The Development of Benzimidazole-Based Clickable Probes for the Efficient Labeling of Cellular Protein Arginine Deiminases (PADs)

Article abstract of DOI:10.1021/acschembio.7b00957

Citrullination is the post-translational hydrolysis of peptidyl-arginines to form peptidyl-citrulline, a reaction that is catalyzed by the protein arginine deiminases (PADs), a family of calcium-regulated enzymes. Aberrantly increased protein citrullinati

Full text of DOI:10.1021/acschembio.7b00957

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Article
The Development of Benzimidazole-Based Clickable Probes for the
Efficient Labeling of Cellular Protein Arginine Deiminases (PADs)
Venkatesh J. Nemmara, Venkataraman Subramanian, Aaron Muth, Santanu Mondal, Ari J
Salinger, Aaron J Maurais, Ronak Tilvawala, Eranthie Weerapana, and Paul R. Thompson
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00957 • Publication Date (Web): 17 Jan 2018
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The Development of Benzimidazole-Based Clickable Probes for the Efficient Labeling of
Cellular Protein Arginine Deiminases (PADs)
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Venkatesh V. Nemmara1,2, Venkataraman Subramanian1,2, Aaron Muth1,2, Santanu Mondal1,2,
Ari J. Salinger1,2, Aaron J. Maurais3, Ronak Tilvawala1,2, Eranthie Weerapana3, and Paul R.
Thompson1,2*
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1Department of Biochemistry and Molecular Pharmacology, UMass Medical School, 364
Plantation Street, Worcester, MA 01605, USA
2Program in Chemical Biology, UMass Medical School, 364 Plantation Street, Worcester, MA,
01605, USA.
3Department of Chemistry, Boston College, Chestnut Hill, MA 02467
Running Title: PAD targeted ABPPs.
*Author to whom correspondence should be addressed: Department of Biochemistry and
Molecular Pharmacology, University of Massachusetts Medical School, LRB 826, 364
Plantation Street,
Worcester MA 01605 tel: 508ꢀ856ꢀ8492; fax: 508ꢀ856ꢀ6215; eꢀmail:
paul.thompson@umassmed.edu.
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ABSTRACT
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Citrullination is the postꢀtranslational hydrolysis of peptidylꢀarginines to form peptidylꢀcitrulline,
a reaction that is catalyzed by the protein arginine deiminases (PADs), a family of calciumꢀ
regulated enzymes. Aberrantly increased protein citrullination is associated with a slew of
autoimmune diseases (e.g., rheumatoid arthritis (RA), multiple sclerosis, lupus, and ulcerative
colitis) and certain cancers. Given the clear link between increased PAD activity and human
disease, the PADs are therapeutically relevant targets. Herein, we report the development of
next generation cell permeable and “Clickable” probes (BBꢀClꢀYne and BBꢀFꢀYne) for covalent
labeling of the PADs both in vitro and in cellꢀbased systems. Using advanced chemoproteomic
technologies, we also report the off targets of both BBꢀClꢀYne and BBꢀFꢀYne. The probes are
highly specific for the PADs, with relatively few off targets, especially BBꢀFꢀYne, suggesting
the preferential use of the fluoroacetamidine warhead in next generation irreversible PAD
inhibitors. Notably, these compounds can be used in a variety of modalities, including the
identification of offꢀtargets of the parent compounds and as ABPPs in target engagement assays
to demonstrate the efficacy of PAD inhibitors.
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INTRODUCTION
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Aberrantly upregulated protein citrullination is associated with a slew of autoimmune
diseases (e.g., rheumatoid arthritis (RA), multiple sclerosis, lupus, and ulcerative colitis), as well
as certain cancers.1, 2 Given these disease links, the protein arginine deiminases (PADs), the
enzymes that catalyze this reaction, have garnered significant recent interest. The most deeply
investigated disease associated with aberrantly increased PAD activity is RA, where these
patients produce autoantibodies targeting numerous citrullinated proteins (e.g., citrullinated
keratin, fibrin, vimentin and enolase).3ꢀ7 Importantly, the presence of these autoantibodies is the
most specific diagnostic test available for RA. Moreover, these autoantibodies are present in
patients’ sera 4ꢀ5 years before clinical onset, and higher titers are associated with a more severe
clinical outcome.7ꢀ9 Thus, the presence of these antiꢀcitrullinated protein antibodies (i.e., ACPA)
is highly predictive of both disease incidence and severity. In addition to ACPAs, PAD2 and
PAD4 are released by immune cells into the synovial joints of patients with RA where they
remain active and citrullinate proteins. Within the joint, these citrullinated proteins then bind
ACPAs,7, 10, 11 thereby setting up a classic positive feedback loop that recruits additional immune
cells into the joint, the release of additional PAD isozymes into the synovium and enhanced
protein citrullination and consequent inflammation.7
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Although the specific cells that release PAD isozymes into the joints of RA patients is
still debated, one likely source is neutrophils. Neutrophils are the predominant white blood cell
in humans and are generally the first responders to signs of infection/inflammation. Depending
on the environmental cues, a subset of these cells will undergo a novel form of cell death known
as Neutrophil Extracellular Trap formation (NET) or NETosis.12ꢀ14 During this process, the
chromatin decondenses and histones and other proteins are hypercitrullinated ultimately resulting
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in the ejection of chromatin fibers from the cell to form a web like structure that can trap
pathogens (e.g. bacteria, fungi, viruses) as well as promote the formation of blood clots.12ꢀ14
Notably, neutrophils have long been known to be important players in the development and
progression of RA as they are a predominant cell type in the synovial fluid of RA patients.15, 16
Enhanced NETosis, as is observed in RA,12 also results in the exposure of citrullinated
autoantigens, which is key to the progression of RA, and is additionally thought to be the source
of extracellular PADs.4, 12 How the PADs contribute to other inflammatory diseases is less well
defined, but characteristic features include enhanced citrullination in the inflamed regions,
suggesting that aberrant NETosis may contribute to these diseases as well. In addition, since the
PADs are histone modifying enzymes that contribute to the epigenetic control of gene
expression, there is emerging evidence to suggest that enhanced PAD activity promotes an
inflamed state by altering the expression and/or activity of key cytokines and chemokines.17, 18
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The role that the PADs play in these diseases is further highlighted by the efficacy of
several PAD inhibitors in a variety of preꢀclinical disease models. Specifically, the firstꢀ
generation irreversible inhibitor Clꢀamidine (1, Figure 1) has demonstrated efficacy in animal
models of rheumatoid arthritis, lupus, ulcerative colitis, breast cancer, and atherosclerosis.12, 19ꢀ27
The therapeutic importance of the PADs was further highlighted by the second generation
inhibitor, BBꢀClꢀamidine (3), which has shown enhanced efficacy over Clꢀamidine (1) in animal
models of lupus and RA.17, 28, 29 Moreover, the allosteric inhibitor GSK199 also shows efficacy
in an RA model.30 Together, these findings have validated the PADs as viable therapeutic
targets for a wide range of inflammatory conditions.
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The PAD reaction involves the hydrolysis of the guanidinium group of arginine to
generate citrullinated proteins.1, 31, 32 This reaction, variably termed citrullination or deimination,
is a calciumꢀdependent process wherein calcium binding triggers a conformational change that
moves a nucleophilic cysteine residue into the active site, resulting in a >10,000ꢀfold increase in
PAD activity.33, 34 There are five PAD isozymes (PADs 1ꢀ4 and 6)1, 31, 32 with unique cellular
and tissue distribution patterns, however, only PADs 1ꢀ4 are catalytically active. It is also
important to note that the PADs are uniquely distributed both within the cell and throughout the
body. Specifically, all the PADs can be found in the cytoplasm while only PADs 1, 2, and 4 are
also localized in the nucleus.35ꢀ37 When it comes to distribution throughout the body, PAD2 is
expressed in most tissues and cell types, PAD1 and PAD3 are principally expressed in the skin
and hair follicles, PAD6 in oocytes, and PAD4 primarily in neutrophils and other myeloid
derived cells.31 While PAD2 and PAD4 play a significant role in the development of RA,3, 38
PAD2 is also overexpressed in luminal breast cancer cells and its expression correlates with the
level of the HER2 protooncogene.27, 36, 39
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Clꢀamidine (1) and the related PAD inhibitor Fꢀamidine (2, Figure 1) irreversibly inhibit
the PADs by covalently modifying an active site cysteine (Cys645 and 647 in PAD4 and PAD2,
respectively). Taking advantage of the conserved nature of this catalytic cysteine, we previously
reported the development of activityꢀbased protein profiling (ABPP) probes targeting the PADs.
These probes include Rhodamineꢀconjugated Fꢀamidine (RFA)40 which labels all 4 active PAD
isozymes (PADs 1ꢀ4) and was used to identify novel inhibitors of both PAD2 and PAD4.41, 42
“Clickable” versions of this probe were also developed and demonstrated their ability to label
and isolate the PADs under a variety of conditions.43
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Despite these early advances, the peptidic nature of these firstꢀgeneration inhibitors and
ABPPs possessed a number of limitations including their susceptibility to proteolysis and poor
membrane permeability.28 This prompted the development of secondꢀgeneration inhibitors
predicated on improving metabolic stability and membrane permeability by substituting the Cꢀ
terminal carboxamide with a benzimidazole moiety.28 This progression led to the discovery of
BBꢀClꢀamidine (3) which has shown enhanced efficacy in a number of cellꢀbased assays as well
as animal models.17, 28, 29, 44 Specifically, BBꢀClꢀamidine (3) demonstrated an ability to block
NETosis while improving efficacy in animal models of lupus and RA.17, 28, 29 In addition to
further validating the PADs as important therapeutic targets, this scaffold also provided a novel
platform for the development of next generation of ABPPs.
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Herein we report the development of BBꢀClꢀYne (5) and BBꢀFꢀYne (6) for labeling the
PADs both in vitro and in cellꢀbased systems (Figure 1). These probes covalently modify the
catalytic cysteine residue conserved in all PAD isozymes and present a handle for a subsequent
copper catalyzed azideꢀalkyne cycloaddition reaction (CuAAC)45 with either TAMRAꢀN3 or
BiotinꢀN3. The probes described herein are highly specific with relatively few off targets. Using
advanced chemoproteomics technology we also report for the first time the off targets of both
BBꢀClꢀYne and BBꢀFꢀYne. Notably, these compounds can be used in a variety of modalities,
including the identification of offꢀtargets of the parent compounds and as ABPPs in target
engagement assays to demonstrate the efficacy of PAD inhibitors. Moreover, we expect that
these probes will aid in furthering our understanding of PAD biology by detecting PADs in
complex biological systems as well as ultimately identifying novel proteins and signaling
pathways that interact with and/or regulate PAD activity.
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RESULTS AND DISCUSSION
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Probe Design. Three key features that we considered during the design of our next generation
PADꢀtargeted ABPPs were: 1) cell permeability; 2) metabolic stability; and 3) alkyne position
for optimal CuAAC. The first two features were implemented by utilizing the next generation
PAD inhibitors, BBꢀClꢀamidine (3) and BBꢀFꢀamidine (4) as the core scaffold; this scaffold
shows improved cell permeability and metabolic stability as compared to the firstꢀgeneration
counterparts, Clꢀamidine (1) and Fꢀamidine (2) (Figure 1A). Considering the structure of BBꢀClꢀ
amidine (3) and BBꢀFꢀamidine (4), we hypothesized that incorporation of an alkyne on either the
benzimidazole moiety or the biphenyl group would optimally facilitate the copper catalyzed
azideꢀalkyne cycloaddition reaction (CuAAC) between the probe and either TAMRAꢀN3 or
BiotinꢀN3.
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Key to developing these “Clickable” probes was the determination of the coꢀcrystal
structure of BBꢀFꢀamidine (4) bound to PAD4 (Figure 1B).46 Close examination of this coꢀ
crystal structure (Figure 1B) shows that the benzimidazole moiety binds deep within the active
site. By contrast, the biphenyl group orients towards the solvent. Figure 1C illustrates the
potential binding of this benzimidazoleꢀbased scaffold to active PAD2 through an overlay of the
PAD4ꢀBBꢀFꢀamidine (4) coꢀcrystal structure with holoPAD2 (PDB: 4N2C).47 This overlay
indicates that BBꢀFꢀamidine (4) binds to PAD2 in a similar fashion to PAD4 where the biphenyl
group is solvent exposed and the benzimidazole moiety is buried within the substrate binding
pocket. These data suggested that placement of the alkyne at the 4ꢀposition of the biphenyl
moiety would allow for optimal CuAAC after covalent modification of the catalytic cysteine
conserved in all active PAD isozymes; PAD6 is not catalytically active.48
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Probe Synthesis. The synthesis of these clickable probes (Scheme 1) commenced with the
generation of key intermediate 16 through a previously published method.28, 49 Briefly, Fmocꢀ
Orn(Boc)ꢀOH underwent HBTUꢀmediated amide coupling with phenylenediamine, followed by
acidꢀcatalyzed cyclization to form the benzimidazole moiety. Subsequent treatment with
piperidine resulted in decarboxylation of the Fmoc protecting group giving 16. This intermediate
was then subjected to HBTUꢀmediated amide coupling with biphenyl carboxylic acid 13 to give
Bocꢀprotected intermediate 17. Subsequent acid catalyzed decarboxylation of the side chain Boc
group followed by installation of either the chloroacetamidine or fluoroacetamidine or warhead
gave desired probes 5 (BBꢀClꢀYne) and 6 (BBꢀFꢀYne) in good yield.
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Probe Validation – Potency and in vitro labeling. To initially establish the utility of these
“clickable” probes, we first determined kinact/KI values to assess their ability to inhibit PADs 1, 2,
3, and 4 (Table1, Figures S1ꢀS2, and Table S1). Importantly, the kinact/KI values for BBꢀClꢀYne
(5) and BBꢀFꢀYne (6) are within 3ꢀfold of the parent compounds BBꢀClꢀamidine and BBꢀFꢀ
amidine indicating that the additional alkyne moiety does not significantly impact potency,
consistent with our modeling (Figure 1BꢀC). Next, we evaluated the ability of BBꢀFꢀYne and
BBꢀClꢀYne to covalently modify recombinant PADs and undergo a subsequent click reaction
with TAMRAꢀN3. Figures 1DꢀE and Figures S3ꢀS4 illustrate the ability of these probes to
effectively label PADs 1ꢀ4 in a doseꢀdependent manner down to probe concentrations as low as
250 nM. Notably, the sensitivity of the probes is as low as 375 fmol with BBꢀFꢀYne (Figure 1F)
and 756 fmol with BBꢀClꢀYne (Figure 1G). Despite the two probes exhibiting similar potencies
against PAD2, the slightly lower limit of detection may be attributable to the higher selectivity of
BBꢀFꢀYne compared to BBꢀClꢀYne (see below).
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Probe validation – Labeling in cells. Having demonstrated their ability to label recombinant
PADs, we next evaluated their ability to label the PADs in cellꢀbased systems. Specifically,
PAD2ꢀoverexpressing HEK293T cell lines were treated with BBꢀFꢀYne and BBꢀClꢀYne in the
presence of a calcium ionophore (i.e., ionomycin) (Figure 2A). Cells were then harvested, lysed
by sonication, and then “Clicked” with TAMRAꢀN3. Notably, BBꢀFꢀYne labeled a single
prominent band at the correct molecular weight for PAD2 (Figure 2B), even when used at
concentrations as high as 25 µM. This level of selectivity is remarkable. In agreement with our
in vitro results, BBꢀClꢀYne did not label PAD2 as efficiently as BBꢀFꢀYne (Figure 2 C), again
indicating the superior selectivity of BBꢀFꢀYne. The diminished labeling ability of BBꢀClꢀYne
may be due to the increased reactivity of the chloroꢀwarhead, compared to the fluoroꢀwarhead,41,
43 which increases the number of off targets such that a smaller amount of the probe is available
to label PAD2.
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To demonstrate their utility in assessing target engagement, BBꢀFꢀYne and BBꢀClꢀYne
were subjected to competition experiments. Briefly, PAD2ꢀovexpressing HEK293T cell lysates
were incubated with increasing concentrations of either BBꢀFꢀamidine or BBꢀClꢀamidine in the
presence of CaCl2 and were then treated with a fixed concentration BBꢀFꢀYne or BBꢀClꢀYne.
Labeled lysates were “Clicked” with TAMRA azide and probe modified proteins were visualized
by fluorescence. Both BBꢀFꢀamidine and BBꢀClꢀamidine dose dependently decrease the labeling
of PAD2 (Figure S6 and S7) by BBꢀFꢀYne and BBꢀClꢀYne, thereby demonstrating their ability
to assess target engagement.
To further illustrate the versatility of these probes, PAD2ꢀovexpressing HEK293T cell
lysates were treated with either BBꢀFꢀYne or BBꢀClꢀYne and then “Clicked” with BiotinꢀN3
(Figure 2D). These lysates were then incubated with streptavidinꢀagarose beads, washed and
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probed for PAD2 or biotinylated proteins as shown in Figure 2D. Both BBꢀFꢀYne and BBꢀClꢀ
Yne show a similar ability to label and isolate PAD2 (Figure 2E and F). The small amount of
PAD2 isolated in the untreated control may be due to the ability of streptavidinꢀagarose beads to
isolate autocitrullinated PAD2; the urea moiety of citrulline is structurally related to biotin and
we consistently observe streptavidin binding to autocitrullinated PADs (data not shown). Note
that PAD2 is expressed at ~0.8% of total proteins in our PAD2 overexpressing cell line (Figure
S8).
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Chemoproteomics experiments. Next, we employed a chemoproteomic approach to identify
the targets of these two probes. Specifically, HEK293T PAD2 overexpressing cell lysates were
treated with BBꢀFꢀYne (25 µM), BBꢀClꢀYne (2.5 µM), or DMSO for one hour (Figure 3A).
These concentrations were chosen because they correspond to the EC50 values of the parent
compounds in cellular citrullination experiments.46 Labeled proteomes were then “Clicked” with
BiotinꢀN3 in the presence of TBTA and CuSO4. The clicked lysates were then isolated on
streptavidin agarose beads followed by onꢀbead trypsin digestion and subsequent LC/LCꢀ
MS/MS. Protein identities were determined by database searches using the SEQUEST
algorithm. Relative quantitation of probe labeled proteins in probe and DMSO treated samples
was achieved by spectral counting.
Consistent with our TamraꢀN3 labeling data, BBꢀFꢀYne selectively enriched PAD2 with
few off targets (Figure 3B and 3D). Figure 3B depicts the difference in spectral counts (ꢁSC)
between DMSO and BBꢀFꢀyneꢀtreated samples for proteins identified in two biological
replicates. Proteins along the diagonal are consistently enriched in the BBꢀFꢀyne sample and
those above one standard deviation (1 SD) are considered statistically significant. Figure 3D
depicts the average spectral count data plotted against the probe/DMSO spectral count ratio,
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which provides a relative measure of probe selectivity. Those proteins with high spectral counts
and high probe/DMSO ratios are significantly enriched. Both plots demonstrate the remarkable
selectivity of BBꢀFꢀYne.
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By contrast, BBꢀClꢀYne reacted with PAD2 and a modest number of off targets in this in
vitro experiment. These proteins include Heterogeneous Nuclear Ribonucleoprotein U
(HNRPU), Clathrin heavy chain 1 (CLTC), Bifunctional glutamate/prolineꢀtRNA ligase (EPRS),
Tubulin beta chain (TUBB) and Actin beta chain (ACTB). Most of these proteins are highly
abundant, contain reactive cysteines that covalently react with cysteineꢀtargeted electrophiles,
and have been found in other proteomic screens.50 Nevertheless, it is important recognize that
PAD2 is amongst the most prominent proteins labeled by BBꢀClꢀYne (Figure 3C and 3E),
thereby indicating the preferential targeting of this protein by both this probe and likely the
parent compound BBꢀClꢀamidine. The lower fold enrichment relates to background binding of
PAD2 to the streptavidin beads, which, as noted above, relates to the ability of autocitrullinated
PADs to bind weakly to streptavidinꢀagarose.
Because the proteomic experiments were performed with cell lysates, we next wished to
confirm that the identified proteins were labeled in cells (Figure 4A). We focused on HNRPU,
CLTC), EPRS), βꢀtubulin, and βꢀActin because they were prominent off targets in our
chemoproteomic screen. GAPDH and RPL were included as negative controls because they
were slightly elevated but the data were not statistically significant. For these experiments, HEK
cells overexpressing PAD2 were treated with BBꢀClꢀYne (2.5 ꢂM) and BBꢀFꢀYne (25 ꢂM) and
then clicked to biotin azide. These concentrations were chosen because they correspond to the
EC50 values for BBꢀFꢀamidine and BBꢀClꢀamidine in cellular citrullination experiments.46
Proteins were then isolated using streptavidin beads and the bound proteins were detected by
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western blotting after SDSꢀPAGE. In parallel we performed similar experiments in cell lysates.
Consistent with our proteomic and labeling studies (see above), BBꢀFꢀYne selectively isolated
PAD2. Remarkably, BBꢀClꢀYne was also selective and failed to isolate βꢀtubulin, βꢀActin,
clathrin, HSP90, GAPDH, and EPRS. By contrast, we did confirm that BBꢀClꢀYne modifies βꢀ
tubulin, βꢀActin, clathirin, HSP90 and EPRS but not GAPDH and RPL3 when cell lysates are
treated with BBꢀClꢀYne at 2.5 ꢂM (Figure 4B). When cells were treated with a higher
concentration of BBꢀClꢀYne (10 ꢂM), we did detect some labeling of βꢀtubulin and HSP90, but
still failed to label βꢀActin and GAPDH (Figure 4C). Overall, these data indicate that the
selectivity of BBꢀClꢀYne in cells is higher than that in cell lysates. The enhanced selectivity is
likely due to a combination of factors including cell permeability which for low permeable
compounds would adversely impact the cellular concentration of the probe and thereby improve
cellular selectivity. Additionally, it is important to recognize that proteins like actin and tubulin
adopt complex structures in cells, as compared to lysates, and as such their reactive cysteines
may not be susceptible to modification.
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Conclusions.
In summary, we describe the development of two new PAD targeted ABPPs, BBꢀFꢀYne
(6) and BBꢀClꢀYne (5), that show enhanced cellular uptake and efficient labeling of the PADs in
cells. We predict that these compounds will be used in a variety of modalities, including as
ABPPs in target engagement assays to demonstrate the efficacy of PAD inhibitors. Moreover,
we expect that these probes will aid in furthering our understanding of PAD biology by detecting
PADs in complex biological systems as well as ultimately identifying novel proteins and
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signaling pathways that interact with and/or regulate PAD activity. Our chemoproteomic data
also highlights several features of the haloacetamidine warheads present in BBꢀFꢀamidine, BBꢀ
Clꢀamidine, and related compounds. First, the fluoroacetamidine warhead is fairly nonꢀreactive
and in the context of the BBꢀFꢀamidine scaffold facilitate the isolation of only a single protein,
i.e. PAD2, despite being tested at 25 µM. Such selectivity is remarkable and unprecedented for
thiol reactive warheads. Second, BBꢀClꢀamidine reacts with a wider array of proteins and this
feature likely explains the generally higher toxicity of chloroacetamidine containing
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compounds.46 Nevertheless, BBꢀClꢀYne still shows high selectivity when applied to intact cells.
This feature, along with its greater cell permeability, likely explains why BBꢀClꢀamidine has
shown remarkable efficacy in animal models of RA and lupus.17, 28, 29 Projecting forward, we
suggest that a primary focus should be placed on developing fluoroacetamidineꢀbased
irreversible inhibitors of the PADs due to the inherent specificity conferred by this warhead.
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Materials. PADs 1, 2, 3 and 4 were purified as reported.22, 51 HEK293T and HEK293T cells
stably expressing human PAD2 (HEK293T/PAD2) were cultured as previously described.41
BioinꢀTEVꢀN3 was synthesized as previously reported.52 TAMRAꢀN3 was obtained from
Lumiprobe.
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Inactivation Kinetics. Inactivation kinetic parameters were determined by incubating PAD1, 2,
or 4 (2.0 µM) or PAD3 (5.0 µM) in a preꢀwarmed (10 min, 37 °C) inactivation mixture (50 mM
HEPES pH 7.6, 10 mM CaCl2, and 2 mM DTT, with a final volume of 60 µL) containing various
concentrations of inhibitor. Aliquots for were removed at various time points and added to a preꢀ
warmed (10 min, 37 °C) reaction mixture (50 mM HEPES pH 7.6, 50 mM NaCl, 10 mM CaCl2,
2 mM DTT, and 10 mM BAEE or 10 mM BAA in the case of PAD3). The final dilution from
the pre-incubation assay to the activity assay was 10-fold. After 15 min, reactions were
quenched in liquid nitrogen and citrulline production quantified using the COLDER assay.34, 53
Data were plotted as a function of time and fit to eq 1,
v = voeꢀkt
eq 1,
using GraFit version 5.0.11, where v is velocity, vo is initial velocity, k (equal to kobs) is the
pseudoꢀfirst order rate constant of inactivation, and t is time. When saturation was reached upon
plotting kobs versus inactivator concentration, the data were fit to eq 2,
kobs=kinact[I]/(KI+[I])
eq 2,
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using GraFit version 5.0.11, where kinact corresponds to the maximal rate of inactivation and KI is
the concentration of inhibitor that gives halfꢀmaximal inactivation. If the plot of kobs versus [I]
was linear and did not saturate, then the value for kinact/KI equaled the slope of the line.
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In vitro labeling of PADs dose dependently with BB-F-Yne (6) and BB-Cl-Yne (5).
Increasing concentrations of BBꢀFꢀYne (6) or BBꢀClꢀYne (5) (0 to 10 µM) were incubated with
recombinant PADs (1 µM) in the presence of CaCl2 (2 mM) in 1x PBS at 37 ºC for 1 h. The
probe labeled enzymes were coupled to TAMRAꢀN3 (20 µM) in the presence of 1X TBTA (0.31
mM), sodium ascorbate (2 mM) and freshly prepared CuSO4 (1 mM). The tubes were gently
tumbled for 1 h. The reactions were quenched with 6x SDS loading buffer and separated by
SDSꢀPAGE (12.5% gel). The bands were visualized by scanning the gel in a typhoon scanner
(approximate excitation/emission maxima ~546/579, respectively).
In vitro labeling of PAD2 with BB-F-Yne (6) and BB-Cl-Yne (5) to determine their limit of
detection (LOD). BBꢀFꢀYne (6) or BBꢀClꢀYne (5) (10 µM) were incubated with decreasing
concentrations of recombinant PAD2 (1.0 to 0.025 µM) in the presence of CaCl2 in 1x PBS (2
mM) at 37 ºC for 1 h. The probe labeled enzymes were coupled to TAMRAꢀN3 (20 µM) in the
presence of 1X TBTA (0.31 mM), sodium ascorbate (2 mM) and freshly prepared CuSO4 (1
mM). The tubes were gently tumbled for 1 h. The reactions were quenched with 6x SDS
loading buffer and separated by SDSꢀPAGE (12.5% gel). The bands were visualized by
scanning the gel in a typhoon scanner (approximate excitation/emission maxima ~546/579,
respectively).
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Cellular labeling of PAD2 with BB-F-Yne and BB-Cl-Yne in ionomycin stimulated
HEK293T/PAD2 cells. HEK293T cells stably expressing human PAD2 (HEK293T PAD2)
were cultured as previously described.41 Cells were grown to ~80% confluence (8 x 106 cells),
trypsinized, and trypsin activity quenched with complete media. The cells were harvested by
centrifugation at 1000 x g for 2 min and washed 4x with 1x PBS. Cells were resuspended in
PBS at 8 x 106 cells/mL and 4 x 105 cells were added to 0.65 mL tubes for subsequent assays.
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HEK293T PAD2 cells were treated with increasing concentrations of BBꢀFꢀYne or BBꢀ
ClꢀYne (0 to 50 µM) in the presence of CaCl2 (2 mM) at 37 ºC. After 30 min, ionomycin (5 µM)
was added and the cells incubated for 1 h followed by sonication at 4 ºC for 1 h. Lysates were
cleared by centrifugation at 21,000 x g for 15 min. The soluble protein fraction was isolated and
quantified by the Detergent Compatible assay (DC assay, Biorad). Lysates (2 ꢂg/ꢂL, 50 ꢂL
total) were “Clicked” with TAMRAꢀN3 (20 µM), 1X TBTA (0.3 mM), sodium ascorbate (2 mM)
and freshly prepared CuSO4 (1 mM). The tubes were gently tumbled for 2 h. The reactions were
quenched by the addition of 6x SDS loading buffer and separated by SDSꢀPAGE (12.5% gel).
The bands were visualized by scanning the gel in a typhoon scanner (approximate
excitation/emission maxima ~546/579, respectively).
Lysates (2 ꢂg/ꢂL, 500 ꢂL total) were also “Clicked” with Biotin azide (100 µM), 1X
TBTA (0.3 mM), sodium ascorbate (2 mM) and freshly prepared CuSO4 (1 mM). The tubes
were gently tumbled for 2 h. The cloudy solution was transferred to a microconcentrator (10
kDa molecular weight cutoff) and centrifuged at 10,000 x g for 5 min at 4 ºC to remove the
excess biotin azide. The protein was then resolubilized in 1X PBS with 0.2% SDS.
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Resolubilized protein samples were incubated with 100 ꢂL streptavidinꢀagarose beads (Thermo
Scientific) at 4 °C for 16 h. The solutions were then incubated at room temperature for 1 h. The
beads were washed with 0.2% SDS/PBS, PBS, and water. The beads were pelleted by
centrifugation (1,600 x g, 3 min) between washes. To the washed beads, 2X SDS loading buffer
was added and heated in a 95 °C heat block for 15 min. The resolubilized protein was separated
by SDSꢀPAGE (4 – 20 % gel) and transferred to PVDF membranes (Biorad) at 150 V for 60
min. The membranes were analyzed for PAD2 and for other proteins.
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Competition experiments. HEK293T PAD2 cell lysates (100 ꢂg) were treated with increasing
concentrations of BBꢀClꢀamidine or BBꢀFꢀamidine (0 – 200 ꢂM) in the presence of CaCl2 (1
mM) at 37 ºC. After 15 minutes, BBꢀClꢀYne or BBꢀFꢀYne (25 ꢂM) was added and the lysates
were incubated for 1 h. Lysates (100 ꢂg, 50 ꢂL total) were “Clicked” with TAMRAꢀN3 (20
µM), 1X TBTA (0.3 mM), and freshly prepared CuSO4 (1 mM). The tubes were gently tumbled
for 2 h. The reactions were quenched by the addition of 6x SDS loading buffer and separated by
SDSꢀPAGE (4ꢀ20 % gel). The bands were visualized by scanning the gel in a typhoon scanner
(approximate excitation/emission maxima ~546/579, respectively). Western blot analysis of the
lysates was performed to detect the presence of PAD2.
Chemoproteomic analysis of cellular PAD2 labeled with BB-F-yne and BB-Cl-yne. Cell
lysates (2 mg lysate per tube) prepared from HEK293T PAD2 expressing cells were labeled with
BBꢀFꢀyne (25 µM) and BBꢀClꢀyne (1 ꢂM) by incubating at 37 °C for 1h. The probes were
‘Clicked’ to biotin azide (100 µM) in the presence of 1X TBTA (0.3 mM) and CuSO4 (1 mM)
for 2 h at room temperature. Samples were mixed well by vortexing them every 15 min. The
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reaction mixture was then centrifuged and the pellet was washed with methanol and resuspended
in 1.2% SDS in PBS (w/v) followed by sonication and heating (5 min, 80 °C). The SDSꢀ
solubilized samples were diluted with PBS (5 mL) for a final SDS concentration of 0.2%. The
solutions were incubated with 100 ꢂL of streptavidin agarose beads (Thermo Fisher Scientific) at
4 °C for 16 h. The solutions were then incubated at room temperature for 3 h. The beads were
washed with 6M urea in PBS (2 × 5 mL), 0.2% SDS/PBS (2 × 5 mL), PBS (3 × 5 mL), and water
(3 × 5 mL). The beads were pelleted by centrifugation (1,400 x g, 3 min) between washes. The
washed beads were suspended in 6 M urea in PBS (500 ꢂL) and 10 mM dithiothreitol (from 20x
stock in water) and placed in a 65 °C heat block for 15 min. Iodoacetamide (20 mM, from 20x
stock in water) was then added to the samples and allowed to react at 37 °C for 30 min.
Following reduction and alkylation, the beads were pelleted by centrifugation (1,400 x g, 3 min)
and resuspended in a premixed solution of 2 M urea in PBS (200 ꢂL), 100 mM CaCl2 in water (2
ꢂL), and trypsin (4 ꢂL of 20 mg reconstituted in 40 ꢂL of trypsin buffer). The digestion was
allowed to proceed overnight at 37 °C. The digested peptides were separated from the beads by
centrifugation and the beads were washed twice with 50 ꢂL of H2O. Formic acid (15 ꢂL) was
added to the samples, and the samples were stored at −80 °C until MS analysis.
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LC/LC-MS/MS and Data Processing. Mass Spectrometry was performed using a Thermo
Fisher LTQ Orbitrap Discovery mass spectrometer in conjunction with Agilent 1200 series
HPLC. Enriched and digested peptide samples were pressureꢀloaded onto 250 µm fused silica
desalting column packed with 4 cm of Aqua C18 reverse phase resin (Phenomenex). Peptides
were eluted onto a biphasic 100 µm fused silica column with a 5 µm tip. The column was
packed with 3 cm Partisphere strong cation exchange resin (SCX, Whatman) followed by 10 cm
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of C18 resin. Elution took place using a gradient from 5% Buffer A (95% water, 5% acetonitrile,
0.1% formic acid), to 100% Buffer B (20% water, 80% acetonitrile, 0.1% formic acid). The
peptides were detached from the SCX onto the C18 resin and into the mass spectrometer using
distinct salt elutions (95% highꢀpurity water, 5% Optimaꢀgrade acetonitrile, 0.1% formic acid
and 500 mM ammonium acetate)52. The flow rate through the column was 250 nL/min, with a
spray voltage of 2.75 kV. With dynamic exclusion enabled, each full MS1 scan (400ꢀ1800 MW)
was followed by 8 highꢀresolution, dataꢀdependent scans of the nth most intense ion.
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The tandem MS data from the multidimensional LC/LCꢀMS/MS protocol (MudPIT) was
analyzed by the SEQUEST algorithm54 using a static Cys modification (+57 Da). Data was
searched against a human reverseꢀconcatenated nonꢀredundant FASTA database applying
Uniprot identifiers. MS2 spectra was assembled using DTASelect 2.055, with trypstat option
applied.
Pulldown experiments using biotin azide. HEK293T cells stably expressing human PAD2
(HEK293T PAD2) were cultured as previously described.41 Cells were grown to ~80%
confluence (8 x 106 cells), trypsinized, and trypsin activity quenched with complete media. The
cells were harvested by centrifugation at 1000 x g for 2 min and washed 4x with 1x PBS. Cells
were resuspended in PBS at 8 x 106 cells/mL and 4 x 105 cells (0.6 mL) were added to 1.5 mL
tubes and were treated with BBꢀFꢀYne (25 µM) or BBꢀClꢀYne (2.5 µM and 10 µM) in the
presence of CaCl2 (2 mM) at 37 ºC. After 30 min, ionomycin (5 µM) was added and the cells
incubated for 1 h followed by sonication at 4 ºC for 1 h. Lysates were cleared by centrifugation
at 21,000 x g for 15 min. The soluble protein fraction was isolated and quantified by the
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Detergent Compatible assay (DC assay, Biorad). Probe labeled proteins present in lysates (2 mg
per tube) were then coupled to biotin azide using the click chemistry protocol described below.
HEK cell lysates (2 mg lysate per tube) were separately labeled with BBꢀFꢀyne (25 µM) and BBꢀ
Clꢀyne (2.5 µM) for 1 h at 37 °C. Click chemistry was performed on all samples using biotin
azide (100 µM), 1X TBTA (0.3 mM) and CuSO4 (1 mM) and incubating at room temperature for
2 h. Samples were mixed well by vortexing them every 15 min. The reaction mixture was then
centrifuged and the pellet was washed with methanol and reꢀsuspended in 1.2% SDS in PBS
(w/v) sonication and heating (5 min, 80 °C). The SDSꢀsolubilized samples were diluted with
PBS (5 mL) for a final SDS concentration of 0.2%. The solutions were incubated with 100 ꢂL of
streptavidin agarose beads (Thermo Fisher Scientific) at 4 °C for 16 h. The solutions were then
incubated at room temperature for 3 h. The beads were washed with PBS (2 × 5 mL), and water
(5 mL). The beads were pelleted by centrifugation (1,400 x g, 3 min) between washes. The
washed beads were suspended in 8 M urea in PBS with 1.2% SDS (with 1X loading dye) and
heated at 95 °C for 30 min and centrifuged. The supernatant was separated by SDSꢀPAGE (4 –
20 % gel) and transferred to PVDF membranes (Biorad) at 150 V for 60 min. The membranes
were analyzed for the presence of PAD2 and other proteins of interest using the following
antibodies: antiꢀclathrin heavy chain antibody (Abcam, ab21679), antiꢀbeta Tubulin antibody
(Abcam, ab604), antiꢀbeta actin antibody (Abcam, ab8227), antiꢀGAPDH antibody (Abcam
ab9484), antiꢀRPL3 antibody (Abcam, ab154882), antiꢀHsp90 antibody (Abcam, ab13495), antiꢀ
glutamyl prolyl tRNA synthetase antibody (Abcam, ab85447), PADI2 antibody (Protein Tech,
Cat No: 12110ꢀ1ꢀAP).
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ASSOCIATED CONTENT
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Supporting information. Full synthetic procedures, Table S1, and Figures S1ꢀS11. This
information is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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Corresponding Author. *Tel.: (508)ꢀ856ꢀ8492. Fax: 508ꢀ856ꢀ6215. Eꢀmail:
paul.thompson@umassmed.edu.
ORCID ID
Paul R. Thompson: 0000ꢀ0002ꢀ1621ꢀ3372
Eranthie Weerapana: 0000ꢀ0002ꢀ0835ꢀ8301
ACKNOWLEDGEMENTS
This work was supported in part by NIH grant GM109767 (P.R.T.) and GM117004 and
GM118431 (E.W.).
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ABBREVIATIONS
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RA, Rheumatoid arthritis; PAD, Protein arginine deiminase; ACPA, Antiꢀcitrullinated protein
antibodies; NET, Neutrophil extracellular trap; ABPP, Activityꢀbased protein profiling; RFA,
Rhodamineꢀconjugated Fꢀamidine; CuAAC, Copper catalyzed azideꢀalkyne cycloaddition;
TAMRAꢀN3, Tetramethylrhodamine azide; BiotinꢀN3, Biotin azide; Orn, Ornithine; LOD, Limit
of detection.
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REFERENCES
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(1) Jones, J. E., Causey, C. P., Knuckley, B., SlackꢀNoyes, J. L., and Thompson, P. R. (2009)
Protein arginine deiminase 4 (PAD4): Current understanding and future therapeutic
potential, Curr Opin Drug Discov Devel 12, 616ꢀ627.
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(2) Bicker, K. L., and Thompson, P. R. (2013) The protein arginine deiminases: Structure,
function, inhibition, and disease, Biopolymers 99, 155ꢀ163.
(3) Van Steendam, K., Tilleman, K., and Deforce, D. (2011) The relevance of citrullinated
vimentin in the production of antibodies against citrullinated proteins and the
pathogenesis of rheumatoid arthritis, Rheumatology 50, 830ꢀ837.
(4) Puszczewicz, M., and Iwaszkiewicz, C. (2011) Role of antiꢀcitrullinated protein antibodies in
diagnosis and prognosis of rheumatoid arthritis, Arch Med Sci 7, 189ꢀ194.
(5) van Boekel, M. A., Vossenaar, E. R., van den Hoogen, F. H., and van Venrooij, W. J. (2002)
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