Academic cross-fertilization by public screening yields a
remarkable class of protein phosphatase methylesterase1 inhibitors
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Bachovchin, D. A. et al. “Organic Synthesis Toward SmallMolecule Probes and Drugs Special Feature: Academic crossfertilization by public screening yields a remarkable class of
protein phosphatase methylesterase-1 inhibitors.” Proceedings of
the National Academy of Sciences 108 (2011): 6811-6816. Web.
9 Nov. 2011. © 2011 National Academy of Sciences
As Published
http://dx.doi.org/10.1073/pnas.1015248108
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SPECIAL FEATURE
Academic cross-fertilization by public screening yields
a remarkable class of protein phosphatase
methylesterase-1 inhibitors
Daniel A. Bachovchina, Justin T. Mohrb, Anna E. Speersa, Chu Wanga, Jacob M. Berlinb, Timothy P. Spicerc,
Virneliz Fernandez-Vegac, Peter Chasec, Peter S. Hodderc, Stephan C. Schürerc, Daniel K. Nomuraa,
Hugh Rosena,d, Gregory C. Fub,1, and Benjamin F. Cravatta,1
a
The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, CA 92037; bDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; cLead Identification Division, Molecular
Screening Center, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458; and dThe Scripps Research Institute Molecular Screening Center,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
P
rotein phosphorylation is a pervasive and dynamic posttranslational protein modification in eukaryotic cells. In mammals, more than 500 protein kinases catalyze the phosphorylation of serine, threonine, and tyrosine residues on proteins (1).
A much more limited number of phosphatases are responsible for
reversing these phosphorylation events (2). For instance, protein
phosphatase 2A (PP2A) and PP1 are thought to be responsible
together for >90% of the total serine/threonine phosphatase
activity in mammalian cells (3). Specificity is imparted on PP2A
activity by multiple mechanisms, including dynamic interactions
between the catalytic subunit (C) and different protein-binding
partners (B subunits), as well as a variety of posttranslational
chemical modifications (2, 4). Within the latter category is an
unusual methylesterification event found at the C terminus of
the catalytic subunit of PP2A that is introduced and removed
by a specific methyltransferase (leucine carbxoylmethyltransferase-1 or LCMT1) (5, 6) and methylesterase (protein phosphatase
methylesterase-1 or PME-1) (7), respectively (Fig. 1A). PP2A
carboxymethylation (hereafter referred to as “methylation”) has
been proposed to regulate PP2A activity, at least in part, by modwww.pnas.org/cgi/doi/10.1073/pnas.1015248108
ulating the binding interaction of the C subunit with various
regulatory B subunits (8–10). A predicted outcome of these
shifts in subunit association is the targeting of PP2A to different
protein substrates in cells. PME-1 has also been hypothesized
to stabilize inactive forms of nuclear PP2A (11), and recent structural studies have shed light on the physical interactions between
PME-1 and the PP2A holoenzyme (12).
Notwithstanding the aforementioned models and findings, the
actual functional consequences of perturbing PP2A methylation
remain largely unexplored. In yeast, LCMT1 deletion caused
severe growth defects under stress conditions, while PME-1
deletion did not result in an observable cellular phenotype (9).
Disruption of the PME-1 gene in mice, on other hand, caused
early postnatal lethality (13), which has limited the experimental
opportunities to explore methylation of PP2A in animals. Recent
studies have found that RNA-interference knockdown of PME-1
in cancer cells leads to activation of PP2A and corresponding
suppression of protumorigenic phosphorylation cascades (14),
indicating that PME-1 could be an attractive drug target in oncology. Changes in PP2A methylation have also been implicated
in Alzheimer’s disease, where this modification may stimulate
PP2A’s ability to promote neural differentiation (15).
Despite the critical role that PME-1 plays in regulating PP2A
structure and function, PME-1 inhibitors have not yet been
described. This deficiency may be due to a lack of PME-1 activity
assays that are compatible with high-throughput screening
(HTS). Assessment of PME-1 activity typically involves either
Western blotting with antibodies that recognize specific methylation states of PP2A (7, 13) or monitoring the release of 3 Hmethanol from radiolabeled-C subunits (16), but neither assay
is easily adapted for HTS. PME-1 is, however, a serine hydrolase
and therefore susceptible to labeling by active-site-directed fluorophosphonate (FP) probes (17). We have recently shown that FP
probes can form the basis for a fluorescence polarization-activitybased protein profiling (fluopol-ABPP) assay suitable for HTS
(18). Here, we apply fluopol-ABPP to screen the 300,000+ National Institutes of Health (NIH) compound library for PME-1
inhibitors. From this screen, we identified a set of aza-β-lactam
(ABL) compounds that act as remarkably potent and selective
Author contributions: D.A.B., J.T.M., H.R., G.C.F., and B.F.C. designed research; D.A.B.,
J.T.M., J.M.B., T.P.S., V.F.-V., P.C., P.S.H., S.C.S., and D.K.N. performed research; D.A.B.,
J.T.M., C.W., J.M.B., and S.C.S. contributed new reagents/analytic tools; D.A.B., J.T.M.,
A.E.S., C.W., T.P.S., P.S.H., S.C.S., H.R., G.C.F., and B.F.C. analyzed data; and D.A.B., J.T.M.,
A.E.S., H.R., G.C.F., and B.F.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence may be addressed. E-mail: cravatt@scripps.edu or gcf@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1015248108/-/DCSupplemental.
PNAS ∣ April 26, 2011 ∣ vol. 108 ∣ no. 17 ∣ 6811–6816
BIOCHEMISTRY
National Institutes of Health (NIH)-sponsored screening centers
provide academic researchers with a special opportunity to pursue
small-molecule probes for protein targets that are outside the
current interest of, or beyond the standard technologies employed
by, the pharmaceutical industry. Here, we describe the outcome
of an inhibitor screen for one such target, the enzyme protein
phosphatase methylesterase-1 (PME-1), which regulates the
methylesterification state of protein phosphatase 2A (PP2A) and is
implicated in cancer and neurodegeneration. Inhibitors of PME-1
have not yet been described, which we attribute, at least in part,
to a dearth of substrate assays compatible with high-throughput
screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use
this platform to screen the 300,000+ member NIH small-molecule
library. This screen identified an unusual class of compounds,
the aza-β-lactams (ABLs), as potent (IC50 values of approximately
10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not
derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that
ABLs are exquisitely selective for PME-1 in living cells and mice,
where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL
inhibitors that can be used to selectively perturb PME-1 activity
in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create
spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class
pharmacological probes for challenging protein targets.
CHEMISTRY
Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved January 13, 2011 (received for review November 24, 2010)
PME-1 inhibitors. We show that these ABLs covalently inactivate
PME-1 with high specificity in living cells and animals, where disruption of this enzyme leads to substantial decreases in demethylated PP2A.
Results
PME-1 Inhibitor Screening by Fluopol-ABPP. Because PME-1 is a
serine hydrolase that is known to interact with reporter-tagged
FP probes (17, 19), we reasoned that this enzyme would be assayable by competitive ABPP methods. However, lower-throughput,
gel-based competitive ABPP screens have not succeeded in identifying lead PME-1 inhibitors (20), indicating the need to survey
larger compound libraries. We therefore asked whether PME-1
could be assayed using the recently introduced, HTS-compatible
fluopol-ABPP platform (18). This technique, where compounds
are tested for their ability to block the increase in fluopol signal
generated by reaction of a fluorescent activity-based probe with a
much larger protein target, has enabled inhibitor screening for a
wide range of probe-reactive enzymes (http://pubchem.ncbi.nlm
.nih.gov/). We confirmed that purified, recombinant wild-type
PME-1, but not a mutant PME-1 in which the serine nucleophile
was replaced with alanine (S156A), labels with a fluorophosphonate rhodamine (FP-Rh) (21) probe (Fig. 1B). This reaction
generates a strong, time-dependent increase in fluopol signal
that is not observed in the absence of enzyme or with the S156A
mutant PME-1 enzyme (Fig. 1C). In collaboration with the Molecular Libraries Probe Production Centers Network (MLPCN),
we screened 315,002 compounds for PME-1 inhibition using fluopol-ABPP (see Fig. 1D for a representative subset of the primary
screening data). Following a confirmation screen on initial hits,
we identified 1,068 compounds as potential PME-1 inhibitors.
As an initial filter, we selected compounds for follow-up studies
that had <5% hit rates in all other bioassays reported in the PubChem database, <30% inhibition in three fluopol-ABPP screens
performed on other enzymes (http://pubchem.ncbi.nlm.nih.gov/),
and >40% inhibition of PME-1 in the confirmation screen. This
filter yielded approximately 300 candidate PME-1 inhibitors.
Discovery of aza-β-lactam (ABL) Inhibitors of PME-1. The approximately 300 hit compounds were next analyzed by gel-based
competitive ABPP (18, 22) in soluble lysates from HEK 293T
cells overexpressing PME-1. This convenient selectivity screen
assessed in parallel the activity of lead compounds against approximately 25 gel-resolvable, FP-Rh-reactive serine hydrolases
expressed in HEK 293Tcells and rapidly eliminated false-positive
and nonselective compounds. Among the compounds that selectively inhibited PME-1 (Fig. S1) were four ABLs (ABL127,
ABL103, ABL105, ABL107) that were similar in structure, all
with a branched alkyl group at R1 and with R stereochemistry
at this position (Fig. 2A). The MLPCN library contained 22 other
ABLs, including the enantiomers of ABL127 (ent-ABL127),
ABL103 (ent-ABL103), and ABL105 (ent-ABL105), that were
all considerably less active toward PME-1 in the primary screen
(Table S1). Intriguingly, the ABLs did not originate from a commercial compound collection but rather were submitted by
the academic chemistry laboratory of our coauthor Gregory Fu,
who generated these compounds as part of an investigation
into the synthetic utility of chiral 4-pyrrolidinopyridine catalysts
(23). We further noted that the ABLs are structurally unusual
compared to the rest of the MLPCN library, lying a considerable
distance in a chemical space plot from the typical structures that
populate the compound collection (Fig. 2B).
The ABL hits were next titrated into the soluble proteome of
MDA-MB-231 cells, a cell line that endogenously expresses
PME-1, to assess their potency (Fig. 2C). The two compounds
bearing cycloalkyl substituents at R1 , ABL127 and ABL103, were
extraordinarily potent inhibitors of PME-1, with IC50 values
of 4.2 and 2.1 nM, respectively (Fig. 2C and Fig. S2). The two
compounds with isopropyl substituents, ABL105 and ABL107,
exhibited lower IC50 values (92 and 24 nM, respectively) but were
still good inhibitors of PME-1. Interestingly, a strong preference
for the R enantiomer of ABLs was observed, as the S enantiomer
of ABL127 (ent-ABL127) was at least two orders of magnitude
less potent at inhibiting PME-1 (Fig. 2 C and D). Indeed, some of
the apparent activity of the S enantiomer may be due to the small
amount of residual R enantiomer in the >99% ee sample.
Before proceeding further, we wanted to confirm that ABLs
could inhibit the ability of PME-1 to demethylate PP2A. We
therefore treated HEK 293T soluble lysates with ABL127
(500 nM, 30 min) or DMSO before adding purified recombinant
PME-1 for an additional hour. In DMSO-treated lysates, we
observed the expected time-dependent increase in demethylated
PP2A and concomitant decrease in methylated PP2A, as determined by immunoblotting with antibodies that specifically recognize either form of the C terminus of PP2A (Fig. 2E). In lysates
containing ABL127, however, we observed little or no change
in the methylation state of PP2A (Fig. 2E), indicating that
ABL127 blocks PME-1’s activity on its physiological substrate.
As ABL127 and ABL103 are highly similar structures and
emerged as virtually indistinguishable in these assays, we selected
one of these compounds, ABL127, for in-depth characterization.
ABLs Covalently Inhibit PME-1. Based on scientific precedent showing that other serine hydrolases can react with and open β-lactam
Fig. 1. A fluopol-ABPP assay for PME-1. (A) Schematic representation of the reversible C-terminal methylation of
PP2A introduced by LCMT1 and removed by PME-1. (B)
FP-Rh (75 nM) labels purified wild-type PME-1 (1 μM) but
not the catalytically dead S156A PME-1 mutant (1 μM).
Top panel: Fluorescent gel image shown in gray scale. (C)
Time course for fluopol signal generated by reactions of
wild-type PME-1 (1 μM) with FP-Rh (75 nM). No signal increase is observed in the absence of enzyme or with the
S156A PME-1 mutant. The indicated 45-min time point
(Z 0 ¼ 0.77), prior to reaction completion, was selected for
HTS. Data are presented as mean values SD. (D) Screening
data for a representative 15,000 compounds of the NIH
small-molecule library. Compounds that reduced the FPRh fluopol signal by >26.13% were designated as hits for
PME-1 (red squares).
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Bachovchin et al.
ABL127 Selectively Inhibits PME-1 in Cells. We next investigated
whether ABLs could inhibit PME-1 in living cells. We incubated
MDA-MB-231 and HEK 293T cells with a concentration range
of ABL127 for 1 h, harvested the soluble proteomes, and then
reacted these lysates with the FP-Rh probe (Fig. 4A and Fig. S3).
In both cell lines, we observed highly potent and selective inhibition of PME-1 with IC50 values of 11.1 nM and 6.4 nM, respectively (Fig. 4B and Fig. S3). Although these gel-based competitive
ABPP studies did not reveal any additional serine hydrolase
targets of ABL127 at concentrations under 10 μM, we wanted
to verify this selectivity profile by higher-resolution LC-MS/MS
methods. To accomplish this, we employed an advanced version
of our competitive ABPP-MudPIT technology (20, 25) that
utilizes stable-isotope labeling of amino acids in cell culture
(SILAC) (26). SILAC involves differential labeling of proteins
with stable isotopes to generate isotopically “light” and “heavy”
samples, which, when pooled and analyzed by MS, yield accurate
quantification by comparing intensities of light and heavy peptide
peaks. SILAC has previously been used to identify enzymes
targets of activity-based probes (27) and small-molecule-binding
proteins in cell lysates (28). In our competitive ABPP-SILAC
experiments, cells grown in light and heavy media were treated
with DMSO or ABL127, respectively, for 1 h. Proteomes were
then harvested, combined at a 1∶1 total protein ratio, and treated
with the activity-based probe FP-biotin (29). FP-biotin-labeled
proteins were then enriched with streptavidin-conjugated beads,
digested on-bead with trypsin, and the resulting peptides analyzed
by liquid chromatography-high-resolution tandem MS using an
LTQ-Orbitrap mass spectrometer. Specifically enriched proteins
were identified and quantified based on analysis of MS2 spectra
and MS1 profiles, respectively. This analysis revealed complete
and selective in situ inhibition of PME-1 by ABL127 with no
activity against >50 other serine hydrolases detected in MDAMB-231 and HEK 293T cells (Fig. 4C and Fig. S4).
We next investigated the impact of ABL127 incubation on the
methylation state of PP2A in cells. As expected, in both MDAMB-231 and HEK 293T cells, we observed significant reductions
Fig. 3. ABLs are covalent inhibitors of PME-1. (A) Proposed
mechanism for covalent PME-1 inhibition by ABL127. (B)
PME-1 (500 nM) was incubated with DMSO or ABL127
(5 μM), and each reaction was split into two fractions.
One fraction was directly labeled with FP-Rh (left panels),
and the other was subjected to gel-filtration to remove free
inhibitor and then reacted with FP-Rh (right panels) to determine the reversibility of inhibition. (C) MS1 traces for the
PME-1 active site peptide not adducted (top panel) or adducted (bottom panel) to ABL127. See SI Materials and
Methods for more information on this experiment. Traces
were obtained from tryptic digests of purified, recombinant
PME-1 (10 μM) treated with ABL127 (50 μM, red trace) or
DMSO (black trace).
Bachovchin et al.
PNAS ∣
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CHEMISTRY
rings by breaking the amide bond, resulting in covalent acylation/
inhibition (24), we hypothesized that the active-site serine (S156)
of PME-1 analogously reacted with the ABL ring of ABL127 to
produce the enzyme-inhibitor adduct shown in Fig. 3A. Consistent with a covalent mode of inhibition, we observed that blockade of FP-Rh labeling of PME-1 by ABL127 was not reversed
by gel filtration (Fig. 3B). The identity of the expected acylation
adduct was confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis of purified, recombinant PME-1 treated
with ABL127 (Fig. 3C).
BIOCHEMISTRY
Fig. 2. Discovery of selective ABL inhibitors of PME-1. (A)
Four ABLs (ABL127, ABL103, ABL105, and ABL107) identified as active in the MLPCN screen. (B) Chemistry space coordinates for compounds (colored red in high occupancy
cells; colored blue in low occupancy cells) that were
screened against PME-1. The 26 ABLs (shown as enlarged
squares; all other compounds are shown as small circles)
are located in a sparsely populated region of chemical
space. This optimized 6-dimensional BCUTs (31) visualization of chemistry space for the compound library was generated using the standard 3D hydrogen suppressed
descriptors with Diverse Solutions (Diverse Solutions, Tripos), with coverage illustrated by compression into an optimized two-dimensional BCUTs space as described in more
detail previously (32). (C) Evaluation of compounds by gelbased competitive ABPP with FP-Rh (2 μM) in the soluble
proteome (1 mg∕mL protein) of MDA-MB-231 cells. (D)
Complete PME-1 IC50 curves for ABL127 (4.2 nM; 95% confidence limits 2.3–7.5 nM) and ent-ABL127 (450 nM; 95%
confidence limits 240–850 nM) in the soluble proteome
of MDA-MB-231 cells (for IC50 curves of ABL103, ABL105,
and ABL107, see Fig. S2). Data are presented as mean
values SEM; n ¼ 3∕group. (E) Pretreatment of HEK 293T
proteomes with ABL127 (500 nM, 30 min) before addition
of purified PME-1 (500 nM, 1 h) blocks PP2A demethylation
as determined by Western blotting with antibodies that recognize specific methylation states of PP2A.
in the levels of demethylated PP2A (35% and 80%, respectively;
Fig. 4 D and E). A trend toward increases in methylated PP2A
was also observed in ABL127-treated HEK 293T cells, but this
change did not reach statistical significance (p ¼ 0.12) (Fig. 4 D
and E). No difference in methylated PP2A was observed in MDAMB-231 cells treated with ABL127 (Fig. 4 D and E). These outcomes might be expected if the vast majority of PP2A is constitutively methylated under standard cell culture conditions. To
investigate this hypothesis, we stably overexpressed PME-1 in
HEK 293T cells (Fig. 4F), which resulted in a dramatic increase
in demethylated PP2A and a significant decrease in methylated
PP2A relative to a control cell line stably expressing GFP (Fig. 4
G and H). Importantly, treatment of PME-1-transfected cells
with ABL127 for only 1 h reduced the amount of demethylated
PP2A back to the level observed in GFP-overexpressing control
cells (Fig. 4 G and H). These ABL127-treated cells also showed
a significant increase in methylated PP2A (Fig. 4 G and H).
Time course studies revealed that a single treatment of ABL127
resulted in sustained inactivation of PME-1 and reductions in
demethylated PP2A for at least 24 h (Fig. 4I). These data, taken
together, indicate that ABL127 selectively inactivates PME-1 in
living cells, which in turn causes significant changes in the methylation state of PP2A.
A Clickable ABL Confirms Proteome-Wide Selectivity for PME-1. Our
competitive ABPP results showed that ABL127 is highly selective
for PME-1 among members of the serine hydrolase family, but
they did not address the possibility that ABL127 might react with
other proteins in the proteome. To investigate this possibility, we
synthesized ABL112, an analog of ABL127 that contains alkyne
groups to serve as latent affinity handles amenable to modification by reporter tags using the copper(I)-catalyzed azide–alkyne
cycloaddition reaction (“click chemistry”) (30) (Fig. 5A). First, we
confirmed that ABL112 retains inhibitory activity for PME-1 by
gel-based competitive ABPP in MDA-MB-231 lysates, where it
showed only a threefold reduction in potency (IC50 ¼ 13.8 nM;
Fig. 5B) compared to the parent inhibitor ABL127 (Fig. S5).
Next, we treated MDA-MB-231 cells with ABL127 (100 nM)
Fig. 4. ABL127 selectively inactivates PME-1 and decreases the demethylated form of PP2A in cells. (A) Gel-based competitive ABPP of the soluble proteomes
from MDA-MB-231 cells treated with ABL127 (0.61–10,000 nM; 1 h) reveals selective blockade of PME-1 with (B) an IC50 value of 11.1 nM. (C) Isotopically “light”
and “heavy” MDA-MB-231 cells were treated with DMSO or ABL127 (100 nM), respectively, for 1 h. Proteomes were combined in a 1∶1 total protein ratio
(0.5 mg each), analyzed by ABPP-MudPIT, and serine hydrolase activities were quantified by comparing intensities of light and heavy peptide peaks (see Fig. S4
for additional SILAC ABPP-MudPIT analyses). Data are presented as mean values SEM for all quantifiable peptides from each serine hydrolase. (D and E) MDAMB-231 and HEK 293T cells treated with ABL127 (500 nM, 1 h) exhibit significant reductions in demethylated PP2A. (F) Stable overexpression of PME-1 in HEK
293T cells compared to control cells overexpressing GFP. PME-1 is completely inhibited by ABL127 (500 nM, 1 h) in both cell lines. (G and H) Cells overexpressing
PME-1 show decreased PP2A methylation, which is reversed by addition of ABL127 (500 nM, 1 h). (I) Time-course for PME-1 inhibition by ABL127 (500 nM) in
PME-1-overexpressing HEK 293T cells. For E and I: *p < 0.05, **p < 0.01 for DMSO-treated versus ABL127-treated cells. #p < 0.05, ##p < 0.01 for cells overexpressing GFP versus PME-1. Data are presented as mean values SEM; n ¼ 3∕group.
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Bachovchin et al.
or DMSO for 30 min before adding ABL112 at a range of concentrations and incubating for another 2 h. Cells were then lysed
and the soluble proteomes were subjected to a “click” reaction
with an azide-Rh tag (RhN3 ), separated by SDS/PAGE, and
ABL112-labeled proteins were visualized by in-gel fluorescence
scanning (Fig. 5C). This analysis identified PME-1 as the only
ABL112-labeled protein that could be competed by pretreatment
with ABL127. One additional ABL112-reactive protein at approximately 80 kDa was also detected, but the labeling of this
protein was not competed by ABL127 or ent-ABL127 (Fig. S5),
indicating that it is exclusively a target of the clickable probe
ABL112 but not ABL127. Consistent with this premise, competitive ABPP analysis identified an 80 kDa FP-Rh-labeled protein
in MDA-MB-231 lysates that was sensitive to inhibition by
ABL112 but not ABL127 (Fig. S5). We performed a similar
“click” analysis with ABL112 in the soluble proteome of mouse
brain, which again revealed selective labeling of PME-1 (Fig. 5D).
Discussion
We report herein a class of ABL inhibitors that show remarkable
selectivity for PME-1 and equipotent activity in both cell-free and
living cell assays. The lead ABL, ABL127 (designated as NIH
Probe ML174), is capable of inactivating PME-1 in both human
cancer cells and mice, suggesting that it should serve as a versatile
pharmacological probe for evaluating PME-1 function in a multitude of living systems. We found that PME-1 inhibition causes a
significant reduction in demethylated PP2A and, in cells with high
levels of PME-1 activity, also a concomitant increase in methylated PP2A. Future studies with ABL127 should facilitate a more
detailed understanding of the role that methylation plays in
regulating PP2A function. Will, for instance, alterations in methylation state impact the composition and/or stability of specific
PP2A complexes? Structural studies have confirmed that
Fig. 6. ABL127 selectively inhibits PME-1 in vivo. (A) Evaluation of mouse brain soluble proteome from mice treated with vehicle or ABL127 (50 mg kg−1 , i.p.,
2 h) by gel-based ABPP. In this analysis, proteomes were also treated ex vivo with DMSO or ABL127 (2 μM) before FP-Rh labeling to confirm complete PME-1
inhibition. (B) ABPP-MudPIT analysis of serine hydrolases from brain proteomes of animals treated with vehicle or ABL127. These data confirm selective inactivation of PME-1 in vivo. (C and D) Mice treated with ABL127 exhibited reductions in demethylated PP2A compared with vehicle-treated mice. *p < 0.01,
**p < 0.001 for DMSO versus ABL127 groups. Data are presented as mean values SEM; n ¼ 3∕group.
Bachovchin et al.
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Fig. 5. A clickable ABL reveals proteome-wide selectivity for PME-1. (A)
Structure of the ABL alkyne probe ABL112. (B) ABL112 inactivates PME-1
(IC50 ¼ 13.8 nM; 95% confidence limits 6.5–29 nM) in the MDA-MB-231 soluble proteome (1 mg∕mL protein) as determined by gel-based competitive
ABPP (see Fig. S5). (C) MDA-MB-231 cells were incubated with DMSO or
ABL127 (100 nM, 30 min) followed by ABL112 (10–200 nM, 2 h). Soluble cell
proteomes (0.5 mg∕mL) were then subjected to a standard “click” reaction
using RhN3 (30) and ABL112-labeled proteins were visualized by in-gel fluorescence scanning. PME-1 was the only protein labeled by ABL112 and competed by ABL127. ABL112 labeled an additional 80 kDa protein that was not
competed by ABL127, suggesting it may be a target for ABL112, but not
ABL127 (see Fig. S5). (D) Mouse brain soluble lysates (1 mg∕mL) were incubated (30 min) with DMSO or ABL127 (100 nM). ABL112 (10 nM) was then
added for an additional 30 minutes before analysis by click chemistry-based
ABPP.
ABL127 Inactivates PME-1 in Mice. As mentioned earlier, PME-1
(−∕−) mice are not viable (13), which has hindered experimental
efforts to characterize this enzyme’s function (and the functional
significance of PP2A methylation) in animals. Pharmacological
inhibition of PME-1 would thus offer a potentially powerful
means to study this enzyme in vivo. With this goal in mind, we
asked whether ABL127 could inhibit PME-1 in mice. C57Bl/6
mice were treated with ABL127 (50 mg∕kg, i.p., 2 h) or vehicle,
sacrificed, and their brain proteomes assayed for PME-1 activity
by competitive ABPP. Gel-based profiles indicated that brain
PME-1 was inactivated by ABL127 (Fig. 6A), but overlapping
serine hydrolase activities precluded a confident assessment of
the extent of inactivation. For enhanced resolution of the activity
state of PME-1 and other brain serine hydrolases, we performed
competitive ABPP-MudPIT studies using FP-biotin. These LCMS profiles confirmed complete inactivation of PME-1 (Fig. 6B)
and no substantial reductions in any of the other approximately
40 brain serine hydrolases detected in this experiment. We also
observed an approximately 35% reduction in the amount of demethylated PP2A in brain tissue from mice treated with ABL127
(Fig. 6 C and D). These results confirm that ABL127 can selectively inhibit PME-1 in mice and this inhibition alters the methylation state of brain PP2A.
BIOCHEMISTRY
These results demonstrate that the remarkable selectivity displayed by ABL127 for PME-1 extends not only across the serine
hydrolase family but also the greater mammalian proteome.
PME-1 is a component of this complex, where its interactions
with the catalytic subunit of PP2A appear to be mediated, at least
in part, by the PME-1 active site (12). It is thus possible that inhibition of PME-1 will affect PP2A complexes not only through
altering methylation but also through disrupting physical interactions between PME-1 and the catalytic subunit of PP2A. Determining how such changes in PP2A complexes affect substrate
interactions and the broader phosphoproteome should represent
an exciting area of research. On this note, recent studies point
to an important role for PME-1 in negatively regulating the tumor-suppressive function of PP2A in cancer cells (14). PME-1
inhibitors may thus have utility as anticancer agents. Finally, we
speculate that the net effect of PME-1 inhibition on PP2A methylation state will be dictated by the expression levels of not only
PME-1 (see Fig. 3G) but also LCMT1. Imbalances in the relative
expression of these two enzymes may thus mark specific cellular
states where PME-1 inhibitors will produce their most dramatic
pharmacological effects.
There were several keys to the success of our probe development effort. First, screening for inhibitors of PME-1 benefited
from the fluopol-ABPP technology, which circumvented the
limited throughput of previously described substrate assays for
this enzyme. Second, we were fortunate that the NIH compound
library contained several members of the ABL class of small molecules. These chiral compounds, which represent an academic
contribution to the NIH library, occupy an unusual portion of
structural space that is poorly accessed by commercial compound
collections. Although at the time of their original synthesis (23)
it may not have been possible to predict whether these ABLs
would show specific biological activity, their incorporation into
the NIH library provided a forum for screening against many
proteins and cellular targets, culminating in their identification
as PME-1 inhibitors. We then used advanced chemoproteomic
assays to confirm the remarkable selectivity displayed by ABLs
for PME-1 across (and beyond) the serine hydrolase superfamily.
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6816 ∣
www.pnas.org/cgi/doi/10.1073/pnas.1015248108
That the mechanism for PME-1 inhibition involves acylation
of the enzyme’s conserved serine nucleophile (Fig. 3) suggests
that exploration of a more structurally diverse set of ABLs might
uncover inhibitors for other serine hydrolases. In this way, the
chemical information gained from a single high-throughput
screen may be leveraged to initiate probe development programs
for additional enzyme targets.
Projecting forward, this research provides an example of how
public small-molecule screening centers can serve as a portal for
spawning academic collaborations between chemical biology and
synthetic chemistry labs. By continuing to develop versatile highthroughput screens and combining them with a small-molecule
library of expanding structural diversity conferred by advanced
synthetic methodologies, academic biologists and chemists are
well-positioned to collaboratively deliver pharmacological probes
for a wide range of proteins and pathways in cell biology.
Materials and Methods
PME-1 Protein Expression and Purification. Human recombinant PME-1 was
expressed in BL21(DE3) Escherichia coli and purified at approximately
5 mg∕L as detailed in SI Materials and Methods.
PME-1 Fluopol-ABPP Assay. See SI Materials and Methods for details.
Competitive ABPP Assays in Proteomes. See SI Materials and Methods for
details.
Synthesis of ABLs. See SI Materials and Methods for details.
ACKNOWLEDGMENTS. We thank Tianyang Ji, David Milliken, Kim Masuda,
Benjamin Kipper, and Jaclyn M. Murphy for technical assistance. This work
was supported by National Institutes of Health grants CA132630 (B.F.C.),
MH084512 (H.R.), GM57034 (G.C.F.), and GM086040 (postdoctoral fellowship
to J.T.M.); the National Science Foundation (predoctoral fellowship to
D.A.B.); the California Breast Cancer Research Program (predoctoral fellowship to D.A.B.); and The Skaggs Institute for Chemical Biology.
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