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hydrolase class,7,14 we wondered if β-lactones derived from
rocaglates would display unique pharmacology and present new
opportunities for probe development for serine hydrolases.
Herein, we report the synthesis of β-lactone 5 and its novel
light-driven di-epimerization to afford an unprecedented
cyclopenta[b]benzofuran scaffold. To identify targets of β-
lactone 5, we profiled this compound against human and mouse
proteomes by competitive activity-based protein profiling
(ABPP) using fluorophosphonate (FP) probes that show
broad-spectrum reactivity with serine hydrolases.15 From this
analysis, we identified several serine hydrolase targets of
rocaglate β-lactones, including α/β hydrolase domain-contain-
ing protein 10 (ABHD10), retinoid-inducible serine carbox-
ypeptidase (SCPEP1), and acyl-coenzyme A thioesterase 1/2
(ACOT1/2), showing that the structural features of rocaglate
β-lactones direct its activity to a unique subset of serine
hydrolases. Finally, we show that (−)-5, the most active
enantiomer, inhibits these enzymes in cells and thus may serve
as a valuable probe for exploring their functions in cells. More
generally, this work underscores the versatility of β-lactones as a
chemotype for serine hydrolase inhibition.
Scheme 2. Extrusion of CO2 Using Microwave Conditions
Scheme 3. Photoisomerization of Rocaglate β-lactone ( )-5
to Di-epi Isomer ( )-10
RESULTS AND DISCUSSION
■
Synthesis. We first evaluated the synthesis of the target
rocaglate β-lactone structure. Using bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride (BOP-Cl)/triethylamine,16 we observed
the transformation of rocagloic acid17 derived from ( )-methyl
rocaglate (2)18 to β-lactone ( )-5 (Scheme 1). Interestingly,
Figure 4. X-ray structure of β-lactone ( )-10. Hydrogens omitted for
clarity. PMP = para-methoxyphenyl.
extrusion of carbon dioxide and observed formation of alkene
( )-9 using microwave conditions (Scheme 2).20,21 Addition-
ally, we explored photochemical methods to extrude carbon
dioxide.6c,22 When exposed to UV light (>280 nm), β-lactone
( )-5 underwent an unexpected rearrangement to form the
corresponding di-epi-β-lactone ( )-10 (Scheme 3). The
relative configuration of isomer 10 was unambiguously
confirmed through X-ray crystal structure analysis (cf. Figure
4).19 The reaction did not go to complete conversion, and
irradiation for more than 4 h led to decomposition. We
considered whether 10 was thermodynamically more stable.
Indeed, computational analysis of 5 and 10 showed that β-
lactone 10 was 7.0 kcal/mol lower in energy.23 This is most
likely due to the release of strain from caged structure 5 to the
staircase structure of stereoisomer 10. Additionally, the
aromatic substituents of 10 are no longer syn.
Further experiments were performed to probe the mecha-
nism of the di-epimerization process. We considered whether
β-lactone 5 could isomerize to 10 in the presence of Brønsted
acid. However, 5 did not undergo the observed rearrangement
when subjected to catalytic amounts of camphorsulfonic acid or
para-toluenesulfonic acid (p-TsOH) without irradiation with
UV light.24 Furthermore, we did not observe a reaction when
aqueous HCl was added. Previously, Ullman and Wan studied
the photosolvolysis of benzylic alcohols in methanol.25 We
considered that 5 may be undergoing a similar transformation
as outlined in Figure 5A. A benzylic cation intermediate may be
formed from excitation of 5 which can then form an ortho-
quinone methide cation.26 The electron-rich para-methoxy-
phenyl moiety of 5 may stabilize the ortho-quinone methide
cation for di-epimerization. Attack by water then gives the di-
epimerized product 10. To test this mechanistic hypothesis, we
conducted photochemical solvolysis experiments. However,
treatment of 5 with methanol (in the presence of heat from the
lamp) ring-opened the β-lactone to form methyl rocaglate (2)
Scheme 1. Synthesis of β-Lactone ( )-5 from ( )-Methyl
Rocaglate (2)
the chemical shifts for H1 and H2 (δ = 5.41 and 4.55 ppm,
respectively) were found to be further downfield from their
respective shifts observed for methyl rocaglate (2) (5.01 and
3.88 ppm, respectively) consistent with deshielding by the β-
lactone. The coupling constants for H1−3 were lower than the
respective coupling constants observed for 2 consistent with
ring strain of the β-lactone. IR analysis (CO stretch, 1830
cm−1)6a and high-resolution mass spectrometry supported the
proposed structure of β-lactone 5 which was later unambigu-
ously confirmed by X-ray analysis (Figure 3).19
With β-lactone 5 in hand, we next studied its reactivity. We
first evaluated thermolysis of β-lactone ( )-5 to facilitate
Figure 3. X-ray structure of β-lactone ( )-5. Hydrogens omitted for
clarity. PMP = para-methoxyphenyl.
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of 5 is 2.9 Å from the β-lactone carbonyl carbon. The resulting
radical ion pair can then undergo C3a−8b cleavage, C8b
inversion, and subsequent recyclization/electron transfer. By
screening different solvents, we found that the reaction does
not proceed when irradiated in toluene (Table 1, entry 3).
However, addition of benzophenone (1 equiv) resulted in a
69% yield of ( )-10 (Table 1, entry 4). Other sensitizing
additives such as 1,4-dicyanonaphthalene and Michler’s ketone
did not afford rearranged 10 (λ > 280 nm, toluene used as
solvent).28c Selective irradiation of benzophenone also
sensitized the transformation when using lower energy light
(Table 1, entries 5 and 6). When the electron-accepting
sensitizer benzophenone was used, we propose that electron
transfer from 5 to triplet-excited benzophenone occurs, thereby
generating a radical cation rocaglate species that can undergo
di-epimerization. Although we cannot rule out the benzylic
cation mechanism (Figure 5A), the reaction is most likely
proceeding via a PET mechanism (Figure 5B) since we
achieved highest yields of 10 employing benzophenone as
sensitizer.
Activity-Based Protein Profiling of β-Lactone 5. With
β-lactone ( )-5 in hand, we next assessed its activity across the
serine hydrolase class using competitive ABPP, a technique that
allows class-wide profiling of serine hydrolase activity in native
proteomes. We treated proteomes derived from human cell
lines (PC3 and LNCaP) and mouse tissues (brain, liver and
testes) with ( )-5 (1.0 μM or 25 μM) or DMSO for 30 min
followed by the serine hydrolase-directed activity-based probe
fluorophosphonate-rhodamine (FP-Rh).29 FP-Rh-labeled pro-
teomes were then resolved by SDS-PAGE and imaged using a
fluorescent gel scanner. This analysis identified multiple serine
hydrolase targets of ( )-5, some of which were prominently
inhibited at 1.0 μM (Figure 6A). We chose to further analyze
the soluble proteome of PC3 cells (a human prostate cancer
cell line) in more detail, as ( )-5 inhibited two enzymes in
these cells that migrated at ∼30 and ∼45 kDa (red asterisks,
Figure 6A). Analyzing ( )-5 over a wider concentration range
(Figure 6B) revealed inhibition of the 30 kDa band (IC50 ∼100
nM) with a ∼10 fold selectivity window over other observable
serine hydrolases. Notably, these targets were not inhibited by
( )-methyl rocaglate (2) and were less sensitive to inhibition
by diastereomeric β-lactone ( )-10 and THL (Supplementary
Figure 1).19
We next applied the quantitative mass spectrometry (MS)-
based platform ABPP-SILAC30 to identify the targets of ( )-5.
PC3 cells grown in media containing isotopically heavy or light
amino acids were harvested and lysed. Whole cell lysates
derived from the “light” cells were treated with DMSO, whereas
the “heavy” cells were treated with ( )-5 (0.01, 0.1, and 1.0
μM), ( )-2, or DMSO. After 30 min, each proteome was
treated with a biotinylated FP probe (FP-biotin)29 and then
combined allowing selective enrichment, identification, and
quantification of serine hydrolase activities by LC/LC-MS/MS
analysis. This analysis identified ∼40 serine hydrolases in PC3
proteomes, four of which were inhibited >75% at 1.0 μM of
( )-5: ABHD10, CTSA, SCPEP1, and ACOT1/2 (Figure 6C).
Notably, these targets were dose dependently inhibited by
( )-5 but not by methyl ester ( )-2 (Figure 6D and
Supplementary Figure 2), indicating that the β-lactone moiety
is essential for serine hydrolase inhibition. Based on the
predicted molecular weights for each enzyme, relative spectral
counts (as an estimate of the relative abundance of serine
hydrolases), and concentration-dependent inhibition profiles
Figure 5. Possible mechanisms of the photochemical diepimerization
via (A) singlet cation or (B) PET.
which further reacted to form multiple products. Use of the
mildly acidic solvent 1,1,1,3,3,3-hexafluoroisopropanol (HFIP)
(Table 1, entry 1) did not afford isomerization product 10. If
Table 1. Evidence for a PET Mechanism Involved in
Transformation of ( )-5 to ( )-10
a
b
entry
λ (nm)
additive
solvent
yield (%)
c
1
2
3
4
5
6
>280
>280
−
−
−
HFIP
CDCl3
PhCH3
PhCH3
CDCl3
CDCl3
−
40
>280
−
69
−
>280
Ph2CO
315−400
315−400
−
Ph2CO
81
a
b
1 equiv of additive used. Isolated yield after column chromatog-
c
raphy. Quantitative recovery of starting material.
the reaction is indeed proceeding through photodehydrox-
ylation, acidic/hydrogen-bonding solvents such as HFIP should
facilitate the reaction outlined in Figure 5A.25d Irradiation of
rocaglaol (3)27 (cf. Figure 1) with and without solvent additives
also led to complex mixtures that we were unable to separate
and characterize.
We also considered an alternative mechanism in which
photoexcited 5 could undergo photochemical electron transfer
(PET)28 from the benzofuran ring oxygen to the β-lactone
carbonyl (Figure 5B). Examination of the X-ray structure of 5
(Figure 3) indicates that the cyclopenta[b]benzofuran oxygen
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noted herein and by List et al.,33 suggests that strained cyclic
esters and amides are particularly well-suited for ABHD10
inhibition. SCPEP1 is a poorly characterized secreted
carboxypeptidase that appears to be involved in vascular
remodeling.34 While lacking selective small-molecule inhibitors,
SCPEP1 has recently been shown to be targeted by the β-
lactones omuralide and vibralactone.33 We are not aware of any
reported inhibitors for ACOT1 or 2. These enzymes appear to
be important for lipid homeostasis through the hydrolysis of
acyl-CoA species; however, the biological consequences of
disrupting ACOT1/2 activity is unknown, warranting the
pursuit of pharmacological inhibitors. Notably, ACOT1 and
ACOT2 were previously screened against a large panel of
carbamates, none of which were found to exhibit inhibitory
activity.35
Having identified several serine hydrolase targets of ( )-5,
we next wondered if its pure enantiomers would exhibit distinct
potency and specificity profiles. Both enantiomers of 5 were
accessed via hydrolysis of ( )-methyl rocaglate (2), menthyl
ester formation/separation, and subsequent hydrolysis/BOP-Cl
coupling (Scheme 4).4 Testing both enantiomers by com-
petitive ABPP revealed that (−)-5 more potently inhibited
ABHD10 and ACOT1/2 than (+)-5 (Figure 7A).
a
Scheme 4. Synthesis of Both Enantiomers of 5
Figure 6. In vitro competitive ABPP of β-lactone ( )-5. (A) Gel-based
ABPP of ( )-5 in various human cancer cell and mouse tissue
proteomes showing inhibition of multiple serine hydrolases (high-
lighted in red boxes). Proteomes were treated with DMSO or ( )-5
(1.0 or 25 μM) for 30 min followed by FP-Rh (30 min). (B)
Concentration-dependent inhibition of serine hydrolase activities in
PC3 cell proteomes treated with DMSO or ( )-5 (0.001−10 μM),
showing significant inhibition of serine hydrolase activities migrating at
30 and 45 kDa (red asterisks). (C) ABPP-SILAC analysis of ( )-5 at
1.0 μM (heavy amino acid-labeled proteome) versus DMSO (light
amino acid-labeled proteome) in PC3 cell proteomes, revealing
inhibition of ABHD10, CTSA, SCPEP1, and ACOT1/2. (D) ABPP-
SILAC analysis of ( )-5 (1.0, 0.1, and 0.01 μM), 2 (10 μM), or
DMSO (heavy) versus DMSO (light) in PC3 cell proteomes showing
dose-dependent inhibition of the four primary targets of ( )-5
observed at 1.0 μM. For C and D, data are presented as the mean
standard deviations of heavy/light ratios for multiple unique peptides
from each serine hydrolase.
a
Conditions: (a) LiOH, THF, H2O, 60 °C; (b) L-menthol, DCC,
DMAP, CH2Cl2, rt (60% combined yield, 2 steps); (c) NaOH,
DMSO, H2O, 60 °C, 53% yield; (d) BOP-Cl, Et3N, CH2Cl2, rt, 65%
yield; (e) NaOH, DMSO, H2O, 60 °C, 55% yield; (f) BOP-Cl, Et3N,
CH2Cl2, rt, 65% yield. DCC = N,N-dicyclohexylcarbodiimide; DMAP
= N,N-dimethylaminopyridine; BOP-Cl = bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride; DMSO = dimethylsulfoxide.
from both gel- and MS-ABPP experiments, we postulate that
the ∼30 and 45 kDa gel bands (Figure 6B) are likely ABHD10
and ACOT1/2, respectively, while CTSA and SCPEP1 did not
appear to be adequately resolved for detection by gel-based
ABPP (see below). It should be noted that ACOT1 and
ACOT2, while distinct enzymes, share very high sequence
identity (>90%), precluding their differentiation in our MS-
based experiments.
Interestingly, CTSA or cathepsin A, one of the primary
targets of ( )-5, is also known to be inhibited by other β-
lactones including omuralide (lactacystin-β-lactone, 6) and
salinosporamide A (8).14b,31 Inhibitors for ABHD10 have only
recently been discovered in competitive ABPP studies of aza-β-
lactams.32 That ABHD10 is also sensitive to β-lactones, as
The activity displayed by 5 for poorly characterized enzymes
that lack selective inhibitors (or even leads) suggests that this
agent, as well as derivatives thereof, could serve as valuable
chemical probes. To achieve this goal, however, 5 would need
to exhibit inhibitory activity in situ. Accordingly, we next treated
PC3 cells with DMSO or increasing concentrations of (−)-5,
and after 2 h, the cells were harvested, lysed, and treated with
FP-Rh for gel-based ABPP. From this analysis, we observed
greater than 90% blockage of ABHD10 activity at 100 nM with
greater than 10-fold selectivity over ACOT1/2, which was also
inhibited at higher in situ concentrations of 5 (Figure 7B).
Notably, a faint band migrating just below ABHD10 was fully
competed at only 10 nM, which is likely the active, 32 kDa
fragment of CTSA36 given its expected migration by gel37 and
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ACKNOWLEDGMENTS
■
We thank the National Institutes of Health (R01 GM073855,
J.A.P., Jr.; DA033760, B.F.C.; and DA032541, M.J.N.) and the
Skaggs Institute for Chemical Biology for research support.
This work was supported in part by a grant from Abide
Therapeutics. We thank Dr. Jeffrey Bacon (Boston University)
for X-ray crystal structure analyses and Professor John Snyder
(Boston University), Professors Kevin Moeller (Washington
University at St. Louis) and David Nicewicz (University of
North Carolina at Chapel Hill), Dr. Kiel Lazarski, and Mr.
Steven Stone (Boston University) for extremely helpful and
stimulating discussions.
Figure 7. In vitro and in situ competitive ABPP of isolated enantiomers
of 5. (A) In vitro gel-based competitive ABPP of pure enantiomers of 5
in PC3 cell proteomes showing that (−)-5 is more potent than (+)-5
for inhibiting ABHD10 and ACOT1/2. (B) In situ gel-based
competitive ABPP of PC3 cells treated with DMSO or (−)-5
(0.001−10 μM) for 2 h, showing that (−)-5 retains its activity in cells.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and characterization data for all new
compounds described herein, including CIF files for com-
pounds 5 and 10. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Authors
cravatt@scripps.edu
porco@bu.edu
Notes
T.; Fremgen, T.; Romo, D. J. Org. Chem. 2010, 76, 2−12. (e) Bottcher,
̈
The authors declare no competing financial interest.
T.; Sieber, S. A. Med. Chem. Commun. 2012, 3, 408−417.
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