Design of Benzoxathiazin-3-one 1,1-Dioxides as a New Class of Irreversible Serine Hydrolase Inhibitors: Discovery of a Uniquely Selective PNPLA4 Inhibitor

Article abstract of DOI:10.1021/jacs.7b02985

The design and examination of 4,1,2-benzoxathiazin-3-one 1,1-dioxides as candidate serine hydrolase inhibitors are disclosed, and represent the synthesis and study of a previously unexplored heterocycle. This new class of activated cyclic carbamates provi

Full text of DOI:10.1021/jacs.7b02985

Article
pubs.acs.org/JACS
Design of Benzoxathiazin-3-one 1,1-Dioxides as a New Class of
Irreversible Serine Hydrolase Inhibitors: Discovery of a Uniquely
Selective PNPLA4 Inhibitor
Anne F. Kornahrens,† Armand B. Cognetta III,‡ Daniel M. Brody,† Megan L. Matthews,‡
Benjamin F. Cravatt,‡ and Dale L. Boger*,†
‡
†Departments of Chemistry, and Chemical Physiology and The Skaggs Institute for Chemical Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: The design and examination of 4,1,2-benzox-
athiazin-3-one 1,1-dioxides as candidate serine hydrolase
inhibitors are disclosed, and represent the synthesis and
study of a previously unexplored heterocycle. This new class of
activated cyclic carbamates provided selective irreversible
inhibition of a small subset of serine hydrolases without
release of a leaving group, does not covalently modify active
site catalytic cysteine and lysine residues of other enzyme
classes, and was found to be amenable to predictable structural
modifications that modulate intrinsic reactivity or active site
recognition. Even more remarkable and within the small pilot
series of candidate inhibitors examined in an initial study, an exquisitely selective inhibitor for a poorly characterized serine
hydrolase (PNPLA4, patatin-like phospholipase domain-containing protein 4) involved in adipocyte triglyceride homeostasis was
discovered.
developed for specific enzyme classes, including fluorophosph-
onate-rhodamine (FP-Rh)21,22 for selective serine hydrolase
labeling, permits the rapid proteome-wide identification of
inhibitor targets, assessment and optimization of inhibitor
selectivity, and functional annotation of uncharacterized
enzymes.23,24 This may be accomplished without recombinant
enzyme expression, protein purification, knowledge of the
endogenous substrate, or the development of specific substrate
assays as required by traditional methods.24−27
INTRODUCTION
■
Serine hydrolases play key roles in human physiology and
disease and are an important class of therapeutic targets. Serine
hydrolases represent more than 1% of predicted proteins in
humans, making it one of the largest and most diverse class of
mammalian enzyme families.1 Not only do they make
fundamental contributions to physiological and pathophysio-
logical processes,2 but a large number of the serine hydrolases
remain uncharacterized or unannotated, lacking a known role,
endogenous substrate, or specific inhibitor. Selective chemical
inhibitors for members of the serine hydrolase family have
uniquely contributed to an understanding of the biological
function of individual enzyme members. They have also led to
new therapeutics, including new treatments for obesity,
diabetes, microbial infections, and Alzheimer’s disease.1 The
active site catalytic triads that contain a serine nucleophile have
inspired the design of many classes of small molecule
inhibitors.3 The inhibitor classes include those that contain
an electrophilic carbonyl such as trifluoromethyl ketones,4−6 α-
ketoamides and esters,5,6 lactones,7,8 lactams,9 α-ketohetero-
cycles,10−12 carbamates,13,14 ureas,15−17 and other activated
carbonyl-containing compounds,18 which act through covalent
modification of the serine nucleophile.
In efforts to interrogate serine hydrolases not yet successfully
targeted by existing inhibitor classes, we have continued to
explore new irreversible covalent inhibitor designs that might
display a unique reaction selectivity among not only classes of
enzymes, but also among a subset of the serine hydrolases.28
A
large body of work on irreversible inhibitors of serine, cysteine,
and threonine proteases is available from which inspiration may
be drawn.3 Tethered reactive moieties that acylate, phospho-
nylate, or sulfonylate active site nucleophiles were of particular
interest, with the saccharin family of serine protease inhibitors
serving as the inspiration for the new inhibitor class detailed
herein.29 The saccharin family of 1,2-benzisothiazol-3-one 1,1-
dioxides has been shown to inhibit serine proteases such as
human leukocyte elastase30−32 and human mast cell tryptase33
through acylation of the nucleophilic serine active site residue
The use of activity-based protein profiling (ABPP)19−21
paired with such selective inhibitor classes has allowed the rapid
analysis of target serine hydrolases and monitoring of enzyme
activity in complex biological systems. The use of ABPP probes
Received: March 25, 2017
© XXXX American Chemical Society
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(Figure 1). The mechanism of inhibition involves serine
nucleophilic attack on the activated amide, collapse of the
release a leaving group, are capable of predictable electronic
modulation of the intrinsic reactivity and selectivity by C7 aryl
substitution (R1), and can be tailored to bind individual enzyme
targets through N-substitution (R2). Their use in the discovery
of the first selective inhibitor of patatin-like phospholipase
domain containing protein 4 (PNPLA4) is disclosed.
RESULTS AND DISCUSSION
■
At the onset of our studies, we elected to probe the aryl
substituent electronic effects (R1 = OMe, H, CN), utilizing the
parent core heterocycles (R2 = H) and those that bear a small
set of N-alkyl substituents that might impart representative
selective enzyme active site targeting (R2 = CH2Ph, (CH2)5Ph
vs H). In particular, the inclusion of the 5-phenylpentyl side
chain might impart binding and inhibition of fatty acid amide
hydrolase (FAAH) for which we were especially well equipped
to examine as part of a pilot examination.10,25 Proteome-wide
evaluation by gel-based ABPP was used to provide an initial
assessment of the impact of expected reactivity trends (X = O >
NH, R1 = CN > H > OMe), to define the utility and
promiscuity of the parent core heterocycles (R2 = H) in
fragment-based screening, and to probe their utility in the
identification of more selective inhibitors for existing (e.g.,
FAAH) or uncharacterized serine hydrolase targets with the N-
substituted heterocycles (R2 = CH2Ph, (CH2)5Ph).
Their synthesis was anticipated to rely on a one-pot addition
and cyclization of anilines (6) and phenols (7) with
chlorosulfonyl isocyanate (8)40 to produce the unsubstituted
benzothiadiazin-3-one 1,1-dioxides (4, X = NH) or benzox-
athiazin-3-one 1,1-dioxides (5, X = O) poised for N-alkylation
(Figure 1). Benzothiadiazin-3-one 1,1-dioxides (4, X = NH)
were targeted first because a two-step synthesis was known for
similar compounds, allowing access to the desired candidate
inhibitor set.34 Interestingly, a number of compounds
containing the substituted benzothiadiazin-3-one 1,1-dioxide
core have been shown to exhibit effective biological
activity,35−38,41 although none are reported to inhibit a
hydrolytic enzyme target (Figure 2).
Figure 1. Saccharin family of protease inhibitors. Proposed mechanism
of inhibition and synthetic design for new inhibitor class.
tetrahedral intermediate, and formation of an acyl enzyme
intermediate in the form of an ester with release of the
sulfonamide as the leaving group.30 The reactivity of such
saccharin derivatives, their intrinsic stability, and inhibitory
potency can be modulated by core substitution. Activation of
the leaving sulfonamide through N-acylation or N-arylation (R)
or electronic modulation of the intrinsic carbonyl reactivity by
C4/C6 aryl substitution has been detailed. However, the acyl
bond formed with the active site residue is a serine ester that is
susceptible to rapid deacylation and enzyme reactivation,
limiting the utility of this inhibitor class for biological studies
where sustained enzyme inhibition is required.
The use of reported conditions for the synthesis of the
benzothiadiazine-3-one 1,1-dioxides (known for R1 = OMe, Cl,
Br),34,40,42,43 enlisting chlorosulfonyl isocyanate (CSI) and
AlCl3 with the electron-rich anilines, provided 9 and 10
The redesigned scaffold (1) detailed herein represents a
modification of the saccharin ring system with insertion of a
heteroatom (O, NH) adjacent to the carbonyl (Figure 1).
Nucleophilic attack of an active site serine on the inhibitor
carbonyl followed by collapse of the tetrahedral intermediate 2
provides a covalently bound inhibitor 3 in the form of a more
stable and potentially irreversible serine carbamate or
carbonate, depending on the choice of heteroatom X (Figure
1). Remarkable in this day and age and while the urea version
of the redesigned scaffold (4, X = NH) represents a well-
established heterocycle class,34−38 the carbamate version (5, X
= O) constitutes a class with only a single report of its
inadvertent synthesis.39 The work detailed herein revealed that
the former N-alkylated ureas may be insufficiently reactive to
irreversibly inhibit serine hydrolases or other enzyme classes.
However, the latter benzoxathiazin-3-one 1,1-dioxides (5, X =
O) proved to be potent and remarkably selective irreversible
serine hydrolase inhibitors. Moreover, these compounds
constitute a class of serine hydrolase inhibitors that do not
Figure 2. Benzothiadiazin-3-one 1,1-dioxides that display biological
activity.
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(Scheme 1), but failed to provide 12 with 4-aminobenzonitrile.
Instead, after successful aniline addition to the isocyanate,
Scheme 1. Synthesis of Benzothiadiazin-3-one 1,1-Dioxide
Cores
attempts to promote electrophilic aromatic substitution led
only to isolation of the 4-cyanophenylurea (13, R1 = CN)
despite a screen of Lewis acids, reaction temperatures, and
reaction solvents. However, the successful use of the method
with p-bromoaniline provided 11 and permitted a subsequent
cyanation reaction44 for accessing 12 (Scheme 1).
Figure 4. FAAH inhibition by benzothiadiazin-3-one 1,1-dioxides.
The four urea-containing core scaffolds were subjected to
core scaffolds (R2 = H, 9A−12A) showed no activity (Ki > 100
μM), and only a limited set of the benzyl substituted
benzothiadiazin-3-one 1,1-dioxides (14A−17A) showed mod-
est activity (16A and 17A). In the phenylpentyl series (18A−
21A), all compounds exhibited modest activity. The most
potent of the inhibitors (20A) was examined in detail.
Compound 20A was found to exhibit simple competitive
inhibition kinetics, and it did not show a time-dependent
increase in the inhibition potency, both of which are indicative
of simple reversible competitive enzyme inhibition. Addition-
ally, the full set of candidate inhibitors (9A−21A) was
incubated with mouse brain membrane and soluble proteome
and labeled with FP-rhodamine or iodoacetamide (IA)-
rhodamine to examine serine and cysteine hydrolase inhibition
(Figures S1 and S2). The reactivity with active site lysine-
containing enzymes was also examined through use of a Kinativ
kit. Ultimately, none of the gel-based ABPP studies showed
evidence of significant target enzyme inhibition, and only weak
reversible inhibition was detected at very high concentrations
for a few compounds. The compounds also displayed a lack of
hydrolytic reactivity of the potentially activated urea, being
stable in methanol, DMSO, and organic solvents as well as in
weakly acidic and basic aqueous media (>24 h). Thus, initial
studies with the benzothiadiazin-3-one 1,1-dioxides, unlike
those with the carbamate variant that followed, suggest they
lack the structure and reactivity to generally act as irreversible
serine hydrolase inhibitors.
alkylation to incorporate the two R2 substituents (Figure 3).
Figure 3. Alkylation of benzothiadiazin-3-one 1,1-dioxide cores and X-
ray crystal structures of 14A and 14B.
Initial studies with 9 (R1 = H), employing NaH and benzyl
bromide, yielded a separable mixture of two products (14A and
14B). They were tentatively assigned to be the products of the
desired N-benzylation (14A, 52%) and O-benzylation (14B,
1
22%) on the basis of the H/13C NMR chemical shifts, and
Remarkably, there is only a single report of the preparation of
a 4,1,2-benzoxathiazin-3-one 1,1-dioxide (5, X = O) with CSI,
and it was isolated as a byproduct in the reaction with p-
cresol.39 This, along with the single report of one N-substituted
derivative (R2 ≠ H),46 made this unexplored carbamate a
compelling class of compounds to prepare, characterize, and
evaluate. p-Bromophenol was chosen as an initial substrate for
examination of its reactivity toward CSI as a route to the
expectedly more reactive carbamate versus urea series. In the
reported synthesis, the cyclized product of p-cresol was isolated
their structures were unambiguously established by single-
crystal X-ray structure determinations (Figure 3).45 Extension
of this method with 10−12 proceeded smoothly to give 15A−
17A in good yields (64−47%). Without optimization, the
synthesis was also used to access the second R2 series, using
NaH and 1-bromo-5-phenylpentane to provide 18A−21A
(Figure 3).
Access to the targeted candidate inhibitors permitted their
examination for inhibition of purified FAAH18 (Figure 4). The
C
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as a byproduct during sulfonylisocyanate formation.39 Attempts
to extend this synthesis to 22 were unsuccessful (Scheme 2),
even upon isolation and resubjection of intermediate 25 to the
Lewis acid-catalyzed cyclization conditions.
Scheme 4. Stepwise Synthesis of Substituted Benzoxathiazin-
3-one 1,1-Dioxides
a
Scheme 2. Direct Reaction of Phenol 22 with CSI
a
Conditions: (i) 1 equiv of NaH, 2 equiv of R2−Br, DMF; (ii) BBr3,
CH2Cl2, −78 to 25 °C; (iii) Pd/C, H2, MeOH; (iv) 5 equiv of NaH, 2
equiv of phosgene, CH2Cl2, 25 °C.
steps, Scheme 4). Hydrogenolysis of 33 and 34 (R1 = Br)
proceeded smoothly to yield the C7-unsubstituted (R1 = H)
precursors 35 and 36 in 92% and 91% yield, respectively, and
subsequent carbamate formation yielded 39 and 40 (Scheme
4).
An alternative approach to 26 via the 2-hydroxybenzene-
sulfonamide 30 was pursued in which aryl chlorosulfonylation
ortho to a protected phenol preceded cyclic carbamate
formation (Scheme 3). Although successful in providing 29
Completion of the series with preparation of the C7-cyano
and C7-methoxy derivatives is summarized in Scheme 5.
Chlorosulfonylation of 4-methoxybenzamide (41) followed by
subsequent dehydration of 42 and chlorosulfonylation of 1,4-
dimethoxybenzene (44) were successfully utilized to access the
sulfonyl chlorides 43 and 45 (Scheme 5). To further streamline
the approach, a direct addition of the desired substituted amine
to the sulfonyl chloride was used. Reaction of 43 and 45 with
benzylamine and 5-phenylpentylamine produced the substi-
tuted sulfonamides 46−49 in high yield. Subjection of the
methyl ethers 48 and 49 to the boron tribromide deprotection
conditions selectively provided 52 and 53, whereas phenol
deprotection in the p-cyano series required stronger reaction
conditions (LiCl, 140 °C, DMF) to form hydroxysulfonamides
50 and 51. Preparation of the four additional benzoxathiazin-3-
one 1,1-dioxides (54−57) proceeded upon treatment of 50−53
with phosgene and NaH. The structure of the candidate
inhibitors was confirmed with a single-crystal X-ray structure
determination45 of 54 (Scheme 5).
Access to the eight benzoxathiazin-3-one 1,1-dioxides
allowed the examination of this carbamate series by gel-based
ABPP. The use of mouse brain proteome (membrane fractions
of proteome) and FP-rhodamine labeling showed clear
inhibition of the serine hydrolase ABHD10 by nearly all
compounds in the series, as well as low micromolar inhibitor
activity against LYPLA1/2 by 55 (Figure 5). Interestingly,
FAAH was not effectively inhibited by members of this class
even with the candidate inhibitors that bear the 5-phenylpentyl
side chain. Although not extensively profiled, IA-rhodamine and
Alexa Fluor labeling did not detect evidence of cysteine
protease/hydrolase or lysine-containing enzyme inhibition
(Figures S3 and S4).
Scheme 3. Attempted Synthesis of Benzoxathiazin-3-one 1,1-
Dioxide Core
and following methyl ether deprotection, attempts to form the
final cyclized product from 30 with triphosgene, phosgene, or
4-nitrobenzoyl chloride were unsuccessful. The highly polar,
acidic, and reactive nature of the product made its isolation
challenging and led to examination of a variation on this route,
introducing the amine substituent prior to cyclization. This
provides nonacidic intermediates with increased organic
solubility, avoiding the challenges of isolation of 26.
N-Alkylation of 29 (NaH, BnBr, DMF), with a methyl ether
serving as a phenol protecting group to prevent O-alkylation,
proceeded to give 31 in 31% yield (Scheme 4), with recovered
starting material and dialkylated product accounting for the
remaining material. Methyl ether deprotection with boron
tribromide provided 33 in 92% yield (Scheme 4). Formation of
benzoxathiazin-3-one 1,1-dioxide was now successful when
conducted with phosgene and NaH to yield 37 in 89% yield
(Scheme 4). This provided a route to N-alkylated benzox-
athiazin-3-one 1,1-dioxides and was applied to the additional
targeted N-substitution product (R2 = (CH2)5Ph), which
proceeded even more smoothly to form 38 (42% over three
The results from this initial gel-based screen displayed
interesting trends. First, the new inhibitor class displayed
surprisingly selective inhibition among the serine hydrolases,
displaying full inhibition of only three enzymes and partial
inhibition of three more (Figure 5). Moreover, even within this
D
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DMEM buffer (1−6 h at 25 or 37 °C, Figure 6), whereas
reaction to produce unknown products was observed in Tris-
HCl buffer (in less than 1 h at 25 °C). Finally, samples stored
in DMSO showed slow conversion to the hydrolysis products
33 and 50 at both 25 °C (50% conversion in approximately 7
or 14 days, respectively) and at −78 °C (50% conversion in
approximately 14 or 21 days, respectively).
Scheme 5. Streamlined Synthesis of Candidate Inhibitors
and X-ray Crystal Structure of Candidate Inhibitor 54
a
Their reactivity toward methanol as solvent provided the
clearest indication of not only the relative reactivity (54 > 37),
but also the reaction course. The reaction product derived from
37 and 54 in methanol was shown to be 58 or 59, and indicates
that the phenol acts as the leaving group (vs −SO2NHR2). In
this respect, the behavior of the benzoxathiazin-3-one 1,1-
dioxides differs from that of the saccharin-based inhibitors
where only the sulfonamide can serve as the leaving group.
Subjection of the full set of phenylpentyl-substituted com-
pounds (R1 = OMe, H, and CN) to the reaction with methanol
allowed a direct comparison of the magnitude of the electronic
effect of the 7-substitution on the carbamate carbonyl reactivity
(Figure 7). The compounds displayed predictable relative
reaction rates (R1 = CN > H > OMe), where compound 55
with the electronic-withdrawing nitrile substituent exhibited the
greatest reactivity observed even at room temperature.
Compound 57 with the electron-donating methoxy substituent
exhibited the lowest reactivity, requiring elevated temperatures
to observe methanol addition, and the differences were of a
magnitude indicative of a pronounced C7 substituent electronic
effect (Figure 7).
The reactivity toward nucleophiles was also examined
through subjection of the same series of compounds to
reactions with benzylamine, benzyl alcohol, and benzyl
mercaptan (1 equiv, Figure 7). The reactions were performed
in deuterated chloroform (0.1 M) with dibromomethane as an
internal standard and analyzed by 1H NMR initially to
determine the reaction time required for 75% conversion to
products 60 (Figure 7). Neither benzyl alcohol nor benzyl
mercaptan reacted with the compounds under these conditions.
However, the reactivity profile with benzylamine exhibited a
clear trend (Figure 7). Very rapid reaction was observed with
the 7-cyano derivative 55 (t1/2 = 1.3 min, k = 5.3 M−1 s−1), and
a slower reaction was seen for the C7 unsubstituted compound
40 (t1/2 = 13 min, k = 1.8 M−1 s−1). The 7-methoxy derivative
57 exhibited a much slower rate of reaction (t1/2 = 66 min, k =
0.24 M−1 s−1) with full conversion observed only after 12 h. A
Hammett plot (log k vs σp) of the results provided a slope (ρ
value) of 1.2, indicating that the electronic effect of the
substituent on this reactivity exceeds even that observed on
benzoic acid acidity.
Because of their cyclic structure, the designed inhibitors are
unique among previously explored carbamates in that there are
not only two potential leaving groups (the sulfonamide or
phenol), but no group is released from the enzyme upon initial
covalent acylation of an active site serine due to the tethered
nature of the carbonyl. Moreover, the compounds are also
potentially capable of a subsequent cross-linking modification at
the active site, where a second nucleophilic residue could attack
the carbonyl of the covalently bound inhibitor, leaving a
carbonyl bound to two residues in the active site with release of
the remainder of the molecule. To probe these two possibilities,
compound 57 was incubated with purified ABHD10 (5-fold
excess of inhibitor) for 30 min at 25 °C and examined by ESI−
MS (Figure 8). Accordingly, the measured mass of ABHD10,
established 1−4 h after the initial 30 min incubation, increased
a
Conditions: (i) 2 equiv of R2−NH2, 0.15 equiv of Et3N, CH2Cl2; (ii)
4 equiv of LiCl, DMF, 140 °C; (iii) BBr3, CH2Cl2, −78 to 25 °C; (iv)
5 equiv of NaH, 2 equiv of phosgene, CH2Cl2.
small set of inhibitors, distinctive selectivity trends were
observed toward these enzymes. Low micromolar inhibition
against ABHD10 was observed for nearly all of the inhibitors
(R2 = (CH2)5Ph > CH2Ph) regardless of core substituent (R1 =
CN, Br, H, OMe). Only the more reactive inhibitor 55 (R1 =
CN) bearing only the phenylpentyl R2 substituent in the set of
eight displayed LYPLA1/2 inhibition. Similarly, only inhibitors
that contain the benzyl substituent (R2 = CH2Ph) inhibited
APEH with core substituent trends reflecting the expected
intrinsic reactivity of the inhibitors (R1 = CN > H > OMe).
Finally, the inhibition was established to be irreversible against
ABHD10 and LYPLA1/2 in studies analogous to those detailed
latter herein (see Figure 12). As a result of these initial
observations, a detailed examination of both the chemical and
the biochemical reactivity of these compounds was conducted
to more comprehensively establish their behavior and the
potential electronic effects of the varied substituents.
The stability and reactivity of the benzoxathiazin-3-one 1,1-
dioxides were briefly and initially surveyed with 37 (R1 = Br, R2
= Bn) and 54 (R1 = CN, R2 = Bn), and both were found to be
stable in DPBS, H2O, THF, and ethanol at 25 °C for over 24 h
(Figure 6). Hydrolysis to provide 33 or 50 was observed in
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Figure 5. Screen of benzoxathiazin-3-one 1,1-dioxides by competitive gel-based ABPP. Mouse brain membrane proteome treated with compounds
(at 1 and 20 μM, 30 min incubation) followed by FP-rhodamine (1 μM, 30 min). Fluorescent images shown in gray.
Figure 7. Reactivity of inhibitors.
activity in cell lysate proteome samples from mouse neuro-
blastomas (N2A cells, Figure 9a) and human embryonic kidney
cells (HEK 293T cells, Figure 9b). Treatment with the three
related compounds 55, 40, and 57 (R2 = (CH2)5Ph), at 1, 50,
Figure 6. Stability and reactivity of 37 and 54.
and 100 μM, showed a clear trend of strongest inhibition
by 376 Da, indicative of only initial acylation of the enzyme
with no evidence of a subsequent cross-linking reaction (no
increase of 26 Da). Thus, 57 serves as an irreversible inhibitor
of ABHD10 that acts by monoacylation of an active site serine
without release of a leaving group under the conditions of our
examinations. Although prolonged incubation conceivably
could lead to cross-linking, these studies suggest it is unlikely
to be observed with ABHD10. However, we would not want to
rule out such behavior against other serine hydrolases with
members of this inhibitor class.
observed with R1 = CN, and progressively less inhibition with
R1 = H and R1 = OMe (Figure 9).
To more comprehensively define the relative selectivity of
the inhibitors, the set comprised of 55, 40, and 57, bearing the
same R2 group ((CH2)5Ph) and the varied R1 group (CN, H,
OMe, respectively), was analyzed by ABPP-SILAC (Stable
Isotopic Labeling with Amino Acids in Cell culture) (Figure
10).14,47 Whole cell proteome samples from isotopically labeled
HEK cell lysates were incubated with the compounds (at 25
μM) or DMSO control, then the activity-based probe FP-
biotin. The FP-biotin-labeled proteins were enriched on
streptavidin beads and then subjected to on-bead trypsin
digestion. The resulting tryptic peptide mixtures were analyzed
Additional proteome-wide screens of the compounds allowed
the further comparison of their reactivity and serine hydrolase
inhibition in vitro, by examining ABHD10 and LYPLA1/2
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identified a larger subset of serine hydrolases that can be
targeted by the new inhibitor class, which most prominently
also includes PNPLA8, AIG1, ABHD6, LIPE, and ABHD11,
but the less reactive members 40 and 57 uniquely identified an
enzyme (PNPLA4) that would have been overlooked by
screening with 55 alone.
PNPLA4 (patatin-like phospholipase domain-containing
protein 4), also known as GS2,49 is present in humans, but is
absent in mouse.50 It constitutes a poorly characterized serine
hydrolase that is known to hydrolyze triglycerides and retinol
esters and is thought to be involved in adipocyte homeostasis.
Thus, the discovery of a selective inhibitor of PNPLA4
constitutes a critical advance for characterizing the physiological
role of PNPLA4. The use of overexpressed PNPLA4 in HEK
cells allowed selective PNPLA4 inhibition by 57 to be observed
in gel-based ABPP assays with whole cell lysates of the soluble
and membrane-bound proteome fractions, with a calculated
IC50 of 1.8 μM determined from the separated membrane
proteome fraction (Figure 11).
Subsequent studies were conducted that demonstrated the
irreversibility of serine hydrolase inhibition through a gel-based
ABPP study. After 30 min of incubation of compound 57 with
the same HEK cell membrane proteome as Figure 11, filtration
through a desalting column removed excess compound and
allowed isolation of the proteome containing the enzyme−
inhibitor complex. Aliquots of the complex in the full proteome
were subjected to FP-rhodamine labeling at various time points
to show the persistence of inhibition of PNPLA4, indicating
both that binding is covalent, because the complex survives
column filtration, and irreversible over the time scale because
little enzyme reactivation was observed (<10% at 8 h
postfiltration, Figure 12).
In preliminary studies, subjection of these same HEK cells to
57 in FBS-free media provided in-cell PNPLA4 inhibition,
albeit at a 10-fold higher concentration (calculated IC50 = 11.7
μM), whereas treatment (50 μM 57) in the presence of fetal
bovine serum (FBS) failed to display activity. At present, we do
not know whether this loss of in-cell activity in the presence of
FBS is due to serum protein binding or serum instability,
although we suspect it is not due to serum instability (see
below). These results are remarkable for a pilot study of a new
serine hydrolase inhibitor chemotype, and further optimization
of 57 can be expected to provide a more potent, cellularly active
inhibitor of PNPLA4 capable of use in defining its physiological
role. Notably, 57 is among the most stable benzoxathiazin-3-
one 1,1-dioxides examined, bearing an aryl electron-donating
substituent that moderates its reactivity. Compound 57 is stable
in DMSO at room temperature for at least 2 weeks, solid
samples could be stored on the bench open to the air for
greater than 6 months, and it was found to be stable to both
Figure 8. ESI-TOF-MS analysis of purified ABHD10 incubated with
57 (red), starting purified ABHD10 (black), and summary of results
(top).
by multidimensional liquid chromatography−tandem mass
spectrometry (LC/LC−MS/MS). The raw data were searched
with the ProLuCID algorithm, and SILAC ratios were
quantified with CIMAGE,48 generating the data graphically
shown in Figure 10. The greatest spectrum of inhibition was
seen with the 7-cyano-substituted inhibitor 55, with 10
enzymes inhibited at greater than 50% (Figure 10a), while
only three enzymes were inhibited at this level by 40 (Figure
10b) and selective inhibition of only a single enzyme
(PNPLA4) was observed for 57 (Figure 10c). These
remarkable results beautifully display the C7 substituent
electronic effect on the reactivity of the carbamate carbonyl
of the benzoxathiazin-3-one 1,1-dioxides and its impact on the
promiscuity of the inhibitor activity (Figure 10d). Additionally,
the selectivity of the inhibition of LYPLA1/2 by 55, observed in
the gel-based ABPP assay, was confirmed by the MudPIT
analysis, as no inhibition of these two enzymes is observed for
40 and 57 (Figure 10). Like most but not all of the identified
enzyme targets, ABHD10 inhibition followed the trends (CN >
H > OMe) first observed by gel-based ABPP (Figure 10d).
Most impressively, the analysis revealed the exquisite selectivity
of 57 for a single human enzyme (not present in mouse):
PNPLA4. Interestingly, both 57 and 40 inhibit PNPLA4
preferentially and completely, whereas the more reactive
inhibitor 55 does so incompletely and with a greater preference
for eight other enzymes. Clearly the more reactive inhibitor 55
Figure 9. In vitro analysis of compounds 55, 40, and 57 (at 1, 50, 100 μM, R2 = (CH2)5Ph) by gel-based ABPP with (a) processed N2A cells and (b)
processed HEK cells showing the electronic effect of aromatic core C7 substituents (R1 = CN, H, OMe) on inhibition of ABHD10 and LYPLA1/2.
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Figure 10. ABPP-SILAC analysis of compounds (55 (a), 40 (b), 57 (c)) using HEK cell lysates showing the effect of substituents on inhibitor
potency and selectivity and (d) heat map showing trends for the three inhibitors and the selectivity for PNPLA4 inhibition.
DPBS (25 °C) and DMEM (25 and 37 °C) buffers over the
time frames used in our assays (1−4 h, longer times not
examined). Finally, and although IA-rhodamine and Alexa
Fluor labeling did not detect evidence of competitive cysteine
protease/hydrolase or lysine-containing enzyme inhibition by
57, it and members of this class have not been examined for
activity against or reactivity toward other cellular targets.
toxicity derived from a released leaving group. In this regard,
the inhibitors more closely parallel irreversible cysteine protease
inhibition derived from halide displacement, conjugate
addition, addition to epoxides or aziridines, or nucleophilic
cleavage of weak bonds.3 Even more remarkable and within the
limited series of candidate inhibitors examined, an exquisitely
selective inhibitor was discovered for an uncharacterized
hydrolase, PNPLA4. This enzyme bears a Ser-Asp catalytic
dyad and is likely involved in adipocyte triglyceride homeo-
stasis.51−53 Also known as protein GS2,49 it is present in a
variety of human tissues, but is absent in mouse.50 PNPLA4 is
known to catalyze the hydrolysis of triglycerides and participate
in retinol ester hydrolytic metabolism.54,55 Further exploration
of this unique PNPLA4-selective inhibition by 57 should allow
the optimization and development of an even more potent and
selective inhibitor suitable for detailed examination of this
underexplored serine hydrolase. Just as significantly, the
benzoxathiazin-3-one 1,1-dioxides represent a new and unex-
plored class of candidate enzyme inhibitors capable of selective
irreversible inhibition of a subclass of serine hydrolases and
amenable to structural modifications that substantially modu-
late intrinsic reactivity and/or active site recognition.
CONCLUSIONS
■
A new class of serine hydrolase inhibitors was discovered,
bearing a previously unexplored chemotype, based on a ring-
expanded saccharin core obtained by introduction of a ring
heteroatom that converted the saccharin amide to a carbamate.
Although the analogous urea core displayed insufficient
reactivity for use as candidate irreversible inhibitors of the
serine hydrolases, the benzoxathiazin-3-one 1,1-dioxides,
containing the carbamate core and a more reactive carbonyl,
were found to be effective and selective irreversible serine
hydrolase inhibitors. The benzoxathiazin-3-one 1,1-dioxides
displayed tunable reactivity capable of C7-substituent electronic
modulation of the inherent reactivity and proved amenable for
active site targeting by variation of the N-substituent.
Additionally and unique among the more traditional carbamate
and urea-based serine hydrolase inhibitors, they do not release a
leaving group upon active site serine acylation.3 This latter
feature avoids unknown or unanticipated pharmacology or
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Full experimental details, characterization data of all
compounds, supplemental figures, and 1H and 13C NMR
spectra of all compounds (PDF)
X-ray crystallographic data for compound 14A (CIF)
X-ray crystallographic data for compound 14B (CIF)
X-ray crystallographic data for compound 54 (CIF)
AUTHOR INFORMATION
Corresponding Author
*boger@scripps.edu
■
ORCID
Benjamin F. Cravatt: 0000-0001-5330-3492
Dale L. Boger: 0000-0002-3966-3317
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(DA015648, D.L.B. and DA033760, B.F.C.) and an NSF
Graduate Research Fellowship (NSF/DGE-1346837, A.F.K.)
and the Skaggs-Oxford Scholarship (A.F.K.). We thank Dr. M.
Gembicky and A. Rheingold of the Crystallography Facility at
the University of California, San Diego for the X-ray structure
determinations of 14A, 14B, and 54 and Minerva Tran at the
TSRI Center for Metabolomics and Mass Spectrometry for
conducting the MS experiment and providing the data
displayed in Figure 8.
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ASSOCIATED CONTENT
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S
* Supporting Information
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